Characterization of the ctgf Promoter from Nibea coibor and its Response to TGF-β1 During Collagen Synthesis in Swim Bladder Cells

Abstract

Connective tissue growth factor (ctgf) is a multifunctional protein that plays a crucial role in promoting collagen (the most abundant protein in organism) synthesis in vertebrates. However, its transcriptional regulation mechanism is poorly understood, especially in teleosts. In this study, we characterized the ctgf promoter (3,185 bp) from a marine teleost, the chu’s croaker (Nibea coibor), and elucidated its response to TGF-β1 during collagen synthesis in swim bladder cells. Bioinformatics analysis revealed conserved cis-elements (TATA box, SP-1, Smad-3, NF-κB) within the promoter. Truncation assays identified a core functional region (-785 ~ + 166 bp). Notably, site-directed mutagenesis showed that mutation of SP-1 binding site enhanced the promoter activity, while mutations in binding sites of Smad-3 (-290 bp) and NF-κB (-262 bp) suppressed its activity. To demonstrate its correlation with collagen synthesis, swim bladder cells stably transfected with ctgf promoter drived EGFP vector (pctgf-EGFP) was constructed. It was further found that the promoter activity was up-regulated in concert with collagen synthesis promoted by TGF-β1 in swim bladder cells. These findings could advance our understanding of the transcriptional regulation of ctgf, and establish a technical platform for further screening natural compounds promoting collagen synthesis in fish.