Recombinant Expression And Functional Characterization Of Grass Carp Squint And Activin A/B

Nodal and activin are the members of the transforming growth factor-(TGF-)superfamily and share a common signal transduction pathway. They play importantroles in the embryogenesis, liver proliferation, bone marrow formation, reproduction,endocrine and immunity of vertebrates.Nodal functions in fish embryogenesis as that in other vertebrates. However, thefunctional role of fish Nodal-related genes in non-embryonic cells is still unclear. Inteleost, three Nodal-related genes, Nodal-related (ndr)1/squint, ndr2/cyclops and ndr3/southpaw have been revealed. In this study, a full-length cDNA for grass carp squintwas cloned and its transcript was detected in all tissues examined, including pituitary,brain, heart, head kidney, kidney, spleen and gonad. To define the functional role ofsquint, recombinant grass carp mature squint (rgcmSQT) was produced in E. coli andpurified by affinity chromatography, and its homodimer form was prepared. Moreover,using the grass carp pituitary cell as the model, the biological activity of rgcmSQT wasinvestigated, showing that it stimulated the mRNA expression of activin A and Bsubunit, as well as activin receptor ActRIB and ActRIIB. These findings not onlycontribute to the understanding of non-embryonic functions of Nodal gene in fish, butprovide new insight into the regulation of activin signaling in vertebrates.Activin is known for its pituitary functions. Recently, much attention has been paidto its role in inflammatory processes. In teleosts, however, it is unknown whetheractivins are involved in immune response. Like its mammalian counterparts, fish activinA and B are homodimers of two activin A and B subunits, respectively. In this study,grass carp activin A subunit mRNA expression shown to be regulated in fish infectedwith Aeromonas hydrophilia and in periphery blood lymphocytes (PBLs) afterlipopolysaccharide (LPS) treatment. Subsquently, recombinant grass carp activin A andB were prepared for elucidating the role of activin in inflammatory response. Asexpected, the recombinant grass carp Activin A and B (rgcACVA/B) significantlyphosphorylated SMAD2in grass carp head kidney leukocytes (HKLs), though theseeffects were less than that of commercial rhActivin A/B. As it failed to obtain the recombinant grass carp activin in Pichia pastoris, rgcACVA was used for examiningfunctional role of activin in fish. In grass carp HKLs, treatment with rgcACVA for6hinhibited the mRNA expression of inducible nitric oxide synthase (inos) andinterleukin-1(il-1) but had no effect on interleukin-6(il-6) mRNA expression, andattenuated LPS-induced increase of these three gene mRNA levels. In grass carp PBLs,rgcACVA also blocked the up-regulation of inos and il-1mRNA expression inducedby LPS. These results suggest that activin A may be involved in inflammatory responsevia regulating immune-related gene expression in grass carp.