Cells

21 pages, 3551 KiB

Open AccessArticle

Proteomic Analysis of miR-195 and miR-497 Replacement Reveals Potential Candidates that Increase Sensitivity to Oxaliplatin in MSI/P53wt Colorectal Cancer Cells

by Dennis Poel, Lenka N.C. Boyd, Robin Beekhof, Tim Schelfhorst, Thang V. Pham, Sander R. Piersma, Jaco C. Knol, Connie R. Jimenez, Henk M.W. Verheul and Tineke E. Buffart

Cells 2019, 8(9), 1111; https://doi.org/10.3390/cells8091111 - 19 Sep 2019

Cited by 25 | Viewed by 4392

Abstract

Most patients with advanced colorectal cancer (CRC) eventually develop resistance to systemic combination therapy. miR-195-5p and miR-497-5p are downregulated in CRC tissues and associated with drug resistance. Sensitization to 5-FU, oxaliplatin, and irinotecan by transfection with miR-195-5p and miR-497-5p mimics was studied using [...] Read more.

Most patients with advanced colorectal cancer (CRC) eventually develop resistance to systemic combination therapy. miR-195-5p and miR-497-5p are downregulated in CRC tissues and associated with drug resistance. Sensitization to 5-FU, oxaliplatin, and irinotecan by transfection with miR-195-5p and miR-497-5p mimics was studied using cell viability and clonogenic assays in cell lines HCT116, RKO, DLD-1, and SW480. In addition, proteomic analysis of transfected cells was implemented to identify potential targets. Significantly altered proteins were subjected to STRING (protein-protein interaction networks) database analysis to study the potential mechanisms of drug resistance. Cell viability analysis of transfected cells revealed increased sensitivity to oxaliplatin in microsatellite instable (MSI)/P53 wild-type HCT116 and RKO cells. HCT116 transfected cells formed significantly fewer colonies when treated with oxaliplatin. In sensitized cells, proteomic analysis showed 158 and 202 proteins with significantly altered expression after transfection with miR-195-5p and miR-497-5p mimics respectively, of which CHUK and LUZP1 proved to be coinciding downregulated proteins. Resistance mechanisms of these proteins may be associated with nuclear factor kappa-B signaling and G1 cell-cycle arrest. In conclusion, miR-195-5p and miR-497-5p replacement enhanced sensitivity to oxaliplatin in treatment naïve MSI/P53 wild-type CRC cells. Proteomic analysis revealed potential miRNA targets associated with the cell-cycle which possibly bare a relation with chemotherapy sensitivity. Full article

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Figure 1
miRNA expression and transfection efficiency in CRC cells. Successful transfection of cel-miR-39-3p in the negative control transfected cells for all four different CRC cell lines (A). y-axis represents raw Cq value of cel-miR-39-3p. Successful transfection of miR-195-5p and miR-497-5p in the four different cell lines HCT116 (B), RKO (C), DLD1 (D) and SW480 (E). y-axis represents log2 expression of miR-195-5p and miR-497-5p relative to miR-16-5p (control). Data is presented as the mean of three independent transfection experiments (measured in duplicate) ± the standard error of the mean. *** p < 0.001, **** p < 0.0001, wt: wild-type, cel: cel-miR-39-3p negative control transfection, mimic: expression level 48 h after transfection. Full article ">Figure 2
HCT116 and RKO cells after transfection with miR-195-5p or miR-497-5p mimic. MTT sensitivity assays for HCT116 cells (A) and RKO cells (D) were performed in triplicate in three independent experiments and are presented as average % proliferation compared to the proliferation of a non-treated control triplicate ± the standard error of the mean (SEM). Presentation of a single clonogenic assay experiment of HCT116 (B) and RKO (E). Bar graphs of the percentage of formed colonies compared to the negative control transfection (cel-miR-39-3p) of HCT116 cells (C) and RKO cells (F) presented as averages of duplicate colony counts from three independent experiments ± SEM. 5-FU; 5-fluorouracil, OHP; oxaliplatin, SN-38; irinotecan, nt; no treatment, ** p < 0.005, *** p < 0.001, **** p < 0.0001. Full article ">Figure 3
miR-195-5p and miR-497-5p target selection and quantification. (A) Venn diagram of the targets selected with miRTarBase, DIANA LAB, and miRDB with the coinciding target mRNAs of the three databases presented in the center. (B) Mature miRNA sequence of miR-195-5p and miR-497-5p matched to the 3′UTR target region of the selected targets with the seed sequence of each miRNA shown in red. Expression levels, presented as log2 relative to GAPDH of the specific mRNA targets CCNE1, E2F3, and WEE1, measured with RT-qPCR in HCT116 cells (C), RKO cells (D) and DLD-1 cells (E). Each target for each transfection is quantified in duplicate in three independent experiments, presented as average ± the SEM. wt: wild-type non-transfected control, neg: negative control transfection (cel-miR-39-3p). * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 4
Number of significantly differential expressed proteins after transfection with miR-195-5p and miR-497-5p miRNA mimics. Venn diagram of the downregulated proteins in the four cell lines after transfection with mimics of miR-195-5p (A) and miR-497-5p (B). Venn diagram of the upregulated proteins in the four cell lines after transfection with mimics of miR-195-5p (C) and miR-497-5p (D). Venn diagram of overlapping downregulated proteins in MSI CRC cells after transfection with mimics of miR-195-5p (E) and miR-497-5p (F). Coinciding significantly differential expressed proteins in MSI/P53 wt CRC cells transfected with miR-195-5p mimic and miR-497-5p mimic are listed in (E) and (F) respectively. In red two proteins downregulated in all four conditions. Full article ">Figure 5
miRNA target evidence of differential expressed proteins from the proteomics screen. Venn diagram of mRNA target evidence found in the different databases for miR-195-5p (A) and miR-497-5p (C). mRNA target sites of potential targets of miR-195-5p (B) and miR-497-5p (D) based on the seed sequence (given in red). Full article ">

28 pages, 1031 KiB

Open AccessEditor’s ChoiceReview

FOXO3a from the Nucleus to the Mitochondria: A Round Trip in Cellular Stress Response

by Candida Fasano, Vittoria Disciglio, Stefania Bertora, Martina Lepore Signorile and Cristiano Simone

Cells 2019, 8(9), 1110; https://doi.org/10.3390/cells8091110 - 19 Sep 2019

Cited by 137 | Viewed by 16163

Abstract

Cellular stress response is a universal mechanism that ensures the survival or negative selection of cells in challenging conditions. The transcription factor Forkhead box protein O3 (FOXO3a) is a core regulator of cellular homeostasis, stress response, and longevity since it can modulate a [...] Read more.

Cellular stress response is a universal mechanism that ensures the survival or negative selection of cells in challenging conditions. The transcription factor Forkhead box protein O3 (FOXO3a) is a core regulator of cellular homeostasis, stress response, and longevity since it can modulate a variety of stress responses upon nutrient shortage, oxidative stress, hypoxia, heat shock, and DNA damage. FOXO3a activity is regulated by post-translational modifications that drive its shuttling between different cellular compartments, thereby determining its inactivation (cytoplasm) or activation (nucleus and mitochondria). Depending on the stress stimulus and subcellular context, activated FOXO3a can induce specific sets of nuclear genes, including cell cycle inhibitors, pro-apoptotic genes, reactive oxygen species (ROS) scavengers, autophagy effectors, gluconeogenic enzymes, and others. On the other hand, upon glucose restriction, 5′-AMP-activated protein kinase (AMPK) and mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -dependent FOXO3a mitochondrial translocation allows the transcription of oxidative phosphorylation (OXPHOS) genes, restoring cellular ATP levels, while in cancer cells, mitochondrial FOXO3a mediates survival upon genotoxic stress induced by chemotherapy. Interestingly, these target genes and their related pathways are diverse and sometimes antagonistic, suggesting that FOXO3a is an adaptable player in the dynamic homeostasis of normal and stressed cells. In this review, we describe the multiple roles of FOXO3a in cellular stress response, with a focus on both its nuclear and mitochondrial functions. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Stress Responses)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Post-translational regulation of FOXO3a. (A) Schematic representation of FOXO3a domains (MPP-MIP motifs, consensus motifs for mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP); forkhead domain, FH; nuclear localization signal, NLS, Kinase-inducible domain interacting domain binding domain, KIX; transactivation domain TAD). (B) Summary of FOXO3a post-translational modifications (PTMs). Depicted are the most important upstream signals (AKT8 virus oncogene cellular homolog, AKT; serum and glucocorticoid-induced kinase, SGK;5′-AMP-activated protein kinase AMPK; c-Jun N-terminal kinase, JNK; extracellular signal-regulated kinase, ERK, SET domain protein 9, SET9) regulating FOXO3a subcellular localization and activity through reversible PTMs, which include phosphorylation (blue circle) and methylation (green circle) on specific amino acid residues (T, threonine; S, serine; K, methionine). Full article ">Figure 2
Schematic representation of Forkhead box O3 (FOXO3a)-mediated stress response. Perturbations of cellular homeostasis, such as nutrient shortage, high concentration of intracellular ROS, or genotoxic stress, activate FOXO3a upstream stress sensors (purple boxes), which in turn modulate FOXO3a subcellular localization and/or activity through various post-translational modifications (PTMs) (green arrows represent activation signals, red bar-headed lines represent inhibitory effects). In the cytoplasm, FOXO3a is inactive and is targeted for poly-ubiquitination, which leads to its further proteasomal degradation. Upon phosphorylation by c-Jun N-terminal kinase (JNK), FOXO3a is shuttled into the nucleus, where its transcriptional activity is further regulated by an activator (e.g., 5′-AMP-activated protein kinase, AMPK; sirtuin 1, SIRT1) or repressor (e.g., AKT8 virus oncogene cellular homolog AKT, CREB binding protein and p300 (CBP/p300) signals. Depending on the PTM pattern, FOXO3a orchestrates different transcriptional programs involved in several cellular processes, including apoptosis, cell cycle progression, DNA repair, reactive oxygen species (ROS) detoxification, and cellular metabolism. Recent evidence showed that metabolic stress or chemotherapy treatment can also promote AMPK- and extracellular signal-regulated kinase (ERK) dependent mitochondrial accumulation of a FOXO3a cleaved form, which activates the expression of mitochondrial oxidative phosphorylation (OXPHOS) genes involved in cell survival. The crosstalk between FOXO3a nuclear and mitochondrial functions is crucial for the restoration and maintenance of cellular homeostasis. Full article ">

13 pages, 6923 KiB

Open AccessArticle

Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis

by Giulia Chiaverina, Laura di Blasio, Valentina Monica, Massimo Accardo, Miriam Palmiero, Barbara Peracino, Marianela Vara-Messler, Alberto Puliafito and Luca Primo

Cells 2019, 8(9), 1109; https://doi.org/10.3390/cells8091109 - 19 Sep 2019

Cited by 47 | Viewed by 5789

Abstract

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that [...] Read more.

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes. Full article

(This article belongs to the Special Issue Angiogenesis in Cancer)

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22 pages, 1839 KiB

Open AccessReview

Environmentally-Induced Transgenerational Epigenetic Inheritance: Implication of PIWI Interacting RNAs

by Karine Casier, Antoine Boivin, Clément Carré and Laure Teysset

Cells 2019, 8(9), 1108; https://doi.org/10.3390/cells8091108 - 19 Sep 2019

Cited by 20 | Viewed by 7353

Abstract

Environmentally-induced transgenerational epigenetic inheritance is an emerging field. The understanding of associated epigenetic mechanisms is currently in progress with open questions still remaining. In this review, we present an overview of the knowledge of environmentally-induced transgenerational inheritance and associated epigenetic mechanisms, mainly in [...] Read more.

Environmentally-induced transgenerational epigenetic inheritance is an emerging field. The understanding of associated epigenetic mechanisms is currently in progress with open questions still remaining. In this review, we present an overview of the knowledge of environmentally-induced transgenerational inheritance and associated epigenetic mechanisms, mainly in animals. The second part focuses on the role of PIWI-interacting RNAs (piRNAs), a class of small RNAs involved in the maintenance of the germline genome, in epigenetic memory to put into perspective cases of environmentally-induced transgenerational inheritance involving piRNA production. Finally, the last part addresses how genomes are facing production of new piRNAs, and from a broader perspective, how this process might have consequences on evolution and on sporadic disease development. Full article

(This article belongs to the Special Issue Evolution of Epigenetic Mechanisms and Signatures)

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Germline PIWI-interacting RNAs (piRNA) biogenesis. In Drosophila melanogaster germ cells, active piRNA clusters are enriched in H3K9me3 and HP1-like protein Rhino (R), which interact with Deadlock (Del), Cutoff (Cuff). Moonshiner (Moon) interacts with Del and recruits transcription initiation factors, allowing transcription of piRNA clusters in the sense and antisense direction. Bootlegger, UAP56, the THO complex, Nxt1, Nxf3 are recruited to the piRNA cluster transcription site. The transcripts are then exported to the cytoplasmic piRNAs biogenesis site via the CRM1 exportin. In the cytoplasm, piRNA precursors are cleaved by Zucchini (Zuc) into primary piRNA and charged by Piwi or Aubergine (Aub) proteins, forming Piwi-piRISC and Aub-piRISC complexes. Piwi-piRISC is translocated into the nucleus and recognizes nascent TE transcripts due to the interaction with Panoramix, the Nxf2, and Nxt1. The association of these factors to nascent TE transcripts causes recruitment of the histone methyltransferase Eggless that transcriptionally silences TEs by local heterochromatinization. In the cytoplasm, Aub-piRISC cleaves TE transcripts into secondary piRNAs, which are loaded onto Ago3, forming Ago3-piRISC complexes. Ago3-piRISC recognizes and cleaves piRNA precursors into new secondary piRNAs, loaded onto Aub. This mechanism, known as the ‘Ping-Pong’ cycle, allows the amplification of piRISC complexes and post-transcriptional regulation of TEs. Full article ">Figure 2
The paramutation process in Drosophila melanogaster relies on maternal piRNA inheritance. At G0, females (grey chromosomes) carrying the BX2 clusters made of repeated P-lacZ-white transgenes (blue arrowheads) producing piRNAs are crossed to males (white chromosomes) carrying the same cluster not producing piRNA. At G1, maternal piRNA deposition in the embryo leads to activation of piRNA production from the paternal cluster allele. The newly activated cluster can activate the paternally-inherited inactive cluster in G2 due to maternal piRNA inheritance (MpI). Then, this process of activation leads to stable piRNAs production across generation. Full article ">Figure 3
Stable, heritable stress-induced de novo piRNAs. In flies, the BX2 cluster, a sequence made of repeated P-lacZ-white transgenes (dark blue arrowheads), is capable of producing piRNA after one generation at 29 °C, then these piRNAs can functionally repress a homologous P-lacZ sequence (light blue arrowheads) in trans. This silencing capacity is maintained at the second generation after return to normal temperature (25 °C) and then up to 50 generations. Hence, when stress is removed, piRNAs can be self-maintained by maternal piRNA inheritance (MpI) at each generation. Full article ">

19 pages, 917 KiB

Open AccessReview

Functions and Regulatory Mechanisms of lncRNAs in Skeletal Myogenesis, Muscle Disease and Meat Production

by Shanshan Wang, Jianjun Jin, Zaiyan Xu and Bo Zuo

Cells 2019, 8(9), 1107; https://doi.org/10.3390/cells8091107 - 19 Sep 2019

Cited by 67 | Viewed by 6201

Abstract

Myogenesis is a complex biological process, and understanding the regulatory network of skeletal myogenesis will contribute to the treatment of human muscle related diseases and improvement of agricultural animal meat production. Long noncoding RNAs (lncRNAs) serve as regulators in gene expression networks, and [...] Read more.

Myogenesis is a complex biological process, and understanding the regulatory network of skeletal myogenesis will contribute to the treatment of human muscle related diseases and improvement of agricultural animal meat production. Long noncoding RNAs (lncRNAs) serve as regulators in gene expression networks, and participate in various biological processes. Recent studies have identified functional lncRNAs involved in skeletal muscle development and disease. These lncRNAs regulate the proliferation, differentiation, and fusion of myoblasts through multiple mechanisms, such as chromatin modification, transcription regulation, and microRNA sponge activity. In this review, we presented the latest advances regarding the functions and regulatory activities of lncRNAs involved in muscle development, muscle disease, and meat production. Moreover, challenges and future perspectives related to the identification of functional lncRNAs were also discussed. Full article

(This article belongs to the Special Issue Regulatory Mechanisms of Skeletal Muscle Stem Cells/Progenitors in Physiological and Pathological Conditions)

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18 pages, 2389 KiB

Open AccessArticle

Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

by Ji Tae Kim, Chang Li, Heidi L. Weiss, Yuning Zhou, Chunming Liu, Qingding Wang and B. Mark Evers

Cells 2019, 8(9), 1106; https://doi.org/10.3390/cells8091106 - 19 Sep 2019

Cited by 48 | Viewed by 7866

Abstract

The Wnt/β-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body β-hydroxybutyrate (βHB), contributes to the regulation of intestinal cell differentiation. Here, we have [...] Read more.

The Wnt/β-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body β-hydroxybutyrate (βHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/β-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and βHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPARγ and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/β-catenin signaling decreased protein and mRNA levels of HMGCS2, βHB production, and expression of PPARγ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPARγ reduced HMGCS2 expression and βHB production, while activation of PPARγ increased HMGCS2 expression and βHB synthesis. Furthermore, PPARγ bound the promoter of HMGCS2 and this binding was enhanced by β-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/β-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/β-catenin/PPARγ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis. Full article

(This article belongs to the Section Cell Signaling)

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18 pages, 2784 KiB

Open AccessReview

Cellular Stress Responses in Radiotherapy

by Wanyeon Kim, Sungmin Lee, Danbi Seo, Dain Kim, Kyeongmin Kim, EunGi Kim, JiHoon Kang, Ki Moon Seong, HyeSook Youn and BuHyun Youn

Cells 2019, 8(9), 1105; https://doi.org/10.3390/cells8091105 - 18 Sep 2019

Cited by 212 | Viewed by 16888

Abstract

Radiotherapy is one of the major cancer treatment strategies. Exposure to penetrating radiation causes cellular stress, directly or indirectly, due to the generation of reactive oxygen species, DNA damage, and subcellular organelle damage and autophagy. These radiation-induced damage responses cooperatively contribute to cancer [...] Read more.

Radiotherapy is one of the major cancer treatment strategies. Exposure to penetrating radiation causes cellular stress, directly or indirectly, due to the generation of reactive oxygen species, DNA damage, and subcellular organelle damage and autophagy. These radiation-induced damage responses cooperatively contribute to cancer cell death, but paradoxically, radiotherapy also causes the activation of damage-repair and survival signaling to alleviate radiation-induced cytotoxic effects in a small percentage of cancer cells, and these activations are responsible for tumor radio-resistance. The present study describes the molecular mechanisms responsible for radiation-induced cellular stress response and radioresistance, and the therapeutic approaches used to overcome radioresistance. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Stress Responses)

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Radiation-induced reactive oxygen species (ROS) response associated with p53 signaling. Irradiation increases intracellular ROS levels facilitated by radiation-mediated mitochondrial damage. In the presence of elevated ROS levels, p53 may importantly ameliorate radiation-induced oxidative stress. Full article ">Figure 2
Radiation-induced double-strand breaks (DSB) response. When DSBs are induced by irradiation, DNA damage-sensing and repair proteins such as ATM, ATR, DNA-PK, H2AX, MDC1, Chk1, and Chk2 are activated. Subsequently, p53 is activated and induces cell cycle arrest for the homologous recombination (HR) or non-homologous end joining (NHEJ) pathways or induces apoptosis by upregulating proapoptotic genes. Full article ">Figure 3
Radiation-induced lipid peroxidation and ceramide signaling. Exposure of the plasma membrane to penetrating radiation leads to the production of homologous recombination (HNE), arachidonic acid-derived lipid metabolites, and ceramide. HNE is associated with the stimulation of unfolded protein response (UPR), and arachidonic acid metabolites promote cell proliferation, inflammation, and protect cells from apoptosis, and thus, contribute to tumor radioresistance. On the other hand, ceramide triggers apoptosis by activating Fas and Bak/Bax signaling and inhibiting PI3K/Akt signaling. Full article ">Figure 4
Radiation-induced mitochondrial response. Radiation induces mitochondrial damage largely via ROS generation. Excessive ROS levels and radiation-induced p53-dependent upregulations of PUMA and Bak/Bax result in mitochondrial membrane permeabilization and subsequent release of cytochrome c into cytosol, and thus, promote intrinsic apoptotic signaling. Full article ">Figure 5
Radiation-induced ER stress response. Radiation can induce ER stress directly or indirectly by generating ROS. Under radiation-induced ER stress, specific signaling by PERK, ATF6, and IRE1 may be activated, and augment the upregulations of UPR-related genes to improve chaperone activity and induce autophagy to recover and recycle misfolded proteins. Full article ">

18 pages, 4489 KiB

Open AccessArticle

Interleukin 21 Receptor/Ligand Interaction Is Linked to Disease Progression in Pancreatic Cancer

by Alica Linnebacher, Philipp Mayer, Nicole Marnet, Frank Bergmann, Esther Herpel, Steffie Revia, Libo Yin, Li Liu, Thilo Hackert, Thomas Giese, Ingrid Herr and Matthias M. Gaida

Cells 2019, 8(9), 1104; https://doi.org/10.3390/cells8091104 - 18 Sep 2019

Cited by 14 | Viewed by 4785

Abstract

Pancreatic ductal adenocarcinoma (PDAC) displays a marked fibro-inflammatory microenvironment in which infiltrated immune cells fail to eliminate the tumor cells and often—rather paradoxically—promote tumor progression. Of special interest are tumor-promoting T cells that assume a Th17-like phenotype because their presence in PDAC tissue [...] Read more.

Pancreatic ductal adenocarcinoma (PDAC) displays a marked fibro-inflammatory microenvironment in which infiltrated immune cells fail to eliminate the tumor cells and often—rather paradoxically—promote tumor progression. Of special interest are tumor-promoting T cells that assume a Th17-like phenotype because their presence in PDAC tissue is associated with a poor prognosis. In that context, the role of IL-21, a major cytokine released by Th17-like cells, was assessed. In all tissue samples (n = 264) IL-21⁺ immune cells were detected by immunohistochemistry and high density of those cells was associated with poor prognosis. In the majority of patients (221/264), tumor cells expressed the receptor for IL-21 (IL-21R) and also a downstream target of IL-21, Blimp-1 (199/264). Blimp-1 expression closely correlated with IL-21R expression and multivariate analysis revealed that expression of both IL-21R and Blimp-1 was associated with shorter survival time of the patients. In vitro data using pancreatic tumor cells lines provided a possible explanation: IL-21 activated ERK and STAT3 pathways and upregulated Blimp-1. Moreover, IL-21 increased invasion of tumor cell lines in a Blimp-1-dependent manner. As an in vivo correlate, an avian xenograft model was used. Here again Blimp-1 expression was significantly upregulated in IL-21 stimulated tumor cells. In summary, our data showed an association of IL-21⁺ immune cell infiltration and IL-21 receptor expression in PDAC with poor survival, most likely due to an IL-21-mediated promotion of tumor cell invasion and enhanced colony formation, supporting the notion of the tumor-promoting abilities of the tumor microenvironment. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Cancers: Pancreatic Cancer)

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18 pages, 3800 KiB

Open AccessArticle

Understanding the Modus Operandi of MicroRNA Regulatory Clusters

by Arthur C. Oliveira, Luiz A. Bovolenta, Lucas Alves, Lucas Figueiredo, Amanda O. Ribeiro, Vinicius F. Campos, Ney Lemke and Danillo Pinhal

Cells 2019, 8(9), 1103; https://doi.org/10.3390/cells8091103 - 18 Sep 2019

Cited by 12 | Viewed by 4422

Abstract

MicroRNAs (miRNAs) are non-coding RNAs that regulate a wide range of biological pathways by post-transcriptionally modulating gene expression levels. Given that even a single miRNA may simultaneously control several genes enrolled in multiple biological functions, one would expect that these tiny RNAs have [...] Read more.

MicroRNAs (miRNAs) are non-coding RNAs that regulate a wide range of biological pathways by post-transcriptionally modulating gene expression levels. Given that even a single miRNA may simultaneously control several genes enrolled in multiple biological functions, one would expect that these tiny RNAs have the ability to properly sort among distinctive cellular processes to drive protein production. To test this hypothesis, we scrutinized previously published microarray datasets and clustered protein-coding gene expression profiles according to the intensity of fold-change levels caused by the exogenous transfection of 10 miRNAs (miR-1, miR-7, miR-9, miR-124, miR-128a, miR-132, miR-133a, miR-142, miR-148b, miR-181a) in a human cell line. Through an in silico functional enrichment analysis, we discovered non-randomic regulatory patterns, proper of each cluster identified. We demonstrated that miRNAs are capable of equivalently modulate the expression signatures of target genes in regulatory clusters according to the biological function they are assigned to. Moreover, target prediction analysis applied to ten vertebrate species, suggest that such miRNA regulatory modus operandi is evolutionarily conserved within vertebrates. Overall, we discovered a complex regulatory cluster-module strategy driven by miRNAs, which relies on the controlled intensity of the repression over distinct targets under specific biological contexts. Our discovery helps to clarify the mechanisms underlying the functional activity of miRNAs and makes it easier to take the fastest and most accurate path in the search for the functions of miRNAs in any distinct biological process of interest. Full article

(This article belongs to the Collection Regulatory Functions of microRNAs)

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Correlation between the number of enriched terms and the number of miRNA-responsive genes in each cluster. Clusters are ordered according to the number of gene members embraced. Full article ">Figure 2
Functional enrichment of messenger RNA (mRNA) clusters after miR-133a transfection. Purple terms belongs to the cluster (−2.156:−0.542), red terms to the cluster (−0.539:−0.119), green terms to the cluster (−0.118:0.119), blue terms to the cluster (0.120:0.666) and yellow terms to the cluster (0.674:4.128). The numbers within brackets represent the minimum and maximum fold change values of each cluster. Terms with different colors inside a cluster indicate that the same term is also present in the cluster of that color. Grey edges connect highly similar terms, and the edge width indicates the similarity degree. Results for other miRNAs can be visualized at <a href="#app1-cells-08-01103" class="html-app">Supplementary Figure S4</a> and a detailed description of the terms found in each cluster can be visualized at <a href="#app1-cells-08-01103" class="html-app">Supplementary Table S3</a>. Full article ">Figure 3
Combined action of distinct regulatory clusters. Each line represents a regulatory cluster connecting miRNAs to the enriched biological term associated with the cluster. The numbers within brackets represent the minimum and maximum fold change values of that cluster. Red lines represent up-regulated gene clusters, green lines represent down-regulated gene clusters, and gray dashed lines represent clusters of genes apparently not regulated by the miRNA. Full article ">Figure 4
Proportion of genes predicted and do not predicted as targeted by miRNAs belonging to each cluster of miRNA-responsive genes for miR-7, miR-128, and miR-181a. Additional data of other miRNAs can be found in the <a href="#app1-cells-08-01103" class="html-app">Supplementary Figure S5</a>. Full article ">Figure 5
miRNA-responsive biological pathways and the presence or absence of interactions between its genes and the transfected miRNA. (A) predicted interactions among miR-9 (yellow ellipses) and the mRNAs of a biological pathway perturbed after miR-9 transfection; (B) miR-1 responsive biological pathway with no predicted interaction between miR-1 and its genes; (C) predicted interactions among miR-142 (yellow ellipses) and genes either up- or down-regulated after miR-142 transfection. Green rectangles represent down-regulated mRNAs. Red rectangles represent up-regulated mRNAs. Gray rectangles represent mRNAs with little-to-no fold change after miRNA transfection. Full article ">Figure 6
Conservation of target responsiveness over miRNA interaction. (A). Heatmaps of Context++Scores for miR-1 and miR-9 predicted targets among 10 vertebrate species. Negative Context++Score values are represented in green, positive values are represented in red, and near zero values are represented in black. Lower Context++Score values represent stronger miRNA regulation levels. Heatmaps for other miRNAs can be found in the <a href="#app1-cells-08-01103" class="html-app">Supplementary Figure S7</a>. (B). Example of evolutionary conservation of the 3’UTR of two genes targeted by miR-1 (PICALM) or miR-9 (GDNF) in the genome of the ten vertebrate species. Yellow marks represent the miRNA binding site at the target mRNA 3’UTR. Full article ">Figure 7
MiRNA-mediated mRNA up-regulation scheme. (A) Interaction network between the miRNA, the up-regulated mRNA, and its suppressor. The miRNA down-regulates mRNA suppressor while fine-tunes the expression of the up-regulated mRNA. The continuous line represents direct interaction and dashed line represents either direct or indirect regulation; (B) Representative expression variation of the network members. Green line represents miRNA expression, the blue line represents up-regulated mRNA expression and the red line represents mRNA suppressor expression. Full article ">

35 pages, 1887 KiB

Open AccessEditor’s ChoiceReview

Trends and Challenges in Tumor Anti-Angiogenic Therapies

by József Jászai and Mirko H.H. Schmidt

Cells 2019, 8(9), 1102; https://doi.org/10.3390/cells8091102 - 18 Sep 2019

Cited by 160 | Viewed by 15277

Abstract

Excessive abnormal angiogenesis plays a pivotal role in tumor progression and is a hallmark of solid tumors. This process is driven by an imbalance between pro- and anti-angiogenic factors dominated by the tissue hypoxia-triggered overproduction of vascular endothelial growth factor (VEGF). VEGF-mediated signaling [...] Read more.

Excessive abnormal angiogenesis plays a pivotal role in tumor progression and is a hallmark of solid tumors. This process is driven by an imbalance between pro- and anti-angiogenic factors dominated by the tissue hypoxia-triggered overproduction of vascular endothelial growth factor (VEGF). VEGF-mediated signaling has quickly become one of the most promising anti-angiogenic therapeutic targets in oncology. Nevertheless, the clinical efficacy of this approach is severely limited in certain tumor types or shows only transient efficacy in patients. Acquired or intrinsic therapy resistance associated with anti-VEGF monotherapeutic approaches indicates the necessity of a paradigm change when targeting neoangiogenesis in solid tumors. In this context, the elaboration of the conceptual framework of “vessel normalization” might be a promising approach to increase the efficacy of anti-angiogenic therapies and the survival rates of patients. Indeed, the promotion of vessel maturation instead of regressing tumors by vaso-obliteration could result in reduced tumor hypoxia and improved drug delivery. The implementation of such anti-angiogenic strategies, however, faces several pitfalls due to the potential involvement of multiple pro-angiogenic factors and modulatory effects of the innate and adaptive immune system. Thus, effective treatments bypassing relapses associated with anti-VEGF monotherapies or breaking the intrinsic therapy resistance of solid tumors might use combination therapies or agents with a multimodal mode of action. This review enumerates some of the current approaches and possible future directions of treating solid tumors by targeting neovascularization. Full article

(This article belongs to the Special Issue Angiogenesis in Cancer)

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Figure 1
Steps of tissue hypoxia-triggered neoangiogenesis and progression of neoplastic lesions. VEGF: vascular endothelial growth factor. The initially avascular tumor cell mass, upon reaching a critical size, cannot ensure its further growth without the trophic support of its own circulation. Hypoxic cells of the tumor mass, via angiogenic factors, initiate the “angiogenic switch” by stimulating nearby endothelial cells of the microvasculature of the host/parental tissue in which they arise. Tumor angiogenesis initiates with a detachment of perivascular cells, the degradation of vessel basal lamina and angiogenic sprouting by the formation of filopodia bearing leading-edge endothelial tip cells from the parental vessels. Newly formed sprouts build anastomoses followed by lumen formation and the recruitment of the pericytic cells necessary for perivascular investment. Angiogenesis will continue as the expansive growth of the tumor mass requires further supply. This process forms a vicious circle that is fueled by the tumor vessel leakiness and the suboptimal tumor perfusion that cannot efficiently relief tissue hypoxia. Recruited inflammatory cells contribute significantly to a hostile microenvironment that boosts further uncontrolled neoangiogenesis and play a role in the evasive resistance of solid tumors. Abbreviations: Treg, regulatory T cell; TAM, tumor-associated macrophage; TEM, Tie-2- expressing monocyte. Full article ">Figure 2
The VEGF/VEGFR signaling axis, its contribution to neoangiogenesis and treatment modalities interfering with its activity. Binding of VEGF ligands to their cognate receptors leads to receptor dimerization and autophosphorylation triggering a down-stream intracellular phosphorylation cascade. In principle, tumor anti-angiogenesis can be achieved (1) by prohibiting ligand binding to their cognate TK receptors (VEGFR1-3) receptors/non-tyrosine kinase (NRPs) co-receptors either by the withdrawal of pro-angiogenic ligands of the VEGF family (Aflibercept, Bevacizumab) or by blocking the accessibility of the binding pocket for ligands on a particular receptor (Ramucirumab); (2) by interfering with the kinase activity of VEGFRs (small molecule multikinase inhibitors: receptor tyrosine kinase (RTK)-inhibitors). Anti-VEGF-targeted therapies result in the inhibition of down-stream signaling mechanisms, governing a range of steps involved in neovessel formation and/or inflammatory infiltration. Abbreviations: EC, endothelial cell; MΦ, macrophage; PlGF, placental growth factor; RTK, receptor tyrosine kinase; TK-domain, tyrosine kinase-domain; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Full article ">Figure 3
Alternative angiogenic pathways playing a role in tumor angiogenesis, refractoriness, evasive resistance or relapses in response to anti-VEGF monotherapies and tools available for targeting their effects. Resistance to anti-angiogenic therapy might be mediated by angiogenic factors that trigger a switch from an initially VEGF-dependent state to a VEGF-independent angiogenic process. In order to interfere with these alternative pathways, besides the small molecule multikinase inhibitors (RTK-inhibitors) blocking down-stream signal cascade activation even in the presence of receptor occupancy, several recombinant biological tools have been developed. The range of these tools includes decoys (“ligand traps”) aimed at withdrawing alternative pro-angiogenic factors either alone (fibroblast growth factor (FGF)-Trap) or simultaneously with VEGF-A (VF-Trap), engineered multivalent monoclonals (CrossMabs) or antibody tools raised against RTK receptors blocking the access of ligands to their cognate receptor (onartuzumab). Abbreviations: EC, endothelial cell; PC, pericyte; TC, tumor cell; Ang-2, angiopoietin-2; FGF-2, fibroblast growth factor-2; FGFR, fibroblast growth factor receptor; HGF, hepatocyte growth factor; MET, mesenchymal-epithelial transition proto-oncogene; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; TIE2, Tyrosine kinase with immunoglobulin-like and EGF-like domains 2; RTK, receptor tyrosine kinase. Full article ">

14 pages, 1904 KiB

Open AccessArticle

Increased Glycolysis and Higher Lactate Production in Hyperglycemic Myotubes

by Jenny Lund, D. Margriet Ouwens, Marianne Wettergreen, Siril S. Bakke, G. Hege Thoresen and Vigdis Aas

Cells 2019, 8(9), 1101; https://doi.org/10.3390/cells8091101 - 18 Sep 2019

Cited by 35 | Viewed by 7104

Abstract

Previous studies have shown that chronic hyperglycemia impairs glucose and fatty acid oxidation in cultured human myotubes. To further study the hyperglycemia-induced suppression of oxidation, lactate oxidation, mitochondrial function and glycolytic rate were evaluated. Further, we examined the intracellular content of reactive oxygen [...] Read more.

Previous studies have shown that chronic hyperglycemia impairs glucose and fatty acid oxidation in cultured human myotubes. To further study the hyperglycemia-induced suppression of oxidation, lactate oxidation, mitochondrial function and glycolytic rate were evaluated. Further, we examined the intracellular content of reactive oxygen species (ROS), production of lactate and conducted pathway-ANOVA analysis on microarray data. In addition, the roles of the pentose phosphate pathway (PPP) and the hexosamine pathway were evaluated. Lactic acid oxidation was suppressed in hyperglycemic versus normoglycaemic myotubes. No changes in mitochondrial function or ROS concentration were observed. Pathway-ANOVA analysis indicated several upregulated pathways in hyperglycemic cells, including glycolysis and PPP. Functional studies showed that glycolysis and lactate production were higher in hyperglycemic than normoglycaemic cells. However, there were no indications of involvement of PPP or the hexosamine pathway. In conclusion, hyperglycemia reduced substrate oxidation while increasing glycolysis and lactate production in cultured human myotubes. Full article

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15 pages, 1545 KiB

Open AccessReview

New Insights into the Liver–Visceral Adipose Axis During Hepatic Resection and Liver Transplantation

by María Eugenia Cornide-Petronio, Mónica B. Jiménez-Castro, Jordi Gracia-Sancho and Carmen Peralta

Cells 2019, 8(9), 1100; https://doi.org/10.3390/cells8091100 - 18 Sep 2019

Cited by 8 | Viewed by 4029

Abstract

In the last decade, adipose tissue has emerged as an endocrine organ with a key role in energy homeostasis. In addition, there is close crosstalk between the adipose tissue and the liver, since pro- and anti-inflammatory substances produced at the visceral adipose tissue [...] Read more.

In the last decade, adipose tissue has emerged as an endocrine organ with a key role in energy homeostasis. In addition, there is close crosstalk between the adipose tissue and the liver, since pro- and anti-inflammatory substances produced at the visceral adipose tissue level directly target the liver through the portal vein. During surgical procedures, including hepatic resection and liver transplantation, ischemia–reperfusion injury induces damage and regenerative failure. It has been suggested that adipose tissue is associated with both pathological or, on the contrary, with protective effects on damage and regenerative response after liver surgery. The present review aims to summarize the current knowledge on the crosstalk between the adipose tissue and the liver during liver surgery. Therapeutic strategies as well as the clinical and scientific controversies in this field are discussed. The different experimental models, such as lipectomy, to evaluate the role of adipose tissue in both steatotic and nonsteatotic livers undergoing surgery, are described. Such information may be useful for the establishment of protective strategies aimed at regulating the liver–visceral adipose tissue axis and improving the postoperative outcomes in clinical liver surgery. Full article

(This article belongs to the Special Issue Adipose Tissue Inflammation 2022)

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15 pages, 3748 KiB

Open AccessArticle

CX₃CR1 Mediates the Development of Monocyte-Derived Dendritic Cells during Hepatic Inflammation

by Salvatore Sutti, Stefania Bruzzì, Felix Heymann, Anke Liepelt, Oliver Krenkel, Alberto Toscani, Naresh Naik Ramavath, Diego Cotella, Emanuele Albano and Frank Tacke

Cells 2019, 8(9), 1099; https://doi.org/10.3390/cells8091099 - 18 Sep 2019

Cited by 28 | Viewed by 5752

Abstract

Recent evidence suggests that hepatic dendritic cells (HDCs) contribute to the evolution of chronic liver diseases. However, the HDC subsets involved and the mechanisms driving these responses are still poorly understood. In this study, we have investigated the role of the fractalkine receptor [...] Read more.

Recent evidence suggests that hepatic dendritic cells (HDCs) contribute to the evolution of chronic liver diseases. However, the HDC subsets involved and the mechanisms driving these responses are still poorly understood. In this study, we have investigated the role of the fractalkine receptor CX₃CR1 in modulating monocyte-derived dendritic cell (moDC) differentiation during liver inflammation. The phenotype of HDC and functional relevance of CX₃CR1 was assessed in mice following necro-inflammatory liver injury induced by the hepatotoxic agent carbon tetrachloride (CCl₄) and in steatohepatitis caused by a methionine/choline-deficient (MCD) diet. In both the experimental models, hepatic inflammation was associated with a massive expansion of CD11c⁺/MHCII^high/CD11b⁺ myeloid HDCs. These cells also expressed the monocyte markers Ly6C, chemokine (C-C Motif) receptor 2 (CCR2), F4/80 and CD88, along with CX₃CR1, allowing their tentative identification as moDCs. Mice defective in CX₃CR1 showed a reduction in liver-moDC recruitment following CCl₄ poisoning in parallel with a defective maturation of monocytes into moDCs. The lack of CX₃CR1 also affected moDC differentiation from bone marrow myeloid cells induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) in vitro. In wild-type mice, treatment with the CX₃CR1 antagonist CX3-AT (150 µg, i.p.) 24 h after CCl₄ administration reduced liver moDC_S and significantly ameliorated hepatic injury and inflammation. Altogether, these results highlight the possible involvement of moDCs in promoting hepatic inflammation following liver injury and indicated a novel role of CX₃CL1/CX₃CR1 dyad in driving the differentiation of hepatic moDCs. Full article

(This article belongs to the Special Issue Recent Advances in Liver Repair Strategies)

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16 pages, 1886 KiB

Open AccessReview

How Cancer Exploits Ribosomal RNA Biogenesis: A Journey beyond the Boundaries of rRNA Transcription

by Marco Gaviraghi, Claudia Vivori and Giovanni Tonon

Cells 2019, 8(9), 1098; https://doi.org/10.3390/cells8091098 - 17 Sep 2019

Cited by 28 | Viewed by 6029

Abstract

The generation of new ribosomes is a coordinated process essential to sustain cell growth. As such, it is tightly regulated according to cell needs. As cancer cells require intense protein translation to ensure their enhanced growth rate, they exploit various mechanisms to boost [...] Read more.

The generation of new ribosomes is a coordinated process essential to sustain cell growth. As such, it is tightly regulated according to cell needs. As cancer cells require intense protein translation to ensure their enhanced growth rate, they exploit various mechanisms to boost ribosome biogenesis. In this review, we will summarize how oncogenes and tumor suppressors modulate the biosynthesis of the RNA component of ribosomes, starting from the description of well-characterized pathways that converge on ribosomal RNA transcription while including novel insights that reveal unexpected regulatory networks hacked by cancer cells to unleash ribosome production. Full article

(This article belongs to the Section Intracellular and Plasma Membranes)

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Transcriptional and post-translational regulation of rRNA transcription by oncogenic pathways. (A) Schematic representation of the main components of the polymerase I (PolI) pre-initiation complex (PIC) and the major activatory (upper panel, orange) and inhibitory (lower panel, purple) post-translational modifications (PTMs) induced by oncogenes or tumor suppressor genes. (B) Schematic representation of the convergent regulatory pathways that boost rRNA transcription upon oncogene activation (left panel, orange) or repress it through tumor suppressor genes (right panel, purple). Full article ">Figure 2
Regulation of rRNA processing in cancer. Schematic representation of the 47S rRNA precursors processing pathways (grey) generating the mature 18S, 5.8S, and 28S molecules (black). The main maturation steps affected by oncogenes (orange ovals) or tumor suppressors (purple ovals) are represented. Full article ">Figure 3
Small nucleolar RNAs (snoRNAs) sustain the oncogenic potential of cancer genes. (A) C/D box snoRNAs are required to sustain oncogene-induced self-renewal and proliferation of acute myeloid leukemia (AML) cells. (B) U3 and U8 snoRNAs are targets of oncogenes (orange ovals) and tumor suppressors (purple ovals) and are essential to promote oncogene-induced cell proliferation. Oncogene stimulation induces U3 stabilization and putatively prevents U3 and U8 decapping, mediated by a DCP1α/DCP2 complex, tethered inside nucleoli by PNRC1 tumor suppressor. Full article ">

22 pages, 8302 KiB

Open AccessArticle

DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88

by Soňa Legartová, Gabriela Lochmanová, Zbyněk Zdráhal, Stanislav Kozubek, Jiří Šponer, Miroslav Krepl, Pavlína Pokorná and Eva Bártová

Cells 2019, 8(9), 1097; https://doi.org/10.3390/cells8091097 - 17 Sep 2019

Cited by 8 | Viewed by 5834

Abstract

The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity [...] Read more.

The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage. Full article

(This article belongs to the Special Issue Nucleolar Organization and Functions in Health and Disease)

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23 pages, 6196 KiB

Open AccessArticle

Reappraisal of Human HOG and MO3.13 Cell Lines as a Model to Study Oligodendrocyte Functioning

by Kim M. A. De Kleijn, Wieteke A. Zuure, Jolien Peijnenborg, Josje M. Heuvelmans and Gerard J. M. Martens

Cells 2019, 8(9), 1096; https://doi.org/10.3390/cells8091096 - 17 Sep 2019

Cited by 21 | Viewed by 7910

Abstract

Myelination of neuronal axons is essential for proper brain functioning and requires mature myelinating oligodendrocytes (myOLs). The human OL cell lines HOG and MO3.13 have been widely used as in vitro models to study OL (dys) functioning. Here we applied a number of [...] Read more.

Myelination of neuronal axons is essential for proper brain functioning and requires mature myelinating oligodendrocytes (myOLs). The human OL cell lines HOG and MO3.13 have been widely used as in vitro models to study OL (dys) functioning. Here we applied a number of protocols aimed at differentiating HOG and MO3.13 cells into myOLs. However, none of the differentiation protocols led to increased expression of terminal OL differentiation or myelin-sheath formation markers. Surprisingly, the applied protocols did cause changes in the expression of markers for early OLs, neurons, astrocytes and Schwann cells. Furthermore, we noticed that mRNA expression levels in HOG and MO3.13 cells may be affected by the density of the cultured cells. Finally, HOG and MO3.13 co-cultured with human neuronal SH-SY5Y cells did not show myelin formation under several pro-OL-differentiation and pro-myelinating conditions. Together, our results illustrate the difficulty of inducing maturation of HOG and MO3.13 cells into myOLs, implying that these oligodendrocytic cell lines may not represent an appropriate model to study the (dys)functioning of human (my)OLs and OL-linked disease mechanisms. Full article

(This article belongs to the Special Issue The Molecular and Cellular Basis for Parkinson's Disease)

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Normalized mRNA expression in undifferentiated HOG cells (uHOG) and HOG cells following differentiation (dHOG) with (A) N2.1 medium, (B) N2.2 medium or (C) T3 medium. OL: oligodendrocyte lineage, OPC: oligodendrocyte precursor, imOL: immature oligodendrocyte, mOL: mature oligodendrocyte, myOL: myelinating oligodendrocyte, NEU: neuronal, NP: neural progenitor, AST: astrocyte, iSC: immature Schwann cell, mSC: mature Schwann cell. Independent samples t-tests are based on triplicates in three independent experiments (n = 3). p-values: * < 0.05, ** < 0.01, *** < 0.001. Full article ">Figure 2
Analysis of protein expression in undifferentiated and differentiated HOG cells. (A) Western blot analyses of myelin basic protein (MBP) (polyclonal), myelin-oligodendrocyte glycoprotein (MOG), CNPase, MBP (monoclonal), class III β-tubulin (TUBB3), proteolipid protein 1 (PLP1) and paraoxonase 2 (PON2) in (+) human motor cortex, (1) undifferentiated HOG cells, (2) differentiated HOG cells following incubation in N2.1 medium, (3) N2.2 medium or (4) T3 medium (normalized to GAPDH expression). Western blot analysis was performed for two independent experiments (n = 2). (B) Example images of immunocytochemistry for CNPase and TUBB3 in undifferentiated HOG cells and HOG cells differentiated with N2.1 medium or N2.2 medium. Scale bar = 50 µm. Full article ">Figure 3
Normalized mRNA expression in undifferentiated MO3.13 cells (uMO3) and MO3.13 cells following differentiation (dMO3) with (A) PMA, (B) N2.1 medium, (C) N2.2 medium or (D) T3 medium. OL: oligodendrocyte lineage, OPC: oligodendrocyte precursor, imOL: immature oligodendrocyte, mOL: mature oligodendrocyte, myOL; myelinating oligodendrocyte, NEU: neuronal, NP: neural progenitor, AST: astrocyte, NP: neural progenitor, iSC: immature Schwann cell, mSC: mature Schwann cell. Independent samples t-tests are based on triplicates in three independent experiments (n = 3). p-values: * < 0.05, ** < 0.01, *** < 0.001. Full article ">Figure 4
Analysis of protein expression in undifferentiated and differentiated MO3.13 cells. (A) Western blot analyses of MBP (polyclonal), MOG, CNPase, MBP (monoclonal), TUBB3, PLP1 and PON2 in (+) human motor cortex, (1) undifferentiated MO3.13 cells, (2) differentiated MO3.13 cells following incubation in N2.1 medium, (3) N2.2 medium or (4) T3 medium (normalized to GAPDH expression). Western blot analyses were performed for two independent experiments (n = 2). (B) Example images of immunocytochemistry for CNPase and TUBB3 in undifferentiated MO3.13 cells and MO3.13 cells differentiated with N2.1 medium or N2.2 medium. Scale bar = 50 µm. Full article ">Figure 5
Normalized mRNA expression of PLP1, CNP, SOX10, vascular endothelial growth factor A (VEGF-A) and VEGF-C at various cell densities in (A) HOG cells and (B) MO3.13 cells. Standard error of the mean (SEM) is based on two technical replicates of one experiment (n = 1). Full article ">Figure 6
Immunocytochemical analysis of neuronal differentiation in co-cultures of HOG or MO3.13 cells with differentiated neuronal SH-SY5Y cells. (A) Representative images of for expression of mature neuronal markers TUBB3 and tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH) in differentiated SH-SY5Y monocultures. (B) Representative images of Jagged1 and L1CAM surface expression and TUBB3 signals in SH-SY5Y monoculture, and SH-SY5Y + HOG and SH-SY5Y + MO3.13 co-cultures. Scale bar = 50 µm. (C) Quantifications of neurite caliber in low density neurite networks of differentiated SH-SY5Y cells (n = 2). Full article ">Figure 7
Immunocytochemical analysis of co-cultures of HOG or MO3.13 cells with differentiated neuronal SH-SY5Y cells. (A) Representative image of neuronal marker neurofilament light chain (NEFL) and TUBB3 stainings in SH-SY5Y + HOG co-culture and FluoroMyelin Red (FLMred) cytoplasmic lipid droplets in living SH-SY5Y + MO3.13 co-cultures. (B) Representative images of TUBB3- and FLMred signals in fixed SH-SY5Y monoculture, and SH-SY5Y + HOG co-culture and SH-SY5Y + MO3.13 co-culture. These images serve as examples for the absence of FLMred overlap with the extensive neurite outgrowth (TUBB3) in our cultures. (C) Representative images of the absence of MBP-signals (polyclonal antibody) and the presence of TUBB3-signals in SH-SY5Y monocultures, and SH-SY5Y + HOG and SH-SY5Y + MO3.13 co-cultures. Scale bar = 50 µm. Full article ">

21 pages, 2515 KiB

Open AccessEditor’s ChoiceArticle

Fatty Acid-Treated Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes Exhibit Adult Cardiomyocyte-Like Energy Metabolism Phenotypes

by Yuichi Horikoshi, Yasheng Yan, Maia Terashvili, Clive Wells, Hisako Horikoshi, Satoshi Fujita, Zeljko J. Bosnjak and Xiaowen Bai

Cells 2019, 8(9), 1095; https://doi.org/10.3390/cells8091095 - 17 Sep 2019

Cited by 105 | Viewed by 12974

Abstract

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. [...] Read more.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. As an example, glycolysis is a major energy source for immature CMs. As CMs mature, the mitochondrial oxidative capacity increases, with fatty acid β-oxidation becoming a key energy source to meet the heart’s high energy demand. The immaturity of iPSC-CMs thereby limits their applications. The aim of this study was to investigate whether the energy substrate fatty acid-treated iPSC-CMs exhibit adult CM-like metabolic properties. After 20 days of differentiation from human iPSCs, iPSC-CMs were sequentially cultured with CM purification medium (lactate+/glucose-) for 7 days and maturation medium (fatty acids+/glucose-) for 3–7 days by mimicking the adult CM’s preference of utilizing fatty acids as a major metabolic substrate. The purity and maturity of iPSC-CMs were characterized via the analysis of: (1) Expression of CM-specific markers (e.g., troponin T, and sodium and potassium channels) using RT-qPCR, Western blot or immunofluorescence staining and electron microscopy imaging; and (2) cell energy metabolic profiles using the XF96 Extracellular Flux Analyzer. iPSCs-CMs (98% purity) cultured in maturation medium exhibited enhanced elongation, increased mitochondrial numbers with more aligned Z-lines, and increased expression of matured CM-related genes, suggesting that fatty acid-contained medium promotes iPSC-CMs to undergo maturation. In addition, the oxygen consumption rate (OCR) linked to basal respiration, ATP production, and maximal respiration and spare respiratory capacity (representing mitochondrial function) was increased in matured iPSC-CMs. Mature iPSC-CMs also displayed a larger change in basal and maximum respirations due to the utilization of exogenous fatty acids (palmitate) compared with non-matured control iPSC-CMs. Etomoxir (a carnitine palmitoyltransferase 1 inhibitor) but not 2-deoxyglucose (an inhibitor of glycolysis) abolished the palmitate pretreatment-mediated OCR increases in mature iPSC-CMs. Collectively, our data demonstrate for the first time that fatty acid treatment promotes metabolic maturation of iPSC-CMs (as evidenced by enhanced mitochondrial oxidative function and strong capacity of utilizing fatty acids as energy source). These matured iPSC-CMs might be a promising human CM source for broad biomedical application. Full article

(This article belongs to the Special Issue Stem Cell-based Therapy and Disease Modeling)

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Graphical abstract

Graphical abstract
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Characterization of human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iPSC-CMs). (A) Schematic depicting the procedure for the generation of cardiomyocytes from iPSCs by temporal modulation of Wnt signaling, purification, and maturation of iPSC-CMs. Note: mTeSR1 and Roswell Park Memorial Institute (RMPI): cell culture medium; B27: culture medium supplement; CHIR-99021: highly selective inhibitor of glycogen synthase kinase 3 (GSK-3); and IWP4: inhibitor of Wnt/β-catenin signaling. (B) Characterization of cultured 1013 iPSCs. Phase contrast image shows that iPSCs grow as colonies (a). Confocal fluorescent images indicate that iPSCs express pluripotent stem cell-specific markers octamer-binding transcription factor (OCT4) (red) (b), and stage-specific embryonic antigen-4 (SSEA4, red) (c). Blue are cell nuclei stained with Hoechst 33342. Scale bar = 50 μm. (C) Characterization of the differentiated cardiomyocytes (1013 iPSC-derived CMs). iPSC-CMs (day 20) grew as a monolayer (a) and expressed cardiomyocyte-specific markers troponin T (green) (b) and sarcomeric α-actinin (red) (c). Blue are cell nuclei. Scale bar = 30 µm. Full article ">Figure 2
Lactate purification of 1013 iPSC-derived CMs. (A) The fluorescent images of iPSC-CMs (day 31) with or without treatment of lactate-contained purification medium (no glucose) for 7 days to eliminate non-cardiomyocytes. Blue are cell nuclei stained with Hoechst 33342 and green are troponin T signals. In the purified cell culture, almost all cells with blue nuclei expressed troponin T. Scale bar = 50 µm. (B) The purification of iPSC-CMs increased from 75% to 98% after culturing in lactate medium. Data are presented as mean ± SEM, n = 4 * p < 0.05 vs. control medium. Full article ">Figure 3
The effect of fatty acid-contained cardiomyocyte maturation medium (no glucose) on the maturation of 1013 iPSC-derived CMs. (A) Representative immunofluorescent images of iPSC-CMs (day 34) cultured with control culture medium (a) and maturation medium for 7 days (b). A-c and A-d are the magnified images marked by yellow rectangles in A-a and A-b, respectively. Scale bar = 20 µm. (B) Analysis of cell area (a), perimeter (b), circularity (c), and elongation (d) of iPSC-CMs using ImageJ software. n = 50–64 * p < 0.05 vs. control medium. (C) Representative electron microscopy images of iPSC-CMs (day 38) treated with control medium (a) and maturation medium (b) for 7 days. Scale bar = 500 nm. Maturation medium-treated iPSC-CMs showed well-aligned visible Z-lines and increased mitochondria within the cells compared with the cells cultured in control medium. Z-lines and mitochondrial are indicated by green and red arrows, respectively. (D) Real time PCR analysis of the effect of maturation medium on the cardiomyocyte maturation-related gene expression. n = 4 * p < 0.05 vs. control medium. (iPSC-CMs day 38) (E) Western blot analysis of the effect of maturation medium on MYL2 (a) and MYH7 (b) protein expression in iPSC-CMs (day 38). n = 4, ** p < 0.01, vs. control medium. Note: TNNI: troponin I; TNNT: troponin T; MYL: myosin light chain; MYH: myosin heavy chain; MYBPC: myosin binding protein C; SCN5A: sodium voltage-gated alpha subunit 5; KCNJ: potassium inwardly rectifying channel subunit J; KCNH: potassium voltage-gated channel subfamily H; KCNQ: potassium voltage-gated channel subfamily Q; KCND: potassium voltage-gated channel, subfamily D; CACNA1C: calcium voltage-gated channel subunit alpha1 C; GJA1: gap junction protein, alpha 1; SERCA2A: sarcoplasmic/endoplasmic reticulum Ca2+ATPase 2a; RYR2: cardiac ryanodine receptor; PPARα: peroxisome proliferator-activator receptor alpha. Full article ">Figure 4
The effect of maturation medium on mitochondrial respiratory capacity of 1013 iPSC-derived CMs. (A) The diagram depicts the trace of oxygen consumption rate (OCR) on cells after sequentially administration of 10 µM oligomycin (ATP synthase inhibitor), 2 µM FCCP (uncoupler of oxidative phosphorylation in mitochondria), and 10 µM antimycin A (electron transport chain blocker) to the culture. The profiles of fundamental parameters of mitochondrial function measured are basal respiration, ATP production, maximal respiration and spare respiratory capacity that were marked with different color in the schematic. (B) Representative two OCR traces of iPSC-CMs (day 34) treated with either control medium or maturation medium for 3 days, respectively, in response to oligomycin, FCCP, and antimycin A. (C) OCR parameters representing mitochondrial function in maturation medium-treated iPSC-CMs were significantly higher. n = 4, * p < 0.05 vs. control medium. Full article ">Figure 5
The effect of exogenous fatty acids (palmitate) pre-treatment on the OCR of 1013 iPSC-derived CMs (day 34) cultured with either control medium or maturation medium. (A) The changed basal and maximum OCR in both control and matured iPSC-CMs in response to exogenous palmitate. iPSC-CMs were pretreated with bovine serum albumin (BSA; as control), palmitate, or etomoxir (ETO, a specific irreversible inhibitor of carnitine palmitoyltransferase 1 inhibitor). Basal and maximum respiration capacity was significantly increased in the matured iPSC-CMs compared to control cells due to the utilization of palmitate. Pretreatment with palmitate-BSA (blue) resulted in the increased basal and maximal respiration in non-matured iPSC-CMs compared with BSA alone treatment group (black) (a). Matured iPSC-CMs exhibit the greater increase in both basal respiration (4.4 fold higher vs. non-matured iPSC-CMs) and maximal respiration (2.5 fold higher vs. non-matured iPSC-CMs) in response to palmitate-BSA (blue) (b and c), indicating the stronger ability to oxidize exogenously added palmitate in the matured iPSC-CMs. n = 4 * p < 0.05. (B) The effect of ETO on the OCR of matured iPSC-CMs pre-treated with BSA or palmitate. Representative OCR traces of iPSC-CMs pre-treated with BSA or palmitate together with or without ETO in response to oligomycin, FCCP, and antimycin A (a). ETO completely inhibited the palmitate pretreatment-mediated increases of mitochondrial respiration in matured iPSC-CMs (b). n = 4 * p < 0.05. (C) The effect of 2-deoxyglucose (2-DG) on the matured iPSC-CMs that were pre-treated with BSA or palmitate. Representative OCR traces of iPSC-CMs pre-treated with BSA or palmitate together with or without 2-DG (glucose analogue, a competitive glycolytic inhibitor) in response to oligomycin, FCCP, and antimycin A (a). 2-DG did not attenuate the palmitate-mediated increase of OCR in matured iPSC-CMs (b), suggesting that the palmitate-mediated increased OCR in palmitate-pretreated iPSC-CMs is resulted from the fatty acid effect. n = 4 * p < 0.05. Full article ">Figure 6
The effect of maturation medium on glycolytic function of iPSC-CMs (day 34) via analysis of the extracellular acidification rate (ECAR) of 1013 iPSC-derived CMs. (A-a) This diagram depicts (1) the representative traces of ECAR on the cells after sequentially adding 5.5 µM glucose, 10 µM oligomycin, and 25 mM 2-DG to the culture and (2) the profiles of key parameters of glycolytic function are glycolysis (blue), glycolytic capacity (green), glycolytic reserve (yellow), and non-glycolytic acidification (red). (A-b) Representative two ECAR traces of iPSC-CMs treated with control and maturation medium for 3 days, respectively. (A-c) The quantified ECAR shows that maturation medium increased glycolysis, glycolytic capacity, and glycolytic reserve of iPSC-CMs, suggesting that matured iPSC-CMs have a higher capacity to switch to glucose utilization when fatty acid oxidation is compromised. n = 4, * p < 0.05 vs. control medium. (B) The effects of different concentrations of glucose on glycolytic function of the control medium-treated iPSC-CMs. There is no significant difference in glycolytic function for each glucose concentration. (C-a) Representative traces of ECAR of matured iPSC-CMs in response to various glucose concentrations. (C-b) The glycolysis and glycolytic capacity exhibited the response to the glucose in a dose-dependent manner. n = 4, * p < 0.05. Full article ">Figure 7
The effect of the maturation medium on protein expression, and metabolism of H3083 iPSC-derived CMs (A) Western blot analysis of the effect of maturation medium treatment for 7 days on the expression of MYL2 and MYH7 proteins in iPSC-CMs (day 38). n = 4 * p < 0.05, vs. control medium. (B) The effect of maturation medium on OCR and ECAR of iPSC-CMs (day 34). * p < 0.05 vs. control medium. Full article ">

18 pages, 3844 KiB

Open AccessArticle

Maternal Protein Restriction Modulates Angiogenesis and AQP9 Expression Leading to a Delay in Postnatal Epididymal Development in Rat

by Talita de Mello Santos, Marilia Martins Cavariani, Dhrielly Natália Pereira, Bruno César Schimming, Luiz Gustavo de Almeida Chuffa and Raquel Fantin Domeniconi

Cells 2019, 8(9), 1094; https://doi.org/10.3390/cells8091094 - 17 Sep 2019

Cited by 5 | Viewed by 3704

Abstract

The maternal nutritional status is essential to the health and well-being of the fetus. Maternal protein restriction during the perinatal stage causes sperm alterations in the offspring that are associated with epididymal dysfunctions. Vascular endothelial growth factor (VEGF) and its receptor, VEGFr-2, as [...] Read more.

The maternal nutritional status is essential to the health and well-being of the fetus. Maternal protein restriction during the perinatal stage causes sperm alterations in the offspring that are associated with epididymal dysfunctions. Vascular endothelial growth factor (VEGF) and its receptor, VEGFr-2, as well as aquaporins (AQPs) are important regulators of angiogenesis and the epididymal microenvironment and are associated with male fertility. We investigated the effects of maternal protein restriction on epididymal angiogenesis and AQP expression in the early stages of postnatal epididymal development. Pregnant rats were divided into two experimental groups that received either a normoprotein (17% protein) or low-protein diet (6% protein) during gestation and lactation. At postnatal day (PND)7 and PND14, male offspring were euthanized, the epididymides were subjected to morphometric and microvascular density analyses and to VEGF-A, VEGF-r2, AQP1 and AQP9 expression analyses. The maternal low-protein diet decreased AQP9 and VEGFr-2 expression, decreased epididymal microvascularity and altered the morphometric features of the epididymal epithelium; no changes in AQP1 expression were observed at the beginning of postnatal epididymal development. Maternal protein restriction alters microvascularization and affects molecules involved in the epidydimal microenvironment, resulting in morphometric alterations related to a delay in the beginning of epididymis postnatal development. Full article

(This article belongs to the Special Issue Aquaporins 2019)

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Full article ">Figure 1
Illustration of the experimental design. Full article ">Figure 2
Expression of AQP1, AQP9, VEGFr-2 and VEGFa in the epididymis of NP and LP animals at PND7. Data are expressed as the mean ± S.E.M. * p < 0.05. Mann–Whitney test. Full article ">Figure 3
Expression of AQP1, AQP9, VEGFr-2 and VEGFa in the epididymis of NP and LP animals at PND14. Data are expressed as the mean ± S.E.M. * p < 0.05. Mann–Whitney test. Full article ">Figure 4
Epididymis sections of the initial segment (IS), caput regions, corpus and cauda from the NP and LP animals at PND14 subjected to AQP1 immunostaining. NP = normoprotein animals; LP = low-protein animals. (A) (IS and Caput from NP group); (B) (IS and Caput from LP group); (C) (Corpus from NP group); (D) (Corpus from LP group); (E) (Cauda from NP group); (F) (Cauda from LP group). Arrow = Positive staining for AQP1 in the vascular endothelium. Full article ">Figure 5
Epididymis sections of the initial segment (IS), caput regions, corpus and cauda from the NP and LP animals at PND14 subjected to AQP9 immunostaining. NP = normoprotein animals; LP = low-protein animals. (A) (IS and Caput from NP group); (B) (IS and Caput from LP group); (C) (Corpus from NP group); (D) (Corpus from LP group); (E) (Cauda from NP group); (F) (Cauda from LP group). Arrow = Positive staining for AQP9. Full article ">Figure 6
The microvascular densities in the epididymis of the NP and LP animals at PND14. Data are expressed as the mean ± S.E.M. * p < 0.05. Mann–Whitney test. Full article ">Figure 7
Representative image that illustrates the impact of maternal protein restriction in the early stage of postnatal epididymal development in rat. Maternal protein restriction decreases microvascular density by decreasing the VEGFr-2 expression in the endothelial cells. With the decrease in the number of vessels, the arrival of growth factors and hormones, including androgens, also decreases. Blood vessels are present around the mesenchymal cells that surround the epithelium, and before the first wave of testicular fluid, the arrival of hormones occurs exclusively through blood supply. When differentiated, the principal cells begin to express AQP9 in the apical region. Because AQP9 is downregulated in these cells, the luminal microenvironment may not be adequately regulated. AQP9 = aquaporin 9; AQP1 = aquaporin 1; VEGFa = vascular endothelial growth factor; VEGFr-2 = vascular endothelial growth factor receptor type 2, LM: luminal microenvironment. Full article ">

19 pages, 3987 KiB

Open AccessArticle

Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In Vivo and Three-Dimensional Models over the Petri-Dish

by Róbert Alföldi, József Á. Balog, Nóra Faragó, Miklós Halmai, Edit Kotogány, Patrícia Neuperger, Lajos I. Nagy, Liliána Z. Fehér, Gábor J. Szebeni and László G. Puskás

Cells 2019, 8(9), 1093; https://doi.org/10.3390/cells8091093 - 16 Sep 2019

Cited by 21 | Viewed by 7420

Abstract

Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing [...] Read more.

Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish. Full article

(This article belongs to the Special Issue Single Cell Analysis)

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Figure 1
Different culture conditions of A549 human adenocarcinoma cells for 4 days (A) and for 9 days (B). Cells were seeded at the same surface/volume ratio regarding the different culture conditions as described in Materials and Methods. Images were taken by the HoloMonitor M3 instrument using phase contrast X10 objective. Scale bar: 150 µm. Full article ">Figure 2
Proliferation rate, viability and apoptosis of A549 cells under different culture conditions. (A) Cells grow slowly in 3D culture than in 2D cultures on culture day 4 (3D spehorids p ≤ 0.0001; RAFT, 3D Cytodex3 and 3D Nutrisphere p ≤ 0.01), but on day 9 only the 3D Spheroid (p ≤ 0.01) and 3D Cytodex3 (p ≤ 0.05) showed significantly lower cell numbers than 2D cultures. (n = 3, paired t-test). The viability of cells (AnnV−/PI− living population) remained around 80–90% at day 4 (B) and day 9 (C), only RAFT culturing resulted in 50% decrease in viability at day 9. Insert shows representative dot plots of single cells for SSC-FSC (Side scatter-Forward scatter, B left insert) and for quadrants detecting living cells (AnnV−/PI−), early apoptotic cells (AnnV+/PI-), late apoptotic cells (AnnV+/PI+) and necrotic cells (AnnV−/PI+) (B right insert). Data are mean ± SD of three replicates. Full article ">Figure 3
Cell cycle phase distribution of A549 cells cultured under different conditions at the 4th (A) and the 9th culturing days (B). Insert shows PI signal of single cells FL2-A (area)-FL2-W (width) gating out aggregates (A left insert) and a representative image of cell cycle phases (A right insert). Data are mean ± SD of three replicates except RAFT where 12 collagen discoids were pooled. Full article ">Figure 4
Gene expression changes after 4 (d4) and 9 days (d9) of culture under different 2D and 3D conditions. Selected genes having cell surface localized protein product and well characterized antibody available on the market: TMEM5A, SLC16A3 (MCT4), CEACAM5, SLC2A1 (GLUT1), and CA9 showed 16–32 times overexpression in 3D Cytodex3 and 3D Nutrisphere compared to 2D monolayer. Data are mean ± SD of three replicates. Full article ">Figure 5
Long-term (day 9, d9) maintenance of 3D cultures (3D Spheroid, 3D Nutrisphere, 3D Cytodex3) mimic the in vivo situation better. The highlighted genes (black squares) were selected for subsequent analysis by single cell mass cytometry. Cluster analysis (hierarchical) was performed by Gene Cluster 3.0 software from expression data (ΔΔCt, log2 values) of presented samples. Full article ">Figure 6
Representative multidimensional visualization of stochastic neighbor embedding (viSNE) analysis of 12 protein markers at single cell resolution in 2D, 3D (3D Cytodex3 or 3D Nutrisphere) cultures and in vivo (early or late stage) tumors. The analysis was performed within 4.5 × 104 HLA-A,B,C positive cells in case of all conditions in order to identify human A549 cells in the xenografts and exclude murine stromal cells. (iterations = 1000, perplexity = 30, theta = 0.5). Full article ">Figure 7
Merging viSNE graphs of multiparametric single cell mass cytometry data (12 parameters) of 2D, 3D and in vivo samples delineates a map with three different islands of 2D, 3D, and in vivo conditions. Full article ">Figure 8
(A) Trajectories tend to localize to early and late stage tumors in the case of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, GAL-3, CD66, and GLUT1. Percentage of HLA-A,B,C positive cells (A549) from a representative experiment were plotted on trajectories among five different conditions: (I) 2D monolayer, (II) 3D Cytodex3, (III) 3D Nutrisphere, (IV) early and (V) late stage in vivo adenocarcinoma. (B) Heatmap of mass cytometry data regarding protein density at single cell resolution among the five different conditions normalized to 2D monolayer (green: low, red: high expression). Full article ">

13 pages, 6062 KiB

Open AccessArticle

Expression of FGF8, FGF18, and FGFR4 in Gastroesophageal Adenocarcinomas

by Gerd Jomrich, Xenia Hudec, Felix Harpain, Daniel Winkler, Gerald Timelthaler, Thomas Mohr, Brigitte Marian and Sebastian F. Schoppmann

Cells 2019, 8(9), 1092; https://doi.org/10.3390/cells8091092 - 16 Sep 2019

Cited by 18 | Viewed by 4106

Abstract

Even though distinctive advances in the field of esophageal cancer therapy have occurred over the last few years, patients’ survival rates remain poor. FGF8, FGF18, and FGFR4 have been identified as promising biomarkers in a number of cancers; however no data exist on [...] Read more.

Even though distinctive advances in the field of esophageal cancer therapy have occurred over the last few years, patients’ survival rates remain poor. FGF8, FGF18, and FGFR4 have been identified as promising biomarkers in a number of cancers; however no data exist on expression of FGF8, FGF18, and FGFR4 in adenocarcinomas of the esophago-gastric junction (AEG). A preliminary analysis of the Cancer Genome Atlas (TCGA) database on FGF8, FGF18, and FGFR4 mRNA expression data of patients with AEG was performed. Furthermore, protein levels of FGF8, FGF18, and FGFR4 in diagnostic biopsies and post-operative specimens in neoadjuvantly treated and primarily resected patients using immunohistochemistry were investigated. A total of 242 patients was analyzed in this study: 87 patients were investigated in the TCGA data set analysis and 155 patients in the analysis of protein expression using immunohistochemistry. High protein levels of FGF8, FGF18, and FGFR4 were detected in 94 (60.7%), 49 (31.6%) and 84 (54.2%) patients, respectively. Multivariable Cox proportional hazard regression models revealed that high expression of FGF8 was an independent prognostic factor for diminished overall survival for all patients and for neoadjuvantly treated patients. By contrast, FGF18 overexpression was significantly associated with longer survival rates in neoadjuvantly treated patients. In addition, FGF8 protein level correlated with Mandard regression due to neoadjuvant therapy, indicating potential as a predictive marker. In summary, FGF8 and FGF18 are promising candidates for prognostic factors in adenocarcinomas of the esophago-gastric junction and new potential targets for new anti-cancer therapies. Full article

(This article belongs to the Special Issue Fibroblast Growth Factor Receptor (FGFR) Signaling Pathway in Tumor)

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14 pages, 1781 KiB

Open AccessFeature PaperArticle

Expression of FGFR1–4 in Malignant Pleural Mesothelioma Tissue and Corresponding Cell Lines and its Relationship to Patient Survival and FGFR Inhibitor Sensitivity

by Gregor Vlacic, Mir A. Hoda, Thomas Klikovits, Katharina Sinn, Elisabeth Gschwandtner, Katja Mohorcic, Karin Schelch, Christine Pirker, Barbara Peter-Vörösmarty, Jelena Brankovic, Balazs Dome, Viktoria Laszlo, Tanja Cufer, Ales Rozman, Walter Klepetko, Bettina Grasl-Kraupp, Balazs Hegedus, Walter Berger, Izidor Kern and Michael Grusch

Cells 2019, 8(9), 1091; https://doi.org/10.3390/cells8091091 - 16 Sep 2019

Cited by 10 | Viewed by 4009

Abstract

Malignant pleural mesothelioma (MPM) is a devastating malignancy with limited therapeutic options. Fibroblast growth factor receptors (FGFR) and their ligands were shown to contribute to MPM aggressiveness and it was suggested that subgroups of MPM patients could benefit from FGFR-targeted inhibitors. In the [...] Read more.

Malignant pleural mesothelioma (MPM) is a devastating malignancy with limited therapeutic options. Fibroblast growth factor receptors (FGFR) and their ligands were shown to contribute to MPM aggressiveness and it was suggested that subgroups of MPM patients could benefit from FGFR-targeted inhibitors. In the current investigation, we determined the expression of all four FGFRs (FGFR1–FGFR4) by immunohistochemistry in tissue samples from 94 MPM patients. From 13 of these patients, we were able to establish stable cell lines, which were subjected to FGFR1–4 staining, transcript analysis by quantitative RT-PCR, and treatment with the FGFR inhibitor infigratinib. While FGFR1 and FGFR2 were widely expressed in MPM tissue and cell lines, FGFR3 and FGFR4 showed more restricted expression. FGFR1 and FGFR2 showed no correlation with clinicopathologic data or patient survival, but presence of FGFR3 in 42% and of FGFR4 in 7% of patients correlated with shorter overall survival. Immunostaining in cell lines was more homogenous than in the corresponding tissue samples. Neither transcript nor protein expression of FGFR1–4 correlated with response to infigratinib treatment in MPM cell lines. We conclude that FGFR3 and FGFR4, but not FGFR1 or FGFR2, have prognostic significance in MPM and that FGFR expression is not sufficient to predict FGFR inhibitor response in MPM cell lines. Full article

(This article belongs to the Special Issue Fibroblast Growth Factor Receptor (FGFR) Signaling Pathway in Tumor)

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Figure 1
Expression of FGFR1–4 in MPM tissue. (A) Representative images of MPM tissue specimens stained for FGFR1 (score 2, epithelioid), FGFR2 (score 2, epithelioid), FGFR3 (score 1, epithelioid), and FGFR4 (score 1, epithelioid). Scale bar = 25 µm. (B) Distribution of staining intensities of FGFR1–4 in 94 MPM tissue specimens. Full article ">Figure 2
Correlation of FGFR expression with histology and patient prognosis. (A) Percentage of epithelioid, biphasic, and sarcomatoid tumors within the different staining groups of FGFR1 (upper panel), FGFR3 (middle panel), and FGFR4 (lower panel). (B) Kaplan–Meier curves for overall survival of MPM patients with different staining scores of FGFR1 (upper panels), FGFR3 (middle panel), and FGFR4 (lower panel). Full article ">Figure 3
Sensitivity of patient-derived cell lines to the FGFR inhibitor infigratinib. (A) MPM cell lines were incubated with increasing concentrations of infigratinib or vehicle (DMSO) as control and cell number was determined after 72 h. Dose–response curves were calculated with GraphPad Prism. Three sensitive cell lines (IC50 < 1 µM), one intermediate cell line (1 µM < IC50 < 10 µM), and three resistant cell lines (IC50 > 10 µM) are shown in green, black, and red, respectively. (B) Infigratinib IC50 values of the cell lines were plotted against the IHC scores for FGFR1, FGFR3, or the sum of FGFR1–3 of the corresponding tumors from which the cell lines were established. Full article ">Figure 4
Expression of FGFR1–4 in patient-derived cell lines. (A) Representative images from cell blocks stained for FGFR1 (Meso208), FGFR2 (Meso161), FGFR3 (VMC45), and FGFR4 (Meso205). Scale bar = 100 µm in overview images and 10 µm in high magnification insets. (B) Total mRNA was isolated from logarithmically growing cell lines and subjected to cDNA synthesis. Quantitative RT-PCR was performed by Taqman assays for FGFR1–4. Expression levels were plotted as 2−dCt × 105 normalized to the house-keeping genes GAPDH and beta-actin. (C) Infigratinib IC50 values were plotted as function of FGFR1 mRNA expression level. (D) BLU9931 IC50 values were plotted as function of FGFR4 mRNA expression levels. Full article ">

20 pages, 886 KiB

Open AccessReview

Therapeutic Approaches Targeting Nucleolus in Cancer

by Pietro Carotenuto, Annalisa Pecoraro, Gaetano Palma, Giulia Russo and Annapina Russo

Cells 2019, 8(9), 1090; https://doi.org/10.3390/cells8091090 - 16 Sep 2019

Cited by 60 | Viewed by 6007

Abstract

The nucleolus is a distinct sub-cellular compartment structure in the nucleus. First observed more than 200 years ago, the nucleolus is detectable by microscopy in eukaryotic cells and visible during the interphase as a sub-nuclear structure immersed in the nucleoplasm, from which it [...] Read more.

The nucleolus is a distinct sub-cellular compartment structure in the nucleus. First observed more than 200 years ago, the nucleolus is detectable by microscopy in eukaryotic cells and visible during the interphase as a sub-nuclear structure immersed in the nucleoplasm, from which it is not separated from any membrane. A huge number of studies, spanning over a century, have identified ribosome biogenesis as the main function of the nucleolus. Recently, novel functions, independent from ribosome biogenesis, have been proposed by several proteomic, genomic, and functional studies. Several works have confirmed the non-canonical role for nucleoli in regulating important cellular processes including genome stability, cell-cycle control, the cellular senescence, stress responses, and biogenesis of ribonucleoprotein particles (RNPs). Many authors have shown that both canonical and non-canonical functions of the nucleolus are associated with several cancer-related processes. The association between the nucleolus and cancer, first proposed by cytological and histopathological studies showing that the number and shape of nucleoli are commonly altered in almost any type of cancer, has been confirmed at the molecular level by several authors who demonstrated that numerous mechanisms occurring in the nucleolus are altered in tumors. Recently, therapeutic approaches targeting the nucleolus in cancer have started to be considered as an emerging “hallmark” of cancer and several therapeutic interventions have been developed. This review proposes an up-to-date overview of available strategies targeting the nucleolus, focusing on novel targeted therapeutic approaches. Finally, a target-based classification of currently available treatment will be proposed. Full article

(This article belongs to the Special Issue Nucleolar Organization and Functions in Health and Disease)

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27 pages, 5139 KiB

Open AccessArticle

Fisetin, a 3,7,3′,4′-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models

by Jean Christopher Chamcheu, Stephane Esnault, Vaqar M. Adhami, Andrea L. Noll, Sergette Banang-Mbeumi, Tithi Roy, Sitanshu S. Singh, Shile Huang, Konstantin G. Kousoulas and Hasan Mukhtar

Cells 2019, 8(9), 1089; https://doi.org/10.3390/cells8091089 - 15 Sep 2019

Cited by 59 | Viewed by 9179

Abstract

Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources [...] Read more.

Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources of therapeutic molecules, which can repair the molecular defects associated with psoriasis and could possibly be developed for its management. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a phytochemical naturally found in pigmented fruits and vegetables, has demonstrated proapoptotic and antioxidant effects in several malignancies. This study utilized biochemical, cellular, pharmacological, and tissue engineering tools to characterize the effects of fisetin on normal human epidermal keratinocytes (NHEKs), peripheral blood mononuclear cells (PBMC), and CD4+ T lymphocytes in 2D and 3D psoriasis-like disease models. Fisetin treatment of NHEKs dose- and time-dependently induced differentiation and inhibited interleukin-22-induced proliferation, as well as activation of the PI3K/Akt/mTOR pathway. Fisetin treatment of TNF-α stimulated NHEKs also significantly inhibited the activation of p38 and JNK, but had enhanced effect on ERK1/2 (MAPK). In addition, fisetin treatment significantly decreased the secretion of Th1/Th-17 pro-inflammatory cytokines, particularly IFN-γ and IL-17A by 12-O-tetradecanolylphorbol 13-acetate (TPA)-stimulated NHEKs and anti-CD3/CD28-activated human PBMCs. Furthermore, we established the in vivo relevance of fisetin functions, using a 3D full-thickness human skin model of psoriasis (FTRHSP) that closely mimics in vivo human psoriatic skin lesions. Herein, fisetin significantly ameliorated psoriasis-like disease features, and decreased the production of IL-17 by CD4+ T lymphocytes co-cultured with FTRHSP. Collectively, our data identify the prodifferentiative, antiproliferative, and anti-inflammatory effects of fisetin, via modulation of the PI3K-Akt-mTOR and p38/JNK pathways and the production of cytokines in 2D and 3D human skin models of psoriasis. These results suggest that fisetin has a great potential to be developed as an effective and inexpensive agent for the treatment of psoriasis and other related inflammatory skin disorders. Full article

(This article belongs to the Special Issue Advances in Human Cell Culture Techniques Towards more Physiological, Differentiated Tissues)

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21 pages, 1518 KiB

Open AccessReview

ER-Mitochondria Communication in Cells of the Innate Immune System

by Dmitry Namgaladze, Vera Khodzhaeva and Bernhard Brüne

Cells 2019, 8(9), 1088; https://doi.org/10.3390/cells8091088 - 15 Sep 2019

Cited by 41 | Viewed by 11470

Abstract

In cells the interorganelle communication comprises vesicular and non-vesicular mechanisms. Non-vesicular material transfer predominantly takes place at regions of close organelle apposition termed membrane contact sites and is facilitated by a growing number of specialized proteins. Contacts of the endoplasmic reticulum (ER) and [...] Read more.

In cells the interorganelle communication comprises vesicular and non-vesicular mechanisms. Non-vesicular material transfer predominantly takes place at regions of close organelle apposition termed membrane contact sites and is facilitated by a growing number of specialized proteins. Contacts of the endoplasmic reticulum (ER) and mitochondria are now recognized to be essential for diverse biological processes such as calcium homeostasis, phospholipid biosynthesis, apoptosis, and autophagy. In addition to these universal roles, ER-mitochondria communication serves also cell type-specific functions. In this review, we summarize the current knowledge on ER-mitochondria contacts in cells of the innate immune system, especially in macrophages. We discuss ER- mitochondria communication in the context of macrophage fatty acid metabolism linked to inflammatory and ER stress responses, its roles in apoptotic cell engulfment, activation of the inflammasome, and antiviral defense. Full article

(This article belongs to the Special Issue Membrane Contact Sites with Mitochondria: Molecular Determinants and Pathophysiological Role)

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Endoplasmic reticulum (ER)-mitochondrial regulation of lipid overload-induced stress responses. Excessive incorporation of saturated fatty acids (SFA) into ER phospholipids or accumulation of free cholesterol in the ER induce ER stress response mediated by activating transcription factor 6 (ATF6), protein kinase RNA-activated (PKR)-like ER kinase (PERK) and inositol requiring enzyme 1 (IRE1) sensor proteins. Concomitantly, SFA undergo fatty acid β-oxidation (FAO), which attenuates SFA incorporation into phosphatidylserine (PS) and phosphatidylethanolamine (PE) and dampens ER stress. Furthermore, fatty acids induce Drp1-dependent mitochondrial fragmentation, which attenuates mitochondrial ROS formation and activation of pro-inflammatory c-Jun N-terminal kinase (JNK) signaling. ER stress also provokes inositol triphosphate receptors (IP3R) activation and mitochondrial Ca2+ overload. Under ER stress conditions, Ca2+/calmodulin-dependent protein kinase IIγ (CAMKIIγ) may translocate to mitochondria and undergo Ca2+- and ROS-dependent activation, promoting apoptosis. CPT: Carnitine palmitoyltransferase; PA: Phosphatidic acid; PSS: Phosphatidylserine synthase; PSD: Phosphatidylserine decarboxylase. Full article ">Figure 2
ER-mitochondrial communication in NOD-like receptor protein 3 (NLRP3) inflammasome regulation. NLRP3 inflammasome activation requires two signals; a priming signal (e.g. toll-like receptor (TLR) activation) induces the expression of pro-IL-1β and NLRP3, which occurs in a mROS-dependent manner. Priming signals also induce mROS-dependent cardiolipin translocation to the outer mitochondrial membrane (OMM), where it recruits NLRP3 and caspase-1. An activation signal via e.g. P2X7 receptor ligation promotes K+ efflux, mitochondrial Ca2+ increase, mROS elevation and release of oxidized mitochondrial DNA (mtDNA). These events contribute to the recruitment of ASC to NLRP3 and caspase-1 and formation of the activated oligomeric inflammasome complex, which cleaves and activates caspase-1, followed by the subsequent cleavage and secretion of interleukin (IL)-1β. Mitochondria-associated membranes (MAM) provide the platform for inflammasome assembly, which may be facilitated by the interaction of NLRP3, mitochondrial antiviral-signaling protein (MAVS) and mitofusin 2 (Mfn2). Movements of mitochondria and ER, driven by a loss of NAD+, SIRT2 inactivation, and tubulin acetylation may also facilitate apposition of NLRP3 and ASC. ER stress activates the NLRP3 inflammasome through IRE1α. Mitophagy may eliminate mROS-producing mitochondria and thus, dampen NLRP3 inflammasome activation. Full article ">Figure 3
Communication of ER and mitochondrial in sensing cytosolic nucleic acids. The response to RNA viruses is orchestrated by the sensor retinoic acid inducible gene I (RIG-I), which upon dsRNA recognition translocates to mitochondria- and the MAM-located adaptor MAVS to activate the interferon regulatory factor 3 (IRF3) transcription factor. MAVS may be negatively regulated by their interaction with Mfn2 and Gp78. Sensing of cytosolic DNA involves activation of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) cascade, whereas activated STING translocates from the ER to Golgi to activate IRF3. The cross-talk between DNA and RNA sensing may involve direct interaction of STING and MAVS. cGAS may also sense mtDNA following mitochondrial damage by translocating to mitochondria. Full article ">

20 pages, 3922 KiB

Open AccessArticle

The Anti-Apoptotic Effect of ASC-Exosomes in an In Vitro ALS Model and Their Proteomic Analysis

by Roberta Bonafede, Jessica Brandi, Marcello Manfredi, Ilaria Scambi, Lorenzo Schiaffino, Flavia Merigo, Ermanna Turano, Bruno Bonetti, Emilio Marengo, Daniela Cecconi and Raffaella Mariotti

Cells 2019, 8(9), 1087; https://doi.org/10.3390/cells8091087 - 14 Sep 2019

Cited by 66 | Viewed by 6521

Abstract

Stem cell therapy represents a promising approach in the treatment of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The beneficial effect of stem cells is exerted by paracrine mediators, as exosomes, suggesting a possible potential use of these extracellular vesicles as non-cell [...] Read more.

Stem cell therapy represents a promising approach in the treatment of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The beneficial effect of stem cells is exerted by paracrine mediators, as exosomes, suggesting a possible potential use of these extracellular vesicles as non-cell based therapy. We demonstrated that exosomes isolated from adipose stem cells (ASC) display a neuroprotective role in an in vitro model of ALS. Moreover, the internalization of ASC-exosomes by the cells was shown and the molecules and the mechanisms by which exosomes could exert their beneficial effect were addressed. We performed for the first time a comprehensive proteomic analysis of exosomes derived from murine ASC. We identified a total of 189 proteins and the shotgun proteomics analysis revealed that the exosomal proteins are mainly involved in cell adhesion and negative regulation of the apoptotic process. We correlated the protein content to the anti-apoptotic effect of exosomes observing a downregulation of pro-apoptotic proteins Bax and cleaved caspase-3 and upregulation of anti-apoptotic protein Bcl-2 α, in an in vitro model of ALS after cell treatment with exosomes. Overall, this study shows the neuroprotective effect of ASC-exosomes after their internalization and their global protein profile, that could be useful to understand how exosomes act, demonstrating that they can be employed as therapy in neurodegenerative diseases. Full article

(This article belongs to the Section Intracellular and Plasma Membranes)

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15 pages, 1410 KiB

Open AccessArticle

Regulation and Function of C-Type Natriuretic Peptide (CNP) in Gonadotrope-Derived Cell Lines

by Samantha M Mirczuk, Andrew J Lessey, Alice R Catterick, Rebecca M Perrett, Christopher J Scudder, Jordan E Read, Victoria J Lipscomb, Stijn J Niessen, Andrew J Childs, Craig A McArdle, Imelda M McGonnell and Robert C Fowkes

Cells 2019, 8(9), 1086; https://doi.org/10.3390/cells8091086 - 14 Sep 2019

Cited by 9 | Viewed by 4139

Abstract

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and [...] Read more.

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LβT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc, Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LβT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun, Egr1, Nr5a1 and Nr0b1, within 8 h in LβT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function. Full article

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Figure 1
Expression profiling of the natriuretic peptide system in primary mouse endocrine tissues. (A) Murine mRNA sequences obtained from NCBI Nucleotide (<a href="https://www.ncbi.nlm.nih.gov/nuccore" target="_blank">https://www.ncbi.nlm.nih.gov/nuccore</a>), were imported into express Designer Software (Beckman Coulter), from which multiplex primers were designed using the following parameters: maximum PCR product = 300 nt, minimum PCR product = 100 nt, minimum separation size = 7 nt. Multiplex PCR reactions were performed, using specific primers for Nppa, Nppb, Nppc, Npr1, Npr2, Npr3, Furin, Corin, ActB, Gapdh and Rpl19 and an internal positive control KanR. As shown (<a href="#cells-08-01086-f001" class="html-fig">Figure 1</a>A), capillary gel electrophoresis was used to separate specific PCR products (blue peaks), and compared alongside the appropriate size standard (red peaks, 140–420 nt). (B) RNA was isolated from range of tissues from 12 week old male and female C57/B6 mice (heart, adrenal, adipose, brain, pituitary, kidney, liver, testis and ovaries; n = 5 to 8). Data shown are means (n = 5 to 8) of relative gene expression (normalized to ActB; red indicates low level expression, green indicates high level expression, white indicates no transcript detected). Full article ">Figure 2
Natriuretic peptide expression profile in gonadotrope-derived cell lines. mRNA expression of natriuretic peptide components in untreated αT3-1 and LβT2 cells (Nppa and Nppb were not detected). Data shown are means ± SEM (n = 5 individual RNA extractions) of relative gene expression (normalized to ActB). Full article ">Figure 3
Effect of continuous or pulsatile GnRH stimulation on natriuretic peptide gene expression in LβT2 and αT3-1 cell lines. (A) Schematic of pulsatile or continuous GnRH treatment protocol, including wash periods. LβT2 cells (B) or αT3-1 cells (C) were treated with 0 or 100 nM GnRH, for either 4 h continuously, or as 5 min pulses every hour for 4 h, before extracting RNA and performing multiplex RT-qPCR to examine alterations in gene expression profiling of natriuretic peptide system (Nppc, Furin, Corin, Npr1, Npr2, Npr3). Data shown are means ± SEM (n = 6 to 9 individual RNA extractions) of relative gene expression (normalized to ActB); *** p < 0.001, ** p < 0.01, significantly different from basal, or continuous vs pulsatile, as indicated). Full article ">Figure 4
Effect of continuous or pulsatile GnRH stimulation on gonadotrope transcription factor gene expression in LβT2 and αT3-1 cell lines. (A) Comparison of gonadotrope transcription factor expression (cFos, cJun, Egr1, Nr5a1, Nr0b1) in LβT2 and αT3-1 cells. Data shown are means (n = 5 to 8 individual RNA extractions) of relative gene expression (normalized to ActB; red indicates low level expression, green indicates high level expression. (B,C) LβT2 cells (B) or αT3-1 cells (C) were treated with 0 or 100 nM GnRH, for either 4 h continuously, or as 5 min pulses every hour for 4 h, before extracting RNA and performing multiplex RT-qPCR for gonadotrope transcription factors. Data shown are means ± SEM (n = 6 to 9 individual RNA extractions) of relative gene expression (normalized to ActB); *** p < 0.001, ** p < 0.01, * p < 0.05, significantly different from basal, or continuous vs pulsatile, as indicated). Full article ">Figure 5
Effect of CNP on gonadotrope transcription factor gene expression in LβT2 and αT3-1 cells. LβT2 (A) and αT3-1 (B) cells were stimulated with 0 or 100 nM CNP for 0, 4, 8, and 24 h, before extracting RNA and performing multiplex RT-qPCR for gonadotrope transcription factors. Data shown are means ± SEM (n = 6 individual RNA extractions) of relative gene expression (normalized to ActB); **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, significantly different from basal (0 h). Full article ">

12 pages, 1741 KiB

Open AccessArticle

Empagliflozin Protects HK-2 Cells from High Glucose-Mediated Injuries via a Mitochondrial Mechanism

by Wen-Chin Lee, You-Ying Chau, Hwee-Yeong Ng, Chiu-Hua Chen, Pei-Wen Wang, Chia-Wei Liou, Tsu-Kung Lin and Jin-Bor Chen

Cells 2019, 8(9), 1085; https://doi.org/10.3390/cells8091085 - 14 Sep 2019

Cited by 53 | Viewed by 10926

Abstract

Empagliflozin is known to retard the progression of kidney disease in diabetic patients. However, the underlying mechanism is incompletely understood. High glucose induces oxidative stress in renal tubules, eventually leading to mitochondrial damage. Here, we investigated whether empagliflozin exhibits protective functions in renal [...] Read more.

Empagliflozin is known to retard the progression of kidney disease in diabetic patients. However, the underlying mechanism is incompletely understood. High glucose induces oxidative stress in renal tubules, eventually leading to mitochondrial damage. Here, we investigated whether empagliflozin exhibits protective functions in renal tubules via a mitochondrial mechanism. We used human proximal tubular cell (PTC) line HK-2 and employed western blotting, terminal deoxynucleotidyl transferase dUTP nick end labelling assay, fluorescence staining, flow cytometry, and enzyme-linked immunosorbent assay to investigate the impact of high glucose and empagliflozin on cellular apoptosis, mitochondrial morphology, and functions including mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and adenosine triphosphate (ATP) generation. We found that PTCs were susceptible to high glucose-induced mitochondrial fragmentation, and empagliflozin ameliorated this effect via the regulation of mitochondrial fission (FIS1 and DRP1) and fusion (MFN1 and MFN2) proteins. Empagliflozin reduced the high glucose-induced cellular apoptosis and improved mitochondrial functions by restoring mitochondrial ROS production, MMP, and ATP generation. Our results suggest that empagliflozin may protect renal PTCs from high glucose-mediated injuries through a mitochondrial mechanism. This could be one of the novel mechanisms explaining the benefits demonstrated in EMPA-REG OUTCOME trial. Full article

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Empagliflozin has no negative effects on the viability of HK-2 cells. Cell viability was measured with crystal violet assay and the absorbance was analyzed at 570 nm using a microplate reader. Data were obtained from three independent experiments and are expressed as mean ± SEM. Only high glucose (30 mM) treatment led to a decrease in cell viability. * P < 0.05 versus the normal glucose (5 mM) treatment group. Glu, glucose; Empa, empagliflozin. Full article ">Figure 2
Empagliflozin rescues high glucose-induced mitochondrial fragmentation in human PTCs. HK-2 cells were cultured in 5 mM glucose (A), 30 mM glucose (B), 30 mM glucose with 100 nM empagliflozin (C), and 30 mM glucose with 500 nM empagliflozin (D). Mitochondria were stained with Mitotracker red. Magnified images of indexed mitochondria (arrow) in each treatment group are shown in (E–H). (I) The mitochondrial fission rate was higher in high glucose condition. Empagliflozin rescued this effect. Data were obtained from three independent experiments and are expressed as mean ± SEM. * P < 0.05, ** P < 0.001. Full article ">Figure 3
Impact of empagliflozin on high glucose-induced alterations in the expression levels of mitochondrial fusion/fission proteins. (A–E) The expression of MFN1, MFN2, DRP1, and FIS1 was analyzed with western blotting and normalized to the level of β-actin. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.001. Full article ">Figure 4
Empagliflozin reduces the high glucose-induced apoptosis of HK-2 cells. (A–D) Fluorescence images show positive TUNEL staining in the four treatment groups. (E) Quantitative analysis of TUNEL assay shows that high glucose markedly induces apoptosis and empagliflozin ameliorates this effect. Data are expressed as mean ± SEM. ** P < 0.001. Full article ">Figure 5
Empagliflozin improves the mitochondrial function of high glucose-treated HK-2 cells. (A) Cellular ROS production, (B) mitochondrial ROS production, (C) MMP, and (D) ATP generation in the four treatment groups. Data were obtained from three independent experiments and are expressed as mean ± SEM. * P < 0.05, ** P < 0.001. Full article ">

12 pages, 2074 KiB

Open AccessArticle

Effect of Adenomatous Polyposis Coli Loss on Tumorigenic Potential in Pancreatic Ductal Adenocarcinoma

by Jennifer M. Cole, Kaitlyn Simmons and Jenifer R. Prosperi

Cells 2019, 8(9), 1084; https://doi.org/10.3390/cells8091084 - 14 Sep 2019

Cited by 9 | Viewed by 3926

Abstract

Loss of the Adenomatous Polyposis Coli (APC) tumor suppressor in colorectal cancer elicits rapid signaling through the Wnt/β-catenin signaling pathway. In contrast to this well-established role of APC, recent studies from our laboratory demonstrated that APC functions through Wnt-independent pathways to [...] Read more.

Loss of the Adenomatous Polyposis Coli (APC) tumor suppressor in colorectal cancer elicits rapid signaling through the Wnt/β-catenin signaling pathway. In contrast to this well-established role of APC, recent studies from our laboratory demonstrated that APC functions through Wnt-independent pathways to mediate in vitro and in vivo models of breast tumorigenesis. Pancreatic ductal adenocarcinoma (PDAC) has an overall median survival of less than one year with a 5-year survival rate of 7.2%. APC is lost in a subset of pancreatic cancers, but the impact on Wnt signaling or tumor development is unclear. Given the lack of effective treatment strategies for pancreatic cancer, it is important to understand the functional implications of APC loss in pancreatic cancer cell lines. Therefore, the goal of this project is to study how APC loss affects Wnt pathway activation and in vitro tumor phenotypes. Using lentiviral shRNA, we successfully knocked down APC expression in six pancreatic cancer cell lines (AsPC-1, BxPC3, L3.6pl, HPAF-II, Hs 766T, MIA PaCa-2). No changes were observed in localization of β-catenin or reporter assays to assess β-catenin/TCF interaction. Despite this lack of Wnt/β-catenin pathway activation, the majority of APC knockdown cell lines exhibit an increase in cell proliferation. Cell migration assays showed that the BxPC-3 and L3.6pl cells were impacted by APC knockdown, showing faster wound healing in scratch wound assays. Interestingly, APC knockdown had no effect on gemcitabine treatment, which is the standard care for pancreatic cancer. It is important to understand the functional implications of APC loss in pancreatic cancer cells lines, which could be used as a target for therapeutics. Full article

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Figure 1
Quantitative PCR demonstrates that APC is knocked down in the cell lines transfected with APC shRNA constructs. Using lentiviral-mediated MISSION shRNA we verified APC knock down in six pancreatic ductal adenocarcinoma cell lines using quantitative PCR and relative expression of APC revealed gene knockdown in AsPC-1, BxPC-3, HPAF-II, Hs 766T, L3.6pl, and MIA PaCa-2 cell lines compared to CTL (<a href="#cells-08-01084-f001" class="html-fig">Figure 1</a>A–F). *p < 0.05. Full article ">Figure 2
APC knockdown did not increase nuclear β-catenin. (A) In AsPC-1, BxPC-3, HPAF-II, Hs 766T, L3.6pl and MIA PaCa-2 CTL cells, β-catenin is localized at the cell–cell junctions. After the knockdown of APC in each of those cell lines: APC shRNA (representative), there were no changes in beta catenin localization. (B) In the APC knockdown PDAC cell lines, we observed no alterations in Wnt/β-catenin signaling using β-catenin/TCF reporter assays. Full article ">Figure 3
Proliferation changes are observed in some cell lines that have been transfected with APC shRNA constructs compared to controls. Proliferation was measured in total cell counts at 24, 48 and 72 h post-plating for each cell line. (B and E) In the BxPC-3 and L3.6pl cells, knockdown of APC resulted in a significant increase in cell proliferation at 48 h. (D) In the Hs 766T cells, knockdown of APC resulted in a significant increase in cell proliferation at 48 and 72 h. (A, C, and F) The AsPC-1, HPAF-II and MIA PaCa-2 cell lines showed no significant differences in cell proliferation of the APC shRNA cells lines compared to CTL. *p < 0.05. Full article ">Figure 4
Migration of cells in wound healing assays revealed faster migration with APC knockdown in some pancreatic cancer cell lines. (A) The AsPC-1 APC shRNA2 knockdown cells migrated slower than the control at 24 and 48 h post wounding. (B and E) Only the BxPC-3 and L3.6pl cells were impacted by APC knockdown, showing faster wound healing. Specifically, at 48 h post-wounding, the APC shRNA1 and APC shRNA2 cell lines were significantly more closed than the parent BxPC-3 cells. (E) In L3.6pl, there were significant differences in percent filled area in APC shRNA1 and APC shRNA2 at 24 h and APC shRNA2 at 48 h compared to CTL. (A, C, D, and F) In AsPC-1, HPAF-II, Hs766T, and MIA PaCa-2 cells, there were no significant differences in percent filled area in APC shRNA cells versus CTL. *p < 0.05. Full article ">Figure 5
Effect of gemcitabine on PDAC cell lines. (A) In AsPC-1 cells, we saw significant decreases in total cell numbers in controls and APC shRNA1, but not in APC shRNA2 when treated with 1 µM gemcitabine for 48 h. (B) In BxPC-3 cells, 1 µM gemcitabine decreased total cell numbers in all cell lines. (C) In HPAF-II cells, 1 µM gemcitabine decreased total cell numbers in APC shRNA1, and APC shRNA2, but not in the CTL. (D) In Hs 766T cells, 48 h treatment with 1 µM gemcitabine resulted in significant decreases in total cell numbers in controls and APC shRNA1, but not in APC shRNA2. (E) In L3.6pl cells, 1 µM gemcitabine decreased total cell numbers in CTL APC shRNA1, and APC shRNA2. (F) In MIA PaCa-2 cells, 1 µM gemcitabine decreased total cell numbers in CTL, APC shRNA1, and APC shRNA2. *p < 0.05. Full article ">

13 pages, 902 KiB

Open AccessFeature PaperReview

Improving Cancer Immunotherapy by Targeting the Hypoxic Tumor Microenvironment: New Opportunities and Challenges

by Muhammad Zaeem Noman, Meriem Hasmim, Audrey Lequeux, Malina Xiao, Caroline Duhem, Salem Chouaib, Guy Berchem and Bassam Janji

Cells 2019, 8(9), 1083; https://doi.org/10.3390/cells8091083 - 14 Sep 2019

Cited by 161 | Viewed by 10149

Abstract

Initially believed to be a disease of deregulated cellular and genetic expression, cancer is now also considered a disease of the tumor microenvironment. Over the past two decades, significant and rapid progress has been made to understand the complexity of the tumor microenvironment [...] Read more.

Initially believed to be a disease of deregulated cellular and genetic expression, cancer is now also considered a disease of the tumor microenvironment. Over the past two decades, significant and rapid progress has been made to understand the complexity of the tumor microenvironment and its contribution to shaping the response to various anti-cancer therapies, including immunotherapy. Nevertheless, it has become clear that the tumor microenvironment is one of the main hallmarks of cancer. Therefore, a major challenge is to identify key druggable factors and pathways in the tumor microenvironment that can be manipulated to improve the efficacy of current cancer therapies. Among the different tumor microenvironmental factors, this review will focus on hypoxia as a key process that evolved in the tumor microenvironment. We will briefly describe our current understanding of the molecular mechanisms by which hypoxia negatively affects tumor immunity and shapes the anti-tumor immune response. We believe that such understanding will provide insight into the therapeutic value of targeting hypoxia and assist in the design of innovative combination approaches to improve the efficacy of current cancer therapies, including immunotherapy. Full article

(This article belongs to the Special Issue Tumor Microenvironment: Interaction and Metabolism)

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The hypoxic tumor microenvironment and its impact on anti-tumor immunity. (A) Hypoxia is established in the tumor microenvironment due to an increase in tumor cell proliferation, and a decrease in oxygen supply. Non-hypoxic tumor regions displayed normal blood vessels covered by well-organized endothelial cells and pericytes. In hypoxic tumor regions, the pressure of oxygen is low which arises from oxygen diffusion limitations due to disorganized, chaotic tumor microvasculature network with leaky vessels. (B) Under hypoxia, the stabilization of hypoxia-inducible factor (HIF)-1α in cells upregulates the expression of PD-L1 in hypoxic tumor cells and PD-L1 and VISTA in hypoxic MDSCs. The increased expression of PD-L1 and VISTA results in an inhibition of T cell proliferation and T cell mediated lysis. (C) HIF-1α is also involved in the upregulation of cluster of differentiation 47 (CD47) on the surface of tumor cells. Following the binding of CD47 to signal regulatory protein α (SIRPα), expressed on the surface of macrophages, tumor cells provide a strong “don’t eat me signal” to block phagocytosis property of macrophages. (D) The activation of autophagy in hypoxic tumor cells impairs tumor cell susceptibility to CTL and NK-mediated lysis by at least two distinct mechanisms involving the degradation of NK-derived Granzyme B and the stabilization of pSTAT3. Other hypoxia-dependent, but autophagy-independent, mechanisms are described in this review including the overexpression of NANOG and miR-210 targeting PTPN1, HOXA1, and TP53I11. (E) Hypoxia upregulates the expression of HLA-G on the surface of tumor cells. The upregulated HLA-G binds to ILT2, ILT4 and KIR2DL4 expressed by several immune cells (B and T cells, NK cells, myelomonocytic cells, dendritic cells, monocytes and macrophages) leading to tumor escape from immune surveillance. Full article ">

20 pages, 4450 KiB

Open AccessArticle

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

by Eunate Gallardo-Vara, Lidia Ruiz-Llorente, Juan Casado-Vela, María J. Ruiz-Rodríguez, Natalia López-Andrés, Asit K. Pattnaik, Miguel Quintanilla and Carmelo Bernabeu

Cells 2019, 8(9), 1082; https://doi.org/10.3390/cells8091082 - 13 Sep 2019

Cited by 25 | Viewed by 6022

Abstract

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin [...] Read more.

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation. Full article

(This article belongs to the Special Issue TGF-beta/BMP Signaling Pathway)

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Figure 1
Protein–protein association between galectin-3 and endoglin. (A–C). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. (A) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin (B) or anti-galectin-3 (C) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b (B) and IgG1 (C) were included. (D) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument. Full article ">Figure 2
Galectin-3 and endoglin co-localize in human endothelial cells. Human umbilical vein-derived endothelial cell (HUVEC) monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed, and incubated with a rabbit polyclonal anti-galectin-3 antibody (PA5-34819). Galectin-3 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. (A) Single staining of galectin-3 (red) and endoglin (green) at the indicated magnifications. (B) Merge images plus DAPI (nuclear staining in blue) show co-localization of galectin-3 and endoglin (yellow color). Representative images of five different experiments are shown. Full article ">Figure 3
Protein–protein association between TRIM21 and endoglin. (A–E) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin (A). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin (B). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (E) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin (C). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included (D). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. (F) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument. Full article ">Figure 4
TRIM21 and endoglin co-localize in human endothelial cells. HUVEC monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed and incubated with a rabbit monoclonal anti-TRIM21 antibody (#92043). TRIM21 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. (A) Single staining of TRIM21 (red) and endoglin (green) at the indicated magnifications. (B) Merge images plus DAPI (nuclear staining in blue) show co-localization of TRIM21 and endoglin (yellow color). Representative images of four different experiments are shown. Full article ">

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Cells, Volume 8, Issue 9 (September 2019) – 170 articles

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