Matrix metalloproteinase-2 stimulates collagen-I expression through phosphorylation of focal adhesion kinase in rat cardiac fibroblasts.
Collagen-I is thought to be the main component of the extracellular matrix in cardiac fibrosis, the accumulation of which occurs with excessive activation of matrix metalloproteinase-2 (MMP-2). MMP-2 degrades the extracellular matrix; however, the relative importance of MMP-2 to collagen-I synthesis in cardiac fibroblasts remains unclear. We investigated whether extracellular activation of MMP-2 regulates collagen-I synthesis and phosphorylation of focal adhesion kinase (FAK) in rat cardiac fibroblasts. Primary cultures of rat cardiac fibroblasts were incubated with purified active MMP-2 to determine whether extracellular MMP-2 affects collagen-I synthesis and FAK phosphorylation in cardiac fibroblasts. Exogenous MMP-2 significantly stimulated FAK (Tyr397) phosphorylation and induced collagen-I expression in a time-dependent manner. Simultaneous treatment with the FAK inhibitor PF573228 abolished exogenous MMP-2-enhanced FAK (Tyr397) phosphorylation and collagen-I expression. Cells were then stimulated with norepinephrine (NE) to investigate whether endogenous MMP-2 could also induce collagen-I expression through FAK (Tyr397) phosphorylation. NE-stimulated endogenous MMP-2 activation in conditioned medium was significantly attenuated by simultaneous treatment with the MMP inhibitor PD166793. Similarly, NE-induced FAK (Tyr397) phosphorylation and collagen-I expression were significantly inhibited by simultaneous treatment with PD166793 or PF573228. Furthermore, MMP-2 knockdown induced by small interfering RNA (siRNA) significantly abolished endogenous MMP-2 expression and activation. MMP-2 siRNA significantly abolished NE-induced FAK (Tyr397) phosphorylation and collagen-I expression. These findings suggest that the extracellular activation of MMP-2 accelerated collagen-I synthesis in rat cardiac fibroblasts and that FAK phosphorylation (Tyr397) plays a pivotal role in MMP-2-stimulated collagen-I synthesis.
Dept. of Small Animal Internal Medicine, School of Veterinary Medicine, Kitasato Univ., 23-35-1 Higashi, Towada, Aomori 034-8628, Japan. hori@vmas.kitasato-u.ac.jp
Yasutomo
Y
Hori
Takashige
T
Kashimoto
Tomohiro
T
Yonezawa
Naoya
N
Sano
Ryuta
R
Saitoh
Shinya
S
Igarashi
Seishiro
S
Chikazawa
Kazutaka
K
Kanai
Fumio
F
Hoshi
Naoyuki
N
Itoh
Sei-Ichi
S
Higuchi
D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020868:Gene Silencing; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000494:pharmacology; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D015363:Quinolones / Q000494:pharmacology; D051381:Rats; D011921:Rats, Inbred WKY; D013450:Sulfones / Q000494:pharmacology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
C370242:(R)-2-(4'-bromo-biphenyl-4-sulfonyl-amino)-3-methyl-butyric acid; C521108:6-(4-(3-(methylsulfonyl)benzylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3,4-dihydroquinolin-2(1H)-one; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D009842:Oligopeptides; D015363:Quinolones; D013450:Sulfones; D014443:Tyrosine; D051417:Focal Adhesion Kinase 1; C496052:Ptk2 protein, rat; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; D009638:Norepinephrine