Regulation of transforming growth factor-beta1 (TGF-β1)-induced pro-fibrotic activities by circadian clock gene BMAL1

Chunmin Dong, Rafael Gongora, Meredith L. Sosulski, Fayong Luo, Cecilia G. Sanchez

BMAL1 is a transcriptional activator of the molecular clock feedback network. Besides its role in generating circadian rhythms, it has also been shown to be involved in the modulation of cell proliferation, autophagy and cancer cell invasion. However, the role of BMAL1 in pulmonary fibrogenesis is still largely unknown. In this study, we investigated the crosstalk between BMAL1 and the signaling transduction and cellular activities of TGF-β1, a key player in lung fibrogenesis. Lungs from wild type and TGF-β1-adenovirus-infected mice were harvested and homogenized for isolation of RNA and protein. RT-PCR and Western Blotting were employed to measure the expression level of clock genes and TGF-β1-induced downstream target genes. siRNA against human BMAL1 gene was transfected by using lipofectamine RNAiMAX to knockdown the endogenous BMAL1 in both lung epithelial cells and fibroblasts. Our results showed that TGF-β1 is able to up-regulate BMAL1 expression in both lung epithelial cells and normal lung fibroblasts. In animal models of pulmonary fibrosis, BMAL1 expression was also significantly higher in adenovirus-TGF-β1-infected mice than in the control group. Interestingly, BMAL1 was mostly found in a deacetylated form in the presence of TGF-β1. Importantly, siRNA-mediated knockdown of BMAL1 significantly attenuated the canonical TGF-β1 signaling pathway and altered TGF-β1-induced epithelial-mesenchymal transition and MMP9 production in lung epithelial cells. In addition, BMAL1 knockdown inhibited the fibroblast to myofibroblast differentiation of normal human lung fibroblasts. Our results indicate that activation of TGF-β1 promotes the transcriptional induction of BMAL1. Furthermore, BMAL1 is required for the TGF-β1-induced signaling transduction and pro-fibrotic activities in the lung.