Transforming growth factor-β-induced CUX1 isoforms are associated with fibrosis in systemic sclerosis lung fibroblasts

In the enhancer region of the human type I collagen alpha 2 (COL1A2) gene, we identified cis-elements for the transcription factor CUX1. However, the role of CUX1 in fibrosis remains unclear. Here we investigated the role of CUX1 in the regulation of COL1 expression and delineated the mechanisms underlying the regulation of COL1A2 expression by CUX1 in systemic sclerosis (SSc) lung fibroblasts. The binding of CUX1 to the COL1A2 enhancer region was assessed using electrophoretic mobility shift assays after treatment with transforming growth factor (TGF)-β. Subsequently, the protein expression levels of CUX1 isoforms were determined using Western blotting. Finally, the expression levels of COL1 and fibrosis-related cytokines, including CTGF, ET-1, Wnt1 and β-catenin were determined. The binding of CUX1 isoforms to the COL1A2 enhancer region increased after TGF-β treatment. TGF-β also increased the protein levels of the CUX1 isoforms p200, p150, p110, p75, p30 and p28. Moreover, SSc lung fibroblasts showed higher levels of CUX1 isoforms than normal lung fibroblasts, and treatment of SSc lung fibroblasts with a cathepsin L inhibitor (IW-CHO) decreased COL1 protein expression and reduced cell size, as measured using immunocytochemistry. In SSc and diffuse alveolar damage lung tissue sections, CUX1 localised within α-smooth muscle actin-positive cells. Our results suggested that CUX1 isoforms play vital roles in connective tissue deposition during wound repair and fibrosis.