Cells

16 pages, 3151 KiB

Open AccessArticle

MEG3 Expression Indicates Lymph Node Metastasis and Presence of Cancer-Associated Fibroblasts in Papillary Thyroid Cancer

by Sina Dadafarin, Tomás C. Rodríguez, Michelle A. Carnazza, Raj K. Tiwari, Augustine Moscatello and Jan Geliebter

Cells 2022, 11(19), 3181; https://doi.org/10.3390/cells11193181 - 10 Oct 2022

Cited by 8 | Viewed by 2947

Abstract

Papillary thyroid cancer is the most common endocrine malignancy, occurring at an incidence rate of 12.9 per 100,000 in the US adult population. While the overall 10-year survival of PTC nears 95%, the presence of lymph node metastasis (LNM) or capsular invasion indicates [...] Read more.

Papillary thyroid cancer is the most common endocrine malignancy, occurring at an incidence rate of 12.9 per 100,000 in the US adult population. While the overall 10-year survival of PTC nears 95%, the presence of lymph node metastasis (LNM) or capsular invasion indicates the need for extensive neck dissection with possible adjuvant radioactive iodine therapy. While imaging modalities such as ultrasound and CT are currently in use for the detection of suspicious cervical lymph nodes, their sensitivities for tumor-positive nodes are low. Therefore, advancements in preoperative detection of LNM may optimize the surgical and medical management of patients with thyroid cancer. To this end, we analyzed bulk RNA-sequencing datasets to identify candidate markers highly predictive of LNM. We identified MEG3, a long-noncoding RNA previously described as a tumor suppressor when expressed in malignant cells, as highly associated with LNM tissue. Furthermore, the expression of MEG3 was highly predictive of tumor infiltration with cancer-associated fibroblasts, and single-cell RNA-sequencing data revealed the expression of MEG3 was isolated to cancer-associated fibroblasts (CAFs) in the most aggressive form of thyroid cancers. Our findings suggest that MEG3 expression, specifically in CAFs, is highly associated with LNM and may be a driver of aggressive disease. Full article

(This article belongs to the Special Issue Regulatory Roles of Non-coding RNAs in Cancer)

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Differential expression analysis between tumor and normal thyroid tissue. (A) Principal component analysis of 44 PTC and matched normal tissue. (B) Proportion of known driver mutations identified in tumor samples. (C) Mutational status, sex, TDS, and ERK score in individual tumors. TDS and ERK scale represent Log2-scaled high and low thyroid differentiation and ERK-activation based on gene signatures [<a href="#B47-cells-11-03181" class="html-bibr">47</a>]. (D) Pathway analysis of differentially expressed genes. (E) Hierarchical clustering of the top 100 differentially expressed lncRNAs (scaled normalized reads by column). FDR, false discovery rate; TDS, thyroid differentiation score. Full article ">Figure 2
LNM-related co-expression analysis of PTC samples. (A) Relationship between WCGNA modules and LNM status. Values represent Pearson correlation coefficient and the correlation p-value (in brackets) between LNM and module eigengene. (B) Hallmark gene set analysis of the module most correlated with LNM status (black). (C) Black module constituent gene expression ranked by gene significance value related to lymph node metastasis (GS.LN) (D) Black module lncRNAs upregulated in LNM+ tissue but not LNM- tissue. LNM+ and LNM−, positive and negative lymph node status. * p < 0.05; ns, non-significant. Full article ">Figure 3
MEG3 expression is increased in PTC with LNM and correlates with poor outcomes (A) TCGA all patients (left) and BRAFV600E cohort (right) survival given top (red) and bottom (blue) quartile MEG3 expression. (B) TCGA Solid Tissue Normal MEG3 expression. (C) MEG3 fold-change expression in NYMC LNM+ and LNM-PTC. Full article ">Figure 4
MEG3 isoform expression and association with infiltration of CAFs. (A) MEG3 transcript isoform usage in normal (non-cancerous) tissue and NYMC PTC (LNM+/−). (B) Scatter plots demonstrating the correlation of MEG3 expression in TCGA THCA project with tumor purity and estimated infiltration level of CAFs using TIMER2.0. EPIC output was used to represent fibroblast infiltration estimates from TIMER2.0. * p < 0.05; ns, non-significant. Full article ">Figure 5
Single-cell RNA-sequencing results identify cell types that express MEG3. Expression profile of MEG3 in 5 ATC patient tumors represented as UMAPs with overlaid heatmaps demonstrating MEG3 expression and expression in different cell subtypes in each sample. Red circles represent clusters of cells that are annotated as fibroblasts. Full article ">

22 pages, 1469 KiB

Open AccessReview

Patient Selection Approaches in FGFR Inhibitor Trials—Many Paths to the Same End?

by Peter Ellinghaus, Daniel Neureiter, Hendrik Nogai, Sebastian Stintzing and Matthias Ocker

Cells 2022, 11(19), 3180; https://doi.org/10.3390/cells11193180 - 10 Oct 2022

Cited by 11 | Viewed by 3332

Abstract

Inhibitors of fibroblast growth factor receptor (FGFR) signaling have been investigated in various human cancer diseases. Recently, the first compounds received FDA approval in biomarker-selected patient populations. Different approaches and technologies have been applied in clinical trials, ranging from protein (immunohistochemistry) to mRNA [...] Read more.

Inhibitors of fibroblast growth factor receptor (FGFR) signaling have been investigated in various human cancer diseases. Recently, the first compounds received FDA approval in biomarker-selected patient populations. Different approaches and technologies have been applied in clinical trials, ranging from protein (immunohistochemistry) to mRNA expression (e.g., RNA in situ hybridization) and to detection of various DNA alterations (e.g., copy number variations, mutations, gene fusions). We review, here, the advantages and limitations of the different technologies and discuss the importance of tissue and disease context in identifying the best predictive biomarker for FGFR targeting therapies. Full article

(This article belongs to the Collection Fibroblast Growth Factor Receptor (FGFR) Signaling Pathway in Tumor 2.0)

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11 pages, 1086 KiB

Open AccessReview

Mitochondrial Distress in Methylmalonic Acidemia: Novel Pathogenic Insights and Therapeutic Perspectives

by Svenja Aline Keller and Alessandro Luciani

Cells 2022, 11(19), 3179; https://doi.org/10.3390/cells11193179 - 10 Oct 2022

Cited by 5 | Viewed by 3090

Abstract

Mitochondria are highly dynamic, double-membrane-enclosed organelles that sustain cellular metabolism and, hence, cellular, and organismal homeostasis. Dysregulation of the mitochondrial network might, therefore, confer a potentially devastating vulnerability to high-energy-requiring cell types, contributing to a broad variety of hereditary and acquired diseases, which [...] Read more.

Mitochondria are highly dynamic, double-membrane-enclosed organelles that sustain cellular metabolism and, hence, cellular, and organismal homeostasis. Dysregulation of the mitochondrial network might, therefore, confer a potentially devastating vulnerability to high-energy-requiring cell types, contributing to a broad variety of hereditary and acquired diseases, which include inborn errors of metabolism, cancer, neurodegeneration, and aging-associated adversities. In this Review, we highlight the biological functions of mitochondria-localized enzymes, from the perspective of understanding the pathophysiology of the inherited disorders destroying mitochondrial homeostasis and cellular metabolism. Using methylmalonic acidemia (MMA) as a paradigm of mitochondrial dysfunction, we discuss how mitochondrial-directed signaling pathways sustain the physiological homeostasis of specialized cell types and how these may be disturbed in disease conditions. This Review also provides a critical analysis of molecular underpinnings, through which defects in the autophagy-mediated quality control and surveillance systems contribute to cellular dysfunction, and indicates potential therapeutic strategies for affected tissues. These insights might, ultimately, advance the discovery and development of new therapeutics, not only for methylmalonic acidemia but also for other currently intractable mitochondrial diseases, thus transforming our ability to modulate health and homeostasis. Full article

(This article belongs to the Special Issue Autophagy in Kidney Homeostasis and Disease)

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The absence of the enzyme MMUT leads to accumulation of organic acids and mitochondrial abnormalities. Mutations in the MMUT gene encoding the mitochondrial enzyme methylmalonyl-coenzyme A mutase, which mediates the terminal step of branched chain amino acid and odd-chain lipid catabolism, trigger the accumulation of metabolites (e.g., methylmalonic acid, propionic acid, and 2-methylcitric acid) and lack of anaplerosis. This leads to morphologically abnormal mitochondria with disorganized cristae, decreased production of ATP (red arrows) and exaggerated generation of ROS/oxidative stress, ultimately causing severe organ dysfunctions that primarily affect brain, liver, and kidney. Full article ">Figure 2
Working model depicting the link between MMUT, mitochondria, mitophagy, and epithelial homeostasis in healthy and MMA-affected kidney cells. In MMA-affected kidney cells and zebrafish, the deficiency of the enzyme MMUT and the resulting accumulation of toxic organic acids trigger mitochondrial abnormalities, which are characterized by a collapse of the mitochondrial membrane potential (ΔΨm, red arrows), impaired ATP production/bioenergetics (red arrows) and augmented generation of mitochondrial ROS and oxidative stress. Faulty execution of PINK1‒Parkin-mediated mitophagy induced by MMUT deficiency impedes the delivery of damaged mitochondria and their dismantling by autophagy–lysosome degradation systems. This, in turn, promotes the accumulation of damaged and/or dysfunctional, ROS-overproducing mitochondria that, ultimately, trigger cellular and kidney damage. The treatments with mitochondria-targeted ROS scavengers mito-TEMPO or MitoQ repair mitochondrial dysfunctions, neutralizes epithelial damage in MMA cells, and improves disease-relevant phenotypes in mmut-deficient zebrafish. Full article ">

22 pages, 4644 KiB

Open AccessArticle

ROCK Inhibitor (Y-27632) Abolishes the Negative Impacts of miR-155 in the Endometrium-Derived Extracellular Vesicles and Supports Embryo Attachment

by Islam M. Saadeldin, Bereket Molla Tanga, Seonggyu Bang, Chaerim Seo, Okjae Koo, Sung Ho Yun, Seung Il Kim, Sanghoon Lee and Jongki Cho

Cells 2022, 11(19), 3178; https://doi.org/10.3390/cells11193178 - 10 Oct 2022

Cited by 6 | Viewed by 2648

Abstract

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment [...] Read more.

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics. Porcine embryos were generated by parthenogenetic electric activation in feeder-free culture conditions supplemented with or without Endo-EVs. The cellular uptake of Endo-EVs was confirmed using the lipophilic dye PKH26. Endo-EVs cargo contained miR-100, miR-132, and miR-155, together with the mRNAs of porcine endogenous retrovirus (PERV) and β-catenin. Targeting PERV with CRISPR/Cas9 resulted in reduced expression of PERV mRNA transcripts and increased miR-155 in the Endo-EVs, and supplementing these in embryos reduced embryo attachment. Supplementing the medium containing Endo-EVs with miR-155 inhibitor significantly improved the embryo attachment with a few outgrowths, while supplementing with Rho-kinase inhibitor (RI, Y-27632) dramatically improved both embryo attachment and outgrowths. Moreover, the expression of miR-100, miR-132, and the mRNA transcripts of BCL2, zinc finger E-box-binding homeobox 1, β-catenin, interferon-γ, protein tyrosine phosphatase non-receptor type 1, PERV, and cyclin-dependent kinase 2 were all increased in embryos supplemented with Endo-EVs + RI compared to those in the control group. Endo-EVs + RI reduced apoptosis and increased the expression of OCT4 and CDX2 and the cell number of embryonic outgrowths. We examined the individual and combined effects of RI compared to those of the miR-155 mimic and found that RI can alleviate the negative effects of the miR-155 mimic on embryo attachment and outgrowths. EVs can improve embryo attachment and the unwanted effects of the de trop cargo contents (miR-155) can be alleviated through anti-apoptotic molecules such as the ROCK inhibitor. Full article

(This article belongs to the Special Issue Molecular and Clinical Advances in Understanding Early Embryo Development)

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Graphical abstract

16 pages, 3794 KiB

Open AccessArticle

Ethylene Activates the EIN2-EIN3/EIL1 Signaling Pathway in Tapetum and Disturbs Anther Development in Arabidopsis

by Ben-Shun Zhu, Ying-Xiu Zhu, Yan-Fei Zhang, Xiang Zhong, Keng-Yu Pan, Yu Jiang, Chi-Kuang Wen, Zhong-Nan Yang and Xiaozhen Yao

Cells 2022, 11(19), 3177; https://doi.org/10.3390/cells11193177 - 10 Oct 2022

Cited by 10 | Viewed by 2792

Abstract

Ethylene was previously reported to repress stamen development in both cucumber and Arabidopsis. Here, we performed a detailed analysis of the effect of ethylene on anther development. After ethylene treatment, stamens but not pistils display obvious developmental defects which lead to sterility. [...] Read more.

Ethylene was previously reported to repress stamen development in both cucumber and Arabidopsis. Here, we performed a detailed analysis of the effect of ethylene on anther development. After ethylene treatment, stamens but not pistils display obvious developmental defects which lead to sterility. Both tapetum and microspores (or microsporocytes) degenerated after ethylene treatment. In ein2-1 and ein3-1 eil1-1 mutants, ethylene treatment did not affect their fertility, indicating the effects of ethylene on anther development are mediated by EIN2 and EIN3/EIL1 in vivo. The transcription of EIN2 and EIN3 are activated by ethylene in the tapetum layer. However, ectopic expression of EIN3 in tapetum did not induce significant anther defects, implying that the expression of EIN3 are regulated post transcriptional level. Consistently, ethylene treatment induced the accumulation of EIN3 in the tapetal cells. Thus, ethylene not only activates the transcription of EIN2 and EIN3, but also stabilizes of EIN3 in the tapetum to disturb its development. The expression of several ethylene related genes was significantly increased, and the expression of the five key transcription factors required for tapetum development was decreased after ethylene treatment. Our results thus point out that ethylene inhibits anther development through the EIN2-EIN3/EIL1 signaling pathway. The activation of this signaling pathway in anther wall, especially in the tapetum, induces the degeneration of the tapetum and leads to pollen abortion. Full article

(This article belongs to the Special Issue Pollen Development)

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11 pages, 448 KiB

Open AccessCommunication

An Inflammatory Signature to Predict the Clinical Benefit of First-Line Cetuximab Plus Platinum-Based Chemotherapy in Recurrent/Metastatic Head and Neck Cancer

by Stefano Cavalieri, Mara Serena Serafini, Andrea Carenzo, Silvana Canevari, Deborah Lenoci, Federico Pistore, Rosalba Miceli, Stefania Vecchio, Daris Ferrari, Cecilia Moro, Andrea Sponghini, Alessia Caldara, Maria Cossu Rocca, Simona Secondino, Gabriella Moretti, Nerina Denaro, Francesco Caponigro, Emanuela Vaccher, Gaetana Rinaldi, Francesco Ferraù, Paolo Bossi, Lisa Licitra and Loris De Cecco Show full author list Hide full author list

Cells 2022, 11(19), 3176; https://doi.org/10.3390/cells11193176 - 10 Oct 2022

Viewed by 2140

Abstract

Epidermal growth factor receptor (EGFR) pathway has been shown to play a crucial role in several inflammatory conditions and host immune-inflammation status is related to tumor prognosis. This study aims to evaluate the prognostic significance of a four-gene inflammatory signature in recurrent/metastatic (R/M) [...] Read more.

Epidermal growth factor receptor (EGFR) pathway has been shown to play a crucial role in several inflammatory conditions and host immune-inflammation status is related to tumor prognosis. This study aims to evaluate the prognostic significance of a four-gene inflammatory signature in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients treated with the EGFR inhibitor cetuximab plus chemotherapy. The inflammatory signature was assessed on 123 R/M HNSCC patients, enrolled in the multicenter trial B490 receiving first-line cetuximab plus platinum-based chemotherapy. The primary endpoint of the study was progression free survival (PFS), while secondary endpoints were overall survival (OS) and objective response rate (ORR). The patient population was subdivided into 3 groups according to the signature score groups. The four-genes-signature proved a significant prognostic value, resulting in a median PFS of 9.2 months in patients with high vs. 6.2 months for intermediate vs. 3.9 months for low values (p = 0.0016). The same findings were confirmed for OS, with median time of 18.4, 13.4, and 7.5 months for high, intermediate, and low values of the score, respectively (p = 0.0001). When ORR was considered, the signature was significantly higher in responders than in non-responders (p = 0.0092), reaching an area under the curve (AUC) of 0.65 (95% CI: 0.55–0.75). Our findings highlight the role of inflammation in the response to cetuximab and chemotherapy in R/M-HNSCC and may have translational implications for improving treatment selection. Full article

(This article belongs to the Special Issue New Trends and Advances in Diagnosis and Prognosis of Head and Neck Cancer from Carcinogenesis to Future Molecular Targeted Therapies)

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13 pages, 640 KiB

Open AccessFeature PaperArticle

KRAS G12D Mutation Subtype in Pancreatic Ductal Adenocarcinoma: Does It Influence Prognosis or Stage of Disease at Presentation?

by Henry Shen, Joanne Lundy, Andrew H. Strickland, Marion Harris, Michael Swan, Christopher Desmond, Brendan J. Jenkins and Daniel Croagh

Cells 2022, 11(19), 3175; https://doi.org/10.3390/cells11193175 - 10 Oct 2022

Cited by 23 | Viewed by 3228

Abstract

Background: KRAS G12D mutation subtype is present in over 40% of pancreatic ductal adenocarcinoma (PDAC), one of the leading global causes of cancer death. This retrospective cohort study aims to investigate whether detection of the KRAS G12D mutation subtype in PDAC patients [...] Read more.

Background: KRAS G12D mutation subtype is present in over 40% of pancreatic ductal adenocarcinoma (PDAC), one of the leading global causes of cancer death. This retrospective cohort study aims to investigate whether detection of the KRAS G12D mutation subtype in PDAC patients is a determinant of prognosis across all stages of disease. Methods: We reviewed the medical records of 231 patients presenting with PDAC at a large tertiary hospital, and compared survival using the Kaplan Meier, log-rank test and Cox proportional hazards regression model. Results: KRAS G12D mutation subtype was not significantly associated with poorer survival compared across the whole population of PDAC patients (p = 0.107; HR 1.293 95% CI (0.946–1.767)). However, KRAS G12D patients who were resectable had a shorter median survival time of 356 days compared to all other genotypes (median survival 810 days) (p = 0.019; HR 1.991 95% CI (1.121–3.537)). Conclusions: KRAS G12D patients who were resectable at diagnosis had shorter survival compared to all other PDAC patients. These data suggest that KRAS G12D may be a clinically useful prognostic biomarker of PDAC. Full article

(This article belongs to the Special Issue Targeting RAS-Dependent Cancers)

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26 pages, 3314 KiB

Open AccessReview

Mitochondrial VDAC1: A Potential Therapeutic Target of Inflammation-Related Diseases and Clinical Opportunities

by Hang Hu, Linlin Guo, Jay Overholser and Xing Wang

Cells 2022, 11(19), 3174; https://doi.org/10.3390/cells11193174 - 10 Oct 2022

Cited by 29 | Viewed by 7060

Abstract

The multifunctional protein, voltage-dependent anion channel 1 (VDAC1), is located on the mitochondrial outer membrane. It is a pivotal protein that maintains mitochondrial function to power cellular bioactivities via energy generation. VDAC1 is involved in regulating energy production, mitochondrial oxidase stress, Ca²⁺ [...] Read more.

The multifunctional protein, voltage-dependent anion channel 1 (VDAC1), is located on the mitochondrial outer membrane. It is a pivotal protein that maintains mitochondrial function to power cellular bioactivities via energy generation. VDAC1 is involved in regulating energy production, mitochondrial oxidase stress, Ca²⁺ transportation, substance metabolism, apoptosis, mitochondrial autophagy (mitophagy), and many other functions. VDAC1 malfunction is associated with mitochondrial disorders that affect inflammatory responses, resulting in an up-regulation of the body’s defensive response to stress stimulation. Overresponses to inflammation may cause chronic diseases. Mitochondrial DNA (mtDNA) acts as a danger signal that can further trigger native immune system activities after its secretion. VDAC1 mediates the release of mtDNA into the cytoplasm to enhance cytokine levels by activating immune responses. VDAC1 regulates mitochondrial Ca²⁺ transportation, lipid metabolism and mitophagy, which are involved in inflammation-related disease pathogenesis. Many scientists have suggested approaches to deal with inflammation overresponse issues via specific targeting therapies. Due to the broad functionality of VDAC1, it may become a useful target for therapy in inflammation-related diseases. The mechanisms of VDAC1 and its role in inflammation require further exploration. We comprehensively and systematically summarized the role of VDAC1 in the inflammatory response, and hope that our research will lead to novel therapeutic strategies that target VDAC1 in order to treat inflammation-related disorders. Full article

(This article belongs to the Collection The Pathomechanism of Mitochondrial Diseases)

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VDAC1 regulates inflammatory pathogenesis. Mitochondria are the center of energy generation, the TCA cycle, glycolysis and lipid metabolism. VDAC1 is the fundamental component that maintains mitochondrial function. VDAC1 plays an important role in the regulation of apoptosis, mtDNA release, Ca2+ signaling, TCA cycle, glycolysis, lipid metabolism and mitophagy. Impaired mitochondrial homeostasis with dysfunctional signal networks results in inflammatory pathogenesis and mitochondrial diseases. Abbreviations: ACSL: long-chain acyl-CoA synthase; BAX: Bcl-2-associated X protein; CPT1a: carnitine palmitoyltransferase 1A; Cyto c: cytochrome c; HK: hexokinase; LC3: microtubule-associated proteins 1A/1B light chain 3; IMM: inner mitochondrial membrane; MOMP: mitochondrial outer membrane permeabilization; mtDNA: mitochondrial DNA; PINK1: PTEN-induced putative kinase 1; TCA cycle: tricarboxylic acid cycle; Ub: ubiquitin; VDAC1: voltage-dependent anion channel 1. Full article ">Figure 2
VDAC1 mediates apoptosis and mtDNA release to promote cytokines expression and inflammatory pathogenesis. (A) Bcl-2 family member, BAX, interacts with VDAC1 to release cytochrome c into the cytoplasm, promoting apoptosis. (B) MOMP induces proteasomal degradation of IAPs, which causes NIK to further induce the pro-inflammatory NF-κB signal and activate caspase-1/8; this in turn results in the maturation of pro-inflammatory factor IL-1β. (C) Mitochondrial overproduced ROS oxidize mtDNA to fomtDNA. The mtDNA and fomtDNA pass the VDAC1 oligomers channel or the oligomerization BAX pore into the cytoplasm. The released mtDNA/fomtDNA induce the cGAS-STING pathway to promote interferon gene expressions via TBK1-IRF3 to up-regulate IFN-α/β, or through TBK1-NF-κB to enhance TNF-α, IL-6 and IL-12. Additionally, mtDNA interacts with TLR9 and promotes TNF-α, IL-6 and IL-12 expression via NF-κB signaling. Moreover, the released mtDNA induces the NLRP3 inflammasome and AIM2 inflammasome to enhance caspase-1/8 activation to promote IL-1β/IL-18 maturation. Abbreviations: AIM2: absent in melanoma 2; BAX: Bcl-2-associated X protein; cGAS: cyclic GMP-AMP synthase; Cyto c: cytochrome c; fomtDNA: oxidized mtDNA fragments; IAP: inhibitors of apoptosis; IFN: interferon; IL: interleukin; IRF3: interferon regulatory factor 3; IMM: inner mitochondrial membrane; MOMP: mitochondrial outer membrane permeabilization; mtDNA: mitochondrial DNA; NF-κB: nuclear factor-κB; NIK: NF-κB induced kinase; NLRP3: nucleotide-binding domain and leucine-rich repeat (LRR) containing P3; ROS: reactive oxygen species; STING: stimulator of interferon genes; TLR9: Toll-like receptor 9; TBK1: TANK-binding kinase 1; TNF: tumor necrosis factor; VDAC1: voltage-dependent anion channel 1. Full article ">Figure 3
VDAC1 is involved in Ca2+ transportation, lipid metabolism, glycolysis, TCA cycleand in inflammatory responses. (A) VDAC1 regulates Ca2+ transportation, glycolysis, TCA cycle and lipid metabolism. VDAC1 transports Ca2+ between the mitochondria and cytoplasm to maintain calcium homeostasis. In the energy generation system, the VDAC1 pore maintains substrates, metabolites, biomolecules, etc., in a balanced manner to sustain salutogenesis. (B) Ca2+ signaling affects the inflammatory responses of neutrophils, macrophages, dendritic cells and CD4+ T cells. (C) Inflammatory responses of macrophages, dendritic cells and CD4+ T cells are promoted by glycolysis/TCA cycle energy generation pathways. Abbreviations: VDAC1: voltage-dependent anion channel 1; TRPML1: also known as MCOLN1, mucolipin TRP cation channel 1; GRP75: glucose-regulated protein 75; IP3R: inositol 1,4,5-trisphosphate receptor; DJ1: deglycase DJ-1, also known as Parkinson disease protein 7, is encoded by the PARK7 gene in human; RyR2: ryanodine receptor 2; CPT1a: carnitine palmitoyltransferase 1A; ACSL: long-chain acyl-CoA synthase; TCA cycle: tricarboxylic acid cycle; HK: hexokinase; ATP: adenosine triphosphate; ADP: adenosine diphosphate; NADH: nicotinamide adenine dinucleotide hydrogen; PEP: phosphoenolpyruvate; Th: T helper. Full article ">Figure 4
VDAC1, PINK1/Parkin signaling and mitophagy. (A) PINK1/Parkin targets damaged mitochondria, ubiquitinates VDAC1, and ultimately degrades damaged mitochondria by promoting mitophagy. (B) Mitophagy modulates NLRP3, MAVS and mtDNA release, affecting the immune response. NLRP3 activates caspase-1 to promote IL-1β/IL-18 maturation. MAVS enhance IFN-α/β expression. Meanwhile, mitophagy suppresses NLRP3, MAVS and mtDNA release, which results in reduced cytokines release. Additionally, mitophagy can also promote mtDNA release, which affects cytokines expression. Abbreviations: IL: interleukin; IFN: interferon; LC3: microtubule-associated proteins 1A/1B light chain 3; MAVS: mitochondrial antiviral signaling protein; NLRP3: nucleotide-binding domain and leucine-rich repeat (LRR) containing P3; PINK1: PTEN-induced putative kinase 1;; Ub: ubiquitin; VDAC1: voltage-dependent anion channel 1. Full article ">

17 pages, 24126 KiB

Open AccessArticle

Captopril Alleviates Chondrocyte Senescence in DOCA-Salt Hypertensive Rats Associated with Gut Microbiome Alteration

by Lok Chun Chan, Yuqi Zhang, Xiaoqing Kuang, Mohamad Koohi-Moghadam, Haicui Wu, Theo Yu Chung Lam, Jiachi Chiou and Chunyi Wen

Cells 2022, 11(19), 3173; https://doi.org/10.3390/cells11193173 - 10 Oct 2022

Cited by 2 | Viewed by 2497

Abstract

Gut microbiota is the key controller of healthy aging. Hypertension and osteoarthritis (OA) are two frequently co-existing age-related pathologies in older adults. Both are associated with gut microbiota dysbiosis. Hereby, we explore gut microbiome alteration in the Deoxycorticosterone acetate (DOCA)-induced hypertensive rat model. [...] Read more.

Gut microbiota is the key controller of healthy aging. Hypertension and osteoarthritis (OA) are two frequently co-existing age-related pathologies in older adults. Both are associated with gut microbiota dysbiosis. Hereby, we explore gut microbiome alteration in the Deoxycorticosterone acetate (DOCA)-induced hypertensive rat model. Captopril, an anti-hypertensive medicine, was chosen to attenuate joint damage. Knee joints were harvested for radiological and histological examination; meanwhile, fecal samples were collected for 16S rRNA and shotgun sequencing. The 16S rRNA data was annotated using Qiime 2 v2019.10, while metagenomic data was functionally profiled with HUMAnN 2.0 database. Differential abundance analyses were adopted to identify the significant bacterial genera and pathways from the gut microbiota. DOCA-induced hypertension induced p16INK4a+ senescent cells (SnCs) accumulation not only in the aorta and kidney (p < 0.05) but also knee joint, which contributed to articular cartilage degradation and subchondral bone disturbance. Captopril removed the p16INK4a + SnCs from different organs, partially lowered blood pressure, and mitigated cartilage damage. Meanwhile, these alterations were found to associate with the reduction of Escherichia-Shigella levels in the gut microbiome. As such, gut microbiota dysbiosis might emerge as a metabolic link in chondrocyte senescence induced by DOCA-triggered hypertension. The underlying molecular mechanism warrants further investigation. Full article

(This article belongs to the Special Issue Cellular Senescence in Aging and Aging-Related Diseases)

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27 pages, 893 KiB

Open AccessEditor’s ChoiceReview

The Extracellular Matrix and Neuroblastoma Cell Communication—A Complex Interplay and Its Therapeutic Implications

by Irena Horwacik

Cells 2022, 11(19), 3172; https://doi.org/10.3390/cells11193172 - 10 Oct 2022

Cited by 10 | Viewed by 3041

Abstract

Neuroblastoma (NB) is a pediatric neuroendocrine neoplasm. It arises from the sympatho-adrenal lineage of neural-crest-derived multipotent progenitor cells that fail to differentiate. NB is the most common extracranial tumor in children, and it manifests undisputed heterogeneity. Unsatisfactory outcomes of high-risk (HR) NB patients [...] Read more.

Neuroblastoma (NB) is a pediatric neuroendocrine neoplasm. It arises from the sympatho-adrenal lineage of neural-crest-derived multipotent progenitor cells that fail to differentiate. NB is the most common extracranial tumor in children, and it manifests undisputed heterogeneity. Unsatisfactory outcomes of high-risk (HR) NB patients call for more research to further inter-relate treatment and molecular features of the disease. In this regard, it is well established that in the tumor microenvironment (TME), malignant cells are engaged in complex and dynamic interactions with the extracellular matrix (ECM) and stromal cells. The ECM can be a source of both pro- and anti-tumorigenic factors to regulate tumor cell fate, such as survival, proliferation, and resistance to therapy. Moreover, the ECM composition, organization, and resulting signaling networks are vastly remodeled during tumor progression and metastasis. This review mainly focuses on the molecular mechanisms and effects of interactions of selected ECM components with their receptors on neuroblastoma cells. Additionally, it describes roles of enzymes modifying and degrading ECM in NB. Finally, the article gives examples on how the knowledge is exploited for prognosis and to yield new treatment options for NB patients. Full article

(This article belongs to the Special Issue Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy)

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14 pages, 2582 KiB

Open AccessArticle

In Vitro Transfection of Up-Regulated Genes Identified in Favorable-Outcome Neuroblastoma into Cell Lines

by Yoko Hiyama, Emi Yamaoka, Takahiro Fukazawa, Masato Kojima, Yusuke Sotomaru and Eiso Hiyama

Cells 2022, 11(19), 3171; https://doi.org/10.3390/cells11193171 - 10 Oct 2022

Viewed by 2474

Abstract

We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology [...] Read more.

We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence. Full article

(This article belongs to the Collection Feature Papers in 'Cell Proliferation and Division')

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Transfection efficiency in SK-N-SH, NH12, and TGW neuroblastoma cell lines. Representative images of transfected, undifferentiated neuroblastoma cell lines. (A) Stable expression levels of transfected gene products were assessed with fluorescent immunostaining in transfected cell lines (a–c). Expression of exogenous proteins was detected with FLAG M2 antibodies and their respective endogenous antibodies. In established cell lines, DHRS3 was expressed mainly in the plasma membrane and endoplasmic reticulum (a,b). The merged photos include nuclear staining by DAPI. (B) DHRS3-transfected strains were lipid-stained using Lipid Tox and visualized by transmission electron microscopy, and we found an accumulation of DHRS3 in small vesicles (arrows). These vesicles were observed shortly after transfection in NH12 and SK-N-SH cells but were absent in TGWs. In addition, CYP26A1 was also expressed in both the nucleus and the endoplasmic reticulum, and NROB1 was expressed in the nucleus (b). (C) The presence of the exogenous proteins was confirmed by their expected molecular weights by Western blotting. Full article ">Figure 2
Cell-cycle analysis of cells by gene transfection or siRNA transduction. (A) Growth curves of neuroblastoma cells transfected with one of these genes selected by the favorable tumors. Overexpression of these factors reduced the growth rates. (B) Growth rates of siRNA transduction for each gene in these cell lines. The growth rates were not affected by siRNA transduction, but some siRNA transduction reduced the growth rates. Data points represent means ± SDs of triplicate wells. The growth rates of transfected cells were reduced, except for DHRS-transfected TGW cells. Native cell lines were confluent at 4–5 days in this culture condition. **: p < 0.01, *: p < 0.05. Full article ">Figure 3
Soft-agar colony-formation assay. (A) The colony sizes in DHRS3-, CYP26A1-, and NROB1-overexpressing clones were significantly reduced (p < 0.05). The CYP26A1-overexpressing TGW cells were hard to obtain, so statistical evaluation was not performed. *: p < 0.05 (B) Cell morphology. The sizes of colonies were obviously large in TGW cells. Full article ">Figure 4
Cell-growth curves of NB cells induced by the Retro-X Tet-On Advanced System. (A) Growth curves of NB cell lines. Gene expression was induced with or without doxycycline. (B) Time course of the DHRS3-gene-expression analysis in SK-N-SH and NH12 cells using qPCR. Gene expression of DHRS3 was up-regulated until 3–6 days after Dox induction. (C) Immunofluorescent imaging of gene expression using the mouse-specific FLAG-M2 monoclonal antibody. Expression of each gene was obvious in all cell lines 5 days after Dox induction, but expression of DHRS3 decreased at 7 and 9 days after Dox induction. Scale bar is 5 µm. (D) Morphological alterations of SK-N-SH and NH-12 cells at 5 days after Dox induction. SK-N-SH and NH12 cells showed cellular enlargement. Expression of DsRed corresponded to inserted gene expression. (E) Cell-cycle analysis using flow cytometry in SK-N-SH cells; G0/G1 arrest was detected in Dox-induced DHRS3-expressing (DHRS3+) and NROB1-expressing (NROB1+) cells, but not in CYP26A1-expressing (CYP26A1+) cells. (F) Percentages of cells in G0/G1, S, and G2/M for the cells with or without Dox induction of each gene. Reductions in the percentages of S and G2/M were detected in the SK-N-SH DHRS3+ and NROB1+ cells, but not in CYP26A1+ cells. Full article ">Figure 5
ATRA-inducing differentiation experiments in Dox-treated NB cells. (A) Expression of senescence markers in Dox-treated SK-N-SH NB cell lines. SA-β-Gal-positive cells (green). SK-N-SH cells were untreated (−Dox) or Dox-treated until day 9 (+Dox). Ectopic Dox-induced SK-N-SH cells were observed to express high levels of a senescence marker. (B) Expression of Dox-treated DHRS3-expressing SK-N-SH NB cells with or without ATRA treatment. After ATRA treatment, SK-N-SH cells were assessed by immunofluorescence. High-dose ATRA induced cellular enlargement, cell death, and strong expression of DHRS3. (C) Expression of a senescence marker in SK-N-SH cell lines after 10 μM of ATRA treatment. SA-β-Gal-positive cells (green). The senescence marker was higher in the Dox-treated SK-N-SH cells. Full article ">

17 pages, 809 KiB

Open AccessReview

Modeling Obesity-Driven Pancreatic Carcinogenesis—A Review of Current In Vivo and In Vitro Models of Obesity and Pancreatic Carcinogenesis

by Sally Kfoury, Patrick Michl and Laura Roth

Cells 2022, 11(19), 3170; https://doi.org/10.3390/cells11193170 - 10 Oct 2022

Cited by 1 | Viewed by 2945

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy with a 5-year survival rate below 10%, thereby exhibiting the worst prognosis of all solid tumors. Increasing incidence together with a continued lack of targeted treatment options will cause PDAC to be the [...] Read more.

Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy with a 5-year survival rate below 10%, thereby exhibiting the worst prognosis of all solid tumors. Increasing incidence together with a continued lack of targeted treatment options will cause PDAC to be the second leading cause of cancer-related deaths in the western world by 2030. Obesity belongs to the predominant risk factors for pancreatic cancer. To improve our understanding of the impact of obesity on pancreatic cancer development and progression, novel laboratory techniques have been developed. In this review, we summarize current in vitro and in vivo models of PDAC and obesity as well as an overview of a variety of models to investigate obesity-driven pancreatic carcinogenesis. We start by giving an overview on different methods to cultivate adipocytes in vitro as well as various in vivo mouse models of obesity. Moreover, established murine and human PDAC cell lines as well as organoids are summarized and the genetically engineered models of PCAC compared to xenograft models are introduced. Finally, we review published in vitro and in vivo models studying the impact of obesity on PDAC, enabling us to decipher the molecular basis of obesity-driven pancreatic carcinogenesis. Full article

(This article belongs to the Special Issue Metabolic Regulation: Cell Growth and Proliferation)

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15 pages, 2948 KiB

Open AccessArticle

The Flavone Cirsiliol from Salvia x jamensis Binds the F₁ Moiety of ATP Synthase, Modulating Free Radical Production

by Lavinia Carlini, Gabriele Tancreda, Valeria Iobbi, Federico Caicci, Silvia Bruno, Alfonso Esposito, Daniela Calzia, Stefano Benini, Angela Bisio, Lucia Manni, Anna Schito, Carlo Enrico Traverso, Silvia Ravera and Isabella Panfoli

Cells 2022, 11(19), 3169; https://doi.org/10.3390/cells11193169 - 9 Oct 2022

Cited by 11 | Viewed by 2161

Abstract

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in [...] Read more.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F₁F_o-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H₂O₂ production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress. Full article

(This article belongs to the Special Issue Recent Advances in Metabolism and Oxidative Stress in Human Diseases)

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Molecular docking analysis of cirsiliol on F1 moiety of the F1Fo-ATP synthase (ATP synthase). (A) Molecular docking (left: conformation 1 using 2JJ2 (see also <a href="#app1-cells-11-03169" class="html-app">Supplementary Video S1</a>); right: conformation 7 using 2JIZ (see also <a href="#app1-cells-11-03169" class="html-app">Supplementary Video S2</a>)). Both conformations displayed hydrogen bonds (highlighted as green dots) with Ser-267.G. (B) Energy parameters assessed for conformations bearing the highest binding energy. The data show the elevated inhibition constants. Full article ">Figure 2
TEM of bovine retina. (A) The retina exhibits photoreceptorinner (IS) and outer (OS) segments. Squared areas B and C are enlarged in panels B and C, respectively. (B) Part of three OS belonging to close photoreceptors. Largest gold particles (40 nm width, arrows) reveal the Ab against anti-rhodopsin; smaller gold particles (10 nm width, arrowheads) evidence the Ab against anti-ATP synthase β-subunit. A gold particle is enlarged in inset. (C) Details of an IS close to a OS. Note the numerous small gold particles in the mitochondrion (mt) of the IS. In the OS, two small gold particles are recognizable. Full article ">Figure 3
Respiratory complexes activity, ATP synthesis and lipid peroxidation in the rod OS. All experiments were carried out on rod OS maintained either in the dark or in ambient light in the presence of 0.6 mM NADH, 10 mM succinate and 0.1 mM ADP. (A–D) Respiratory complexes activity. (E) ATP synthesis. (F) MDA levels as a lipid peroxidation marker. Data are representative of at least three independent experiments. *, *** and **** indicate a significant difference for p < 0.05, 0.001 and 0.0001, respectively, between the rod OS kept in the dark or in ambient light. Full article ">Figure 4
Effect of cirsiliol on F1Fo -ATP synthase activity in rod OS. ATP synthesis in the rod OS after preincubation of the sample with the indicated cirsiliol concentrations (see Materials and Methods section). Data are representative of at least three independent experiments and are indicated as mean ± SD. * and **** indicate a significant difference for p < 0.005 or p < 0.0001, respectively, between the subsequent samples. ns indicates a non-significant statistical difference. Full article ">Figure 5
Effect of cirsiliol on the respiratory complex activity in the rod OS in coupled conditions. Figure shows the activity of the four respiratory complexes. Panels (A–D) report the activity of Complexes I to IV in coupled conditions in the absence or in the presence of cirsiliol. Data are from n = 5 independent experiments and are expressed as mean ± SD. Results show a non-statistically significant difference in activity. **** indicates a significant difference for p < 0.0001 between treated and untreated samples in coupled conditions. Full article ">Figure 6
Effect of cirsiliol on the respiratory complex activity in the rod OS in uncoupled conditions. Figure shows the activity of the four respiratory complexes in the absence or in the presence of cirsiliol. Panels (A–D) report the activity of Complexes I to IV in uncoupled conditions (i.e., after addition of 0.01 mM nigericin plus 0.01 mM valinomycin) in the absence or in the presence of cirsiliol. Data are expressed as mean ± SD and are from n = 5 independent experiments. A non-statistically significant difference is observed. Full article ">Figure 7
Effect of cirsiliol on H2O2 production and rod OS integrity. (A) H2O2 production in rod OS treated (black squares) or untreated (black circles) with 50 μM cirsiliol during 40 min of ambient light exposure. (B) Percentage of structure integrity of rod OS in the same condition of Panel A. *, **, **** indicate a significant difference for p < 0.05, 0.01, 0.0001, respectively, between treated and untreated samples in coupled conditions. Data are expressed as mean ± SD. Full article ">

10 pages, 935 KiB

Open AccessEditor’s ChoiceHypothesis

AIRE in Male Fertility: A New Hypothesis

by Jana Petrusová, Jasper Manning and Dominik Filipp

Cells 2022, 11(19), 3168; https://doi.org/10.3390/cells11193168 - 9 Oct 2022

Cited by 1 | Viewed by 1915

Abstract

Male infertility affects approximately 14% of all European men, of which ~44% are characterized as idiopathic. There is an urgency to identify the factors that affect male fertility. One such factor, Autoimmune Regulator (AIRE), a protein found in the thymus, has been studied [...] Read more.

Male infertility affects approximately 14% of all European men, of which ~44% are characterized as idiopathic. There is an urgency to identify the factors that affect male fertility. One such factor, Autoimmune Regulator (AIRE), a protein found in the thymus, has been studied in the context of central tolerance functioning as a nuclear transcription modulator, responsible for the expression of tissue-restricted antigens in specialized thymic cells that prevent autoimmunity. While its expression in the testes remains enigmatic, we recently observed that sterility in mice correlates with the absence of Aire in the testes, regardless of the deficient expression in medullary thymic epithelial cells or cells of the hematopoietic system. By assessing the Aire transcript levels, we discovered that Sertoli cells are the exclusive source of Aire in the testes, where it most likely plays a non-immune role, suggesting an unknown mechanism by which testicular Aire regulates fertility. Here, we discuss these results in the context of previous reports which have suggested that infertility observed in Aire deficient mice is of an autoimmune aetiology. We present an alternative point of view for the role of Aire in testes in respect to fertility altering the perspective of how Aire’s function in the testes is currently perceived. Full article

(This article belongs to the Section Reproductive Cells and Development)

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Depletion of Aire in spermatogonia stem cells and spermatocytes does not affect male fertility. (A) Male mice with conditionally depleted Aire in spermatogonia stem cells (VasaCreAirefl/fl) and primary spermatocytes (Smc1βiCreAirefl/fl) exhibit fertility that is comparable to wild-type males in terms of the number of pups per litter. In contrast, whole-body Aire knock-out (Aire−/−) mice produced only a few pups at the beginning of their reproduction period. We established five independent breeding pairs (A–E) and monitored them for six-consecutive months (1–6, gray continuous arrows). (B) The size of the testes in VasaCreAirefl/fl as well as Smc1βiCreAirefl/fl males was fully comparable to WT controls. In contrast, six–week–old Aire−/− males had significantly smaller testes. (C) The decrease in testes size was confirmed by assessing their weight. p < 0.0001 = **** and ns = not significant. Full article ">Figure 2
Sertoli cells express Aire-coding mRNA. (A) The cell population from within the seminiferous tubule was stained with Hoechst 33342 and visualized in Hoechst red and blue channels. A color code was applied to the following cell types: turquoise spermatogonia stem cell (SSC), green primary spermatocytes (SCI), orange secondary spermatocytes (SCII), red round spermatids and sperm (RS+S), magenta Sertoli cells (Sertoli). (B) A single cell suspension from (A) was FACS-sorted and the indicated cell-types were prepared for mRNA isolation and qRT–PCR. Only Sertoli cells expressed physiologically relevant levels of Aire mRNA. The y axis represents the relative transcript levels of Aire and marker genes normalized to the Casc3 gene. (C) Protein immunodetection on the testicular tissue-sections showed the localization of AIRE in the nuclei of Sertoli cells (upper right panel) identified by their positivity to SOX9 (white color, upper left panel). SCP3 is a marker of primary spermatocytes (SCI). Spermatogonia stem cells (SSC), typically positioned between two Sertoli cells at the base of seminiferous tubule and round spermatid (RS), are identified as DAPI+SOX9−SCP3−, exhibiting a typical nuclear morphology [<a href="#B49-cells-11-03168" class="html-bibr">49</a>]. The dashed line demarcates the edge of the seminiferous tubule. Cell types within the diagram (lower right panel) correspond to the color scheme used in qPCR. Full article ">

17 pages, 8269 KiB

Open AccessArticle

Cellular Sources and Neuroprotective Roles of Interleukin-10 in the Facial Motor Nucleus after Axotomy

by Elizabeth M. Runge, Deborah O. Setter, Abhirami K. Iyer, Eric J. Regele, Felicia M. Kennedy, Virginia M. Sanders and Kathryn J. Jones

Cells 2022, 11(19), 3167; https://doi.org/10.3390/cells11193167 - 9 Oct 2022

Cited by 3 | Viewed by 2664

Abstract

Facial motoneuron (FMN) survival is mediated by CD4+ T cells in an interleukin-10 (IL-10)-dependent manner after facial nerve axotomy (FNA), but CD4+ T cells themselves are not the source of this neuroprotective IL-10. The aims of this study were to (1) identify the [...] Read more.

Facial motoneuron (FMN) survival is mediated by CD4+ T cells in an interleukin-10 (IL-10)-dependent manner after facial nerve axotomy (FNA), but CD4+ T cells themselves are not the source of this neuroprotective IL-10. The aims of this study were to (1) identify the temporal and cell-specific induction of IL-10 expression in the facial motor nucleus and (2) elucidate the neuroprotective capacity of this expression after axotomy. Immunohistochemistry revealed that FMN constitutively produced IL-10, whereas astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 was critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 alone could provide neuroprotection after FNA. These findings suggest that coordinated neuronal and astrocytic IL-10 production is necessary for FMN survival and has roles in neuronal homeostasis, as well as neuroprotective trophism after axotomy. Full article

(This article belongs to the Collection Retrograde Responses of Neurons and Associated Glial Cells to Axon Injury)

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21 pages, 3788 KiB

Open AccessArticle

Dynamics of Actin Cytoskeleton and Their Signaling Pathways during Cellular Wound Repair

by Shigehiko Yumura, Md. Shahabe Uddin Talukder, Mst. Shaela Pervin, Md. Istiaq Obaidi Tanvir, Takashi Matsumura, Koushiro Fujimoto, Masahito Tanaka and Go Itoh

Cells 2022, 11(19), 3166; https://doi.org/10.3390/cells11193166 - 9 Oct 2022

Cited by 4 | Viewed by 2730

Abstract

The repair of wounded cell membranes is essential for cell survival. Upon wounding, actin transiently accumulates at the wound site. The loss of actin accumulation leads to cell death. The mechanism by which actin accumulates at the wound site, the types of actin-related [...] Read more.

The repair of wounded cell membranes is essential for cell survival. Upon wounding, actin transiently accumulates at the wound site. The loss of actin accumulation leads to cell death. The mechanism by which actin accumulates at the wound site, the types of actin-related proteins participating in the actin remodeling, and their signaling pathways are unclear. We firstly examined how actin accumulates at a wound site in Dictyostelium cells. Actin assembled de novo at the wound site, independent of cortical flow. Next, we searched for actin- and signal-related proteins targeting the wound site. Fourteen of the examined proteins transiently accumulated at different times. Thirdly, we performed functional analyses using gene knockout mutants or specific inhibitors. Rac, WASP, formin, the Arp2/3 complex, profilin, and coronin contribute to the actin dynamics. Finally, we found that multiple signaling pathways related to TORC2, the Elmo/Doc complex, PIP2-derived products, PLA2, and calmodulin are involved in the actin dynamics for wound repair. Full article

(This article belongs to the Special Issue Toward Understanding Wound Repair Mechanism)

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Actin accumulation at wound sites is essential for wound repair. (A) Schema for the improved laserporation using gold coating. The wound diameter was set as 0.5 µm. (B) When the laser beam was focused on the surface of the coated coverslip, the gold coating peeled off, leaving behind a small white spot (arrow). (C) A typical sequence of fluorescence images of PI influx after laserporation. The wound laser beam was applied at 0 s and the duration was set at 8 ms. (D) Time courses of PI influx in the presence (LatA) and absence (BSS) of latrunculin A (n = 25 each). (E) A typical sequence of fluorescence images of a cell expressing GFP-lifeact after laserporation. The arrow shows the wound site. (F) Enlarged images of the rectangle in panel E 0.5 s before wounding (−0.5 s, red) and 0.5 or 3.0 s after wounding (0.5 s in upper panel and 3.0 s in lower panel, green), and the merged images. Arrows show the wound sites. (G) Time courses of fluorescence intensities at the wound site in the presence (LatA) and absence (BSS) of latrunculin A. The fluorescence intensities were normalized by setting the value before wounding to 1 after subtracting the background (n = 25 each). Scale bars: 10 µm for panels B, C, and D, and 1 µm for panel F. Full article ">Figure 2
Actin polymerizes de novo at wound sites. (A) Typical sequences of fluorescence images of cells expressing GFP-lifeact in the presence of latrunculin A and/or jasplakinolide. Arrows and arrowheads show the wound sites and large actin aggregates, respectively. (B) Time courses of fluorescence intensities of GFP-lifeact at the wound site in the presence (Jasp) and absence (BSS) of jasplakinolide, respectively. Note that the time courses in the presence of latrunculin A are shown in <a href="#cells-11-03166-f001" class="html-fig">Figure 1</a>G. (C) The initiation, peak, and termination times of wound-induced actin accumulation are compared in the presence and absence of jasplakinolide (n = 25 each). Data are presented as means ± SD. ** p ≤ 0.0001; ns: not significant, p > 0.05. (D) Time courses of PI influx in the presence and absence of jasplakinolide (n = 25 each). (E) Time courses of fluorescence intensities of GFP-lifeact at the wound site in the presence and absence of both inhibitors (Jasp + LatA) (n = 25 each). (F) Time courses of fluorescence intensities of GFP-lifeact at the wound site in annexin C1-null cells. The graphs with different colors show time courses of six different cells. (G) Time courses of fluorescence intensities of GFP-lifeact at the wound site in wild-type cells in the presence and absence of W7 (n = 25 each). Full article ">Figure 3
Dynamics of ARPs during wound repair. (A) Typical sequences of fluorescence images of cells expressing various (13 kinds) GFP-ARPs at wound sites. (B) Time courses of fluorescence intensities of GFP-ARPs and GFP-actin at wound sites (n = 25 each). (C) Normalized curves of panel B when each peak value is set as 100%. (D) Duration of appearance of individual ARPs at wound sites (red). Duration of actin dynamics (green), Ca2+ influx (blue), calmodulin dynamics (blue), ESCRT component vps4 dynamics (blue), and annexin C1 dynamics (blue) are also plotted. In addition, the duration of Efa1A disappearance (orange) is plotted. Asterisks in the duration bars indicate the peak times. Full article ">Figure 4
Durations of wound-induced actin dynamics in null cells or wild-type cells in the presence of inhibitors. Durations of wound-induced actin dynamics in null cells or wild-type cells in the presence of inhibitors: actin-related (red), signal-related (purple), membrane dynamics-related (blue), and microtubule-related (gray) proteins. Actual time courses of the fluorescence intensities are shown in <a href="#app1-cells-11-03166" class="html-app">Supplementary Figures S3 and S4</a>. Red asterisks indicate the cases showing significantly retarded initiation time compared with the control, and black asterisks indicate the cases showing significantly retarded termination time. * p ≤ 0.001, ** p ≤ 0.0001 (n ≥ 25 each). Full article ">Figure 5
Peak amplitudes of wound-induced actin dynamics in null cells or wild-type cells in the presence of inhibitors. The amplitudes at the peaks of the actin dynamics under the same conditions as shown in <a href="#cells-11-03166-f004" class="html-fig">Figure 4</a>. Actual time courses of the fluorescence intensities are shown in <a href="#app1-cells-11-03166" class="html-app">Supplementary Figures S3 and S4</a>. Asterisks show cases with significant differences from wild-type cells in the absence of inhibitors. Data are presented as means ± SD. * p ≤ 0.001, ** p ≤ 0.0001 (n ≥ 25 each). Full article ">Figure 6
Signal pathways for actin dynamics in wound repair. With reference to the signal cascades for chemotaxis, potent candidates for wound-related signals, ARPs, or other proteins we found in the present and previous studies are marked (red). Some proteins (yellow) accumulate or disappear at the wound site, but their contribution remains to be clarified using knockout or inhibitor experiments. Most of the examined proteins (green) do not contribute to wound repair. Full article ">

19 pages, 4761 KiB

Open AccessArticle

Investigating Two Modes of Cancer-Associated Antigen Heterogeneity in an Agent-Based Model of Chimeric Antigen Receptor T-Cell Therapy

by Tina Giorgadze, Henning Fischel, Ansel Tessier and Kerri-Ann Norton

Cells 2022, 11(19), 3165; https://doi.org/10.3390/cells11193165 - 9 Oct 2022

Cited by 4 | Viewed by 2329

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has been successful in treating liquid tumors but has had limited success in solid tumors. This work examines unanswered questions regarding CAR T-cell therapy using computational modeling, such as, what percentage of the tumor must express cancer-associated [...] Read more.

Chimeric antigen receptor (CAR) T-cell therapy has been successful in treating liquid tumors but has had limited success in solid tumors. This work examines unanswered questions regarding CAR T-cell therapy using computational modeling, such as, what percentage of the tumor must express cancer-associated antigens for treatment to be successful? The model includes cancer cell and vascular and CAR T-cell modules that interact with each other. We compare two different models of antigen expression on tumor cells, binary (in which cancer cells are either susceptible or are immune to CAR T-cell therapy) and gradated (where each cancer cell has a probability of being killed by a CAR T-cell). We vary the antigen expression levels within the tumor and determine how effective each treatment is for the two models. The simulations show that the gradated antigen model eliminates the tumor under more parameter values than the binary model. Under both models, shielding, in which the low/non-antigen-expressing cells protect high antigen-expressing cells, reduced the efficacy of CAR T-cell therapy. One prediction is that a combination of CAR T-cell therapies that targets the general population of cells as well as one that specifically targets cancer stem cells should increase its efficacy. Full article

(This article belongs to the Special Issue In Silico Models of Cell–Microenvironment Interactions in Healthy and Tumor Tissues)

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Flowchart of the CAR T-cell TNBC model. Full article ">Figure 2
3D plots of tumors with gradated (a) and binary (b) heterogeneity plotted over time with 12.5-day intervals starting from day 12.5 and ending on day 75, the last day of our simulations. Tumor cells are plotted in blue, CAR T-cells are plotted in green, and the vasculature is plotted as red capillaries growing across the grid. The data in the gradated heterogeneity tumor progression (a) is from a run with 12.5% mean antigen expression tumor cells. The data in the binary heterogeneity tumor progression (b) is from a run with 75% antigen presenting tumor cells. CAR T-cells are introduced halfway through the simulation, on day 37.5. Full article ">Figure 3
Tumor metrics over time for gradated heterogeneity (A) and binary heterogeneity (B). In both cases, the metrics include total number of tumor cells over time (a), total number of CAR T-cells (b), total number of cancer cell deaths (c), and total number of stem cells over time (d). Each iteration represents 6 h of real time. For the gradated heterogeneity metrics, 0% mean antigen expression is plotted in yellow, 12.5% mean antigen expression is plotted in light blue, 25% mean antigen expression is plotted in red, and 50% mean antigen expression is plotted in dark blue. For the binary heterogeneity metrics, 25% antigen presentation is plotted in yellow, 50% antigen presentation is plotted in light blue, 75% antigen expression is plotted in red, and 98% antigen expression is plotted in dark blue. Note: the parameters in which the line does not reach day 75 indicate that one of the tumors died out at this point and no further data was collected. Full article ">Figure 4
3D plots of tumors on the last day of our simulation, day 75, with different antigen presentations. For tumor with gradated heterogeneity (a), the data comes from 0%, 12.5%, 25%, and 50% mean antigen presentation (left to right). Vasculature is plotted as red capillaries growing across the plane. Tumor cells with 100% chance of presenting the antigen are plotted in yellow. Tumor cells with 0% chance of presenting the antigen are plotted in red. Every cell with a chance of antigen presentation between 0% and 100% is plotted on a gradient from red to yellow. CAR T-cells are plotted in green and are introduced on day 37.5 of the simulation. For the tumors with binary heterogeneity (b), the data comes from runs with 25%, 50%, 75%, and 98% antigen presentation. Tumor cells with 100% antigen presentation are plotted in yellow; cells with 0% antigen presentation are plotted in red. Full article ">Figure 5
Examples of antigen shielding occurring in tumors with gradated heterogeneity (a) and binary heterogeneity (b). For gradated heterogeneity, the 3D plot shows tumor cells with 100% of antigen presentation in yellow, tumor cells with 0% antigen presentation in red, and tumor cells with intermediate probabilities of antigen presentation on a gradient. For binary heterogeneity, antigen-presenting tumor cells are plotted in yellow and antigen non-presenting tumor cells in red. In both cases, the figure shows a layer (shield) of antigen non/low-presenting cells forming a shield with high antigen-presenting cells. In both cases, such a shield makes it difficult for CAR T-cells to break through that layer, as the cells making up that layer are either fully or highly immune to CAR T-cell therapy. Full article ">

19 pages, 3650 KiB

Open AccessArticle

Profound Effects of Dexamethasone on the Immunological State, Synthesis and Secretion Capacity of Human Testicular Peritubular Cells

by Youli Konstantinovitch Stepanov, Jan Dominik Speidel, Carola Herrmann, Nina Schmid, Rüdiger Behr, Frank-Michael Köhn, Jan Bernd Stöckl, Ulrich Pickl, Matthias Trottmann, Thomas Fröhlich, Artur Mayerhofer and Harald Welter

Cells 2022, 11(19), 3164; https://doi.org/10.3390/cells11193164 - 9 Oct 2022

Cited by 5 | Viewed by 2405

Abstract

The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid [...] Read more.

The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they may be regulated by glucocorticoids (GCs). Herein, we studied the consequences of the GC dexamethasone (Dex) in cultured HTPCs, which serves as a unique window into the human testis. We examined changes in cytokines, mainly by qPCR and ELISA. A holistic mass-spectrometry-based proteome analysis of cellular and secreted proteins was also performed. Dex, used in a therapeutic concentration, decreased the transcript level of proinflammatory cytokines, e.g., IL6, IL8 and MCP1. An siRNA-mediated knockdown of GR reduced the actions on IL6. Changes in IL6 were confirmed by ELISA measurements. Of note, Dex also lowered GR levels. The proteomic results revealed strong responses after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h of Dex exposure (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were extracellular matrix (ECM) and basement membrane components (e.g., FBLN2, COL1A2 and COL3A1), as well as PTX3 and StAR. These results pinpoint novel, profound effects of Dex in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may occur in men upon treatment with Dex for medical reasons. Full article

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Consequences of Dex on cytokine levels. (A) HTPCs treated with 1 µM Dex for 48 h responded with the decreased secretion of IL6, IL8, MCP1 and CXCL1 compared to untreated cells (n = 1), as demonstrated by a human cytokine profiler assay. The upper panel shows the corresponding membrane spots of untreated control, while the lower panel depicts Dex (1 µM)-treated HTPCs and reveals a reduction of the signal intensity of IL6, IL8, MCP1 and CXCL1. (B) Using an ELISA, highly significantly decreased IL6 levels could be detected in the culture media of HTPCs from four individual patients treated for 48 h with 1 µM Dex compared to the corresponding control condition. Results were normalized to the total protein amount and statistically analyzed using a paired t-test (two-tailed paired t-test: ** p < 0.01; n = 4). (C) Quantitative PCR revealed significantly decreased mRNA expression levels of IL6, IL8, MCP1, MCP3 and IL1B in HTPCs after 24 h stimulation with 1 µM Dex. CXCL1 transcripts did not change significantly compared to control (ns: not significant, * p < 0.05; n = 4). Full article ">Figure 2
Regulation of GR by siRNA and Dex. (A) Silencing of GR by siRNA: Two representative GR Western blots from two patients (P1 and P2, n = 2) show the results of HTPCs transfected with non-targeting (NT) control siRNA and GR siRNA (siGR) at two different time points (48 h and 72 h) each and emphasize the efficiency of the transfection with siGR RNA. (B) Transfection of HTPCs with a GR siRNA abolished the observed effect on IL6 and FKBP5 mRNA. Quantitative PCR data of HTPCs from three different patients (n = 3) transfected with a non-targeting control siRNA (siNT) or GR siRNA (siGR) and treated with Dex (1 µM; siNT + Dex and siGR + Dex) or an equal volume of EtOH (Basal Co and Dex) for 24 h are depicted, ## p ≤ 0.01 vs. Co cells; * p ≤ 0.05, ** p ≤ 0.01 vs. siNT + dex cells. (C) Stimulation of HTPCs with Dex led to the downregulation of GR expression. In the presence of 1 µM Dex for 24 h and 48 h, GR protein in HTPCs from two different donors (P1 and P2, n = 2) was downregulated compared to control (Co), as shown by Western blot. In addition, significantly diminished GR (n = 7; upper left picture) but not AR mRNA (n = 9; lower left picture) was detected after 24 h incubation with 1 µM Dex. Full article ">Figure 3
Volcano plot analysis of cellular proteomes treated with 1 µM dexamethasone for 24 h (A) and 72 h (B). The data was analyzed with a paired Student’s t-test; false-discovery rate (FDR): 0.05. Each colored dot represents a protein fulfilling the significance criteria (q-value < 0.05, log2 fold change >|0.6|): blue–proteins less abundant in the treated group; red–proteins more abundant in the treated group. For selected significant hits, gene names are displayed. (C) = log2 fold changes of significantly differentially abundant proteins of the 24 h-treatment and 72 h-treatment datasets. For the overlap, the 72 proteins (fulfilling the above significance criteria) from 72 h dataset were used as “base”. This was overlayed by the proteins from the 24 h dataset fulfilling only the “overlap criterion” of q-value < 0.05, which additionally includes potential trends, given the lower size of this dataset. Full article ">Figure 4
Volcano plot of cellular secretomes treated with 1 µM dexamethasone for 24 h (A) and a 72 h (B). The data was analyzed with a paired Student’s t-test; false-discovery rate (FDR): 0.05. Each colored dot represents a protein fulfilling the significance criteria (q-value < 0.05, log2 fold change > |0.6|): blue—proteins less abundant in the treated group; red—proteins more abundant in the treated group. For selected significant hits, gene names are displayed. Full article ">Figure 5
Bioinformatic analysis based on the functional enrichment of all merged (proteome + secretome) significantly differentially abundant proteins (A,B). 24 h-treatment, PROTEOMAPS-based analysis. (a,b): 24 h-treatment, DAVID-based analysis. (C,D): 72 h-treatment, PROTEOMAPS-based analysis. (c,d): 72 h-treatment, DAVID-based analysis. For PROTEOMAPS, proteins were annotated using GO “biological process”. The terms are shown in the legend and comprise all tiles of similar color. Annotated individual proteins are displayed in the mosaic plots. For DAVID analysis, functional enrichment terms GO_BP_all, GO_CC_all, GO_MF_all, KEGG and Reactome were used. Full article ">Figure A1
Regulation of PDGFRalpha (PDGFRa) by Dex. In the presence of 1 µM Dex for 72 h, PDGFRa protein in HTPCs from three donors (P1-P3) was increased, compared to controls (Co), as shown by Western blot. Full article ">

24 pages, 1216 KiB

Open AccessReview

Multi- and Transgenerational Effects of Environmental Toxicants on Mammalian Reproduction

by Paola Rebuzzini, Gemma Fabozzi, Danilo Cimadomo, Filippo Maria Ubaldi, Laura Rienzi, Maurizio Zuccotti and Silvia Garagna

Cells 2022, 11(19), 3163; https://doi.org/10.3390/cells11193163 - 9 Oct 2022

Cited by 14 | Viewed by 3224

Abstract

Environmental toxicants (ETs) are an exogenous chemical group diffused in the environment that contaminate food, water, air and soil, and through the food chain, they bioaccumulate into the organisms. In mammals, the exposure to ETs can affect both male and female fertility and [...] Read more.

Environmental toxicants (ETs) are an exogenous chemical group diffused in the environment that contaminate food, water, air and soil, and through the food chain, they bioaccumulate into the organisms. In mammals, the exposure to ETs can affect both male and female fertility and their reproductive health through complex alterations that impact both gametogeneses, among other processes. In humans, direct exposure to ETs concurs to the declining of fertility, and its transmission across generations has been recently proposed. However, multi- and transgenerational inheritances of ET reprotoxicity have only been demonstrated in animals. Here, we review recent studies performed on laboratory model animals investigating the effects of ETs, such as BPA, phthalates, pesticides and persistent contaminants, on the reproductive system transmitted through generations. This includes multigenerational effects, where exposure to the compounds cannot be excluded, and transgenerational effects in unexposed animals. Additionally, we report on epigenetic mechanisms, such as DNA methylation, histone tails and noncoding RNAs, which may play a mechanistic role in a nongenetic transmission of environmental information exposure through the germline across generations. Full article

(This article belongs to the Special Issue Effects and Mechanisms of Endocrine Disruptors on Germ Cells, Gonads and Embryos)

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14 pages, 1131 KiB

Open AccessArticle

Crosstalk between Extracellular Matrix Stiffness and ROS Drives Endometrial Repair via the HIF-1α/YAP Axis during Menstruation

by Tao Zhang, Yan Wang, Yingnan Wang, Cuiyan Liu and Chunyang Han

Cells 2022, 11(19), 3162; https://doi.org/10.3390/cells11193162 - 9 Oct 2022

Cited by 5 | Viewed by 2554

Abstract

Although the menstrual cycle driven by sex steroid hormones is an uncomplicated physiological process, it is important for female health, fertility and regenerative biology. However, our understanding of this unique type of tissue homeostasis remains unclear. Here, we examined the biological effects of [...] Read more.

Although the menstrual cycle driven by sex steroid hormones is an uncomplicated physiological process, it is important for female health, fertility and regenerative biology. However, our understanding of this unique type of tissue homeostasis remains unclear. Here, we examined the biological effects of mechanical force by evaluating the changing trend of extracellular matrix (ECM) stiffness, and the results suggested that ECM stiffness was reduced and that breaking of mechanotransduction delayed endometrium repair in a mouse model of simulated menses. We constructed an ECM stiffness interference model in vitro to explain the mechanical force conduction mechanism during endometrial regeneration. We discovered that ECM stiffness increased the expression and nuclear transfer of YAP, which improved the creation of a microenvironment, in a manner that induced proliferation and angiogenesis for endometrial repair by activating YAP. In addition, we observed that physiological endometrial hypoxia occurs during the menstrual cycle and that the expression of HIF-1α was increased. Mechanistically, in addition to the classical F-actin/YAP pathway, we also found that the ROS/HIF-1α/YAP axis was involved in the transmission of mechanical signals. This study provides novel insights into the essential menstrual cycle and presents an effective, nonhormonal treatment for menstrual disorders. Full article

(This article belongs to the Topic Cell Signaling Pathways)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
ECM stiffening features of the endometrium in a mouse model of simulated menses. (a) Diagram depicting the murine model of simulated menstruation. E2 17-β-estradiol, P4 progesterone. Timed to 0 h, 24 h, 48 h and 72 h based on P4 withdrawal time. (b) Representative whole uterus images (top) and histopathological images of paraffin-embedded mouse uterine sections (bottom, n = 2). Scale bar: 400 μm. (c,d) Representative images of uterine sections stained with TUNEL (top, n = 2) and BrdU (bottom, n = 2) and quantification of TUNEL+ and BrdU+ positive cells ((d), n = 5). Scale bars, 200 μm. The data are presented as mean ± SEM. ** p < 0.01; *** p < 0.001. (e) Western blot analysis of pFAK, FAK, pPMLC2 and pMLC2 expression. GAPDH was used as a loading control. (f) Representative images of vinculin and F-actin in mouse uterine sections. Scale bar, 20 μm. n = 4. (g) Endometrial histological breakdown/repair score at 24 h and 72 h in mice treated with DMSO and Cyto. B. Graphs represent the percentage of mice at each histological grade per experimental group. n = 9. Experiments were repeated n times with duplicate biological replicates. Full article ">Figure 2
Pharmacological inhibition of YAP at menses delays repair. (a,b) Representative immunofluorescence images ((a), n = 2) and quantification of nuclear and cytoplasmic subcellular localization ((b), n = 9) of YAP in uterine sections. Scale bar: 100 μm. (c) Western blotting for YAP in nuclear and cytoplasmic protein fractions from mouse endometrial tissue. n = 3. Lamin B1 and GAPDH were used as loading controls for nuclear and cytoplasmic proteins, respectively. (d) RT-qPCR analysis of CYR61 and CTGF mRNA levels. n = 3. (e) Endometrial histological breakdown/repair score at 24 h and 72 h in mice treated with DMSO and VP. The graphs represent the percentage of mice at each histological grade per experimental group. (f) Representative immunofluorescence images (left, n = 2) and quantification of YAP- and BrdU-the positive cells ((f), n = 5) in the uterus of mice uterine treated with DMSO or VP at 24 h and 48 h. Scale bar: 100 μm. Enlarge: 10 μm. The data are presented as the mean ± SEM. Experiments were repeated n times with duplicate biological replicates. * p < 0.05, *** p < 0.001. Full article ">Figure 3
Hypoxia sustains HIF-1α protein stability to activate YAP. (a) Analysis of the antioxidant indices T-AOC activity and SOD activity in endometrial tissue. n =3. (b) The protein levels of VEGF, CXCR4, and HIF-1α were evaluated in the uterus by Western blotting. n = 3. (c) Comparison of the mRNA expression levels of VEGF and CXCR4 in isolated mouse endometrial tissue during menstruation. n = 3. (d) Lysates of ESCs transfected with siYAP and siNC after withdrawing P4 were analyzed for the presence of the indicated proteins. n = 3. (e) Confocal immunofluorescence images (left, n = 2) and quantification of nuclear and cytoplasmic subcellular localization (right, n = 15) of YAP in decidualized cells transfected with siYAP. Scale bar: 100 μm. The data are presented as the mean ± SEM. Experiments were repeated n times with duplicate biological replicates. * p < 0.05, ** p < 0.01; *** p < 0.001. Full article ">Figure 4
ECM stiffness regulates the activity of YAP/HIF-1α and ROS production in ESCs. (a) Western blotting for YAP in nuclear and cytoplasmic protein fractions from ESCs plated on 1 kPa or 40 kPa fibronectin-coated hydrogels for 24 h, n = 3. (b) Confocal immunofluorescence images (left, n = 2) and quantification of nuclear and cytoplasmic subcellular localization (right, n = 15) of YAP in ESCs plated on hydrogels with different rigidities. Scale bar, 10 μm. (c) Western blotting for HIF-1α from decidualized cells plated on 1 kPa or 40 kPa hydrogels for 0 h and 24 h, n = 3. (d,e) Western blot analysis of HIF-1α and YAP expression in decidualized cells treated with siYAP (d) or VP (e) plated on 1 kPa or 40 kPa hydrogels for 24 h, n =3. (f) The intracellular ROS levels were measured by staining with DCFH-DA and then determined by flow cytometry. n = 2. The data are presented as the mean ± SEM. Experiments were repeated n times with duplicate biological replicates. * p < 0.05 and ** p < 0.01. Full article ">Figure 5
YAP activation provides the physiological environment needed for endometrial repair. (a) Confocal immunofluorescence images (left n = 2) and quantification of Ki67 positive cells (right, n = 9) in ESCs transfected with siYAP and pcDNA 3.1 (+) YAP. Scale bars, 200 μm. (b,c) Schematic diagram of cell cycle detection results. n = 3. (d,e) Protein and mRNA expression levels of p21, cyclin D1 and CDK4 were detected in ESCs transfected with si YAP or pcDNA 3.1 (+) YAP, n = 3. (f) RT-qPCR analysis of Ang-2 expression levels in ESCs transfected with siYAP or pcDNA3.1(+) YAP, n = 3. The data are presented as the mean ± SEM. Experiments were repeated n times with duplicate biological replicates. ** p < 0.01, *** p < 0.001. (g) Schematic images depicting upregulation of ECM stiffness and ROS upon nuclear translocation and activation of YAP/HIF-1α and subsequent binding to TEAD, Ang2, and CTGFdriving endometrium repair after in the menstrual cycle. Full article ">

22 pages, 3065 KiB

Open AccessReview

Pathological Role of HDAC8: Cancer and Beyond

by Ji Yoon Kim, Hayoung Cho, Jung Yoo, Go Woon Kim, Yu Hyun Jeon, Sang Wu Lee and So Hee Kwon

Cells 2022, 11(19), 3161; https://doi.org/10.3390/cells11193161 - 9 Oct 2022

Cited by 38 | Viewed by 3920

Abstract

Histone deacetylase 8 (HDAC8) is a class I HDAC that catalyzes the deacetylation of histone and non-histone proteins. As one of the best-characterized isoforms, numerous studies have identified interacting partners of HDAC8 pertaining to diverse molecular mechanisms. Consequently, deregulation and overexpression of HDAC8 [...] Read more.

Histone deacetylase 8 (HDAC8) is a class I HDAC that catalyzes the deacetylation of histone and non-histone proteins. As one of the best-characterized isoforms, numerous studies have identified interacting partners of HDAC8 pertaining to diverse molecular mechanisms. Consequently, deregulation and overexpression of HDAC8 give rise to diseases. HDAC8 is especially involved in various aspects of cancer progression, such as cancer cell proliferation, metastasis, immune evasion, and drug resistance. HDAC8 is also associated with the development of non-cancer diseases such as Cornelia de Lange Syndrome (CdLS), infectious diseases, cardiovascular diseases, pulmonary diseases, and myopathy. Therefore, HDAC8 is an attractive therapeutic target and various HDAC8 selective inhibitors (HDAC8is) have been developed. Here, we address the pathological function of HDAC8 in cancer and other diseases, as well as illustrate several HDAC8is that have shown anti-cancer effects. Full article

(This article belongs to the Special Issue Transcription and Chromatin Dysregulation in Cancer)

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Figure 1
Crystal structure of HDAC8. (A) Head-to-head dimeric arrangement of HDAC8 (PDB ID: 2V5W [<a href="#B27-cells-11-03161" class="html-bibr">27</a>]). Two monomers of HDAC8 are shown in light and dark yellow. Zinc and potassium are represented as grey and purple spheres, respectively (B) Liganded form of HDAC8 (PDB ID: 1T67 [<a href="#B15-cells-11-03161" class="html-bibr">15</a>]). L2 loop and MS-344 are colored grey and green, respectively. (C) Catalytic machinery of HDAC8 (PDB ID: 1T64 [<a href="#B15-cells-11-03161" class="html-bibr">15</a>]). Zinc ion is coordinated to three HDAC8 residues and two oxygen atoms of TSA. The zinc coordination shell is indicated by dashed lines. (D) Active site of HDAC8 (PDB ID: 1T64 [<a href="#B15-cells-11-03161" class="html-bibr">15</a>]). Six key residues form the hydrophobic tunnel that occupies two TSA molecules. Full article ">Figure 2
Schematic representation of human HDAC8 and its regulation. HDAC8 is upregulated by transcription factors and downregulated by miRNA, autophagy, and proteasomal degradation. Full article ">Figure 3
Schematic representation of histone and non-histone substrates of HDAC8. HDAC8 deacetylates histones and suppresses transcription of target genes. HDAC8 also deacetylates non-histone substrates such as SMC3, α-tubulin, cortactin, HSP20, p53, PKM2, AKT, ERRα, and c-Jun. Full article ">Figure 4
Schematic representation of pathological functions of HDAC8 in cancer. HDAC8 promotes tumor growth by enhancing tumor cell proliferation and suppressing apoptosis. HDAC8 also enhances metastasis and is involved in drug resistance and immune evasion. Full article ">

10 pages, 3458 KiB

Open AccessArticle

Stereological Estimation of Myocardial Fat and Its Associations with Obesity, Epicardial, and Visceral Adipose Tissue

by Pernille Heimdal Holm, Louise Hindsø, Kristine Boisen Olsen and Jytte Banner

Cells 2022, 11(19), 3160; https://doi.org/10.3390/cells11193160 - 8 Oct 2022

Viewed by 1841

Abstract

The normal human heart contains epicardial adipose tissue (EAT) and myocardial fat. The associations between obesity, myocardial fat, visceral adipose tissue (VAT), and cardiovascular disease are not fully understood. The objective of this study was to estimate myocardial fat using stereological methods and [...] Read more.

The normal human heart contains epicardial adipose tissue (EAT) and myocardial fat. The associations between obesity, myocardial fat, visceral adipose tissue (VAT), and cardiovascular disease are not fully understood. The objective of this study was to estimate myocardial fat using stereological methods and investigate its relations with obesity, EAT, and VAT. To establish the EAT volume, 115 deceased individuals were included, and postmortem computed tomography was conducted on their eviscerated hearts. Six samples from the left and right ventricles (LV and RV) of the heart were stereologically examined to calculate the percentage of myocardial fat. Kidney and omental fat were weighed at autopsy, and the waist–hip ratio was calculated. Females had a slightly non-significantly (p = 0.054) larger proportion of RV fat (13.2% ± 4.4) compared to that in men (11.5% ± 2.7). We found a significant positive correlation between body mass index (BMI) and LV myocardial fat (p = 0.033). In the RV, this correlation was only at the borderline of significance (p = 0.052). The EAT volume was positively correlated with both RV and LV myocardial fat. We found no association with the waist–hip ratio (WHR) or the omental or kidney fat as measures of VAT. The myocardial fat was normal, most prominent in the RV, and correlated with the EAT and, partly, BMI. We found no association with VAT. Full article

(This article belongs to the Special Issue Adipose Tissue in Cardiovascular Health)

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11 pages, 606 KiB

Open AccessReview

Cell-Enriched Lipotransfer (CELT) Improves Tissue Regeneration and Rejuvenation without Substantial Manipulation of the Adipose Tissue Graft

by Lukas Prantl, Andreas Eigenberger, Ruben Reinhard, Andreas Siegmund, Kerstin Heumann and Oliver Felthaus

Cells 2022, 11(19), 3159; https://doi.org/10.3390/cells11193159 - 8 Oct 2022

Cited by 6 | Viewed by 2180

Abstract

The good availability and the large content of adult stem cells in adipose tissue has made it one of the most interesting tissues in regenerative medicine. Although lipofilling is one of the most frequent procedures in plastic surgery, the method still struggles with [...] Read more.

The good availability and the large content of adult stem cells in adipose tissue has made it one of the most interesting tissues in regenerative medicine. Although lipofilling is one of the most frequent procedures in plastic surgery, the method still struggles with high absorption rates and volume losses of up to 70%. Therefore, many efforts have been made to optimize liposuction and to process the harvested tissue in order to increase fat graft retention. Because of their immunomodulatory properties, their cytokine secretory activity, and their differentiation potential, enrichment with adipose tissue-derived stem cells was identified as a promising tool to promote transplant survival. Here, we review the important parameters for lipofilling optimization. Finally, we present a new method for the enrichment of lipoaspirate with adipose tissue-derived stem cells and discuss the parameters that contribute to fat graft survival. Full article

(This article belongs to the Special Issue Regeneration of Tissue with Mesenchymal Stem Cells)

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3 pages, 226 KiB

Open AccessEditorial

Mesenchymal Stem/Stromal Cells as a Therapeutic Tool in Cell-Based Therapy and Regenerative Medicine: An Introduction Expertise to the Topical Collection

by Makram Merimi, Hassan Fahmi, Joery De Kock, Charline Beguin, Arsène Burny, Guido Moll, Alessandro Poggi and Mehdi Najar

Cells 2022, 11(19), 3158; https://doi.org/10.3390/cells11193158 - 8 Oct 2022

Cited by 7 | Viewed by 1781

Abstract

We are pleased to present this opening editorial, introducing our topical collection, “The New Era of Mesenchymal Stromal/Stem Cell Functional Application: State of the Art, Therapeutic Challenges and Future Directions” [...] Full article

(This article belongs to the Collection The New Era of Mesenchymal Stromal/Stem Cell Functional Application: State of the Art, Therapeutic Challenges and Future Directions)

26 pages, 940 KiB

Open AccessReview

Left Atrial Myocardium in Arterial Hypertension

by Jens Kockskämper and Florentina Pluteanu

Cells 2022, 11(19), 3157; https://doi.org/10.3390/cells11193157 - 8 Oct 2022

Cited by 17 | Viewed by 3836

Abstract

Arterial hypertension affects ≈ 1 billion people worldwide. It is associated with increased morbidity and mortality and responsible for millions of deaths each year. Hypertension mediates damage of target organs including the heart. In addition to eliciting left ventricular hypertrophy, dysfunction and heart [...] Read more.

Arterial hypertension affects ≈ 1 billion people worldwide. It is associated with increased morbidity and mortality and responsible for millions of deaths each year. Hypertension mediates damage of target organs including the heart. In addition to eliciting left ventricular hypertrophy, dysfunction and heart failure, hypertension also causes left atrial remodeling that may culminate in atrial contractile dysfunction and atrial fibrillation. Here, we will summarize data on the various aspects of left atrial remodeling in (essential) hypertension gathered from studies on patients with hypertension and from spontaneously hypertensive rats, an animal model that closely mimics cardiac remodeling in human hypertension. Analyzing the timeline of remodeling processes, i.e., distinguishing between alterations occurring in prehypertension, in early hypertension and during advanced hypertensive heart disease, we will derive the potential mechanisms underlying left atrial remodeling in (essential) hypertension. Finally, we will discuss the consequences of these remodeling processes for atrial and ventricular function. The data imply that left atrial remodeling is multifactorial, starts early in hypertension and is an important contributor to the progression of hypertensive heart disease, including the development of atrial fibrillation and heart failure. Full article

(This article belongs to the Special Issue Molecular Biology of Atrial Myocardium)

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17 pages, 3070 KiB

Open AccessArticle

by Ting Huang, Xuan Feng, Jiaqi Wang, Jingyi Zhou and Jianliu Wang

Cells 2022, 11(19), 3156; https://doi.org/10.3390/cells11193156 - 8 Oct 2022

Cited by 3 | Viewed by 2429

Abstract

Endometrial cancer (EC) is the most common gynecologic cancer with increasing incidence. The dysregulation of intracellular calcium plays a crucial role in cancer progression. However, the relationship between calcium-related genes and prognosis remains unclear. In this study, we aimed to establish a risk [...] Read more.

Endometrial cancer (EC) is the most common gynecologic cancer with increasing incidence. The dysregulation of intracellular calcium plays a crucial role in cancer progression. However, the relationship between calcium-related genes and prognosis remains unclear. In this study, we aimed to establish a risk model based on calcium-related genes for prognosis prediction in patients with EC. The TCGA-total set was divided into a training set and a testing set (1:1). The four-gene prognostic signature (CACNA2D1, SLC8A1, TRPM4 and CCL2) was established and classified all EC patients into a low-risk or high-risk group. This model was validated in both the testing dataset and the total set. The EC patients with high RiskScores showed significantly shorter overall survival than those with low RiskScores, and this trend was consistent among most subgroups. Moreover, an enrichment analysis confirmed that calcium-related and estrogen-response signalings were significantly enriched in the high-risk group. The knockdown of CACNA2D1 by siRNA or its blocker, amlodipine (AM) inhibited cell proliferation and induced cycle arrest in vitro. The calcium channel blocker AM inhibited cell proliferation and induced cycle arrest in vitro. AM also showed marked tumor inhibition effects in vivo. In summary, the prognostic model constructed by four calcium-related genes can reliably predict the outcomes of EC patients, and a calcium channel blocker, AM, has significant potential for EC treatment. Full article

(This article belongs to the Special Issue Advances in Calcium Signaling)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Prognostic analysis of differentially expressed calcium-related genes and the construction of a risk score signature. (A) Venn diagram to identify the common genes of calcium-related genes and DEGs. (B) Volcano plot for DEGs. A total of 158 calcium-related DEGs are highlighted in boxes. (C,D) Visualization of the protein–protein interaction (PPI) network formed by all 158 genes. The MCODE complexes are colored according to their clustering. (E) Significant genes of each MCODE complex are labeled and visualized. (F) Forest plots showing the results of the univariate Cox regression analysis of 6 prognostic genes. (G,H) The cvfit and lambda curves in LASSO regression. (I) The coefficients of four genes. (J) Forest plots showing the results of the multivariate Cox regression analysis of 4 prognostic genes. Full article ">Figure 2
Validation of the calcium-related signature model in the training cohort, testing cohort and total cohort. (A–C) Risk curves, survival statuses and gene expression levels for high- and low-risk groups in the TCGA-training set, TCGA-test set and TCGA-total set. (D–F) ROC curves showing the potential of the prognostic 4-gene calcium-related gene model in predicting 1-, 3- and 5-year OS in the TCGA-training, TCGA-testing and TCGA-total sets. (G–I) Kaplan–Meier curves for survival status and survival time in the TCGA-training, TCGA-testing and TCGA-total sets. (J–L) The nomograms to predict the 1-year, 3-year and 5-year overall survival rates of EC patients in the TCGA-training, TCGA-testing and TCGA-total sets. * p < 0.05, ** p < 0.01 and *** p < 0.001. Full article ">Figure 3
Correlation analysis between the prognostic signature and different clinicopathological characteristics in the TCGA-total cohort. (A–D) The histogram depicting the significant differences in the RiskScores in EC patients stratified by age, histopathological type, stage, and grade. (E,F) Subgroup analysis of Kaplan–Meier curves in different ages ≤60 and >60. (G,H) Subgroup analysis of Kaplan–Meier curves in different histopathological types. (I,J) Subgroup analysis of Kaplan–Meier curves in different stages I–II and III–IV. (K,L) Subgroup analysis of Kaplan–Meier curves in different grades 1–2 and 3. * p < 0.05, and *** p < 0.001. Full article ">Figure 4
Enrichment analysis of the high-risk group and low-risk group based on the 4-gene prognostic signature. (A) Volcano map of DEGs between the high-risk and low-risk groups. (B–D) GSEA against Hallmark showing significant enrichment of xenobiotic metabolism, estrogen response early and estrogen response late, respectively, in the low-risk endometrial cancer patients. (E) KEGG and GO analyses showing that calcium-related biological processes were significantly enriched. Full article ">Figure 5
Silencing CACNA2D1 inhibited the proliferation and migration of EC cells. (A) Representative images of Western blotting of endogenous expression of CACNA2D1 in eight endometrial cancer cell lines. (B) Immunofluorescence staining of CACNA2D1 and phalloidin in Ishikawa (upper) and HEC-108 (lower) cells. Nuclei were stained with DAPI. Scale bar, 25 µm (Magnification: 40×). (C,D) Transfection efficiency of CACNA2D1 via small interfering RNA in Ishikawa (C) and HEC-108 cells (D). (E,F) CCK8 assay showing the effect of CACNA2D1 silencing on Ishikawa (E) and HEC-108 (F) cell proliferation. (G) EdU staining showing the effect of CACNA2D1 silencing on Ishikawa and HEC-108 cells. (H,I) The statistical data of the EdU-positive rate after interfering with CACNA2D1. (J) Transwell assay detecting the effect of CACNA2D1 silencing on Ishikawa and HEC-108 cells. (K,L) The statistical data of the migrating cells after interfering with CACNA2D1. Data are shown as the mean ± SD. T-test. * p < 0.05, ** p < 0.01 and *** p < 0.001. These experiments were conducted in triplicate independently with similar results. Full article ">Figure 6
AM inhibited EC cell proliferation both in vitro and in vivo. (A) A CCK-8 assay was used to detect the effect of AMs on the proliferation of Ishikawa cells. (B) EdU staining showing the effect of AMs on Ishikawa cells. (C) The statistical data of the EdU-positive rate after treatment with AMs. (D) Cell apoptosis rate was detected by flow cytometry using Annexin-V/PI staining. (E) Representative images of tumor samples (n = 6). (F,G) The statistical data of tumor weight are shown as the mean ± SD. One-way ANOVA. ** p < 0.01, *** p < 0.001. (H) H&E and PCNA staining of sections from mice treated with AMs (0, 15, 20 mg/kg). Data are shown as the mean ± SD. (C) t-Test. (E) One-way ANOVA. ** p < 0.01 and *** p < 0.001. These experiments were conducted in triplicate independently with similar results. Full article ">

20 pages, 1078 KiB

Open AccessReview

Nasal Microbiome and Its Interaction with the Host in Childhood Asthma

by Yao Zeng and Jessie Qiaoyi Liang

Cells 2022, 11(19), 3155; https://doi.org/10.3390/cells11193155 - 7 Oct 2022

Cited by 6 | Viewed by 3131

Abstract

Childhood asthma is a major chronic non-communicable disease in infants and children, often triggered by respiratory tract infections. The nasal cavity is a reservoir for a broad variety of commensal microbes and potential pathogens associated with respiratory illnesses including asthma. A healthy nasal [...] Read more.

Childhood asthma is a major chronic non-communicable disease in infants and children, often triggered by respiratory tract infections. The nasal cavity is a reservoir for a broad variety of commensal microbes and potential pathogens associated with respiratory illnesses including asthma. A healthy nasal microenvironment has protective effects against respiratory tract infections. The first microbial colonisation in the nasal region is initiated immediately after birth. Subsequently, colonisation by nasal microbiota during infancy plays important roles in rapidly establishing immune homeostasis and the development and maturation of the immune system. Dysbiosis of microbiota residing in the mucosal surfaces, such as the nasopharynx and guts, triggers immune modulation, severe infection, and exacerbation events. Nasal microbiome dysbiosis is related to the onset of symptomatic infections. Dynamic interactions between viral infections and the nasal microbiota in early life affect the later development of respiratory infections. In this review, we summarise the existing findings related to nasal microbiota colonisation, dynamic variations, and host–microbiome interactions in childhood health and respiratory illness with a particular examination of asthma. We also discuss our current understanding of biases produced by environmental factors and technical concerns, the importance of standardised research methods, and microbiome modification for the prevention or treatment of childhood asthma. This review lays the groundwork for paying attention to an essential but less emphasized topic and improves the understanding of the overall composition, dynamic changes, and influence of the nasal microbiome associated with childhood asthma. Full article

(This article belongs to the Special Issue The Multifaceted Microbiome in Health and Disease)

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15 pages, 3593 KiB

Open AccessArticle

A Novel Homozygous Founder Variant of RTN4IP1 in Two Consanguineous Saudi Families

by Mazhor Aldosary, Maysoon Alsagob, Hanan AlQudairy, Ana C. González-Álvarez, Stefan T. Arold, Mohammad Anas Dababo, Omar A. Alharbi, Rawan Almass, AlBandary AlBakheet, Dalia AlSarar, Alya Qari, Mysoon M. Al-Ansari, Monika Oláhová, Saif A. Al-Shahrani, Moeenaldeen AlSayed, Dilek Colak, Robert W. Taylor, Mohammed AlOwain and Namik Kaya

Cells 2022, 11(19), 3154; https://doi.org/10.3390/cells11193154 - 7 Oct 2022

Cited by 1 | Viewed by 2074

Abstract

The genetic architecture of mitochondrial disease continues to expand and currently exceeds more than 350 disease-causing genes. Bi-allelic variants in RTN4IP1, also known as Optic Atrophy-10 (OPA10), lead to early-onset recessive optic neuropathy, atrophy, and encephalopathy in the afflicted patients. The gene [...] Read more.

The genetic architecture of mitochondrial disease continues to expand and currently exceeds more than 350 disease-causing genes. Bi-allelic variants in RTN4IP1, also known as Optic Atrophy-10 (OPA10), lead to early-onset recessive optic neuropathy, atrophy, and encephalopathy in the afflicted patients. The gene is known to encode a mitochondrial ubiquinol oxidoreductase that interacts with reticulon 4 and is thought to be a mitochondrial antioxidant NADPH oxidoreductase. Here, we describe two unrelated consanguineous families from the northern region of Saudi Arabia harboring a missense variant (RTN4IP1:NM_032730.5; c.475G<T, p.Val159Phe) in the gene. Clinically affected individuals presented with intellectual disability, encephalopathy, ataxia, optic atrophy, and seizures. Based on whole exome sequencing and confirmatory Sanger sequencing, the variant was fully segregated with the phenotype in the families, absent among large ethnically matching controls as well as numerous in-house exomes, and predicted to be pathogenic by different in silico classifiers. Structural modeling and immunoblot analyses strongly indicated this variant to be pathogenic. Since the families belong to one of the tribal inhabitants of Saudi Arabia, we postulate that the variant is likely to be a founder. We provide the estimated age of the variant and present data confirming the disease-causality of this founder variant. Full article

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

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Figure 1
Histochemical and immunohistochemical analyses of muscle biopsy. (A) Gomori trichrome staining shows prominent subsarcolemmal accumulation of mitochondria in most fibers with ragged red-like fibers. (B) COX staining shows no COX-negative fibers, but there is marked subsarcolemmal staining. (C) SDH staining shows marked subsarcolemmal staining. (D) Oil red staining indicates moderate increase in lipid stores. Full article ">Figure 2
Brain MRI results. Selected MRI sequences and planes showing optic chiasm atrophy (white/red/black arrows) in patient 1 (image (1B) coronal CISS), Patient 2 (image (1C) coronal T2), Patient 4 (image (1E) sagittal T1), and Patient 5 (image (1F) coronal T2). Other selected images also show bilateral optic nerve atrophy (white arrows) in patient 1 (image (1A) coronal T2), Patient 3 (image (1D) axial T2), and Patient 6 (image (1G) axial T2; image (1H) coronal T2). Full article ">Figure 3
Brain MRS findings of the patients. Multiple single-voxel MRS studies showed small lactate doublets at 1.3 ppm (white arrows) in Patient 1 (image (2A)), Patient 3 (image (2B)), Patient 5 (image (2D)), and Patient 6 (image (2E)). There is no obvious lactate doublet seen in Patient 4 (image (2C)). An MRS study was not performed for Patient 2. Full article ">Figure 4
Genetic analysis results. (A) Pedigree of the Family 1. Affected individuals are labeled with black-colored (filled) symbols. Carriers are represented with half-filled symbols; squares represent males, circles represent females, and double line indicates consanguinity. (B) Pedigree for the family 2. Brief Sanger sequencing results of the c.475 G>T variant under the pedigrees show the results for the Families. (C) Homozygosity results for all the affected individuals in both families. The patients are aligned with the normal individuals in both families. The shared ROH block is shown in the red bracket on chromosome 6 where RTN4IP1 is located. Numbers 1-6 indicate family members from Family 1, individuals 1 to Family 2, and individual 3, (F1-II-1, F1-II-2, F1-II-3, F2-II-1, F2-II-2, and F2-II-3), respectively. (D) Protein sequence alignment of RTN4IP1, the blue arrow shows the affected Valine amino acids at position 159 by the novel variant. The alignment showed that the amino acid in this region is highly conserved among different species. Full article ">Figure 5
Structural analysis and immunoblotting experiment results. (A) The location of the variant within the 3-D crystal structure of RTN4IP1 (PDB ID: 2VN8). RTN4IP1 forms a dimer (green and blue chains). Val159 is shown as a red stick model. The cofactor NADPH is shown as a stick model with carbon atoms in white. The zoom view shows the effect of the p.Val159Phe substitution. Clashes of the Phenylalanine side chain (yellow) are shown as red discs. Key side chains in the vicinity are highlighted as stick models. (B) Immunoblotting analysis results. The Western blotting analysis revealed significantly decreased RTN4IP1 protein levels in the patient’s extracts from cultured fibroblasts in comparison to controls. A polyclonal antibody was targeted against the protein, and beta-actin was used as a control-loading marker. (C) Schematic presentation of RTN4IP1. The top panel shows localization of all previously reported variants (black) and the newly identified novel variant (red) distributed among the nine exons (blue). The bottom panel shows both the alcohol dehydrogenase domain (ADH_N) and the zinc-binding motif (ADH_Zinc). Full article ">

20 pages, 2269 KiB

Open AccessReview

TFEB; Beyond Its Role as an Autophagy and Lysosomes Regulator

by Berenice Franco-Juárez, Cristina Coronel-Cruz, Beatriz Hernández-Ochoa, Saúl Gómez-Manzo, Noemi Cárdenas-Rodríguez, Roberto Arreguin-Espinosa, Cindy Bandala, Luis Miguel Canseco-Ávila and Daniel Ortega-Cuellar

Cells 2022, 11(19), 3153; https://doi.org/10.3390/cells11193153 - 7 Oct 2022

Cited by 48 | Viewed by 9407

Abstract

Transcription factor EB (TFEB) is considered the master transcriptional regulator of autophagy and lysosomal biogenesis, which regulates target gene expression through binding to CLEAR motifs. TFEB dysregulation has been linked to the development of numerous pathological conditions; however, several other lines of evidence [...] Read more.

Transcription factor EB (TFEB) is considered the master transcriptional regulator of autophagy and lysosomal biogenesis, which regulates target gene expression through binding to CLEAR motifs. TFEB dysregulation has been linked to the development of numerous pathological conditions; however, several other lines of evidence show that TFEB might be a point of convergence of diverse signaling pathways and might therefore modulate other important biological processes such as cellular senescence, DNA repair, ER stress, carbohydrates, and lipid metabolism and WNT signaling-related processes. The regulation of TFEB occurs predominantly at the post-translational level, including phosphorylation, acetylation, SUMOylating, PARsylation, and glycosylation. It is noteworthy that TFEB activation is context-dependent; therefore, its regulation is subjected to coordinated mechanisms that respond not only to nutrient fluctuations but also to stress cell programs to ensure proper cell homeostasis and organismal health. In this review, we provide updated insights into novel post-translational modifications that regulate TFEB activity and give an overview of TFEB beyond its widely known role in autophagy and the lysosomal pathway, thus opening the possibility of considering TFEB as a potential therapeutic target. Full article

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The TFEB activity is regulated by post-translational modifications. TFEB is mainly phosphorylated to avoid its nuclear translocation; however, depending on the cellular context, TFEB can be dephosphorylated, acetylated, glycosylated, or PARsylated. Full article ">Figure 2
The TFEB activation can influence the induction of senescence. Decreased nuclear TFEB localization may be one of many causes of senescence; however, restoring TFEB nuclear activity could contribute to avoiding cellular senescence, perhaps through the induction of autophagy or DNA repair genes. Full article ">Figure 3
The TFEB regulates the expression of several regulators of the ER stress response. ER stress, triggered by several compounds, promotes the nuclear localization of TFEB in a PERK-, ATF6-, IRE1α-, and calcineurin-dependent manner that consequently induces the transcription of UPR-related genes to restore ER homeostasis and support cell survival or apoptosis, if the stress is persistent. Full article ">Figure 4
The TFEB regulates genes involved in carbohydrate metabolism, lipid metabolism, and insulin signaling. Once active, TFEB may promote energy utilization through the activation of several related genes. Some of the proposed metabolic processes regulated by TFEB are shown, together with the relevant substrates. Full article ">Figure 5
The TFEB is expressed in several tissues, including the liver, intestine, and white adipose tissue. In these cell types, in response to several stimuli, TFEB is activated and induces the expression of several genes, as indicated in the figure. In the liver, cholesterol accumulation causes lysosomal stress, which consequently induces TFEB nuclear translocation, activating the expression of cholesterol 7α-hydroxylase (CYP7A1) to produce hepatic bile acid synthesis that, in the intestine cells, promotes the production of the hormone fibroblast growth factor-15/19 (FGF15/19), and this consequently activates both the activate orphan nuclear receptor, the small heterodimer partner (SHP/NR0B2) and TFEB, to activates genes (Ulk1 and Atgl) essential for lipophagy. TFEB activation by overexpression or the occurrence of lysosomal stress in fat-resident macrophages protects against diet-induced weight gain and adiposity through the induction of growth differentiation factor 15 (GDF15)-enhanced adipose lipid catabolism, and it reduces inflammation. Full article ">

17 pages, 8208 KiB

Open AccessArticle

Immunomodulatory Effect and Bone Homeostasis Regulation in Osteoblasts Differentiated from hADMSCs via the PD-1/PD-L1 Axis

by Seung-Cheol Lee, Min Kyoung Shin, Bo-Young Jang, Seung-Ho Lee, Min Kim and Jung-Suk Sung

Cells 2022, 11(19), 3152; https://doi.org/10.3390/cells11193152 - 7 Oct 2022

Cited by 3 | Viewed by 2386

Abstract

Human mesenchymal stem cells (hMSCs) are promising candidates for stem cell therapy and are known to secrete programmed death-1 (PD-1) ligand 1 (PD-L1) regulating T cell-mediated immunosuppression. Given the limitations of current stem cell therapy approaches, improvements in immunomodulatory capacity and stem cell [...] Read more.

Human mesenchymal stem cells (hMSCs) are promising candidates for stem cell therapy and are known to secrete programmed death-1 (PD-1) ligand 1 (PD-L1) regulating T cell-mediated immunosuppression. Given the limitations of current stem cell therapy approaches, improvements in immunomodulatory capacity and stem cell differentiation efficacy are needed. In this study, we propose novel strategies to overcome the challenges that remain in hMSC-mediated bone regeneration. We found that PD-1 is highly expressed in osteoblasts, and the PD-1/PD-L1 axis mediated the decreased proinflammatory cytokine expressions in differentiated osteoblasts cocultured with human adipose derived mesenchymal stem cells (hADMSCs). Moreover, the decrease was attenuated by PD-1/PD-L1 pathway inhibition. Osteogenic properties including osteogenic gene expression and calcium deposits were increased in osteoblasts cocultured with hADMSCs compared with those that were monocultured. Osteoblasts treated with PD-L1 and exosomes from hADMSCs also exhibited enhanced osteogenic properties, including calcium deposits and osteogenic gene expression. In our cocultured system that mimics the physiological conditions of the bone matrix, the PD-1/PD-L1 axis mediated the increased expression of osteogenic genes, thereby enhancing the osteogenic properties, while the calcium deposits of osteoblasts were maintained. Our results provide the therapeutic potentials and novel roles of the PD-1/PD-L1 axis in bone matrix for modulating the bone properties and immunosuppressive potentials that can aid in the prevention of bone diseases via maintaining bone homeostasis. Full article

(This article belongs to the Special Issue Bone Cell Function and Interplay in Physiological and Pathological Conditions)

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Cells, Volume 11, Issue 19 (October-1 2022) – 253 articles

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