TGF-β1 induces alveolar epithelial to mesenchymal transition in vitro

Abstract

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) 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 α-smooth muscle actin (α-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-β1 were measured with ELISA. Incubation of AECs with TGF-β1 (0.1-10 ng/mL) induced abundant expression of α-SMA protein, and α-SMA expression in AECs reached a plateau when TGF-β1 was > 3 ng/mL. Furthermore, we found that TGF-β1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of α-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-β1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-β1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-β1 released from AECs was observed. At time points 72 h, TGF-β1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-β1, underwent a conversion process into myofibroblasts in vitro. © 2004 Elsevier Inc. All rights reserved.