Characterization of Sma- and mad-related protein 3 (Smad3) of Chu's croaker (Nibea coibor) and its role in collagen synthesis in the swim bladder.

The Sma- and Mad-related protein 3 (SMAD3) is a pivotal downstream mediator of the transforming growth factor-β (TGF-β) signaling pathway, playing a central role in collagen synthesis and fibrotic processes in mammals. However, its function is tissue context-specific and remains poorly elucidated in teleosts. In this study, we identified and functionally characterized smad3 from Nibea coibor (designated as Ncsmad3), an important marine economic fish valued for its collagen-rich swim bladder. The Ncsmad3 gene encodes a 425-amino acid protein with conserved Mad Homology 1 (MH1) and Mad Homology 2 (MH2) domains, showing the highest identity with other fish Smad3 orthologs. Tissue expression profiling revealed ubiquitous Ncsmad3 expression. The factors promoting collagen synthesis (Tgf-β1, leucine, proline, hydroxyproline) significantly upregulated the expression of Ncsmad3 in swim bladder cells. Furthermore, bioinformatics analysis revealed multiple Smad-binding elements in the collagen alpha 1 chain (col1a1) and collagen I alpha 2 chain (col1a2) promoters, and dual-luciferase assays confirmed that Ncsmad3 enhances their promoter activity. These results underscore the role of Ncsmad3 in regulating collagen synthesis in teleosts, providing a potential molecular target for improving collagen deposition in aquaculture species.