HSP72 inhibits Smad3 activation and nuclear translocation 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-DeltaPBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (HSP72-DeltaNLS). Overexpression of wild-type HSP72 or HSP72-DeltaNLS inhibited TGF-beta1-induced EMT, but HSP72-DeltaPBD did not, suggesting a critical role for the PBD in this inhibition. HSP72 overexpression inhibited TGF-beta1-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-beta1-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.
Zhou
Yi
Y
Department of Nephrology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China, 510080.
Mao
Haiping
H
Li
Shu
S
Cao
Shirong
S
Li
Zhijian
Z
Zhuang
Shougang
S
Fan
Jinjin
J
Dong
Xiuqing
X
Borkan
Steven C
SC
Wang
Yihan
Y
Yu
Xueqing
X
D000818:Animals; D002454:Cell Differentiation; D004847:Epithelial Cells / Q000166:cytology*; D050884:HSP72 Heat-Shock Proteins / Q000502:physiology*; D007668:Kidney; D008297:Male; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D051900:Smad3 Protein / Q000502:physiology*; D019459:Urothelium / Q000166:cytology*
D050884:HSP72 Heat-Shock Proteins; D051900:Smad3 Protein