TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro.
The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.
Yao
Hong-Wei
HW
Zhejiang Respiratory Drugs Research Laboratory of State Food Drugs Administration of China, School of Medicine, Zhejiang University, Hangzhou 310031, China. yhgwei@hotmail.com
Xie
Qiang-Min
QM
Chen
Ji-Qiang
JQ
Deng
Yang-Mei
YM
Tang
Hui-Fang
HF
D000199:Actins / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000502:physiology; D015151:Immunoblotting; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D008648:Mesoderm / Q000502:physiology; D009130:Muscle, Smooth / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000503:physiopathology*; D011658:Pulmonary Fibrosis / Q000503:physiopathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000502:physiology
D000199:Actins; D015820:Cadherins; D024042:Collagen Type I; D016212:Transforming Growth Factor beta