UCH-L5 Regulates TGF?-1 Signaling By De-ubiquitinating And Stabilizing Smad2/Smad3 In Pulmonary Fibrosis

BackgroundTransforming growth factor ?-1(TGF?-1)-induced signaling pathway plays a crucial role in the pathogenesis of idiopathic pulmonary fibrosis(IPF).Smad2 and Smad3 are downstream transcription factors.However,the molecular regulation of Smad2/Smad3 proteins stability remains a mystery.UCH-L5,a DUBs,can reverse polyubiquitination of substance protein and degaradation through UPS and is of great impotance in the regulation of protein function and signaling pathway.UCH-L5 has been reported to interact with Smad7,potentially reverses Smurf-mediated ubiquitination of T?RI and stabilizes T?RI,therefore enhance TGF?-1-induced pathway.However,the effcet of UCH-L5 on regulation of Smad2/Smad3 and pathogenesis of IPF is unclear.Our studies provide a molecular mechanism by which UCH-L5 regultes TGF?-1 signaling by stabilizing Smad2/Smad3.These data indicate that UCH-L5 may contribute to the pathogenesis of IPF and may be a potential therapeutic target.Methods(1)The effects of b-AP15 on TGF?-1-induced signaling pathway in HLF cells were determined by immunoblotting with antibodies to UCH-L5,ALK5,Smad2,Smad3,Smad7,FN and ?-SMA after TGF?-1 treatment.Immunofluorescence staining with antibodies to FN and ?-SMA was performed to confirm the results of immunoblotting.(2)To compare the effects of IU1 and b-AP15 on TGF?-1 signaling,HLF or Mrc5 cells were treated with IU1 or different doses of b-AP15.Cell lysates were collected and immunoblotting was performed with antibodies to ALK5,Smad2,Smad3,Smad7.(3)Expression levels of m RNA of Smad2 and Smad3 after b-AP15 treatment in HLF cells were examined by q PCR.(4)HLF cells were transfected with vector or HA-Ubi K0 plasmid,immunoblotting was performed to determine whether the effect of b-AP15 on Smad2 and Smad3 was affected by protein polyubiquitination.Ubiquitin immunoprecipitation was used to detect the polyubiquitination of Smad2 or Smad3 in the b-AP15 treated cells.(5)HLF cells were pretreated with Leupeptin,MG-132 or bafilomycin A1.Expressions of Smad2 and Smad3 after b-AP15 treatment were examined by immunoblotting.(6)HLF cells were pretreated with bafilomycin A1,then immunofluorescence staining using lyso-tracker was performed to detect cellular location of Smad2,Smad3 and lysosome.(7)HLF cells were transfected with vector or UCH-L5 plasmid followed by CHX treatment,the stabilities of Smad2 and Smad3 were examined by immunoblotting.(8)Overexpression of UCH-L5 with UCH-L5 plasmid transfection or knockdown with UCH-L5 sh RNA in HLF cells,ubiquitin immunoprecipitation was performed to detect the polyubiquitination of Smad2 and Smad3.(9)Co-immunoprecipitation was performed to detect whether UCH-L5 binds to Smad2 and Smad3.HLF cells were transfected with wild type Flag-Smad3 or Flag-Smad3T66A?Flag-Smad3T66 D mutation plasmid,co-immunoprecipitation was performed to detect the binding site.(10)Bleomycin(0.045 U)was administered to C57BL/6 mice by intranasal injection.To examine the therapeutic effect of b-AP15,on day 11 after bleomycin injury or saline controls,b-AP15(5 mg/kg)was given intraperitoneally every other day until the mice were sacrificed on day 21.Routine hematoxylin and eosin(H&E)and Masson's trichrome staining was performed.Partial right lungs were homogenized in cell lysis buffer.Protein levels were analyzed by Western blotting using antibodies to Smad2,Smad3,p38,UCH-L5,FN,collagen.Results(1)b-AP15 attenuates TGF?-1 signaling.(2)b-AP15 reduces Smad2/Smad3 levels through enhancing their poly-ubiquitination and lysosomal degradation.(3)UCH-L5 stabilizes Smad2/Smad3 and promotes TGF?-1 signaling.(4)UCH-L5 regulates Smad3 degradation through binding to Smad3.(5)UCH-L5 is a potential therapeutic target for drug treatment.ConclusionsIn conclusion,we demonstrate that UCH-L5 de-ubiquitinates and stabilizes Smad2 and Smad3,promotes TGF? signaling,and contributes to the pathogenesis of pulmonary fibrosis.b-AP15,an inhibitor of UCH-L5,attenuates TGF?-1 signaling and diminishes pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis model.This study indicates that targeting UCH-L5 may be a new potential therapeutic strategy to treat IPF.