Submitted on September 10, 2007
Accepted on April 17, 2008
TGF
-Induced RhoA Activation and Fibronectin Production in Mesangial Cells Requires Caveolae
Fangfang Peng1, Baifang Zhang2, Dongcheng Wu3, Alistair J Ingram3, Bo Gao3, and Joan C Krepinsky1*
1 Nephrology, McMaster University, Hamilton, Canada
2 Hamilton, Canada; Nephrology, McMaster University, Hamilton, Canada
3 Nephrology, McMaster University, Hamilton, Canada; Hamilton, Canada
* To whom correspondence should be addressed. E-mail: krepinj{at}mcmaster.ca.
Glomerular sclerosis of diverse etiologies is characterized by mesangial matrix accumulation, with TGF
an important pathogenic factor. The GTPase RhoA mediates TGF-induced matrix accumulation in some settings. Here we study the role of the membrane microdomains caveolae in TGF-induced RhoA activation and fibronectin upregulation in mesangial cells (MC).
In primary rat MC, TGF1 time-dependently increased RhoA and downstream Rho-kinase activation. Rho pathway inhibition blocked TGF1-induced upregulation of fibronectin transcript and protein. TGF1-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. Compared to wild-type, 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 TGF1-induced RhoA activation. TGF1 also activated Src, and its inhibition blocked RhoA activation. Furthermore, TGF1 led to association of RhoA and caveolin-1. This was prevented by Src or TGF receptor I inhibition, and by caveolin-1 Y14A overexpression. Last, fibronectin upregulation by TGF1 was blocked by Src inhibition, not seen in caveolin-1 knockout MC, and restored by caveolin-1 reexpression in the latter. TGF1-induced collagenI accumulation also required caveolae. TGF1-mediated Smad2/3 activation, however, did not require caveolae.
We conclude that RhoA/Rho-kinase mediate TGF-induced fibronectin upregulation. This requires caveolae and caveolin-1 interaction with RhoA. Interference with caveolin/-ae or RhoA signaling thus represents a potential target for the treatment of fibrotic renal disease.
