The Signal Effects Of SMAD3 And SMAD7 On The TGF-β/SMADs Signal Transduction Of Fibroblasts Derived From Keloid

It is generally agreed that keloid is characterized by excessive deposition of extracellular matrix and over-proliferated fibroblast. TGF- 3 plays an crucial role in keliod development.TGF- 3 initiates signaling through their interaction wiJi TGF- 3 receptor complexes. The SMAD complex, which has been activated by TGF-3 receptors, translocates from the cytoplasm into the nucleus and stimulates gene transcription. But its mechanism ,such as how TGF- 3 regulates the expression of SMADs ,what kinds of cellular gene expression may be activated and how this activation occurs, is still incompletely characterized, especially in the fields of keloid researches.The expression and regulation of TGF- 3 I / II receptor and SMAD3 and SMAD7 of keloid-derived fibroblasts(KFB) has been studied. Theeffects of the Smad3 and Smad4 antisense oligonucleotides, which were used to inhibit endogenous Smad expression, on the proliferation and synthesis of collagen of KFB also have been observed.Firstly, the expression of type I and II TGF- P receptor of fibroblasts in vitro has been observed using immunohistochemistry and imaging analysis techniques. The results show that the expression of type I and II TGF- P receptor of KFB is higher than that of NFB(p<0.05).The same results can be obtained between treated and untreated KFB with TGF- P i.Secondly, the expression and regulation of SMAD3 , SMAD7 and their rnRNA of KFB have been studied by applying RT-PCR and Western blot and cellular immunofluorescence. The results show that the expression of SMAD3 and its mRNA between KFB and NFB has no difference, But SMAD3-associated fluorescence in nuclear of KFB shows a high degree compared with that of NFB. The Smad3 mRNA expression was reduced in a dose- and time-dependent manner and the levels of SMAD3 were reduced by 70% in KFB exposed to TGF- P ,.The induction of Smad? mRNA were markedly up-regulated by 3 fold of control exposed to 5 pmol/L TGF- P i for 90 min, and By 24 h, it returned to control levels. The expression of SMAD7 were increased maximal value upon incubation with TGF- PI for 2 h. Inhibition of de novo protein synthesis with CHX caused down-regulated Smad3 mRNA levels by TGF- P i In contrast, TGF- P i induction of SmadTmRNA was unaffected by pretreatment with CFDC These results suggest that SmadV gene is a direct target of receptor-activated SMAD signals. Thirdly, the localization of SMAD3 and SMAD7 has been detected. The results indicate that TGF- P i induces a translocation of SMAD3 from thecytoplasm into the nucleus in a dose- and time- dependent manner and a accumulation of SMAD7-specific immunofluorescence in the cytoplasm of KFB.Fourthly, the opposite effects of IFN- y on TGF- P /SMAD signal transduction pathway of KFB also has been investigated. We found that IFN-Y could increase Smad? mRNA levels at least 8 fold over the basal level . This increase was maximal 30 min after IFN- Y addition and still present after 8h. It was unaffected by the protein-synthesis inhibitor CHX. IFN- Y also inhibited the translocation of SMAD3 caused by TGF- 3 i.It indicates IFN-Y may cause the inhibition of TGF- P /SMAD signaling by increasing the expression of Smad7 mRNA directly.Finally, the SmadS mRNA antisense oligonucleotide were added directly to the monolayer cultures of KFB, both of the MTT reaction A values and 3H-proline incorporation were decreased. The similar effects were found in the Smad4 mRNA antisense oligonucleotide. These results suggest the disruption of TGF- P /SMAD signaling by the SmadS or Sad4 mRNA antisense oligonucleotide can inhibit the proliferation and the synthesis of collagen of KFB. Conclusion:1. The levels of type I> II TGF- P receptors expression is higher in KFB.2. The activated-SMAD3 may be in a higher level in KFB than in NFB.3. The similar mechanism of TGF- P /SMDA is present in KFB , NFB and others.4. The inhibition of TGF- P /SMAD signaling can be caused by IFN- Y , which can increase the expression of Smad? mRNA directly.5. The disruption of T...