[The mechanism of transforming growth factor beta1 in myofibroblast differentiation].

OBJECTIVE: To investigate the mechanism underlying myofibroblast differentiation induced by transforming growth factor (TGF) beta1 in obliterative bronchiolitis following lung transplantation. METHODS: Heterotopic tracheal transplantation was performed in Smad3 wild-type and knock-out mice to simulate the lung transplantation in human. Murine tracheal fibroblasts cultivated in primary culture were used for in vitro study. Immunohistochemistry, immunocytochemistry, Western Blotting, RT-PCR and DNA electrophoresis mobility gel shift assay were conducted to detect the expression of alpha-smooth muscle actin (alphaSMA), the marker of fibroblast-myofibroblast differentiation, and the activation of Smad3, p38 and ERK1/2. RESULTS: In affected airways of experimental obliterative bronchiolitis, abundant expression of alphaSMA were found. In vitro study for tracheal fibroblasts, the activation of Smad3 by TGF-beta1 presents as three major forms, phosphorylation, nuclear translocation and DNA binding. In Smad3 wild-type fibroblasts, TGF-beta1 induces the increase of the myofibroblasts transformation, characterized by the elevation of alphaSMA, both at transcription and protein level. While in Smad3 knock-out fibroblasts, the transformation of myofibroblasts induced by TGF-beta1 is significantly decreased (t = 2.080, P = 0.027; t = 1.982, P = 0.032), but not completely abolished. Further study in Smad3-deficient fibroblasts demonstrates that p38 and ERK1/2 could be activated by TGF-beta1 and result in fibroblast differentiation. CONCLUSIONS: TGF-beta1 could promote the transformation of fibroblasts into myofibroblasts in Smad3 dependent and independent signal pathways, especially the Smad3 dependent path, and result in the development of obliterative bronchiolitis.

[The role of transforming growth factor-beta1/Smad3 signaling in bronchiolitis obliterans following lung transplantation].

OBJECTIVE: To investigate the mechanism underlying bronchiolitis obliterans (OB) following lung transplantation and the significance of transforming growth factor (TGF)-beta1/Smad3 signal pathway in this pathological process. METHODS: The tracheas of BALB/c mice were transplanted into the subcutaneous tissues of a Smad3ex8/ex8 gene knock-out Swiss black mouse and a Smad3 wild-type Swiss black mouse. Forty-two days later the tracheas were taken out. Immunocytochemistry was used to detect the alpha-smooth muscle actin (alphaSMA), a marker of fibroblast-myofibroblast differentiation. The tracheas of Smad3 knock-out and wild type mice were taken out, broken to pieces, and cultured to obtain the fibroblasts. The tracheal fibroblasts in primary culture were treated with TGF-beta1. The activation of Smad3 molecules was investigated with immunocytochemistry, Western blotting and DNA electrophoresis mobility gel shift assay (EMSA). Immunocytochemistry staining was also employed to detect the cytoskeletal polymerization and alphaSMA immunofluorescence after incubation with TGF-beta1; Western blotting and RT-PCR was conducted to detect the difference of alphaSMA at transcriptional and protein level. RESULTS: The number of alphaSMA positive myofibroblasts was great in the experimental OB models produced be transplantation of heterogeneous trachea from Smad3 wild type mice and was very small in the OB model produced be transplantation of heterogeneous trachea from Smad3 knock-out mice (t = 2.125, P = 0.040). Western blotting showed that in vitro experiment showed that phosphorylation of Smad3 protein was increased in the fibroblasts treated with TGF-beta1 and was almost absent in those not treated with TGF-beta1. EMSA showed that DNA binding was increased in the fibroblasts treated with TGF-beta1 and was almost absent in those not treated with TGF-beta1. Immunofluorescence staining showed that the cytoplasm of the fibroblasts not treated with TGF-beta1 was Smad3 positive, however, the nuclei of the fibroblasts treated with TGF-beta1 was Smad3 positive. RT-PCR showed that the alphaSMA mRNA expression level in the Smad3 wild-type fibroblasts was increased after treated with TGF-beta1, and was significantly higher than in the Smad3 knock-out fibroblasts treated with TGF-beta1 (t = 2.080, P = 0.027). Western blotting showed that the alphaSMA protein expression level in the Smad3 wild-type fibroblasts was increased after treatment with TGF-beta1, and was significantly higher than that of the Smad3 knock-out fibroblasts (t = 1.982, P = 0.032). CONCLUSION: TGFbeta1 promotes the production of alphaSMA protein and transformation of fibroblasts into myofibroblasts through the Smad3 dependent signal pathway, thus resulting in the development of bronchiolitis obliterans.