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Focal adhesions (FAs) provide the cells linkages to extracellular matrix (ECM) at sites of integrins binding and transmit mechanical forces between the ECM and the actin cytoskeleton. Cells sense and respond to physical stimuli from their surrounding environment through the activation of mechanosensitive signaling pathways, a process called mechanotransduction. In this study, we used RGD-peptide conjugated DNA tension gauge tethers (TGTs) with different tension tolerance (T tol ) to determine the molecular forces required for FA maturation in different sizes and YAP nuclear translocation. We found that the limitation of FA sizes in cells seeded on TGTs with different T tol were less than 1 μm, 2 μm, 3 μm, and 6 μm for T tol values of 43 pN, 50 pN, 54 pN, and 56 pN, respectively. This suggests that the molecular tension across integrins increases gradually as FA size increases throughout FA maturation. For YAP nuclear translocation, significant YAP nuclear localization was observed only in the cells seeded on the TGTs with T tol ≥ 54 pN, but not on TGTs with T tol ≤ 50 pN, suggesting a threshold of molecular force across integrins for YAP nuclear translocation lies in the range of 50 pN-54 pN., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Mesangial cells are the major extracellular matrix (ECM)-producing cells in the kidney glomerulus and, when exposed to elevated glucose levels, they up-regulate assembly of fibronectin (FN) and other ECM proteins. Increases in glucose concentration are known to alter gene expression; here we investigated the connection between increased ECM production and changes in gene expression in mesangial cells. Comparison of mesangial cells grown in normal or high glucose conditions by RNA-sequencing showed significant expression changes in over 6000 genes and, when grouped by KEGG pathway analysis, identified the ECM-receptor interaction and focal adhesion pathways among the top 5 upregulated pathways. Of note was the significant increase in expression of tenascin-C (TN-C), a known regulator of FN matrix assembly. Mouse TN-C has multiple isoforms due to alternative splicing of 6 FNIII repeat exons. In addition to the transcriptional increase with high glucose, exon inclusion via alternative splicing was also changed resulting in production of higher molecular weight isoforms of TN-C. Mesangial cells grown in normal glucose secreted small isoforms with 1-2 variable repeats included whereas in high glucose large isoforms estimated to include 5 repeats were secreted. Unlike the smaller isoforms, the larger TN-C was not detected in the FN matrix. This change in TN-C isoforms may affect the regulation of FN matrix assembly and in this way may contribute to increased ECM accumulation under high glucose conditions., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2020 The Author(s).)

Emerging evidence suggests that dysfunction of the ubiquitin-proteasome system is involved in the pathogenesis of numerous senile degenerative diseases including retinal disorders. The aim of this study was to assess whether there is a link between proteasome regulation and retinal pigment epithelium (RPE)-mediated expression of extracellular matrix genes. For this purpose, human retinal pigment epithelial cells (ARPE-19) were treated with different concentrations of transforming growth factor-β (TGFβ), connective tissue growth factor (CTGF), interferon-γ (IFNγ) and the irreversible proteasome inhibitor epoxomicin. First, cytotoxicity and proliferation assays were carried out. The expression of proteasome-related genes and proteins was assessed and proteasome activity was determined. Then, expression of fibrosis-associated factors fibronectin (FN), fibronectin EDA domain (FN EDA), metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinases-1 (TIMP-1) and peroxisome proliferator-associated receptor-γ (PPARγ) was assessed. The proteasome inhibitor epoxomicin strongly arrested cell cycle progression and down-regulated TGFβ gene expression, which in turn was shown to induce expression of pro-fibrogenic genes in ARPE-19 cells. Furthermore, epoxomicin induced a directional shift in the balance between MMP-2 and TIMP-1 and was associated with down-regulation of transcription of extracellular matrix genes FN and FN-EDA and up-regulation of the anti-fibrogenic factor PPARγ. In addition, both CTGF and TGFβ were shown to affect expression of proteasome-associated mRNA and protein levels. Our results suggest a link between proteasome activity and pro-fibrogenic mechanisms in the RPE, which could imply a role for proteasome-modulating agents in the treatment of retinal disorders characterized by RPE-mediated fibrogenic responses.