Search

Fibroblasts remodel extracellular matrix collagen, in part, through phagocytosis. This process requires formation of cell extensions, which in turn involves interaction of the actin-binding protein flightless-1 (FliI) with non-muscle myosin IIA (NMMIIA; heavy chain encoded by MYH9) at cell-matrix adhesion sites. As Ca2+ plays a central role in controlling actomyosin-dependent functions, we examined how Ca2+ controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca2+ influx at the extensions of fibroblasts. Western blotting and quantitative (q)PCR showed high expression levels of the Ca2+-permeable transient receptor potential vanilloid-4 (TRPV4) channel, which co-immunoprecipitated with β1 integrin and localized to adhesions. Treatment with β2β1-integrin-blocking antibody or the TRPV4-specific antagonist AB159908, as well as reduction of TRPV4 expression through means of siRNA, blocked Ca2+ influx. These treatments also inhibited the interaction of FliI with NMMIIA, reduced the number and length of cell extensions, and blocked collagen remodeling. Pulldown assays showed that Ca2+ depletion inhibited the interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed that FliI-NMMIIA interactions require Ca2+ influx.We conclude that Ca2+ influx through the TRPV4 channel regulates FliI-NMMIIA interaction, which in turn enables generation of the cell extensions essential for collagen remodeling. [ABSTRACT FROM AUTHOR]

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α-smooth muscle actin expression. Compared with native collagen, binding of purified α11β1 integrin to glycated collagen was reduced by >fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-β2 but not TGF-β1 or TGF-β3. The increased expression of TGF-β2 was inhibited by triple helical collagen peptides that mimic the α11β1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between −809 and −1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-β2 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis. J. Cell. Physiol. 230: 327-336, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

During the development of pressure-induced cardiac hypertrophy, fibroblasts are activated to become myofibroblasts, which exhibit actin-cytoskeletal remodeling and express α-smooth muscle actin (SMA; encoded by ACTA2). Currently, the mechanosensing signaling pathways that regulate SMA expression are not defined. Because focal-adhesion complexes are putative mechanosensing organelles, we examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin in the regulation of SMA expression. We subjected NIH3T3 cells to tensile forces (0.65 pN/μm²) by using collagen-coated magnetite beads attached to integrins. After stimulation by mechanical force, FAK and gelsolin were recruited to magnetite beads and there was increased phosphorylation of Tyr397FAK. Mechanical force enhanced SMA promoter activity by twofold; this increased activity was blocked by FAK knockdown using siRNA and by deletion of gelsolin. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA that is regulated by actin filaments, was also reduced by FAK knockdown. Phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], which uncaps gelsolin from actin filaments, was enriched at sites of force application. Type-I phosphatidylinositol 4-phosphate 5 kinase-γ(PIP5KI), which generates PtdIns(4,5)P2, associated with FAK and was required for force-mediated SMA-promoter activity and actin assembly. Catalytically inactive PIP5KIγinhibited force-induced phosphorylation of FAK at Tyr397. These data suggest a novel pathway in which mechanosensing by FAK regulates actin assembly via gelsolin and the activity of PIP5KIγ actin assembly in turn controls SMA expression via MRTF-A. [ABSTRACT FROM AUTHOR]