P27 Promotes TGF- β-Mediated Pulmonary Fibrosis via Interacting with MTORC2

Abstract

Pulmonary fibrosis (PF), a progressive and life-threatening pulmonary disease, is the main pathological basis of interstitial lung disease (ILD) which includes the idiopathic pulmonary fibrosis (IPF). No effective therapeutic strategy for pulmonary fibrosis has been established. TGF-β signaling has emerged as the vital regulator of PF; however, the detailed molecular mechanisms of TGF-β in PF were uncertain. In the present study, we proved that inhibition of MTORC2 suppresses the expression of P27 in MRC5 and HLF cells. And in bleomycin-induced PF model, the expression of α-SMA and P27 was upregulated. Moreover, TGF-β application increased the level of α-SMA, vimentin, and P27 in MRC5 and HLF cells. Furthermore, P27 overexpression advanced the cell cycle process and promoted the proliferation of MRC5 and HLF cells. Finally, the rescue experiment showed that MTORC2 knockdown reversed P27 overexpression-induced cell cycle acceleration and proliferation. Thus, our results suggest that P27 is involved in TGF-β-mediated PF, which was regulated by MTORC2, providing a novel insight into the development of PF.

Figure 1

Expression of a-SAM, fibronectin, and P27 in bleomycin mouse lung fibrosis model. (a) H&E staining of lung fibrotic tissue in the NC and the bleomycin group. (b-c) Expression of a-SAM and fibronectin in the NC and the bleomycin group by IHC. (d) The relative expression of a-SAM, fibronectin, and P27 in the NC and the bleomycin group. The results of a-SAM and fibronectin expression were from the IHC staining density, and the results of P27 expression were from RT-PCR.

Figure 2

The role of P27 on the cell cycle of proliferation in MRC5 and HLF cells. (a, b) The role of P27 on the cell cycle by flow cytometry. (c, d) The role of P27 on the cell proliferation by CCK-8 assay.

Figure 3

MTORC2 inhibition decreased the level of P27. (a, b) Western blot analysis of expression of mTORC-p, RICTOR, and P27 in MRC5 and HLF cells transfected with RICTOR siRNA.

Figure 4

TGF-β promoted the expression of P27. (a, b) Western blot analysis of expression of a-SAM, vimentin, and P27 in MRC5 and HLF cells treated with TGF-β.

Figure 5

Rictor knockdown reversed the role of P27 on cell cycle and proliferation. (a, b) The cell cycle assay in MRC5 and HLF cells transfected with P27 siRNA plus Rictor siRNA or P27 ovexpression vector plus Rictor siRNA. (c, d) The cell proliferation assay in MRC5 and HLF cells transfected with P27 siRNA plus Rictor siRNA or P27 ovexpression vector plus Rictor siRNA.