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Increased transcriptional activities of transforming growth factor beta receptors in scleroderma fibroblasts
Kenichi Yamane1, Hironobu Ihn, Masahide Kubo
1University of Tokyo, Tokyo, Japan.
Objective:
To investigate the molecular mechanism of the overexpression of transforming growth factor beta receptors (TGF(beta)Rs) in dermal fibroblasts from patients with systemic sclerosis (SSc).
Methods:
Dermal fibroblasts from 7 patients with diffuse SSc of recent onset and from 7 healthy individuals were studied. The expression of TGF(beta)R type I (TGF(beta)RI), TGF(beta)RII, and type I collagen proteins in dermal fibroblasts was determined by immunoblotting. TGF(beta)RI, TGF(beta)RII, and alpha2(I) collagen messenger RNA (mRNA) were evaluated by Northern blot analysis. The transcriptional activities of the TGF(beta)RI and TGF(beta)RII genes were examined by luciferase assay.
Results:
SSc fibroblasts expressed increased levels of TGF(beta)RI and TGF(beta)RII protein and mRNA, as well as increased levels of type I collagen protein and alpha2(I) collagen mRNA. Moreover, the half-lives of TGF(beta)RI and TGF(beta)RII mRNA in SSc fibroblasts did not change compared with those in control dermal fibroblasts. The promoter activities of the TGF(beta)RI and TGF(beta)RII genes were both significantly increased in SSc fibroblasts compared with those in control fibroblasts. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited TGF(beta)RI promoter activity in SSc fibroblasts, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), inhibited TGF(beta)RII promoter activity in SSc fibroblasts. Moreover, calphostin C and LY294002 inhibited the up-regulation of TGF(beta)RI and TGF(beta)RII mRNA, respectively, in SSc fibroblasts.
Conclusion:
These results suggest that increased levels of TGF(beta)Rs in SSc fibroblasts play a role in excessive collagen production, and that up-regulation of TGF(beta)R expression might occur at the transcriptional levels. PKC and/or PI 3-kinase might contribute to the up-regulation of TGF(beta)R expression in SSc fibroblasts.
Insights
Systemic sclerosis (SSc) fibroblasts show increased transforming growth factor beta receptors (TGF(beta)Rs), leading to excessive collagen production. This up-regulation occurs at the transcriptional level, potentially involving protein kinase C (PKC) and phosphoinositide 3-kinase (PI 3-kinase).
Area of Science:
- Dermatology
- Molecular Biology
- Rheumatology
Background:
- Systemic sclerosis (SSc) is characterized by excessive collagen deposition in the skin.
- Transforming growth factor beta receptors (TGF(beta)Rs) play a crucial role in regulating extracellular matrix production.
Purpose of the Study:
- To elucidate the molecular mechanisms behind the overexpression of TGF(beta)Rs in dermal fibroblasts from SSc patients.
- To investigate the role of TGF(beta)R up-regulation in the pathogenesis of SSc, particularly in collagen production.
Main Methods:
- Dermal fibroblasts were isolated from SSc patients and healthy controls.
- Expression levels of TGF(beta)R types I and II (TGF(beta)RI, TGF(beta)RII), and type I collagen were assessed at protein and mRNA levels using immunoblotting and Northern blot analysis.
- Transcriptional activity of TGF(beta)R genes was evaluated using luciferase assays, with specific inhibitors of protein kinase C (PKC) and phosphoinositide 3-kinase (PI 3-kinase) employed.
Main Results:
- SSc fibroblasts exhibited significantly higher levels of TGF(beta)RI and TGF(beta)RII protein and mRNA compared to controls.
- Increased expression of type I collagen protein and alpha2(I) collagen mRNA was observed in SSc fibroblasts.
- Promoter activities of TGF(beta)RI and TGF(beta)RII genes were elevated in SSc fibroblasts, indicating transcriptional up-regulation.
- Inhibition of PKC and PI 3-kinase pathways reduced TGF(beta)R promoter activity and mRNA levels in SSc fibroblasts.
Conclusions:
- Overexpression of TGF(beta)Rs in SSc fibroblasts contributes to excessive collagen production.
- The up-regulation of TGF(beta)R expression in SSc occurs at the transcriptional level.
- PKC and PI 3-kinase signaling pathways are implicated in the increased TGF(beta)R expression observed in SSc fibroblasts.