The trans‐differentiation of alveolar epithelial type II cells to type I cells involves autocrine signaling through Smad‐mediated TGF beta1 pathway

The Type II Alveolar Epithelial Cells (AEC II) trans‐differentiate into Type I‐like AEC when being cultured in vitro on a plastic dish for several days. We hypothesized that the signaling by Transforming Growth Factor β1 (TGF β1) through its down stream Smads has a role in this process. TGF β1 and co‐Smad 4 were highly expressed in AEC II, but not in AEC I in the lung. Using BrdU and cell‐specific marker labeling, we found that there was a proliferation phase (day 1 to day 3), followed by a trans‐differentiation phase (day 3 to day 5) in our culture system. The mRNA and protein levels of TGF β1 were lower during the proliferative phase and higher during the differentiation phase. TGF β1 secreted into the media showed a gradual increase as the trans‐differentiation progressed. The mRNA levels of down stream R‐Smads 2 and 3 and their protein phosphorylation increased during the differentiation phase. The expression of cell cycle inhibitors, p15 Ink4b and p21 Cip1 showed the same trend as TGF β1 while the cyclin‐dependent kinases 2, 4 and 6 showed an opposite trend of expression. Further, when co‐Smad 4 was silenced by RNAi, AEC II cultured on plastic plates retained their AEC II characteristics. These results suggest that the initial proliferation and later differentiation of AEC II to AEC I‐like cells is modulated by altering the expression of cell cycle proteins that control the G1 to S phase entry via Smad‐dependent TGF β1 pathway (supported by NIH HL‐052146 and HL‐071628 to LL).