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

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-beta signaling from the cytosol to the nucleus and HSP72 assists in protein folding and facilitates nuclear translocation, we investigated whether HSP72 inhibits TGF-beta-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-Delta PBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (H5P72-Delta NLS). Overexpression of wild-type HSP72 or HSP72-Delta NLS inhibited TGF-beta 1-induced EMT, but HSP72-Delta PBD did not, suggesting a critical role for the PBD in this inhibition. H5P72 overexpression inhibited TGF-beta 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-beta 1-induced Smad3 phosphorylation and EMT and confirmed the interaction of H5P72 with both Smad3 and p-Smad3. in vivo, induction of H5P72 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.