Contribution of Src-FAK signaling to the induction of connective tissue growth factor in renal fibroblasts.
Expression of connective tissue growth factor (CTGF) is sensitive to reorganization of the actin cytoskeleton, but also to alterations in cell morphology due to extracellular forces, for example, cyclic stretching or mechanical loading. Dynamic alterations of focal adhesion proteins were thus proposed to modulate CTGF induction. Immortalized human renal fibroblasts were cultured in or on top of preformed collagen-1 gels. Proteins were detected by immunofluorescence and quantified by Western blotting. Fibroblasts cultured in/on collagen gels resembled cells in vivo by their spindle-like morphology, absence of actin stress fibers, small punctiform focal contacts, and low levels of CTGF expression. Disassembly of microtubules by short-term treatment with colchicine induced cell rounding, cortical recruitment of patchy F-actin, reorganization of focal contacts into strong clusters, and upregulation of CTGF, all of which were dependent on RhoA-Rho-kinase signaling. Clustering of focal adhesion sites activated Src-family kinases and focal adhesion kinase (FAK). Interference with Src activity by PP2 had no effect on the morphological alterations but decreased tyrosine phosphorylation of focal adhesion proteins and almost completely prevented upregulation of CTGF. Furthermore, inhibition of phosphatidylinositol 3-kinase reduced CTGF expression. On the other hand, when the fibroblasts were cultured on a rigid matrix, that is collagen-coated plates, strong focal complexes prevented the dynamic alterations, and RhoA-mediated upregulation of CTGF expression was independent of Src-FAK signaling. Assembly of focal adhesion proteins regulates CTGF expression, providing a link between actin network, adhesion receptors, and CTGF-mediated functions such as synthesis of extracellular matrix proteins.
Department of Nephrology, University of Erlangen-Nuremberg, Erlangen, Germany.
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Graness
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Cicha
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Goppelt-Struebe
D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D002867:Chromones / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D024042:Collagen Type I / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010590:Phalloidine; D012235:Rhodamines; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D000077191:Wortmannin; D019061:src-Family Kinases / Q000378:metabolism*
D000730:Androstadienes; C526317:CCN2 protein, human; D002867:Chromones; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005456:Fluorescent Dyes; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009025:Morpholines; D012235:Rhodamines; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D010590:Phalloidine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017239:Paclitaxel; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D003078:Colchicine; D000077191:Wortmannin