TGFbeta-induced RhoA activation and fibronectin production in mesangial cells require caveolae.
Glomerular sclerosis of diverse etiologies is characterized by mesangial matrix accumulation, with transforming growth factor-beta (TGFbeta) an important pathogenic factor. The GTPase RhoA mediates TGFbeta-induced matrix accumulation in some settings. Here we study the role of the membrane microdomain caveolae in TGFbeta-induced RhoA activation and fibronectin upregulation in mesangial cells (MC). In primary rat MC, TGFbeta1 time dependently increased RhoA and downstream Rho kinase activation. Rho pathway inhibition blocked TGFbeta1-induced upregulation of fibronectin transcript and protein. TGFbeta1-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. Compared with wild types, RhoA/Rho kinase activation was absent in MC lacking caveolae. Reexpression of caveolin-1 (and caveolae) restored these responses. Phosphorylation of caveolin-1 on Y14, effected by Src kinases, has been implicated in signaling responses. Overexpression of nonphosphorylatable caveolin-1 Y14A prevented TGFbeta1-induced RhoA activation. TGFbeta1 also activated Src, and its inhibition blocked RhoA activation. Furthermore, TGFbeta1 led to association of RhoA and caveolin-1. This was prevented by Src or TGFbeta receptor I inhibition, and by caveolin-1 Y14A overexpression. Last, fibronectin upregulation by TGFbeta1 was blocked by Src inhibition, not seen in caveolin-1 knockout MC, and restored by caveolin-1 reexpression in the latter. TGFbeta1-induced collagen I accumulation also required caveolae. TGFbeta1-mediated Smad2/3 activation, however, did not require caveolae. We conclude that RhoA/Rho kinase mediates TGFbeta-induced fibronectin upregulation. This requires caveolae and caveolin-1 interaction with RhoA. Interference with caveolin/caveolae or RhoA signaling thus represents a potential target for the treatment of fibrotic renal disease.
Peng
Fangfang
F
Division of Nephrology, McMaster University, Hamilton, Ontario, Canada.
Zhang
Baifang
B
Wu
Dongcheng
D
Ingram
Alistair J
AJ
Gao
Bo
B
Krepinsky
Joan C
JC
D000818:Animals; D021941:Caveolae / Q000502:physiology*; D004789:Enzyme Activation; D005353:Fibronectins / Q000096:biosynthesis*; D007211:Indoles / Q000494:pharmacology; D050527:Mesangial Cells / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D051381:Rats; D017207:Rats, Sprague-Dawley; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000502:physiology*; D015854:Up-Regulation; D054460:rho-Associated Kinases / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D005353:Fibronectins; D007211:Indoles; D018125:Receptors, Transforming Growth Factor beta; C416927:SU 6656; D013449:Sulfonamides; D016212:Transforming Growth Factor beta; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein