TGFβ-induced RhoA activation and fibronectin production in mesangial cells require caveolae

Abstract

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.

Early Epithelial Restitution by Nonsteroidal Anti-Inflammatory Drug–Activated Gene 1 Counteracts Intestinal Ulcerative Injuries

“…Along with cancer metastasis studies, the current study suggested that RhoA-inducing TAK1 was a modulator of epithelial motility and contributes to normal tissue integrity by promoting cell migration and restitution of the injured tissues. In terms of mechanism, the current study addressed the effects of NAG-1-linked TAK1 on the induction of RhoA expression, supporting some connections between the TGF-b superfamily and RhoA signaling activation have been reported (24,28). In these previous studies, RhoA expression was not altered, but only RhoA activity was enhanced.…”

“…As the central downstream mediators of signaling pathway of TGF-b superfamily NAG-1, smads at convergent points of TGF-b receptor-linked pathway can be activated to RhoA GTPase. However, it has been well documented that RhoA activation is independent of smad-linked signaling pathways (23,24). Therefore, we focused on another target of TGF-b receptor-linked pathway, TAK1, as a mediator of NAG-1-induced RhoA activity.…”

Caveolin-1 is involved in reactive oxygen species-induced SHP-2 activation in astrocytes

“…This regulation is known to act most commonly in an inhibitory fashion, with release from caveolin-1 enabling activation (Razani et al, 2002). However, positive regulatory functions have also been demonstrated, as are seen in insulin receptor signaling and transforming growth factor-beta (TGFβ)-induced Rho A activation processes (Yamamoto et al, 1998;Peng et al, 2008). In our studies, H 2 O 2 -mediated SHP-2/ERK phosphorylation was reduced in astrocytes by caveolin-1 siRNA, suggesting that caveolin-1 may function as a positive regulator in SHP-2/ERK signaling pathways in response to H 2 O 2 in astrocytes.…”

Blocking VEGF/Caveolin-1 signaling contributes to renal protection of fasudil in streptozotocin-induced diabetic rats

“…Effects of fasudil on the phosphorylation of Src and caveolin-1 in diabetic glomeruli Src-family kinases are the only known caveolin-1 Y14 kinases [24,25] , and our previous studies demonstrated that TGF-β-and VEGF-induced Src activation mediated caveolin-1 phosphorylation at Y14 followed by fibronectin synthesis in mesangial cells [23,26] . Because fasudil prevented VEGF upregulation in diabetic kidneys, we investigated the effect of fasudil on Src and caveolin-1 phosphorylation levels.…”