The functional differentiation of four smad4 paralogs in TGF-β signaling pathway of Japanese flounder (Paralichthys olivaceus)

As a classical signaling pathway, transforming growth factor β (TGF-β) has been studied in various animals for more than decade years. However, the members of TGF-β were markedly expanded in teleost specific third and fourth rounds of whole genome duplication (WGD). Here, four smad4s named Posmad4a, Posmad4b, Posmad4c and Posmad4d were identified in Japanese flounder. Our study showed that four flounder smad4s had distinct properties in terms of their protein structure, expression pattern, protein interaction and subcellular localization. PoSMAD4a/b were mainly located in the cytoplasm, and could co-localize in the nucleus with PoSMAD3a after TGF-β activator stimulation. PoSMAD4c was mainly located in nucleus, whereas PoSMAD4d distributed in the whole cell. Both PoSMAD4c and PoSMAD4d could co-localize in the nucleus with PoSMAD3b after TGF-β activator stimulation. Furthermore, Posmad4c responded most strongly to TGF-β signal stimulation. Dual-luciferase reporter assay also showed that Posmad4c could specifically up-regulate the TGF-β signal luciferase reporter gene, Posmad4b could enhance Wnt signal luciferase reporter gene, while both Posmad4b and Posmad4d could markedly up-regulate Notch signal reporter gene. All results indicated that Posmad4a/b/c/d had significantly functional differences among TGF-β, Notch and Wnt signaling pathways. Our study provided important understanding to the biology of smad4s and its pathway crosstalk in teleost.