Kindlin-2 Mediates Activation of TGF-β/Smad Signaling and Renal Fibrosis

Abstract

Activation of TGF-b/Smad signaling plays a central role in the pathogenesis of tubulointerstitial fibrosis, but the mechanisms underlying the initial interaction of the TGF-b receptor with Smads, leading to their activation, remain unclear. Here, we found that Kindlin-2, an integrin-binding protein, physically mediated the interaction of the TGF-b type I receptor (TbRI) with Smad3 in human kidney tubular epithelial cells. Kindlin-2 bound to TbRI through its FERM domain and to Smad3 through its N terminus. Overexpression of Kindlin-2 increased TGF-b-induced Smad3 activation. Knockdown of Kindlin-2 significantly suppressed the engagement of TbRI with Smad3 and inhibited TGF-b-induced Smad3 activation, as well as the expression of its target genes. Neither transfection of a Kindlin-2 mutant incapable of binding to b1 integrin nor knockdown of b1 integrin influenced the effect of Kindlin-2 on TGF-b1-induced Smad3 activation, indicating that this effect is independent of integrin. Kindlin-2 expression was markedly increased, predominantly in renal tubular epithelial cells, both in the unilateral ureteral obstruction model of kidney fibrosis and in human tissue exhibiting tubulointerstitial fibrosis. Furthermore, in the unilateral ureteral obstruction model, knocking down Kindlin-2 significantly inhibited activation of TGF-b/Smad signaling, decreased the expression of matrix genes, and ameliorated fibrosis. In summary, Kindlin-2 physically interacts with both TbRI and Smad3, promoting the activation of TGF-b/Smad signaling and contributing to the pathogenesis of tubulointerstitial fibrosis. Blockade of Kindlin-2 might be a rational therapeutic strategy for the treatment of fibrotic kidney diseases.

Kindlin-2

“…In Figure 3, A and B, in their article, these investigators show data from co-immunoprecipitation experiments. 2 The findings indicate that Kindlin-2 also associates with smad2 but perhaps quantitatively less or more weakly compared with smad3. There also appears to be less or weaker co-localization of Kindlin-2 with smad2 than with smad3, as may be deduced from immunofluorescence staining ( Figure 3C in their article).…”

“…2 Moreover, these investigators demonstrate in vivo that Kindlin-2 positively regulates TGF-b/smad3-dependent profibrogenic signals independent of its interactions with b-integrins. In a commonly used rodent model of TGF-b-mediated renal fibrosis, unilateral obstructive nephropathy in mice, knock-down of Kindlin-2 reduces interstitial fibrosis.…”

“…Taken together, Kindlin-2 serves as a TBR1/ smad3 adapter protein, directs and augments TGF-b signals toward the smad3 pathway, and amplifies profibrogenic effects of this cytokine in tubular cells in vitro and in a mouse model of renal fibrogenesis in vivo. 2 The experiments by Wei et al show in considerable detail how Kindlin-2 interacts with the TBR1 receptor: It binds to the receptor with its FERM (4.1 protein, ezrin, radixin, moesin) domain (consisting of the F1, F2, and F3 subdomains). Interestingly, the F3 subdomain in the Kindlins is also an important domain for their binding to b-integrins.…”

“…3 This subdomain is located near the N-terminus, which was determined by Wei et al as the site of interaction of Kindlin-2 with smad3. 2 Hence, it appears to be likely that at a given time Kindlin-2 interacts with either b-integrins or TBR1/smad3, but not both. Indeed, this notion is consistent with the finding by Wei and colleagues that augmentation of smad3 signaling by Kindlin-2 is independent of b-integrins.…”

“…2 It appears that TGF-b upregulates expression of Kindlin-2, likely by transcriptional activation. This is inferred from the finding that Kindlin-2 is induced by TGF-b 4 and from findings in podocytes where TGF-b 1 increases Kindlin-2 levels.…”