Isolation of the canine inhibin beta B subunit gene and characterization of signalling mediated by canine inhibin beta B

Activin B, a homodimer of the inhibin beta B subunit, acts as a regulator of gonadal function and as an adipokine. To clarify the role of activin B in dogs, we characterized the canine inhibin beta B gene and signalling pathways regulated by the canine inhibin beta B. Using 5 '- and 3 '-rapid amplification of cDNA end (RACE) and RT-PCR on RNA isolated from the ovary of dogs, we identified short and long forms of the inhibin beta B gene. Immunoreactive inhibin beta B molecules were detected at similar to 25 and similar to 14 kDa under nonreducing and reducing conditions, respectively, in culture supernatants from HEK293 cells transfected with a plasmid containing the long form of the inhibin beta B gene, indicating activin B production and secretion. Similar to human and murine activin B, the canine activin B-stimulated transcriptions of reporter genes, CAGA-luc and Hepcidin-luc, regulated by the canonical activin/transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) pathway, respectively. Activin B-induced CAGA-luc transcription was not detected in ALK7-deficient MDCK canine-derived cells; however, the forced expression of ALK7 resulted in the activin B-dependent expression in MDCK cells. Unexpectedly, the activin B-induced activation of the BMP pathway was partially blocked by the inhibition of endogenous activin/TGF-beta receptor activity. The present study identified an experimentally isolated long form of the canine inhibin beta B gene producing activin B that transactivates BMP- and activin/TGF-beta-regulated gene expression.