[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).


CONCLUSIONS
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.
Liu
Hong-xu
HX
Department of Thoracic Surgery, First Hospital, China Medical University, Shenyang 110001, China.
Li
Yu
Y
Zhao
Cheng-hai
CH
Liu
Yang
Y
Zhang
Qi-gang
QG
Cong
Wei
W
Lan
Xin-gang
XG
Xu
Shun
S
Han
Li-bo
LB
Zhang
Lin
L
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001989:Bronchiolitis Obliterans / Q000209:etiology / Q000378:metabolism / Q000503:physiopathology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D016040:Lung Transplantation / Q000009:adverse effects / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014132:Trachea / Q000166:cytology / Q000637:transplantation; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology / Q000502:physiology*; D016042:Transplantation, Heterotopic
D000199:Actins; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1