FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of the mTOR complex.
OBJECTIVE
Cardiac fibroblasts are activated by mechanical stress, but the underlying mechanisms involved remain poorly understood. In this study, we investigated whether focal adhesion kinase (FAK) plays a role in the activation of cardiac fibroblasts in response to cyclic stretch.


RESULTS
Neonatal (NF-P3/80--third passage, 80% confluence) and adult (AF-P1/80--first passage, 80% confluence) rat cardiac fibroblasts were exposed to cyclic stretch (biaxial, 1 Hz), which enhanced FAK phosphorylation at Tyr397. Proliferation (anti-5-bromo-2'-deoxyuridine and anti-Ki67 nuclear labelling), differentiation into myofibroblasts (expression of alpha-smooth muscle actin--alpha-SMA), and the activity of matrix metalloproteinase-2 were equally enhanced in stretched NF-P3/80 and AF-P1/80. Treatment with the integrin inhibitor RGD peptide impaired FAK phosphorylation and increased apoptosis (TUNEL) in non-stretched and stretched NF-P3/80, whereas FAK silencing induced by small interfering RNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF-P3/80 invoked by cyclic stretch. In addition, NF-P3/80 depleted of FAK were defective in AKT Ser473, TSC-2 Thr1462, and S6 kinase Thr389 phosphorylation induced by cyclic stretch. The activation of NF-P3/80 invoked by cyclic stretch was prevented by pre-treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin, whereas supplementation with the amino acid, leucine, activated S6K and rescued the stretch-induced activation of NF-P3/80 depleted of FAK.


CONCLUSIONS
These findings demonstrate a critical role for the mTOR complex, downstream from FAK, in mediating the activation of cardiac fibroblasts in response to mechanical stress.
Dalla Costa
Ana Paula
AP
Department of Internal Medicine, School of Medicine and Department of Cell Biology, State University of Campinas, Campinas, SP, Brazil.
Clemente
Carolina F M Z
CF
Carvalho
Hernandes F
HF
Carvalheira
José B
JB
Nadruz
Wilson
W
Franchini
Kleber G
KG
D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D012694:Serine; D020123:Sirolimus / Q000494:pharmacology; D013314:Stress, Mechanical; D058570:TOR Serine-Threonine Kinases; D013912:Threonine; D013997:Time Factors; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014443:Tyrosine
D000199:Actins; D024042:Collagen Type I; D051417:Focal Adhesion Kinase 1; D047908:Intracellular Signaling Peptides and Proteins; D007930:Leucine; D020778:Matrix Metalloproteinase 2; D009842:Oligopeptides; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D012694:Serine; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D013912:Threonine; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; C498862:Acta2 protein, rat; C047981:arginyl-glycyl-aspartic acid; C546843:mTOR protein, mouse; C546845:mTOR protein, rat; C496052:Ptk2 protein, rat; C522362:Mmp2 protein, mouse