Cells

14 pages, 3059 KiB

Open AccessEditor’s ChoiceArticle

Dental Epithelial Stem Cells as a Source for Mammary Gland Regeneration and Milk Producing Cells In Vivo

by Lucia Jimenez-Rojo, Pierfrancesco Pagella, Hidemitsu Harada and Thimios A. Mitsiadis

Cells 2019, 8(10), 1302; https://doi.org/10.3390/cells8101302 - 22 Oct 2019

Cited by 9 | Viewed by 31879

The continuous growth of rodent incisors is ensured by clusters of mesenchymal and epithelial stem cells that are located at the posterior part of these teeth. Genetic lineage tracing studies have shown that dental epithelial stem cells (DESCs) are able to generate all [...] Read more.

The continuous growth of rodent incisors is ensured by clusters of mesenchymal and epithelial stem cells that are located at the posterior part of these teeth. Genetic lineage tracing studies have shown that dental epithelial stem cells (DESCs) are able to generate all epithelial cell populations within incisors during homeostasis. However, it remains unclear whether these cells have the ability to adopt alternative fates in response to extrinsic factors. Here, we have studied the plasticity of DESCs in the context of mammary gland regeneration. Transplantation of DESCs together with mammary epithelial cells into the mammary stroma resulted in the formation of chimeric ductal epithelial structures in which DESCs adopted all the possible mammary fates including milk-producing alveolar cells. In addition, when transplanted without mammary epithelial cells, DESCs developed branching rudiments and cysts. These in vivo findings demonstrate that when outside their niche, DESCs redirect their fates according to their new microenvironment and thus can contribute to the regeneration of non-dental tissues. Full article

(This article belongs to the Special Issue Stem Cell Therapy in Oral and Maxillofacial Region)

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Figure 1
Injection of DESCs and MECs into mammary fat pads results in the formation of a chimeric ductal epithelium. (A) GFP-DESCs and DsRed-mammary epithelial cells (MECs) were mixed and injected into the mammary fat pads of immunocompromised mice. (B) Before injecting them into the mammary microenvironment, DESCs expressed epithelial markers such as keratin 14 (Krt14) and E-cadherin (E-cad); the dental epithelial stem cell marker Sox2 and the incisor epithelium marker Islet1. (C–K) Whole mount fluorescent imaging of epithelial outgrowths from virgin (C–H) and pregnancy day 16.5 (J,K) chimeric mammary glands. Boxes in (G) and (J) represent the areas of high magnifications in (H) and (K), respectively. Scale bars: 25 μm (B); 2 mm (C,J); 400 μm (D–I,K). Abbreviations: cl, cervical loop; de, dental epithelium; DESCs, dental epithelial stem cells; dm, dental mesenchyme; fp, fat pad; me, mammary epithelium; MECs, mammary epithelial cells. Full article ">Figure 2
DESCs give rise to different cell lineages of mammary epithelium. (A,B) Hematoxylin-eosin staining of the chimeric ducts (A) and schematic representation (B). (C–N) Double immunofluorescence against Krt14 and GFP (C,D,F,G,I,J) and against oestrogen receptor alpha (ERα) and GFP (L,M), and schematic representations of the various types of alveolar cells (E,H,K,N) showing the integration of GFP positive cells (DESC-derived) within the different compartments of the chimeric mammary ducts. Boxes in C,F,I,L represent high magnifications shown in D,G,J and M. (O–R) Immunofluorescent staining against GFP and β-casein. (O–Q) Single channels; (R) merged image. Scale bars: 50 μm (A); 40 μm (C,F,I,L); 10 μm (D,G,J,M); 20 μm (O–R). Abbreviation: ld, lipid droplet. Full article ">Figure 3
Injection of DESCs alone in mammary fat pads. (A) GFP+ DESCs injected alone in mammary fat pads managed to form epithelial branched structures (green colour). (C,D) Hematoxylin-eosin staining showing DESCs-originated ducts. Notice the presence of secretions within the ducts (asterisk in D). (E) Immunofluorescent staining showing that ducts are originated exclusively by GFP+ DESCs (green colour). (F,G) Immunofluorescent staining showing Krt14-expressing myoepithelial cells (F) and Krt8-expressing luminal cells (G) originated from DESCs. (H–J) Immunofluorescent staining against GFP (green colour) and ERα (H), β-casein (I), or amelogenin (J; red colour). Arrowheads indicate double-positive cells for each combination of staining. (K–M) Fluorescent imaging (K), H and E staining (L), and immunofluorescent staining against GFP (M) showing cyst-like structures originated from DESCs. Scale bars: 400 μm (B,K), 100 μm (C–E,H–J,L,M); 50 μm (F,G). Abbreviations: le, luminal epithelium; m, myoepithelium. Full article ">Figure 4
DESCs integrate in epithelial rudiments and potentially surrounding stroma. (A,B) Hematoxylin-eosin staining showing the formation of dense fibrotic tissue upon transplantation of DESCs. Black rectangular box in A indicates the region shown in B. Notice the presence of secretions within the ducts (asterisk in B). (C,D) Masson’s trichrome staining showing the composition of the fibrotic tissue surrounding the ducts. Black rectangular box in C indicates the region shown in D. (E,F) Double immunofluorescent staining against GFP and αSMA. White arrowheads indicate some of the double GFP+/α-SMA+ cells. (G,H) Double immunofluorescent staining against GFP and Fibronectin. White arrowheads indicate double GFP+/Fibronectin+ cells. Scale bars: 100 μm (B); 200 μm (A,C,E,G); 20 μm (F,H). Full article ">Figure 5
Schematic representation recapitulating the experimental approach and the main results showing the plasticity of dental epithelial stem cells and their potential to adopt mammary epithelial cell fates. Full article ">

19 pages, 3211 KiB

Open AccessArticle

Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System

by Caroline D. Pena, Stephanie Zhang, Robert Majeska, Tadmiri Venkatesh and Maribel Vazquez

Cells 2019, 8(10), 1301; https://doi.org/10.3390/cells8101301 - 22 Oct 2019

Cited by 12 | Viewed by 3982

Abstract

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by [...] Read more.

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insufficient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies. Full article

(This article belongs to the Special Issue Cell Biological Techniques and Cell-Biomaterial Interactions)

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Drosophila melanogaster model. (A) Image of third instar larva. (B) Dissected eye-brain complex with GFP+ glia (Scale bar: 100 µm). (C) Dissection arrangement via microscope within a laminar flow hood. (D) Schematic of key steps in the dissection process, where third instar larvae are segmented using tweezers, and mouth hooks with excess tissue are removed to isolate eye-brain complexes (green cartoon). Full article ">Figure 2
Overview of the µLane system. (A) Schematic of microfluidic system comprised of two volumetric reservoirs connected by a 200-micron-diameter channel. (B) Image of fabricated device loaded with red dye. (C) The distribution of FGF concentration achieved within the µLane, normalized to the input concentration, Co. Transport within the µLane is defined by the convective-diffusion equation shown, where areas of mathematically-distinct changes in concentration gradients are defined along different lengths of the microchannel as marked: G1, G2 and G3. Full article ">Figure 3
Distributions of collective RPC clusters and individual cells post-dissection. Average surface areas of individually adhered cells and adhered RPC clusters of small and large size. A representative small cluster of approximately 3 cells is shown alongside a singleton cell to demonstrate consistency with the size and shape of single cells. Error bars the denote standard deviation. Full article ">Figure 4
Average ratio of neuronal progenitors to total cells, RN, in third instar larvae. (A) Average ratio of neuronal cells to total cells (RN) in large clusters, small clusters and single cells, obtained from immunostaining. Error bars denote the standard deviation. (B,C) Representative confocal images of RFP+ neurons and GFP+ glia at mid-plane. Full article ">Figure 5
Viability and adhesion of disassociated cells upon extracellular substrates. Primary RPCs upon (A) uncoated Petri dish, (B) Concanavalin A (Con-A) with neurite extensions highlighted by arrows, (C) Laminin (LM) with outlined RPC clusters and (D) Poly-L-Lysine (PLL) with both RPC clusters and neurite extensions highlighted. (E) Normalized cell viability at 24 h and 48 h time points. (F) Average RPC cluster surface area (RC SA) on Con-A, LM and PLL. (G) Average values of cell shape index (CSI) measured at 24 h time point for cells adhered on Con-A, LM and PLL. Error bars denote standard deviation. (Scale bar = 20 µm for all images.). Statistical significance (p < 0.05) against control is denoted by *. Full article ">Figure 6
Adhesion of retinal progenitor groups within the µLane system. The three populations of cells observed in suspension and on ECM-treated plates were also seen within the µLane system. Representative images of (A) Small RPC clusters and individual cells, (B) Large RPC clusters and individual cells and (C) Individual cells within the µLane system (Scale bar = 50 µm). Full article ">Figure 7
Bee swarm plots of average path length and chemotactic index. (A) Average path length of single cells in control and gradient fields, G1, G2 and G3. Statistical significance ** p < 0.01 between control groups and experimental groups. (B) Average path length of small and large RPC clusters in control (i.e., Schneider’s medium only) and gradient fields, G1, G2 and G3, generated within µLane. Statistical significance ** p < 0.01 between control and experimental groups. Statistical significance * p < 0.05 between medium and high gradient fields in large clusters. (C) Chemotactic index, CI, of single cells, small and large RPC clusters in control and gradient fields, G1, G2 and G3. No statistical significance amongst groups for single cells. Statistical significance ** p < 0.01 between control groups and experimental groups for RPC clusters. Full article ">

10 pages, 1441 KiB

Open AccessArticle

Association of the Rheumatoid Arthritis Severity Variant rs26232 with the Invasive Activity of Synovial Fibroblasts

by Emma R Dorris, Eimear Linehan, Michelle Trenkmann, Douglas J Veale, Ursula Fearon and Anthony G. Wilson

Cells 2019, 8(10), 1300; https://doi.org/10.3390/cells8101300 - 22 Oct 2019

Cited by 6 | Viewed by 3390

Abstract

rs26232, located in intron one of C5orf30, is associated with the susceptibility to and severity of rheumatoid arthritis (RA). Here, we investigate the relationship between this variant and the biological activities of rheumatoid arthritis synovial fibroblasts (RASFs). RASFs were isolated from the [...] Read more.

rs26232, located in intron one of C5orf30, is associated with the susceptibility to and severity of rheumatoid arthritis (RA). Here, we investigate the relationship between this variant and the biological activities of rheumatoid arthritis synovial fibroblasts (RASFs). RASFs were isolated from the knee joints of 33 RA patients. The rs26232 genotype was determined and cellular migration, invasion, and apoptosis were compared using in vitro techniques. The production of adhesion molecules, chemokines, and proteases was measured by ELISA or flow cytometry. Cohort genotypes were CC n = 16; CT n = 14; TT n = 3. In comparison with the RASFs of the CT genotype, the CC genotype showed a 1.48-fold greater invasiveness in vitro (p = 0.02), 1.6-fold higher expression intracellular adhesion molecule (ICAM)-1 (p = 0.001), and 5-fold IFN-γ inducible protein-10 (IP-10) (p = 0.01). There was no association of the rs26232 genotype with the expression levels of either total C5orf30 mRNA or any of the three transcript variants. The rs26232 C allele, which has previously been associated with both the risk and severity of RA, is associated with greater invasive activity of RASFs in vitro, and with higher expression of ICAM-1 and IP-10. In resting RASFs, rs26232 is not a quantitative trait locus for C5orf30 mRNA, indicating a more complex mechanism underlying the genotype‒phenotype relationship. Full article

(This article belongs to the Special Issue The Molecular and Cellular Basis for Rheumatoid Arthritis)

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The rs26232 genotype is associated with RASF invasiveness. (A) Proliferation rates of RASFs are not associated with rs26232 genotype (p = 0.980). (B) The capacity for cells to migrate was not associated with genotype (p = 0.783). (C) RASF of the CC genotype are more invasive than CT (p = 0.020). Representative images of RASF in vitro (D) migration and (E) invasive assays of rs26232 genotypes. Each circle represents the average of an individual donor. Horizontal black bars represent the cohort mean. Statistical significance: * p < 0.05. Full article ">Figure 2
The rs26232 genotype is associated with RASF expression of ICAM1, MMP14 and IP-10. (A) Representative histogram showing ICAM-1 expression by RASFs of different rs26232 genotypes. (B) Expression of ICAM-1 protein is greater in RASFs of the CC compared to CT genotype (1.5-fold, p = 0.039). (C) ICAM1 relative gene expression is higher in CC compared to CT RASFs (2-fold, p = 0.044). (D) MMP14 relative gene expression is higher in CC compared to CT RASFs (1.6-fold, p = 0.021) (E) RASFs of CC genotype produce greater IP10 (CXCL10) compared to CT genotype (5-fold, p = 0.011). Each circle represents an individual donor. The black bar represents the mean. Statistical significance: * p < 0.05. Full article ">Figure 3
rs26232 is not an eQTL for C5orf30. rs26232 genotype was not associated with (A) total C5orf30 mRNA (all variants) (p = 0.506), (B) variant 1 (p = 0.469), (C) variant 2 (p = 0.352), or (D) variant 3 (p = 0.482). Gene expression is analysed using relative quantitation (RQ) to the endogenous control HPRT1. Y-axis is mean Log2 transformed RQ values per patient RASF line. Each circle represents the average of an individual donor. Horizontal black bars represent the cohort mean. Full article ">

17 pages, 2820 KiB

Open AccessArticle

Opposite Effects of Moderate and Extreme Cx43 Deficiency in Conditional Cx43-Deficient Mice on Angiotensin II-Induced Cardiac Fibrosis

by Laura Valls-Lacalle, Corall Negre-Pujol, Cristina Rodríguez, Saray Varona, Antoni Valera-Cañellas, Marta Consegal, Jose Martínez-González and Antonio Rodríguez-Sinovas

Cells 2019, 8(10), 1299; https://doi.org/10.3390/cells8101299 - 22 Oct 2019

Cited by 14 | Viewed by 4568

Abstract

Connexin 43 (Cx43) is essential for cardiac electrical coupling, but its effects on myocardial fibrosis is controversial. Here, we analyzed the role of Cx43 in myocardial fibrosis caused by angiotensin II (AngII) using Cx43^fl/fl and Cx43^Cre-ER(T)/fl inducible knock-out (Cx43 content: 50%) [...] Read more.

Connexin 43 (Cx43) is essential for cardiac electrical coupling, but its effects on myocardial fibrosis is controversial. Here, we analyzed the role of Cx43 in myocardial fibrosis caused by angiotensin II (AngII) using Cx43^fl/fl and Cx43^Cre-ER(T)/fl inducible knock-out (Cx43 content: 50%) mice treated with vehicle or 4-hydroxytamoxifen (4-OHT) to induce a Cre-ER(T)-mediated global deletion of the Cx43 floxed allele. Myocardial collagen content was enhanced by AngII in all groups (n = 8–10/group, p < 0.05). However, animals with partial Cx43 deficiency (vehicle-treated Cx43^Cre-ER(T)/fl) had a significantly higher AngII-induced collagen accumulation that reverted when treated with 4-OHT, which abolished Cx43 expression. The exaggerated fibrotic response to AngII in partially deficient Cx43^Cre-ER(T)/fl mice was associated with enhanced p38 MAPK activation and was not evident in Cx43 heterozygous (Cx43^+/-) mice. In contrast, normalization of interstitial collagen in 4-OHT-treated Cx43^Cre-ER(T)/fl animals correlated with enhanced MMP-9 activity, IL-6 and NOX2 mRNA expression, and macrophage content, and with reduced α-SMA and SM22α in isolated fibroblasts. In conclusion, our data demonstrates an exaggerated, p38 MAPK-dependent, fibrotic response to AngII in partially deficient Cx43^Cre-ER(T)/fl mice, and a paradoxical normalization of collagen deposition in animals with an almost complete Cx43 ablation, an effect associated with increased MMP-9 activity and inflammatory response and reduced fibroblasts differentiation. Full article

(This article belongs to the Special Issue Cells in Cardiovascular Disease)

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17 pages, 5279 KiB

Open AccessArticle

MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

by Hung-Yu Lin, Feng-Sheng Wang, Ya-Ling Yang and Ying-Hsien Huang

Cells 2019, 8(10), 1298; https://doi.org/10.3390/cells8101298 - 22 Oct 2019

Cited by 77 | Viewed by 8075

Abstract

MicroRNA-29 (miR-29) has been shown to play a critical role in reducing inflammation and fibrosis following liver injury. Non-alcoholic fatty liver disease (NAFLD) occurs when fat is deposited (steatosis) in the liver due to causes other than excessive alcohol use and is associated [...] Read more.

MicroRNA-29 (miR-29) has been shown to play a critical role in reducing inflammation and fibrosis following liver injury. Non-alcoholic fatty liver disease (NAFLD) occurs when fat is deposited (steatosis) in the liver due to causes other than excessive alcohol use and is associated with liver fibrosis. In this study, we asked whether miR-29a could reduce experimental high fat diet (HFD)-induced obesity and liver fibrosis in mice. We performed systematical expression analyses of miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates subjected to HFD-induced NAFLD. The results demonstrated that increased miR-29a not only alleviated HFD-induced body weight gain but also subcutaneous, visceral, and intestinal fat accumulation and hepatocellular steatosis in mice. Furthermore, hepatic tissue in the miR-29aTg mice displayed a weak fibrotic matrix concomitant with low fibrotic collagen1α1 expression within the affected tissues compared to the wild-type (WT) mice fed the HFD diet. Increased miR-29a signaling also resulted in the downregulation of expression of the epithelial mesenchymal transition-executing transcription factor snail, mesenchymal markers vimentin, and such pro-inflammation markers as il6 and mcp1 within the liver tissue. Meanwhile, miR-29aTg-HFD mice exhibited significantly lower levels of peroxisome proliferator-activated receptor γ (PPARγ), mitochondrial transcription factor A TFAM, and mitochondria DNA content in the liver than the WT-HFD mice. An in vitro luciferase reporter assay further confirmed that miR-29a mimic transfection reduced fatty acid translocase CD36 expression in HepG2 cells. Conclusion: Our data provide new insights that miR-29a can improve HDF-induced obesity, hepatocellular steatosis, and fibrosis, as well as highlight the role of miR-29a in regulation of NAFLD. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms Underlying the Pathogenesis of Hepatic Fibrosis)

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26 pages, 7021 KiB

Open AccessArticle

Inhibition of Non-Small Cell Lung Cancer Cells by Oxy210, an Oxysterol-Derivative that Antagonizes TGFβ and Hedgehog Signaling

by Frank Stappenbeck, Feng Wang, Liu-Ya Tang, Ying E. Zhang and Farhad Parhami

Cells 2019, 8(10), 1297; https://doi.org/10.3390/cells8101297 - 22 Oct 2019

Cited by 14 | Viewed by 5193

Abstract

Non-Small Cell Lung Cancer (NSCLC) is a common malignancy and leading cause of death by cancer. Metastasis and drug resistance are serious clinical problems encountered in NSCLC therapy. Aberrant activation of the Transforming Growth Factor beta (TGFβ) and Hedgehog (Hh) signal transduction cascades [...] Read more.

Non-Small Cell Lung Cancer (NSCLC) is a common malignancy and leading cause of death by cancer. Metastasis and drug resistance are serious clinical problems encountered in NSCLC therapy. Aberrant activation of the Transforming Growth Factor beta (TGFβ) and Hedgehog (Hh) signal transduction cascades often associate with poor prognosis and aggressive disease progression in NSCLC, as these signals can drive cell proliferation, angiogenesis, metastasis, immune evasion and emergence of drug resistance. Therefore, simultaneous inhibition of TGFβ and Hh signaling, by a single agent, or in combination with other drugs, could yield therapeutic benefits in NSCLC and other cancers. In the current study, we report on the biological and pharmacological evaluation of Oxy210, an oxysterol-based dual inhibitor of TGFβ and Hh signaling. In NSCLC cells, Oxy210 inhibits proliferation, epithelial-mesenchymal transition (EMT) and invasive activity. Combining Oxy210 with Carboplatin (CP) increases the anti-proliferative response to CP and inhibits TGFβ-induced resistance to CP in A549 NSCLC cells. In addition, Oxy210 displays encouraging drug-like properties, including chemical scalability, metabolic stability and oral bioavailability in mice. Unlike other known inhibitors, Oxy210 antagonizes TGFβ and Hh signaling independently of TGFβ receptor kinase inhibition and downstream of Smoothened, respectively. Full article

(This article belongs to the Special Issue Hedgehog Signaling in Development and Cancer)

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19 pages, 3017 KiB

Open AccessArticle

Hepatitis C Virus Improves Human Tregs Suppressive Function and Promotes Their Recruitment to the Liver

by Laurissa Ouaguia, Olivier Moralès, Lynda Aoudjehane, Czeslaw Wychowski, Abhishek Kumar, Jean Dubuisson, Yvon Calmus, Filomena Conti and Nadira Delhem

Cells 2019, 8(10), 1296; https://doi.org/10.3390/cells8101296 - 22 Oct 2019

Cited by 10 | Viewed by 3457

Abstract

Background: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we [...] Read more.

Background: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we have investigated the ability of Tregs to hang HCV on and the subsequent effect on their suppressive function and phenotype. Moreover, we have evaluated how HCV could promote the recruitment of Tregs by infected primary human hepatocytes. Methods: Tregs of healthy donors were incubated with JFH-1/HCVcc. Viral inoculation was assessed using adapted assays (RT-qPCR, Flow Citometry (FACS) and Western Blot (WB). Expression of Tregs phenotypic (CD4, CD25, CD127 and Foxp3) and functional (IL-10, GZMB, TGF-β1 and IL-2) markers was monitored by RT-qPCR, FACS and ELISA. Suppressive activity was validated by suppressive assays. Tregs recruitment by infected primary hepatic cells was evaluated using Boyden Chamber. Results: Tregs express the classical HCV receptors (CD81, CLDN1 and LDLR) and some co-receptors (CD5). HCV inoculation significantly increases the suppressive phenotype and activity of Tregs, and raises their anergy by inducing an unexpected IL-2 production. Moreover, HCV infection induces the expression of chemokines (CCL17, CXCL16, and CCL20) by primary hepatic human hepatocytes and chemokine receptors (CCR4, CXCR6 and CCR6) by Tregs. Finally, infected hepatocytes have a significantly higher potential to recruit Tregs in a seemingly CCL20-dependent manner. Conclusions: Direct interaction between HCV and Tregs represents a newly defined mechanism that could potentiate HCV immune evasion and favor intratumoral recruitment contributing to HCC progression. Full article

(This article belongs to the Special Issue Regulatory T (Treg) Cells in Health and Diseases)

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17 pages, 1806 KiB

Open AccessArticle

Tumor Cells Develop Defined Cellular Phenotypes After 3D-Bioprinting in Different Bioinks

by Sonja K. Schmidt, Rafael Schmid, Andreas Arkudas, Annika Kengelbach-Weigand and Anja K. Bosserhoff

Cells 2019, 8(10), 1295; https://doi.org/10.3390/cells8101295 - 22 Oct 2019

Cited by 41 | Viewed by 5611

Abstract

Malignant melanoma is often used as a model tumor for the establishment of novel therapies. It is known that two-dimensional (2D) culture methods are not sufficient to elucidate the various processes during cancer development and progression. Therefore, it is of major interest to [...] Read more.

Malignant melanoma is often used as a model tumor for the establishment of novel therapies. It is known that two-dimensional (2D) culture methods are not sufficient to elucidate the various processes during cancer development and progression. Therefore, it is of major interest to establish defined biofabricated three-dimensional (3D) models, which help to decipher complex cellular interactions. To get an impression of their printability and subsequent behavior, we printed fluorescently labeled melanoma cell lines with Matrigel and two different types of commercially available bioinks, without or with modification (RGD (Arginine-Glycine-Aspartate)-sequence/laminin-mixture) for increased cell-matrix communication. In general, we demonstrated the printability of melanoma cells in all tested biomaterials and survival of the printed cells throughout 14 days of cultivation. Melanoma cell lines revealed specific differential behavior in the respective inks. Whereas in Matrigel, the cells were able to spread, proliferate and form dense networks throughout the construct, the cells showed no proliferation at all in alginate-based bioink. In gelatin methacrylate-based bioink, the cells proliferated in clusters. Surprisingly, the modifications of the bioinks with RGD or the laminin blend did not affect the analyzed cellular behavior. Our results underline the importance of precisely adapting extracellular matrices to individual requirements of specific 3D bioprinting applications. Full article

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Printability of bioink and cell distribution. Constructs with an area of 1 cm2, three layers high, containing 105 cells/mL were printed with Cellink Bioink, Cellink RGD, GelXA, GelXA Laminink+ or Matrigel, respectively, using the Cellink+ bioprinter. (A) Representative macroscopic images of cell-loaded 3D printed constructs at time points d0, d7, and d14. (B) Representative fluorescence microscope images of melanoma cell lines Mel Im GFP (green) and MV3dc (red/green) in the respective inks 1 day after 3D printing. Scale bars represent 200 µm. Full article ">Figure 2
Survival of melanoma cells in the bioinks. (A) Two representative fluorescence microscope images of each of the cell lines Mel Im GFP and MV3dc one day after 3D printing. Both melanoma cell lines survived the bioprinting and crosslinking process in all bioinks. Scale bars represent 100 µm. (B) Quantification of living cells per mm2 in the bioinks on the day Mel Im GFP showed low amounts of living cells in both Cellink-based inks, a higher rate in GelXA-based inks, and revealed the significantly highest amount of viable cells in Matrigel. MV3dc revealed appropriate amounts of living cells in all materials, with the lowest amount in the Cellinks and GelXA Laminink+, and the significantly highest amount in Matrigel. * p ≤ 0.05 (One-way ANOVA). Full article ">Figure 3
Morphology of melanoma cells in the different bioinks. (A) Fluorescence microscope images revealing the morphology of each three representative Mel Im GFP or MV3dc single cells on day 4, cultured in the different 3D matrices. The scale bars represent 20 µm. (B) Quantification of protrusion lengths (in 2D) of single cells at time points d1, d2, and d4 in all bioinks. Mel Im GFP spread and revealed increasing protrusion lengths over the observation period. Most distinct protrusions were observed in Matrigel. MV3dc cells revealed a tendency to form shorter protrusions in all bioinks, without clear time dependency of their lengths. Protrusion lengths on day 4 are not significantly different in the different materials for both cell lines (One-way ANOVA). (C) Fluorescence images of MCAT-eGFP transfected MV3 cells in printed constructs on day 4 and 7, the respective fluorescence/brightfield overlay images are shown for day 7. In all materials, some cells activate YAP/TAZ-TEAD, indicating cell–material interactions. The scale bars represent 100 µm. Full article ">Figure 4
Tumor cell proliferation in bioinks over 14 days of culture. (A) Representative fluorescence images showing the proliferation of Mel Im GFP and MV3dc on d4, d7, and d14 after being printed with Cellink Bioink, Cellink RGD, GelXA, GelXA Laminink+ and Matrigel. Mel Im GFP and MV3dc showed hardly any growth or proliferation in Cellink Bioink and Cellink RGD but formed irregular clusters in GelXA and GelXA Laminink+. In Matrigel, cells proliferated quickly, Mel Im GFP grew in clusters, which fused together with time, whereas MV3dc cells formed dense chain-like networks throughout the material. Scale bars represent 200 µm. (B) Quantification of proliferation by determination of the mean gray value intensities of at least three fluorescence images per time point plotted logarithmically to emphasize the strongest proliferation of both cell types in Matrigel. The statistical comparison presents the significant difference of mean gray value intensities in Matrigel on day 14 with the other materials on that day. The same significance was observed in comparing day 4 or day 7; however, due to clarity reasons, these significances are not shown in the graph. * p ≤ 0.05 (One-way ANOVA). Full article ">

7 pages, 238 KiB

Open AccessEditorial

Tubulin: Structure, Functions and Roles in Disease

by Pavla Binarová and Jack Tuszynski

Cells 2019, 8(10), 1294; https://doi.org/10.3390/cells8101294 - 22 Oct 2019

Cited by 83 | Viewed by 9826

Abstract

Highly conserved α- and β-tubulin heterodimers assemble into dynamic microtubules and perform multiple important cellular functions such as structural support, pathway for transport and force generation in cell division. Tubulin exists in different forms of isotypes expressed by specific genes with spatially- and [...] Read more.

Highly conserved α- and β-tubulin heterodimers assemble into dynamic microtubules and perform multiple important cellular functions such as structural support, pathway for transport and force generation in cell division. Tubulin exists in different forms of isotypes expressed by specific genes with spatially- and temporally-regulated expression levels. Some tubulin isotypes are differentially expressed in normal and neoplastic cells, providing a basis for cancer chemotherapy drug development. Moreover, specific tubulin isotypes are overexpressed and localized in the nuclei of cancer cells and/or show bioenergetic functions through the regulation of the permeability of mitochondrial ion channels. It has also become clear that tubulin isotypes are involved in multiple cellular functions without being incorporated into microtubule structures. Understanding the mutations of tubulin isotypes specifically expressed in tumors and their post-translational modifications might help to identify precise molecular targets for the design of novel anti-microtubular drugs. Knowledge of tubulin mutations present in tubulinopathies brings into focus cellular functions of tubulin in brain pathologies such as Alzheimer’s disease. Uncovering signaling pathways which affect tubulin functions during antigen-mediated activation of mast cells presents a major challenge in developing new strategies for the treatment of inflammatory and allergic diseases. γ-tubulin, a conserved member of the eukaryotic tubulin superfamily specialized for microtubule nucleation is a target of cell cycle and stress signaling. Besides its microtubule nucleation role, γ-tubulin functions in nuclear and cell cycle related processes. This special issue “Tubulin: Structure, Functions and Roles in Disease” contains eight articles, five of which are original research papers and three are review papers that cover diverse areas of tubulin biology and functions under normal and pathological conditions. Full article

(This article belongs to the Special Issue Tubulin: Structure, Functions and Roles in Disease)

17 pages, 695 KiB

Open AccessReview

What Do Microglia Really Do in Healthy Adult Brain?

by Marcus Augusto-Oliveira, Gabriela P. Arrifano, Amanda Lopes-Araújo, Leticia Santos-Sacramento, Priscila Y. Takeda, Daniel C. Anthony, João O. Malva and Maria Elena Crespo-Lopez

Cells 2019, 8(10), 1293; https://doi.org/10.3390/cells8101293 - 22 Oct 2019

Cited by 101 | Viewed by 16328

Abstract

Microglia originate from yolk sac-primitive macrophages and auto-proliferate into adulthood without replacement by bone marrow-derived circulating cells. In inflammation, stroke, aging, or infection, microglia have been shown to contribute to brain pathology in both deleterious and beneficial ways, which have been studied extensively. [...] Read more.

Microglia originate from yolk sac-primitive macrophages and auto-proliferate into adulthood without replacement by bone marrow-derived circulating cells. In inflammation, stroke, aging, or infection, microglia have been shown to contribute to brain pathology in both deleterious and beneficial ways, which have been studied extensively. However, less is known about their role in the healthy adult brain. Astrocytes and oligodendrocytes are widely accepted to strongly contribute to the maintenance of brain homeostasis and to modulate neuronal function. On the other hand, contribution of microglia to cognition and behavior is only beginning to be understood. The ability to probe their function has become possible using microglial depletion assays and conditional mutants. Studies have shown that the absence of microglia results in cognitive and learning deficits in rodents during development, but this effect is less pronounced in adults. However, evidence suggests that microglia play a role in cognition and learning in adulthood and, at a cellular level, may modulate adult neurogenesis. This review presents the case for repositioning microglia as key contributors to the maintenance of homeostasis and cognitive processes in the healthy adult brain, in addition to their classical role as sentinels coordinating the neuroinflammatory response to tissue damage and disease. Full article

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13 pages, 3711 KiB

Open AccessProtocol

Turning the World Upside-Down in Cellulose for Improved Culturing and Imaging of Respiratory Challenges within a Human 3D Model

by Viktoria Zaderer, Martin Hermann, Cornelia Lass-Flörl, Wilfried Posch and Doris Wilflingseder

Cells 2019, 8(10), 1292; https://doi.org/10.3390/cells8101292 - 21 Oct 2019

Cited by 19 | Viewed by 11515

Abstract

Polarized growth of human-derived respiratory epithelial cells on hydrogel-coated filters offers big advantages concerning detailed experiments with respect to drug screening or host pathogen interactions. Different microscopic approaches, such as confocal analyses and high content screening, help to examine such 3D respiratory samples, [...] Read more.

Polarized growth of human-derived respiratory epithelial cells on hydrogel-coated filters offers big advantages concerning detailed experiments with respect to drug screening or host pathogen interactions. Different microscopic approaches, such as confocal analyses and high content screening, help to examine such 3D respiratory samples, resulting in high-resolution pictures and enabling quantitative analyses of high cell numbers. A major problem employing these techniques relates to single-use instead of multiple-use of Transwell filters and difficulties in the digestion of collagen if subsequent analyses are needed. Up to date, cells are seeded in collagen-based matrices to the inner field of Transwell inserts, which makes it impossible to image due to the design of the inserts and hard to perform other analyses since digestion of the collagen matrix also affects Transwell grown cells. To overcome these problems, we optimized culturing conditions for monitoring cell differentiation or repeated dose experiments over a long time period. For this, cells are seeded upside-down to the bottom side of filters within an animal-free cellulose hydrogel. These cells were then grown inverted under static conditions and were differentiated in air-liquid interphase (ALI). Full differentiation of goblet (Normal Human Bronchial Epithelial (NHBE))/Club (small airway epithelia (SAE)) cells and ciliated cells was detected after 12 days in ALI. Inverted cell cultures could then be used for ‘follow-up’ live cell imaging experiments, as well as, flow-cytometric analyses due to easy digestion of the cellulose compared to classical collagen matrices. Additionally, this culture technique also enables easy addition of immune cells, such as dendritic cells (DCs), macrophages, neutrophils, T or B cells alone or in combination, to the inner field of the Transwell to monitor immune cell behavior after repeated respiratory challenge. Our detailed protocol offers the possibility of culturing human primary polarized cells into a fully differentiated, thick epithelium without any animal components over >700 days. Furthermore, this animal-free, inverted system allows investigation of the same inserts, because the complete Transwell can be readily transferred to glass-bottom dishes for live cell imaging analyses and then returned to its original plate for further cultivation. Full article

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

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Graphical abstract

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Normal seeding (left) of primary, respiratory epithelial cells (Normal Human Bronchial Epithelial (NHBE), small airway epithelia (SAE)) in cellulose-based GrowDex® does not allow multiple uses of 3D grown tissue, since Transwell inserts have to be cut out or paraffin-embedded for imaging analyses. Imaging is not possible from below due to the thickness of the filter-grown tissue and flipping of normally grown wells is not possible due to the side walls of the Transwell. Upside-down seeding of respiratory cells grown in GrowDex® allows an easy transfer of the Transwell to a glass-bottom dish by lifting the Transwell insert to the other dish under sterile conditions. After-life cell imaging analyses are done, the Transwell insert is transferred back into the original well. Therefore, among many other applications, cell differentiation and mucociliary clearance can be monitored using the same cells grown in an animal-free cellulose hydrogel. Full article ">Figure 2
NHBE cells, day 15 (d15) in air-liquid interphase (ALI) and seeded upside-down. Left panel: cells grown on the animal-free, birch-based hydrogel. Right panel: cells grown on rat tail collagen-coated membranes. Images show faster proliferation, due to more cell layers and an increased cell number when the hydrogel was applied. Nuclei were stained using Höchst (blue), cilia using wheat germ agglutinin (green), mitochondria using mitotracker (red). An overlay is illustrated in the bottom right panels. Two representative images from at least 3 independent experiments are depicted for cells cultured upside-down in GrowDex® or rat-tail collagen. Full article ">Figure 3
Quantitative analyses of nuclei within GrowDex® and rat-tail collagen. Differentiated cells revealed a significantly higher cell number within the birch-based hydrogel on day 15 post-ALI. Five independent regions of two Transwell inserts were quantified using the Operetta CLS™ HCS, and cells were automatically quantified using the Harmony software. Full article ">Figure 4
Analysis of mucociliary clearance of NHBE cells cultured in birch-based cellulose hydrogel and upside-down. High content screening of the mucociliary clearance was performed after the addition of fluorescently labeled beads (green) and an overview of the bead distribution is depicted. The experiment was repeated three times in independent experiments. Full article ">Figure 5
Analysis of epithelial integrity of upside-down cultured NHBE cells on d700 in ALI. Cilia of NHBE cells cultured in GrowDex® for 700 days in ALI and upside-down were stained using WGA-488 (green), cytoskeleton using Phalloidin-Alexa555 (yellow) and nuclei using Höchst (blue). Full article ">Figure 6
Seeding procedure of primary respiratory cells in cellulose for upside-down cell culture in Transwell chambers. Primary cells (NHBE/SAE) are mixed in GrowDex® as described below and then transferred to an inverted Transwell chamber fixed with tape in a 6-well plate. Cells are allowed to adhere overnight, and the next day, the Transwell chamber is flipped into a 24-Transwell plate. Cells are cultured submerged for 3 days and then transferred into ALI during the differentiation process. Full article ">

25 pages, 1143 KiB

Open AccessReview

Current Paradigms of Tolerogenic Dendritic Cells and Clinical Implications for Systemic Lupus Erythematosus

by Patcharee Ritprajak, Chamraj Kaewraemruaen and Nattiya Hirankarn

Cells 2019, 8(10), 1291; https://doi.org/10.3390/cells8101291 - 21 Oct 2019

Cited by 25 | Viewed by 5787

Abstract

Tolerogenic dendritic cells (tolDCs) are central players in the initiation and maintenance of immune tolerance and subsequent prevention of autoimmunity. Recent advances in treatment of autoimmune diseases including systemic lupus erythematosus (SLE) have focused on inducing specific tolerance to avoid long-term use of [...] Read more.

Tolerogenic dendritic cells (tolDCs) are central players in the initiation and maintenance of immune tolerance and subsequent prevention of autoimmunity. Recent advances in treatment of autoimmune diseases including systemic lupus erythematosus (SLE) have focused on inducing specific tolerance to avoid long-term use of immunosuppressive drugs. Therefore, DC-targeted therapies to either suppress DC immunogenicity or to promote DC tolerogenicity are of high interest. This review describes details of the typical characteristics of in vivo and ex vivo tolDC, which will help to select a protocol that can generate tolDC with high functional quality for clinical treatment of autoimmune disease in individual patients. In addition, we discuss the recent studies uncovering metabolic pathways and their interrelation intertwined with DC tolerogenicity. This review also highlights the clinical implications of tolDC-based therapy for SLE treatment, examines the current clinical therapeutics in patients with SLE, which can generate tolDC in vivo, and further discusses on possibility and limitation on each strategy. This synthesis provides new perspectives on development of novel therapeutic approaches for SLE and other autoimmune diseases. Full article

(This article belongs to the Special Issue The Molecular and Cellular Basis for Lupus)

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13 pages, 2838 KiB

Open AccessArticle

Helicobacter pylori Induces IL-33 Production and Recruits ST-2 to Lipid Rafts to Exacerbate Inflammation

by Chia-Jung Kuo, Chun-Ya Chen, Horng-Ren Lo, Chun-Lung Feng, Hui-Yu Wu, Mei-Zi Huang, Tung-Nan Liao, Yu-An Chen and Chih-Ho Lai

Cells 2019, 8(10), 1290; https://doi.org/10.3390/cells8101290 - 21 Oct 2019

Cited by 16 | Viewed by 5201

Abstract

Helicobacter pylori colonizes human gastric epithelial cells and contributes to the development of several gastrointestinal disorders. Interleukin (IL)-33 is involved in various immune responses, with reported proinflammatory and anti-inflammatory effects, which may be associated with colitis and colitis-associated cancer. IL-33 induces the inflammatory [...] Read more.

Helicobacter pylori colonizes human gastric epithelial cells and contributes to the development of several gastrointestinal disorders. Interleukin (IL)-33 is involved in various immune responses, with reported proinflammatory and anti-inflammatory effects, which may be associated with colitis and colitis-associated cancer. IL-33 induces the inflammatory cascade through its receptor, suppression of tumorigenicity-2 (ST-2). Binding of IL-33 to membrane-bound ST-2 (mST-2) recruits the IL-1 receptor accessory protein (IL-1RAcP) and activates intracellular signaling pathways. However, whether IL-33/ST-2 is triggered by H. pylori infection and whether this interaction occurs in lipid rafts remain unclear. Our study showed that both IL-33 and ST-2 expression levels were significantly elevated in H. pylori-infected cells. Confocal microscopy showed that ST-2 mobilized into the membrane lipid rafts during infection. Depletion of membrane cholesterol dampened H. pylori-induced IL-33 and IL-8 production. Furthermore, in vivo studies revealed IL-33/ST-2 upregulation, and severe leukocyte infiltration was observed in gastric tissues infected with H. pylori. Together, these results demonstrate that ST-2 recruitment into the lipid rafts serves as a platform for IL-33-dependent H. pylori infection, which aggravates inflammation in the stomach. Full article

(This article belongs to the Special Issue Immunomodulatory Factors in Host Defense)

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H. pylori induces Interleukin (IL)-33 and suppression of tumorigenicity-2 (ST-2) expression in gastric epithelial cells. AGS cells were infected with H. pylori (A) at a multiplication of infection (MOI) of 100 for the indicated times or (B) at different MOIs for 9 h. Total cell lysates were prepared to analyze the expression levels of IL-33 and ST-2 by Western blot. Molecular weights of full-length IL-33 and processed IL-33 were 36 KDa and 18 KDa, respectively. β-actin was used as an internal control. The expression levels of processed IL-33 and ST-2 were quantified by the signal intensity and indicated at the bottom of each lane. Full article ">Figure 2
IL-33 translocates from the nucleus to cytoplasm in response to H. pylori infection. AGS cells were left untreated or infected with H. pylori at an MOI of 100 for 9 h. Cells were fixed and stained for IL-33 (red), then probed with Hoechst 33342 (blue) to identify the cell nucleus. (A) The stained cells were analyzed by confocal microscopy. Scale bar, 5 μm. (B) IL-33 (red) signal was quantified and normalized with Hoechst 33342. (C) The cytoplasmic IL-33 was quantified and normalized with nuclear fluorescence. Imaging data of arithmetic mean intensity were analyzed by using the ZEN-blue edition software (Carl Zeiss). (D) Cytoplasmic and nuclear fractions were analyzed to determine IL-33 levels by ELISA. *, p < 0.05. Full article ">Figure 3
H. pylori induces IL-1 receptor accessory protein (IL-1RAcP) expression but not in lipid rafts. AGS cells were left untreated or infected with H. pylori at an MOI of 100 for 9 h. (A) Expression levels of IL-1RAcP and β-actin were assessed using Western blot. (B) Cells were stained for IL-1RAcP (green), Hoechst 33342 (blue), and cholera toxin subunit B (CTX-B) to label lipid rafts (red). The stained cells were analyzed by confocal microscopy. Scale bar, 5 μm. Full article ">Figure 4
H. pylori elicits ST-2 mobilization into lipid rafts. AGS cells were left untreated or infected with H. pylori at an MOI of 100 for 9 h. Cells were subsequently stained for ST-2 (green), CTX-B (red), and Hoechst 33342 (blue). The stained cells were analyzed by confocal microscopy. The co-localization of ST-2 with CTX-B appears yellow in the merged image. Scale bar, 5 μm. Full article ">Figure 5
Recruitment of ST-2 into the lipid rafts is induced by IL-33. AGS cells were pretreated with or without 5 mM MβCD followed by incubation with recombinant IL-33 (100 ng/mL) at 11 °C for 1 h. Cells were then stained for ST-2 (green), CTX-B (red), and Hoechst 33342 (blue). Fluorescence distributions of ST-2 (green) and CTX-B (red) signals across the white lines were analyzed and exhibited as a line intensity histogram in the right panel. Scale bar, 10 µm. Full article ">Figure 6
Sufficient cholesterol in the membrane rafts is crucial for H. pylori-induced IL-33 and IL-8 production. AGS cells were left untreated or pretreated with nystatin (50 μg/mL), simvastatin (10 μM), or MβCD (5.0 mM), or treated MβCD followed by replenishment of water-soluble cholesterol (400 μg/mL). After H. pylori infection at an MOI of 100 for 9 h, the expression levels of (A) IL-8 and (B) IL-33 were determined by ELISA. *, P < 0.05. Full article ">Figure 7
H. pylori infection induces IL-33 and ST-2 expression in mouse gastric epithelial cells. (A) Mice were infected with H. pylori (1 × 108) by intragastric gavage once every 2 days for a total of six administrations. (B) Tissue sections of the stomach were fixed and stained with H&E, or prepared for IHC staining with specific antibodies against IL-33 and ST-2. The magnified images are shown in the right panel of each cropped image. Inflammatory cell infiltration in the gastric epithelium was observed (black arrowheads), along with evidence of both IL-33 and ST-2 expression in the gastric tissues (red arrowheads). Scale bars in left panels, 20 μm, and in magnified right panels, 60 μm. Full article ">

19 pages, 2969 KiB

Open AccessArticle

The Charcot–Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

by Christina Wolf, Rahel Zimmermann, Osamah Thaher, Diones Bueno, Verena Wüllner, Michael K.E. Schäfer, Philipp Albrecht and Axel Methner

Cells 2019, 8(10), 1289; https://doi.org/10.3390/cells8101289 - 21 Oct 2019

Cited by 16 | Viewed by 4780

Abstract

Charcot–Marie tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which [...] Read more.

Charcot–Marie tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic, and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential, and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 µM hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an up-regulation of hexokinase 1 and pyruvate kinase M2, suggesting increased pyruvate shuttling into mitochondria. Interestingly, these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells. We conclude that mitochondria harboring the disease-causing R94Q mutation in MFN2 are more susceptible to oxidative stress, which causes uncoupling of respiration and ATP production possibly by a less efficient mitochondrial quality control. Full article

(This article belongs to the Special Issue Roles and Functions of ROS and RNS in Cellular Physiology and Pathology)

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23 pages, 9489 KiB

Open AccessArticle

Subcutaneous and Visceral Adipose-Derived Mesenchymal Stem Cells: Commonality and Diversity

by Andreas Ritter, Alexandra Friemel, Susanne Roth, Nina-Naomi Kreis, Samira Catharina Hoock, Babek Khan Safdar, Kyra Fischer, Charlotte Möllmann, Christine Solbach, Frank Louwen and Juping Yuan

Cells 2019, 8(10), 1288; https://doi.org/10.3390/cells8101288 - 21 Oct 2019

Cited by 41 | Viewed by 4259

Abstract

Adipose-derived mesenchymal stem cells (ASCs) are considered to be a useful tool for regenerative medicine, owing to their capabilities in differentiation, self-renewal, and immunomodulation. These cells have become a focus in the clinical setting due to their abundance and easy isolation. However, ASCs [...] Read more.

Adipose-derived mesenchymal stem cells (ASCs) are considered to be a useful tool for regenerative medicine, owing to their capabilities in differentiation, self-renewal, and immunomodulation. These cells have become a focus in the clinical setting due to their abundance and easy isolation. However, ASCs from different depots are not well characterized. Here, we analyzed the functional similarities and differences of subcutaneous and visceral ASCs. Subcutaneous ASCs have an extraordinarily directed mode of motility and a highly dynamic focal adhesion turnover, even though they share similar surface markers, whereas visceral ASCs move in an undirected random pattern with more stable focal adhesions. Visceral ASCs have a higher potential to differentiate into adipogenic and osteogenic cells when compared to subcutaneous ASCs. In line with these observations, visceral ASCs demonstrate a more active sonic hedgehog pathway that is linked to a high expression of cilia/differentiation related genes. Moreover, visceral ASCs secrete higher levels of inflammatory cytokines interleukin-6, interleukin-8, and tumor necrosis factor α relative to subcutaneous ASCs. These findings highlight, that both ASC subpopulations share multiple cellular features, but significantly differ in their functions. The functional diversity of ASCs depends on their origin, cellular context and surrounding microenvironment within adipose tissues. The data provide important insight into the biology of ASCs, which might be useful in choosing the adequate ASC subpopulation for regenerative therapies. Full article

(This article belongs to the Special Issue Adipose-Derived Stromal/Stem Cells)

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17 pages, 8243 KiB

Open AccessArticle

Estimating Dynamic Cellular Morphological Properties via the Combination of the RTCA System and a Hough-Transform-Based Algorithm

by Lejun Zhang, Yang Ye, Rana Dhar, Jinsong Deng and Huifang Tang

Cells 2019, 8(10), 1287; https://doi.org/10.3390/cells8101287 - 21 Oct 2019

Cited by 10 | Viewed by 3397

Abstract

The xCELLigence real-time cell analysis (RTCA) system has the potential to detect cellular proliferation, migration, cytotoxicity, adherence, and remodeling. Although the RTCA system is widely recognized as a noninvasive and efficient tool for real-time monitoring of cellular fate, it cannot describe detailed cell [...] Read more.

The xCELLigence real-time cell analysis (RTCA) system has the potential to detect cellular proliferation, migration, cytotoxicity, adherence, and remodeling. Although the RTCA system is widely recognized as a noninvasive and efficient tool for real-time monitoring of cellular fate, it cannot describe detailed cell morphological parameters, such as length and intensity. Transforming growth factor beta(TGF-β) induced the epithelial–mesenchymal transition (EMT), which produces significant changes in cellular morphology, so we used TGF-β to treat A549 epithelial cells in this study. We compared it with lipopolysaccharide (LPS) and cigarette smoke extract (CSE) as stimulators. We developed an efficient algorithm to quantify the morphological cell changes. This algorithm is comprised of three major parts: image preprocessing, Hough transform (HT), and post-processing. We used the RTCA system to record the A549 cell index. Western blot was used to confirm the EMT. The RTCA system showed that different stimulators produce different cell index curves. The algorithm determined the lengths of the detected lines of cells, and the results were similar to the RTCA system in the TGF-β group. The Western blot results show that TGF-β changed the EMT markers, but the other stimulator remained unchanged. Optics-based computer vision techniques can supply the requisite information for the RTCA system based on good correspondence between the results. Full article

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17 pages, 2101 KiB

Open AccessFeature PaperArticle

A Machine Learning-Based Prediction Platform for P-Glycoprotein Modulators and Its Validation by Molecular Docking

by Onat Kadioglu and Thomas Efferth

Cells 2019, 8(10), 1286; https://doi.org/10.3390/cells8101286 - 21 Oct 2019

Cited by 23 | Viewed by 4966

Abstract

P-glycoprotein (P-gp) is an important determinant of multidrug resistance (MDR) because its overexpression is associated with increased efflux of various established chemotherapy drugs in many clinically resistant and refractory tumors. This leads to insufficient therapeutic targeting of tumor populations, representing a major drawback [...] Read more.

P-glycoprotein (P-gp) is an important determinant of multidrug resistance (MDR) because its overexpression is associated with increased efflux of various established chemotherapy drugs in many clinically resistant and refractory tumors. This leads to insufficient therapeutic targeting of tumor populations, representing a major drawback of cancer chemotherapy. Therefore, P-gp is a target for pharmacological inhibitors to overcome MDR. In the present study, we utilized machine learning strategies to establish a model for P-gp modulators to predict whether a given compound would behave as substrate or inhibitor of P-gp. Random forest feature selection algorithm-based leave-one-out random sampling was used. Testing the model with an external validation set revealed high performance scores. A P-gp modulator list of compounds from the ChEMBL database was used to test the performance, and predictions from both substrate and inhibitor classes were selected for the last step of validation with molecular docking. Predicted substrates revealed similar docking poses than that of doxorubicin, and predicted inhibitors revealed similar docking poses than that of the known P-gp inhibitor elacridar, implying the validity of the predictions. We conclude that the machine-learning approach introduced in this investigation may serve as a tool for the rapid detection of P-gp substrates and inhibitors in large chemical libraries. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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14 pages, 1399 KiB

Open AccessReview

Nuclear Mechanics in the Fission Yeast

by Paola Gallardo, Ramón R. Barrales, Rafael R. Daga and Silvia Salas-Pino

Cells 2019, 8(10), 1285; https://doi.org/10.3390/cells8101285 - 20 Oct 2019

Cited by 6 | Viewed by 5656

Abstract

In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in [...] Read more.

In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in compacted conformation at sites of cytoskeleton contacts seem to be key for withstanding nuclear mechanical stress. Mechanical perturbations of the nucleus normally occur during nuclear positioning and migration. In addition, cell contraction or expansion occurring for instance during cell migration or upon changes in osmotic conditions also result innuclear mechanical stress. Recent studies in Schizosaccharomyces pombe (fission yeast) have revealed unexpected functions of cytoplasmic microtubules in nuclear architecture and chromosome behavior, and have pointed to NE-chromatin tethers as protective elements during nuclear mechanics. Here, we review and discuss how fission yeast cells can be used to understand principles underlying the dynamic interplay between genome organization and function and the effect of forces applied to the nucleus by the microtubule cytoskeleton. Full article

(This article belongs to the Special Issue Nuclear Organisation)

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Schematic representation of the fission yeast nucleus. Schematic representation of a fission yeast cell (above). Magnification of the area marked by dashed lines (below). Global chromosome organization with centromeres attached underneath the spindle pole body (SPB) and telomeres and nucleolus distantly positioned. Chromatin is linked to the nuclear envelope (NE) by the interaction of different genomic elements with inner nuclear membrane (INM) proteins and linker of nucleoskeleton and cytoskeleton (LINC) complexes. Note that the interaction of Lem2 protein with chromatin might be indirect. The NE is continuous with the perinuclear endoplasmic reticulum. The SBP and other microtubule organizing centers (MTOCs) organize antiparallel bundles of microtubules (MTs). Full article ">Figure 2
NE microdomains and their association with different genomic regions. Schematic representation of the linkage between centromeres, telomeres, double strand breaks (DSBs) and other genomic loci and INM proteins. DSB repair factories are linked to the NE by the LINC complex and can be moved by cytoplasmic MTs and positioned in close proximity of the SPB. The different elements are depicted as in <a href="#cells-08-01285-f001" class="html-fig">Figure 1</a>. Full article ">Figure 3
Fission yeast nucleus-SPB and NE under the forces produced by MTs. (A) Schematic representation of a fission yeast cell over time. The nucleus suffers periodic oscillations. Chromosomes are depicted as dark lines. SPB/centromeres are depicted in orange. MTs are depicted in green. The red color at MT ends represent stronger forces. (B) Image of the SPB (marked with GFP) showing regular oscillations around the cell center. Numbers correspond to those marked in A. Nuclear and SPB oscillations depend on alternative MT pushing (by polymerization) at each cell tip. (C) Schematic representation of the transmission of forces produced by cytoplasmic MTs to the chromatin, coupled to MT dynamics. (D) Schematic representation of a fission yeast nucleus under MT-dependent forces applied to the SPB, NE, and centromeric regions. Tethering of chromatin to the NE through INM proteins contributes to support nuclear mechanics. Notice that forces produced by other non-SPB MT bundles at other sites of the NE are not shown. Full article ">

18 pages, 4480 KiB

Open AccessArticle

Transcriptional Regulator TonEBP Mediates Oxidative Damages in Ischemic Kidney Injury

by Eun Jin Yoo, Sun Woo Lim, Hyun Je Kang, Hyun Park, Sora Yoon, Dougu Nam, Satoru Sanada, Mi Jin Kwon, Whaseon Lee-Kwon, Soo Youn Choi and Hyug Moo Kwon

Cells 2019, 8(10), 1284; https://doi.org/10.3390/cells8101284 - 20 Oct 2019

Cited by 2 | Viewed by 3753

Abstract

TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional regulator whose expression is elevated in response to various forms of stress including hyperglycemia, inflammation, and hypoxia. Here we investigated the role of TonEBP in acute kidney injury (AKI) using a line of TonEBP haplo-deficient [...] Read more.

TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional regulator whose expression is elevated in response to various forms of stress including hyperglycemia, inflammation, and hypoxia. Here we investigated the role of TonEBP in acute kidney injury (AKI) using a line of TonEBP haplo-deficient mice subjected to bilateral renal ischemia followed by reperfusion (I/R). In the TonEBP haplo-deficient animals, induction of TonEBP, oxidative stress, inflammation, cell death, and functional injury in the kidney in response to I/R were all reduced. Analyses of renal transcriptome revealed that genes in several cellular pathways including peroxisome and mitochondrial inner membrane were suppressed in response to I/R, and the suppression was relieved in the TonEBP deficiency. Production of reactive oxygen species (ROS) and the cellular injury was reproduced in a renal epithelial cell line in response to hypoxia, ATP depletion, or hydrogen peroxide. The knockdown of TonEBP reduced ROS production and cellular injury in correlation with increased expression of the suppressed genes. The cellular injury was also blocked by inhibitors of necrosis. These results demonstrate that ischemic insult suppresses many genes involved in cellular metabolism leading to local oxidative stress by way of TonEBP induction. Thus, TonEBP is a promising target to prevent AKI. Full article

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15 pages, 1676 KiB

Open AccessArticle

Increased Expression of Meteorin-Like Hormone in Type 2 Diabetes and Obesity and Its Association with Irisin

by Irina AlKhairi, Preethi Cherian, Mohamed Abu-Farha, Ashraf Al Madhoun, Rasheeba Nizam, Motasem Melhem, Mohamed Jamal, Suleiman Al-Sabah, Hamad Ali, Jaakko Tuomilehto, Fahd Al-Mulla and Jehad Abubaker

Cells 2019, 8(10), 1283; https://doi.org/10.3390/cells8101283 - 19 Oct 2019

Cited by 58 | Viewed by 5721

Abstract

Type 2 diabetes (T2D) is a growing pandemic associated with metabolic dysregulation and chronic inflammation. Meteorin-like hormone (METRNL) is an adipomyokine that is linked to T2D. Our objective was to evaluate the changes in METRNL levels in T2D and obesity and assess the [...] Read more.

Type 2 diabetes (T2D) is a growing pandemic associated with metabolic dysregulation and chronic inflammation. Meteorin-like hormone (METRNL) is an adipomyokine that is linked to T2D. Our objective was to evaluate the changes in METRNL levels in T2D and obesity and assess the association of METRNL levels with irisin. Overall, 228 Arab individuals were enrolled. Plasma levels of METRNL and irisin were assessed using immunoassay. Plasma levels of METRNL and irisin were significantly higher in T2D patients than in non-diabetic patients (p < 0.05). When the population was stratified based on obesity, METRNL and irisin levels were significantly higher in obese than in non-obese individuals (p < 0.05). We found a significant positive correlation between METRNL and irisin (r = 0.233 and p = 0.001). Additionally, METRNL and irisin showed significant correlation with various metabolic biomarkers associated with T2D and Obesity. Our data shows elevated METRNL plasma levels in individuals with T2D, further exacerbated with obesity. Additionally, a strong positive association was observed between METRNL and irisin. Further studies are necessary to examine the role of these proteins in T2D and obesity, against their ethnic background and to understand the mechanistic significance of their possible interplay. Full article

(This article belongs to the Special Issue Adipocytes and Metabolic Health)

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15 pages, 3727 KiB

Open AccessArticle

Differentiation of Baboon (Papio anubis) Induced-Pluripotent Stem Cells into Enucleated Red Blood Cells

by Emmanuel N. Olivier, Kai Wang, Joshua Grossman, Nadim Mahmud and Eric E. Bouhassira

Cells 2019, 8(10), 1282; https://doi.org/10.3390/cells8101282 - 19 Oct 2019

Cited by 3 | Viewed by 5002

Abstract

As cell culture methods and stem cell biology have progressed, the in vitro production of cultured RBCs (cRBCs) has emerged as a viable option to produce cells for transfusion or to carry therapeutic cargoes. RBCs produced in culture can be quality-tested either by [...] Read more.

As cell culture methods and stem cell biology have progressed, the in vitro production of cultured RBCs (cRBCs) has emerged as a viable option to produce cells for transfusion or to carry therapeutic cargoes. RBCs produced in culture can be quality-tested either by xeno-transfusion of human cells into immuno-deficient animals, or by transfusion of autologous cells in immuno-competent models. Although murine xeno-transfusion methods have improved, they must be complemented by studies in immuno-competent models. Non-human primates (NHPs) are important pre-clinical, large animal models due to their high biological and developmental similarities with humans, including their comparable hematopoietic and immune systems. Among NHPs, baboons are particularly attractive to validate cRBCs because of the wealth of data available on the characteristics of RBCs in this species that have been generated by past blood transfusion studies. We report here that we have developed a method to produce enucleated cRBCs by differentiation of baboon induced pluripotent stem cells (iPSCs). This method will enable the use of baboons to evaluate therapeutic cRBCs and generate essential pre-clinical data in an immuno-competent, large animal model. Production of the enucleated baboon cRBCs was achieved by adapting the PSC-RED protocol that we previously developed for human cells. Baboon-PSC-RED is an efficient chemically-defined method to differentiate iPSCs into cRBCs that are about 40% to 50% enucleated. PSC-RED is relatively low cost because it requires no albumin and only small amounts of recombinant transferrin. Full article

(This article belongs to the Special Issue iPS Cells for Disease Modeling)

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Production of baboon iPSCs: (a) FACS analysis of human and baboon iPSCs grown in chemically-defined-conditions. Blue histogram: human iPSCs; red histograms: baboon iPSCs; grey histograms: isotype controls. Baboon iPSCs express pluripotency markers albeit at lower levels than human iPSCs. (b) Teratoma analysis. 1 × 106 baboon iPSCs were injected intramuscularly into the hind leg of a 6–8 week old NSG mouse. Six weeks later, tumors were fixed in 10% formalin, paraffin embedded, sectioned, and stained with hematoxylin/eosin. Tumors from two different iPSC clones are shown. Structures originating from all three germ layers were found in most tumors analyzed; (c) embryoid bodies were formed using the hanging drop method in 20% FBS for 10 days. Cells were fixed with paraformaldehyde, stained with indicated antibodies, and counterstained with DAPI. Cells expressing α-feto-protein (endoderm), α-smooth muscle actin (mesoderm) and β-III-tubulin were detectable in 10-day EBs. Baboon iPSCs maintained in chemically-defined conditions are pluripotent; (d) karyotyping: two clones of iPSCs were analyzed using standard karyotyping methods. Full article ">Figure 2
PSC-RED protocols: (a) Scheme illustrating the long PSC-RED protocol to produce enucleated cultured RBCs (cRBCs) from iPSC. In the short version of the protocol, the expansion step in S4 from day 10 to day 16 is omitted. Media and cytokine supplements are described in the Methods section; (b) graphs illustrating the number of cRBCs/iPSCs observed during the erythroid differentiation of two different baboon iPSC clones (clone 1 and clone 3) using the short or long PSC-RED protocols. Data are expressed as average ± SEM of 3 independent experiments. Full article ">Figure 3
Morphological analysis of baboon iPSCs differentiating into cRBCs using the short PSC-RED protocol. 630x magnification micrograph illustrating the typical morphology of the erythroid cells observed at days 17, 24, 27, 31, and 35 of culture after rapid Romanowsky staining. The majority of cells were pro or basophilic erythroblasts at day 17, and orthochromatic erythroblasts at day 35. Full article ">Figure 4
Morphological analysis of baboon iPSCs differentiating into cRBCs using the long PSC-RED protocol: 630x magnification micrograph illustrating the typical morphology of the erythroid cells observed at days 22, 31, 34, 38, and 42 of culture after rapid Romanowsky staining. The majority of cells were pro-erythroblasts at day 22, and orthochromatic erythroblasts and reticulocytes at day 42. Full article ">Figure 5
Differentiation analysis: (a) Cells were spun on microscope slides, stained using the rapid Romanowsky methods and classified as proE (pro-erythroblasts); basoE (basophilic erythroblasts); polyE (polychromatophilic erythroblasts); orthoE (orthochromatophilic erythroblasts) or retic. (reticulocytes (enucleated cRBCs)). Graphs illustrate the average (±SD) number of erythroid precursors during differentiation using either the short (left) or long (right) PSC-RED protocols (n = 2); (b) FACS plots representative of the enucleation rates as determined by DNA content measurement using Draq5. The rate of enucleation is much higher using the long protocol (Student’s t-test p-value between the rate of enucleation short and long protocol in s: <0.00001) (c) FACS plots illustrating the proportion of erythroid cells during the culture using anti-baboon RBCs antibody E34-731. Full article ">Figure 6
Globin expression. (a) HPLC chromatograms illustrating globin expression. Cultured RBCs obtained at day 32 (short-protocol) or 36 (long-protocol) were lysed and analyzed by HPLCs. RBCs from an adult baboon were used as controls (lower chromatogram). ζ and ε-globins are expressed at lower levels in cells obtained with the short protocol; (b) histograms summarizing the results of the HPLC analysis. Percentage of a-like = 100 × (α or ζ)/(α + ζ). Percentage of β-like = 100 × (ε( Gα + Aγ) or β)/(ε + Gγ + Aγ + β). Data are the average ± SEM of four independent experiments. The long PSC-RED protocol yields more developmentally mature cells. Full article ">

19 pages, 1934 KiB

Open AccessReview

Enhancer Dysfunction in 3D Genome and Disease

by Ji-Han Xia and Gong-Hong Wei

Cells 2019, 8(10), 1281; https://doi.org/10.3390/cells8101281 - 19 Oct 2019

Cited by 13 | Viewed by 9342

Abstract

Spatiotemporal patterns of gene expression depend on enhancer elements and other factors during individual development and disease progression. The rapid progress of high-throughput techniques has led to well-defined enhancer chromatin properties. Various genome-wide methods have revealed a large number of enhancers and the [...] Read more.

Spatiotemporal patterns of gene expression depend on enhancer elements and other factors during individual development and disease progression. The rapid progress of high-throughput techniques has led to well-defined enhancer chromatin properties. Various genome-wide methods have revealed a large number of enhancers and the discovery of three-dimensional (3D) genome architecture showing the distant interacting mechanisms of enhancers that loop to target gene promoters. Whole genome sequencing projects directed at cancer have led to the discovery of substantial enhancer dysfunction in misregulating gene expression and in tumor initiation and progression. Results from genome-wide association studies (GWAS) combined with functional genomics analyses have elucidated the functional impacts of many cancer risk-associated variants that are enriched within the enhancer regions of chromatin. Risk variants dysregulate the expression of enhancer variant-associated genes via 3D genomic interactions. Moreover, these enhancer variants often alter the chromatin binding affinity for cancer-relevant transcription factors, which in turn leads to aberrant expression of the genes associated with cancer susceptibility. In this review, we investigate the extent to which these genetic regulatory circuits affect cancer predisposition and how the recent development of genome-editing methods have enabled the determination of the impacts of genomic variation and alteration on cancer phenotype, which will eventually lead to better management plans and treatment responses to human cancer in the clinic. Full article

(This article belongs to the Special Issue Nuclear Organisation)

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Active enhancers promote high-level gene expression. Epigenetic marks for active enhancers usually include H3K4me1 (monomethylation of H3 lysine 4) and H3K27ac whereas the trimethylation of histone H3 lysine 4 (H3K4me3) is often enriched at gene promoters. The active enhancers regulate gene transcription through chromatin looping with the promoters of target genes. Thus, looping formation eventually contributes to the recruitment of transcription factors, coactivators, and RNA polymerase, promoting high levels of target gene expressions. Full article ">Figure 2
Genome-wide identification of cis-regulatory drivers, the enhancers. The cells were cross-linked with formaldehyde, and glycine was added to stop the reaction. Cell pellets were collected and suspended to isolate the nuclei. Chromatin was prepared by sonication into certain size, and the fragments were incubated with antibodies against target proteins. Then, extraction buffer was added to extract and purify the DNA from the complexes. The target DNA fragments were enriched and sequenced by using ChIP-seq in combination with bioinformatics analysis. According to the called ChIP-seq peaks, the enhancer elements in the chromatin can be identified. Full article ">Figure 3
Looping between clusters of activators, mediators and transcription factors in enhancer and promoter regions. The enhancer region of chromatin is bound by the transcriptional complexes including various activators, mediators and transcription factors, which form enhancer clusters. The enhancer interacts with the promoter region, which combines with transcription factors and RNA polymerase to increase the rate of gene transcription. Full article ">Figure 4
Enhancers in gene regulation and disease. (A) The aggressive G allele of rs11672691 enhances the binding of transcription factor HOXA2, thus increasing the expression level of the plausible candidate genes PCAT19 and CEACAM21, which results in prostate cancer cell growth and progression. (B) In wild-type cells, the driver of transcription factor binding with other factors in the enhancer region contributes to promoter interaction with the promoter of target genes to upregulate gene expression. However, in disease conditions, genetic variation and chromosomal aberrations affect the binding affinity of clusters of activators, mediators, and transcription factors, thereby dysregulating the expression of target genes. Full article ">Figure 5
Integrated analysis of germline and somatic mutations. The mutation in germline and somatic cells can be tested by high-throughput sequencing techniques at the genetic or transcript level. Integrated analyses of disease-related gene expression or pathways combined with histological data might contribute to the identification of biomarkers for disease diagnoses and treatment. Full article ">

21 pages, 2338 KiB

Open AccessReview

The Adaptive Immune System in Multiple Sclerosis: An Estrogen-Mediated Point of View

by Alessandro Maglione, Simona Rolla, Stefania Federica De Mercanti, Santina Cutrupi and Marinella Clerico

Cells 2019, 8(10), 1280; https://doi.org/10.3390/cells8101280 - 19 Oct 2019

Cited by 27 | Viewed by 5160

Abstract

Multiple sclerosis (MS) is a chronic central nervous system inflammatory disease that leads to demyelination and neurodegeneration. The third trimester of pregnancy, which is characterized by high levels of estrogens, has been shown to be associated with reduced relapse rates compared with the [...] Read more.

Multiple sclerosis (MS) is a chronic central nervous system inflammatory disease that leads to demyelination and neurodegeneration. The third trimester of pregnancy, which is characterized by high levels of estrogens, has been shown to be associated with reduced relapse rates compared with the rates before pregnancy. These effects could be related to the anti-inflammatory properties of estrogens, which orchestrate the reshuffling of the immune system toward immunotolerance to allow for fetal growth. The action of these hormones is mediated by the transcriptional regulation activity of estrogen receptors (ERs). Estrogen levels and ER expression define a specific balance of immune cell types. In this review, we explore the role of estradiol (E2) and ERs in the adaptive immune system, with a focus on estrogen-mediated cellular, molecular, and epigenetic mechanisms related to immune tolerance and neuroprotection in MS. The epigenome dynamics of immune systems are described as key molecular mechanisms that act on the regulation of immune cell identity. This is a completely unexplored field, suggesting a future path for more extensive research on estrogen-induced coregulatory complexes and molecular circuitry as targets for therapeutics in MS. Full article

(This article belongs to the Special Issue The Molecular and Cellular Basis for Multiple Sclerosis)

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26 pages, 1395 KiB

Open AccessReview

Magnetic-Assisted Treatment of Liver Fibrosis

by Kateryna Levada, Alexander Omelyanchik, Valeria Rodionova, Ralf Weiskirchen and Matthias Bartneck

Cells 2019, 8(10), 1279; https://doi.org/10.3390/cells8101279 - 19 Oct 2019

Cited by 28 | Viewed by 6835

Abstract

Chronic liver injury can be induced by viruses, toxins, cellular activation, and metabolic dysregulation and can lead to liver fibrosis. Hepatic fibrosis still remains a major burden on the global health systems. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are considered [...] Read more.

Chronic liver injury can be induced by viruses, toxins, cellular activation, and metabolic dysregulation and can lead to liver fibrosis. Hepatic fibrosis still remains a major burden on the global health systems. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are considered the main cause of liver fibrosis. Hepatic stellate cells are key targets in antifibrotic treatment, but selective engagement of these cells is an unresolved issue. Current strategies for antifibrotic drugs, which are at the critical stage 3 clinical trials, target metabolic regulation, immune cell activation, and cell death. Here, we report on the critical factors for liver fibrosis, and on prospective novel drugs, which might soon enter the market. Apart from the current clinical trials, novel perspectives for anti-fibrotic treatment may arise from magnetic particles and controlled magnetic forces in various different fields. Magnetic-assisted techniques can, for instance, enable cell engineering and cell therapy to fight cancer, might enable to control the shape or orientation of single cells or tissues mechanically. Furthermore, magnetic forces may improve localized drug delivery mediated by magnetism-induced conformational changes, and they may also enhance non-invasive imaging applications. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms Underlying the Pathogenesis of Hepatic Fibrosis)

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21 pages, 3076 KiB

Open AccessArticle

Intrinsically Disordered SRC-3/AIB1 Protein Undergoes Homeostatic Nuclear Extrusion by Nuclear Budding While Ectopic Expression Induces Nucleophagy

by Miguel A. Cabrita, L. Isabel Renart, Rosanna Lau and M. A. Christine Pratt

Cells 2019, 8(10), 1278; https://doi.org/10.3390/cells8101278 - 19 Oct 2019

Cited by 2 | Viewed by 4278

Abstract

SRC-3/AIB1 (Amplified in Breast Cancer-1) is a nuclear receptor coactivator for the estrogen receptor in breast cancer cells. It is also an intrinsically disordered protein when not engaged with transcriptional binding partners and degraded upon transcriptional coactivation. Given the amplified expression of SRC-3 [...] Read more.

SRC-3/AIB1 (Amplified in Breast Cancer-1) is a nuclear receptor coactivator for the estrogen receptor in breast cancer cells. It is also an intrinsically disordered protein when not engaged with transcriptional binding partners and degraded upon transcriptional coactivation. Given the amplified expression of SRC-3 in breast cancers, the objective of this study was to determine how increasing SRC-3 protein levels are regulated in MCF-7 breast cancer cells. We found that endogenous SRC-3 was expelled from the nucleus in vesicle-like spheres under normal growth conditions suggesting that this form of nuclear exclusion of SRC-3 is a homeostatic mechanism for regulating nuclear SRC-3 protein. Only SRC-3 not associated with CREB-binding protein (CBP) was extruded from the nucleus. We found that overexpression in MCF-7 cells results in aneuploid senescence and cell death with frequent formation of nuclear aggregates which were consistently juxtaposed to perinuclear microtubules. Transfected SRC-3 was SUMOylated and caused redistribution of nuclear promyelocytic leukemia (PML) bodies and perturbation of the nuclear membrane lamin B1, hallmarks of nucleophagy. Increased SRC-3 protein-induced autophagy and resulted in SUMO-1 localization to the nuclear membrane and formation of protrusions variously containing SRC-3 and chromatin. Aspects of SRC-3 overexpression and toxicity were recapitulated following treatment with clinically relevant agents that stabilize SRC-3 in breast cancer cells. We conclude that amplified SRC-3 levels have major impacts on nuclear protein quality control pathways and may mark cancer cells for sensitivity to protein stabilizing therapeutics. Full article

(This article belongs to the Section Cell Nuclei: Function, Transport and Receptors)

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14 pages, 1738 KiB

Open AccessArticle

Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH)

by Savneet Kaur, Preety Rawal, Hamda Siddiqui, Sumati Rohilla, Shvetank Sharma, Dinesh M Tripathi, Sukriti Baweja, Mohsin Hassan, Sebastian Vlaic, Reinhard Guthke, Maria Thomas, Rania Dayoub, Chaggan Bihari, Shiv K. Sarin and Thomas S. Weiss

Cells 2019, 8(10), 1277; https://doi.org/10.3390/cells8101277 - 18 Oct 2019

Cited by 28 | Viewed by 5864

Abstract

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially [...] Read more.

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF-β significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms Underlying the Pathogenesis of Hepatic Fibrosis)

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Correlation between RUNX1 mRNA expression and histopathological parameters. Expression of RUNX1 mRNA was analyzed by qRT-PCR in liver tissue samples from patients with NASH (n = 43), hepatic steatosis (n = 46) and normal liver tissue (n = 33) and correlated to histopathologic proven (A) NASH activity score (B) steatosis grade (C) inflammation grade and (D) fibrosis grade. HPRT mRNA expression was determined for normalization, statistical differences between several grades were analyzed by Kruskal-Wallis Test (p < 0.05 was considered significant) and ‘r’ denotes the Pearson’s correlation coefficient. Full article ">Figure 2
Immunohistochemical (IHC) analysis of RUNX1 expression in NASH patients. (A) RUNX1 immunostained images (20× objective) with an increasing number of brown nuclear immuno-positive cells (score 1–4). RUNX1 positivity was mostly observed in the non-parenchymal cells. Hematoxylin stained nuclei were distinguishable from RUNX1-positive brown nuclei. (B) Correlation between RUNX1 IHC and NASH activity score (n = 16), (C) RUNX1 IHC score and fibrosis grade (n = 16), and (D) RUNX1 IHC score and inflammatory grade (n = 16) in NASH patients. (E) Correlation between RUNX1 mRNA and its IHC score in liver tissues of patients (n = 16). The Pearson correlation (r) and statistical significance (p) were calculated. Full article ">Figure 3
RUNX1 and angiogenic gene expression in endothelial cells maintained in conditioned medium (CM) from palmitic acid (PA) treated hepatoma (Huh7) cells. (A) Huh7 cells treated with 0.2 mM PA for 48 h were analyzed for mRNA expression of RUNX1 and genes identified as angiogenesis associated DEGs in human NASH samples. The dotted line represents the control showing gene expression in Huh7 cells treated with BSA (n = 4). (B) HUVECs incubated with CM from PA-Huh7 cells were analyzed for mRNA expression of RUNX1, its target and angiogenic genes. The dotted line represents control, showing gene expression in HUVECs treated with CM from BSA-Huh7 cells (n = 4). 18S RNA expression was used for normalization. (C) Huh7 cells were treated with BSA or PA or CM alone and analyzed for the release of VEGF, PDGF-BB and TGF-β (pg/mL) (n = 3). (D) Relative RUNX1 mRNA expression in HUVECs treated with VEGF and TGF-β (10 ng/mL each) for 24 h. Un-induced cells without any manipulation were used as respective controls (Dotted line) (n = 3). 18S RNA expression was used for normalization. Data represent mean ± SD. * p < 0.05; ** p < 0.001. Full article ">Figure 4
RUNX1 alters expression of angiogenic and adhesion molecules in endothelial cells (A) HUVECs, treated with RUNX1 siRNA or NC siRNA and incubated with CM from PA-Huh7 cells, were analyzed for mRNA expression (fold change) of angiogenic, adhesion molecule and RUNX1 target genes (n = 3). (B) HUVECs transfected with RUNX1 expression plasmid (pRUNX1), control plasmid (pControl, i.e., empty vector) and/or incubated with VEGF (10 ng/mL) were analyzed for mRNA expression of adhesion molecule and chemotactic genes. HUVECs without any treatment were used as respective controls (Dotted line) (n = 3). 18S RNA expression was used for normalization. (C) Quantitative analysis of flow cytometry from (C) is shown (n = 3). (D) CCL2 levels (pg/mL) in culture media of HUVECs transfected with RUNX1 expression plasmid (pRUNX1), control plasmid (pControl, empty vector) and/or incubated with VEGF (10 ng/mL) (n = 4). Data represent mean ± SD. * p < 0.05 and ** p < 0.001. Full article ">Figure 5
RUNX1 enhances the angiogenic activitiy of endothelial cells. (A) Representative tube formation images of HUVECs on matrigel (4× objective) transfected with RUNX1 expression plasmid (pRUNX1), control plasmid (pControl, empty vector) and/or incubated with VEGF (10 ng/mL). (B) Average number of branch points per field and (C) tube length per field formed by HUVECs on matrigel under conditions described in (A) (n = 3). Data represent mean ± SD. * p < 0.05. Full article ">

20 pages, 3118 KiB

Open AccessArticle

Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells

by Chang Liu, Rouven Arnold, Gonçalo Henriques and Karima Djabali

Cells 2019, 8(10), 1276; https://doi.org/10.3390/cells8101276 - 18 Oct 2019

Cited by 55 | Viewed by 6013

Abstract

Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke, is caused by mutations in the LMNA gene. Nearly 90% of HGPS cases carry the G608G mutation [...] Read more.

Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke, is caused by mutations in the LMNA gene. Nearly 90% of HGPS cases carry the G608G mutation within exon 11 that generates a truncated prelamin A protein “progerin”. Progerin accumulates in HGPS cells and induces premature senescence at the cellular and organismal levels. Children suffering from HGPS develop numerous clinical features that overlap with normal aging, including atherosclerosis, arthritis, hair loss and lipodystrophy. To determine whether an aberrant signaling pathway might underlie the development of these four diseases (atherosclerosis, arthritis, hair loss and lipodystrophy), we performed a text mining analysis of scientific literature and databases. We found a total of 17 genes associated with all four pathologies, 14 of which were linked to the JAK1/2-STAT1/3 signaling pathway. We report that the inhibition of the JAK-STAT pathway with baricitinib, a Food and Drug Administration-approved JAK1/2 inhibitor, restored cellular homeostasis, delayed senescence and decreased proinflammatory markers in HGPS cells. Our ex vivo data using human cell models indicate that the overactivation of JAK-STAT signaling mediates premature senescence and that the inhibition of this pathway could show promise for the treatment of HGPS and age-related pathologies. Full article

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Text mining analysis to identify genes associated with vascular disease, arthritis, alopecia and lipodystrophy, and characterization of the cell-based aging model. (a) Venn diagram of the 2778 genes linked to the four diseases as determined by text mining. (b) Interaction network of the 17 gene products visualized by STRING. Nodes are proteins, and lines represent functional associations between proteins. A green line indicates neighborhood evidence; a blue line indicates cooccurrence evidence; a purple line indicates experimental evidence; a light blue line indicates database evidence; a black line indicates coexpression evidence. (c) Venn diagram of the signal transducer and activator of transcription (STAT) gene family showing the number of genes that are regulated by different STATs as indicated by the corresponding circles. Each gene was searched in the TRRUST version 2 (<a href="https://www.grnpedia.org/trrust/" target="_blank">https://www.grnpedia.org/trrust/</a>), TfactS (<a href="http://www.tfacts.org/" target="_blank">http://www.tfacts.org/</a>) and Regulatory Circuits databases (<a href="http://regulatorycircuits.org/" target="_blank">http://regulatorycircuits.org/</a>). Altogether, a total of 269 genes were found and curated to prevent repetitions due to aliases. (d) Growth curves of four independent control cell strains (purple, blue, orange, gray) and three Hutchinson-Gilford progeria syndrome (HGPS) cell strains (light blue, red, and turquoise). All cultures started at passage 13, and at this passage, the percentage of SA-ß-gal-positive cells was less than 5% in all cell strains. Proliferation rates were determined over 26 passages over 104 days. (e) The percentage of SA-ß-gal-positive cells was scored every other passage in cultures from panel d. (f) Representative images of SA-ß-gal-positive cells in cultures exhibiting <5% SNS, 15% SNS and 30% SNS. GMO1651c corresponds to normal fibroblasts and P003 corresponds to HGADFN003, an HGPS cell strain. (g) Relative percentage of cells in the G0/G1, S and G2/M phases of the cell cycle are shown for normal (1651c) and HGPS (P003) cultures with a senescence index of <5% and 30%. PI was used for DNA staining. (h) Representative western blot of control (1651c, 1652c) and HGPS (P003, P127) fibroblasts from cultures at the indicated percentages of senescence. Antibodies directed against the indicated proteins were used. All experiments were performed at least three times (n > 3). Full article ">Figure 2
Quantitative real-time PCR analysis of the 17 genes identified by text mining in normal and HGPS cells during replicative senescence. (a) Table showing the cell strains and the passages corresponding percentage of senescence (SNS). (b–r) mRNA levels of the indicated genes were determined in controls (GMO1651c, and GMO1652c) and HGPS (HGADFN003 and HGADFN127) cell strains at the indicated percentage of senescence (SNS). Relative expression was normalized to the expression of GAPDH. Graphs show the mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, n > 3). The mean fold changes between 5% and 30% SNS for control and HGPS are indicated. Full article ">Figure 3
Status of the JAK-STAT signaling pathway in control and HGPS cells during replicative senescence. (a) Table showing the cell strains and the passages corresponding percentages of senescence (SNS). (b,e,h) Representative western blot images for JAK1/2, STAT1/3, p-STAT1 (tyr701), p-STAT3 (tyr705) and β-actin in the control (GMO1651c, and GMO1652c) and HGPS (HGADFN003, and HGADFN127) cell strains at the indicated percentages of senescence. (c) Quantification of JAK1, (d) JAK2, (f) STAT1, (g) p-STAT1, (i) STAT3, and (j) p-STAT3. Graphs show the mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, n > 3). Fold changes between the samples with 5% and 30% senescence are indicated. Full article ">Figure 4
Status of the JAK-STAT signaling pathway in normal and HGPS cells treated with 1 μM Bar. (a,b) Representative images of western blots for JAK1/2, STAT1/3, P-STAT1/3 and β-actin in normal (GMO1651c, and GMO1652c) and HGPS (HGADFN003, and HGADFN127) cells treated as indicated. Cultures exhibiting <5% senescence were treated with Bar or DMSO for a period of one month. (c) Quantification of JAK1, (d) JAK2, (e) P-STAT3, (f) STAT3, (g) P-STAT1, and (h) STAT1. Graphs show the mean ± SD. Protein levels were compared by two-tailed t test (* p < 0.05, ** p < 0.01, *** p < 0.001, n > 3). Full article ">Figure 5
Bar treatment ameliorates normal and HGPS cellular functions during long-term treatment. (a) Graph shows the population doublings of control (GMO1651c, and GMO1652c) and HGPS (HGADFN003, and HGADFN127) cells. Bar (1 μM) or DMSO treatment was administrated for one week or one month as indicated. The percentage of senescence (SNS) in cultures prior to treatment is indicated. (b) Graph shows the percentage of SA-ß-gal-positive cells measured after treatment. (c) Autophagy activity was determined by measuring MDC levels using fluorescence photometry of the same cultures as in (a). (d) Proteasome activity was determined by measuring chymotrypsin-like proteasome activity using Suc-LLVY-AMC as a substrate. (e) Intracellular ROS levels were determined by measuring oxidized dichlorofluorescein (DCF) levels using a DCFDA cellular ROS detection assay. (f) Cellular ATP levels were measured using a CellTiter-Glo luminescence ATP assay. (c–f) The percent change in Bar-treated cells relative to mock-treated counterparts is indicated. (g) Representative images of a western blot for progerin in HGPS (HGADFN127) cells from cultures at 5% and 15% senescence that were administrated the mock or Bar treatment for one week or one month as indicated. (h) Quantification of the progerin signal. The percent change between Bar-treated cells and mock-treated counterparts are indicated. Graphs show the mean ± SD. Comparisons were performed by two-tailed t test (* p < 0.05, ** p < 0.01, *** p < 0.01, (n > 3)). Full article ">Figure 6
Etoposide treatment activates the JAK-STAT signaling pathway in HGPS and normal cells. (a) Schematic representation of the etoposide treatment protocol. All treatments started with cultures exhibiting <5% senescence (SNS). Cells were either pretreated with or without 1 µM Bar for two days and then expose to medium with or without etoposide in the presence or absence of Bar for a period of six days. Next, cells were grown for four days with or without Bar as indicated. (b) Representative images of SA-ß-gal-positive cells in mock-, etoposide- and etoposide + Bar-treated cultures (GMO1651c and HGADFN003) are shown. The percentages of senescence (SNS) after treatment are indicated. (c) Representative images of western blots for progerin, p21 and ß-actin in normal (GMO1651c, and GMO1652c) and HGPS (HGADFN003, and HGADFN127) cells treated as indicated. (d) and (g) Representative images of western blots for JAK1/2, STAT1/3, P-STAT1/3 and ß-actin in normal (GMO1651c, and GMO1652c) and HGPS (HGADFN003, and HGADFN127) cells treated as indicated. (e) Quantification of STAT1, (f) p-STAT1, (h) STAT3, and (i) p-STAT3. The percent change between etoposide- and etoposide+Bar-treated cells is indicated. (j–n) Quantitative real-time PCR analysis of CCL2, CXCL8, IFNG, IL6, and TNFα in cells treated as indicated. Relative expression was normalized to the expression of GAPDH. Graphs show the mean ± SD. Comparisons were done by two-tailed t test (* p < 0.05, ** p < 0.01, *** p < 0.001). All experiments were repeated at least three times (n > 3). Full article ">

12 pages, 2351 KiB

Open AccessArticle

Capsaicin Targets tNOX (ENOX2) to Inhibit G1 Cyclin/CDK Complex, as Assessed by the Cellular Thermal Shift Assay (CETSA)

by Atikul Islam, Ally J. Su, Zih-Ming Zeng, Pin Ju Chueh and Ming-Hung Lin

Cells 2019, 8(10), 1275; https://doi.org/10.3390/cells8101275 - 18 Oct 2019

Cited by 26 | Viewed by 6785

Abstract

Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed [...] Read more.

Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed in cancer but not in non-transformed cells. This attenuates cancer cell growth by inducing apoptosis. The capsaicin-mediated inhibition of tNOX was recently shown to prolong the cell cycle. However, the molecular events underlying this regulation have not yet been investigated. In the present study, we used a cellular thermal shift assay (CETSA) to detect “target engagement” of capsaicin and its consequent impact on cell cycle progression. Our results indicated that capsaicin engaged with tNOX and triggered the proteasomal degradation of tNOX, which leads to the inhibition of NAD⁺-dependent SIRT1 deacetylase. Ultimately, the acetylation levels of c-Myc and p53 were enhanced, which suppressed the activation of G1 cyclin/Cyclin-dependent kinase complexes and triggered cell cycle arrest in cancer cells. The results obtained when tNOX was overexpressed in non-cancer cells validated its importance in cell cycle progression. These findings provide the first molecular insights into the regulatory role of tNOX and the anti-proliferative property of capsaicin in regulating the cell cycle of bladder cancer cells. Full article

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

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Figure 1

Figure 1
CETSA-based determination of binding between capsaicin and tNOX. (A) CETSA was performed as described in the Materials and Methods section. Cell lysates were separated by SDS-PAGE and analyzed by Western blotting. β-Actin was detected as an internal control. Representative images are shown. (B) CETSA curves of tNOX in T24 cells were determined in the absence and presence of capsaicin. Each band intensity of tNOX was normalized with respect to that obtained at 43 °C. The graphs are an average of three independent experiments. (C) The quantification of relative intensity of tNOX protein versus increased temperature from three independent experiments (* p < 0.001). Full article ">Figure 2
Capsaicin suppresses tNOX expression through proteasome-mediated degradation. Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting. β-Actin was detected as an internal control. Representative images are shown. Values (mean ± S. E.) are from at least three independent experiments (* p < 0.05, *** p < 0.001 for capsaicin-treated cells vs. controls). (A) tNOX expression was significantly decreased by capsaicin at 100 and 200 μM. (B) Capsaicin (200 μM) clearly reduces tNOX stability as assessed with a cycloheximide-chase assay. (C) The proteasome inhibitor, MG132, reverses the capsaicin-induced suppression of tNOX expression. Full article ">Figure 3
Capsaicin induces cell cycle arrest at the G0/G1 phases. T24 cells were treated with capsaicin or ethanol for 24 h. Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting. β-Actin was detected as an internal control. Representative images are shown. Full article ">Figure 4
Effect of capsaicin on the proliferation and cell cycle of T24 cells. (A) Dynamic monitoring of cell proliferation was performed using impedance technology, as described in the Materials and Methods section. Normalized cell index values measured over 96 h are shown. (B) Cells were exposed to different concentrations of capsaicin for 24 or 48 h and cell viability was measured using MTT assays. Values (means ± SDs) are from three independent experiments. There was a significant decrease in cell viability in cells treated with capsaicin when compared with controls (** p <0.01, *** p <0.001). (C,D) T24 cells were exposed to different concentrations of capsaicin for 24 h and flow cytometry was used to assess their cell-cycle phase. The graphs are representative of three independent experiments. Full article ">Figure 5
tNOX overexpression in NIH3T3 cells enhances cell growth. NIH3T3 cells were transfected with GST, GST–tNOX wild-type, GST–tNOXS504D, or GST–tNOXS504A. (A,B) Overexpression of GST–tNOX wild-type, GST–tNOXS504D, or GST–tNOX S504A fusion proteins was analyzed using anti-GST, anti-tNOX, anti-phospho-ERK, and anti-ERK antibodies. β-Actin was detected as a loading control. Representative images are shown. (C) The cell-doubling time was determined by CMFDA staining of NIH3T3 cells. The presented values (mean ± S.E.) represent at least three independent experiments (** p < 0.001). (C) Dynamic monitoring of cell proliferation was performed using impedance technology, as described in the Materials and Methods section. Normalized cell index values measured over 138 h are shown. Full article ">

18 pages, 2070 KiB

Open AccessArticle

The Oncogenic Signaling Pathways in BRAF-Mutant Melanoma Cells Are Modulated by Naphthalene Diimide-Like G-Quadruplex Ligands

by Marta Recagni, Martina Tassinari, Filippo Doria, Graziella Cimino-Reale, Nadia Zaffaroni, Mauro Freccero, Marco Folini and Sara N. Richter

Cells 2019, 8(10), 1274; https://doi.org/10.3390/cells8101274 - 18 Oct 2019

Cited by 12 | Viewed by 3642

Abstract

Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of [...] Read more.

Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of the non-canonical nucleic acid G-quadruplex structures within oncogene promoters has stood out as a promising approach to interfere with oncogenic signaling pathways in cancer cells, paving the way toward the development of G-quadruplex ligands as antitumor drugs. Here, we present the synthesis and screening of a library of differently functionalized core-extended naphthalene diimides for their activity against the BRAFV600E-mutant melanoma cell line. The most promising compound was able to stabilize G-quadruplexes that formed in the promoter regions of two target genes relevant to melanoma, KIT and BCL-2. This activity led to the suppression of protein expression and thus to interference with oncogenic signaling pathways involved in BRAF-mutant melanoma cell survival, apoptosis, and resistance to drugs. This G-quadruplex ligand thus represents a suitable candidate for the development of melanoma treatment options based on a new mechanism of action and could reveal particular significance in the context of resistance to targeted therapies of BRAF-mutant melanoma cells. Full article

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21 pages, 5570 KiB

Open AccessArticle

Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling

by Christopher Differ, Franka Klatte-Schulz, Nicole Bormann, Susann Minkwitz, Petra Knaus and Britt Wildemann

Cells 2019, 8(10), 1273; https://doi.org/10.3390/cells8101273 - 18 Oct 2019

Cited by 8 | Viewed by 5396

Abstract

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large [...] Read more.

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling. Full article

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

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Cells, Volume 8, Issue 10 (October 2019) – 191 articles

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