Mechanisms Of MAPK Pathway Mediating Signal Through TGF-β₁/Smads In Keloid Fibroblasts

Keloid is recognized as a fibrotic disease characterized by accumulation of extracellular matrix(ECM),especially collagen deposition,with unclear pathogenesis.Transforming growth factor beta 1(TGF-β₁) is known as the critical factor in keloid,which promotes fibroblasts proliferation and product collagen that leads to ECM accumulation and fibrosis.TGF-β₁ transmits signal mainly through Smad family,and also activates mitogen-activated protein kinases(MAPK) including extracellular signal-regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK) and p38 subfamilies.Although studies have indicated that TGF-β₁/Smad pathway plays an important role in the pathogenesis of keloid,but the specific phosphorylation sites of Smads have not been identified.Meanwhile.the mechanisms of activated MAPK pathway mediating TGF-β₁/Smad signal in keloid still need to be studied.In order to elucidate the mechanisms of TGF-β₁-induced Smad3 phosphorylation at C-terminal and linker region in keloid pathogenesis,and MAPK pathway regulating TGF-β₁-induced Smad3 activation,translocation and downstream target gene plasminogen activator inhibitor-1(PAI-1) transcription in KFs,we adopted molecular biological techniques such as immunoprecipitation and Western blot analysis, immunofluorescence study and real-time RT-PCR to perform the main researches as follows:1.Inhibitory effects of three MAPK inhibitors on TGF-β₁-induced MAPK pathway activation The results showed TGF-β₁ enhanced MAPK phosphorylation in KFs,and further promoted pERK,pJNK and pp38 nuclear accumulation.Meanwhile,ERK inhibitor,JNK inhibitor and p38 inhibitor significantly inhibited MAPK phosphorylation and nuclear accumulation respectively.2.Effects of three MAPK inhibitors on the phosphorylation of Smad3 at C-terminal and linker region induced by TGF-β₁TGF-β₁ obviously induced Smad3 phosphorylation at both C-terminal and linker region(C-terminally phosphorylated Smad3,pSmad3C;linker phosphorylated Smad3, pSmad3L),p38 inhibitor manifestly suppressed pSmad3C,and almost thoroughly blocked pSmad3L expression.Besides,ERK and JNK inhibitors showed weaker inhibitory effects on Smad3 phosphorylation than p38 inhibitor.3.Effects of three MAPK inhibitors on TGF-β₁-mediated Smad2/3/4 complex formation and nuclear translocationTGF-β₁ significantly induced Smad2/3/4 complex formation and nuclear translocation; p38 inhibitor almost entirely inhibited the complex formation,whereas ERK and JNK inhibitors presented few inhibitory effects.However,the complex's localization into nucleus was significantly blocked at the presence of three MAPK inhibitors.These results preferred that p38 inhibitor might be mainly through inhibiting Smad2/3/4 complex formation to block the complex's translocation;whereas ERK and JNK inhibitors might mainly block the complex's translocation into nucleus rather than its formation.4.Effects of three MAPK inhibitors on the expression of PAI-1mRNA induced by TGF-β₁Three MAPK inhibitors all obviously decreased TGF-β₁-induced PAI-1mRNA expression in KFs.The results suggest that TGF-β₁-induced PAI-1 transcription activity in KFs was depending on ERK,JNK and p38 pathway activation. In conclusion,TGF-β₁ obviously induced Smad3 phosphorylation at both C-terminal and linker region,thus promoting Smad2/3/4 complex formation and nuclear translocation and target gene PAI-1 expression,p38 pathway mainly regulated Smad3 phosphorylation,especially at linker region,and furthermore Smad2/3/4 complex formation, thus regulate PAI-1 mRNA expression.ERK and JNK pathways are mainly through modulating Smad2/3/4 complex's translocation into nucleus to regulate PAI-1 mRNA expression.These results identified the molecular mechanism of MAPK pathway mediating TGF-β₁/Smad signal in KFs,and imply specific targets of drug therapy for keloid.