HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

Abstract

Although heat shock protein 72 (HSP72) ameliorates renal tubulointerstitial fibrosis by inhibiting epithelial-to-mesenchymal transition (EMT), the underlying mechanism is unknown. Because Smad proteins transduce TGF-␤ signaling from the cytosol to the nucleus and HSP72 assists in protein folding and facilitates nuclear translocation, we investigated whether HSP72 inhibits TGF-␤-induced EMT by modulating Smad expression, activation, and nuclear translocation. To evaluate the roles of distinct HSP72 structural domains in these processes, we constructed vectors that expressed wild-type HSP72 or mutants lacking either the peptide-binding domain (HSP72-⌬PBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (HSP72-⌬NLS). Overexpression of wild-type HSP72 or HSP72-⌬NLS inhibited TGF-␤1-induced EMT, but HSP72-⌬PBD did not, suggesting a critical role for the PBD in this inhibition. HSP72 overexpression inhibited TGF-␤1-induced phosphorylation and nuclear translocation of Smad3 and p-Smad3, but not Smad2; these inhibitory effects required the PBD but not the NLS. Coimmunoprecipitation assays suggested a physical interaction between Smad3 and the PBD. siRNA knockdown of endogenous HSP72 enhanced both TGF-␤1-induced Smad3 phosphorylation and EMT and confirmed the interaction of HSP72 with both Smad3 and p-Smad3. In vivo, induction of HSP72 by geranylgeranylacetone suppressed Smad3 phosphorylation in renal tubular cells after unilateral ureteral obstruction. In conclusion, HSP72 inhibits EMT in renal epithelial cells primarily by exerting domain-specific effects on Smad3 activation and nuclear translocation. 21: 598 -609, 201021: 598 -609, . doi: 10.1681 Transforming growth factor ␤ (TGF-␤) and related factors regulate a wide variety of biologic activities. 1 Furthermore, abnormalities in TGF-␤ signaling lead to various human diseases. 2 Signals from TGF-␤ are mainly transduced by the Smad family of transcription factors. Upon TGF-␤ stimulation, Smad2 and Smad3 are phosphorylated at their carboxy-tails by the activated TGF-␤ type I receptor kinase, 3 forming a stable complex with Smad4 in cytoplasm and then accumulating in the nucleus to regulate transcription of target genes. In contrast, Smad7 inhibits the TGF-␤ receptor type I-dependent Smad2/3 activation. It has been demonstrated that Smad3 phosphorylation plays a predominant role in guiding the complex through the nuclear pore. 4 cytoplasm. 5 Blockade of TGF-␤ signaling events, specifically the phosphorylation of Smad2 and Smad3 and/or nuclear translocation, prevent epithelial-to-mesenchymal transition (EMT) and tissue fibrosis. 6,7 Heat shock protein 72 (HSP72) exerts cytoprotective effects by assisting in protein folding, assembly/disassembly and translocation of client proteins across membranes, protein degradation, and signal transduction. 8,9 However, some cytoprotective effects do not require the HSP72 chaperone function. 10 HSP72 contains distinct domains: a C-terminal peptide-binding domain (PBD) responsible...

Heat Shock Protein 72 Enhances Autophagy as a Protective Mechanism in Lipopolysaccharide-Induced Peritonitis in Rats

“…25 Densitometric quantification was performed with the image analysis program (FluorChem 8900; Alpha Innotech Corp, San Leandro, CA).…”

“…25,31,34,47 To evaluate the effects of HSP72 on LPS-induced cell autophagy and apoptosis in vivo, GGA and quercetin were used to regulate HSP72 expression in rats. In line with our previous observation, oral administration of GGA markedly increased the expression of HSP72, but not HSP90 and HSP27, of the peritoneum in the presence or absence of LPS treatment.…”

Oxymatrine inhibits renal fibrosis of obstructive nephropathy by downregulating the TGF-β1-Smad3 pathway

“…Smad3 is pathogenic, and inhibition of Smad3 phosphorylation and translocation attenuates renal fibrosis. 23 Smad3 can directly bind to the promoter region of collagens to promote their production. At the same time, activated fibroblasts can produce more ECM, such as COI-1 and FN.…”

Molecular Mechanisms of TGF-β Signaling in Renal Fibrosis

“…Ectopic expression of Smad3 stimulates TGF-b-induced fibronectin expression [52], and deletion of Smad3 in mice suppresses in mouse models of diabetic nephropathy [39,41], obstructive nephropathy [32], and chronic aristolochic acid nephropathy [45], confirming the critical role of TGF-b/Smad3 signaling during fibrosis. In addition, treatment of heat-shock protein 72 (HSP72) is capable of inhibiting tubulointerstitial fibrosis in a rat model of UUO, which is associated with its ability to suppress TGF-b1-induced phosphorylation and nuclear translocation of Smad3, but not Smad2 [53,54].…”

“…Our laboratory and others have demonstrated that restoration of renal Smad7 expression is capable of suppressing renal fibrosis in numerous rodent models, including diabetes, obstructive nephropathy, remnant kidney, and autoimmune disease [3, 37, 43,64]. Furthermore, gene transfer of Smad7 is capable of protecting kidneys from fibrosis by regulating TGF-b/Smad3-mediated expression of miR-21, miR-192, and miR-29b in diseased kidneys [79,82] Recent studies have demonstrated that a specific inhibitor of Smad3 (SIS3), HSP72, and inhibitors of HSP90 are able to reduce renal fibrosis [31, 53,99 • ]. On the other hand, a traditional Chinese herb, Asiatic acid, acts as an agonist of Smad7 to inhibit liver fibrosis by upregulating Smad7 expression [100 • ].…”