Novel pituitary actions of TAC3 gene products in grass carp

Abstract

TAC3 is a member of tachykinins and its gene products neurokinin B (NKB) through activation of its receptor NK3R has recently emerged as a key regulator for LH release through modulation of kisspeptin/GnRH system within the hypothalamus. However, the current studies on NKB are mostly based on mammals with focus on GnRH regulation within the hypothalamus, little/no information is available regarding its functional role at the pituitary level. Using grass carp pituitary cells as a model, we sought to (i) investigate the pituitary actions of TAC3 gene products in fish model, and (ii) elucidate the mechanisms underlying the regulatory functions of NKB/NK3R system at the pituitary level. As a first step, two TAC3 isoforms, TAC3a and TAC3b, were cloned in grass carp and confirmed to be single copy genes in the carp genome. Interestingly, individual TAC3 isoforms were found to encode its own version of the mature peptide for NKB together with a novel tachykinin-like peptide called NKBrelated peptide (NKBRP). For nomenclature purpose, the gene products encoded by TAC3a were designated as NKBa and NKBRPa while those encoded by TAC3b were referred to as NKBb and NKBRPb, respectively. The gene products of carp TAC3a and TAC3b were synthesized and their biological actions were tested in carp pituitary cells. Although these TAC3 gene products did not have direct effects on LH secretion and gene expression at the pituitary cell level, NKBa, NKBRa and NKBRPb, and to a lesser extent for NKBb, were effective in inducing prolactin (PRL) and somatolactin α (SLα) synthesis and secretion via activation of NK2R and NK3R, respectively. The stimulatory actions of TAC3a and TAC3b gene products on SLα secretion and mRNA expression were mediated by AC/cAMP/PKA, PLC/IP3/PKC and 〖Ca〗^(2+)/CaM/CaMK-II pathways coupled to NK3R activation. The signaling mechanisms for PRL responses were also similar, except that the PKC comment was not involved. In our studies, NKBb, the gene product of TAC3b carrying a “FXGLL” motif with a M to L mutation in the signature motif “FXGLM” of tachykinin family, consistently displayed a partial agonistic effect for NK3R with lower bioactivity in terms of PRL and SLα stimulation. With co-treatment of NKBa, NKBRPa and NKBRPb respectively, NKBb could also serve as a competitive antagonist and suppress the stimulation by other TAC3 gene products on SLα gene expression. Subsequent mutagenesis also revealed that the M to L mutation in “FXGLM” motif could reduce the NK3R binding affinity of NKBa, NKBRPa and NKBRPb and inhibit their stimulation on the cAMP-, PKC- and 〖Ca〗^(2+) - depended cascades while the opposite was true for L to M mutation in the “FXGLL” motif of NKBb. These results, as a whole, shed light on the molecular determinant within the signature motif of fish tachykinins involved in NK3R binding and unravel the novel function of NKBb as a negative modulator for NK3R activation in the carp pituitary. In mammals, no information is available yet regarding NK3R regulation at the pituitary level. In carp pituitary cells, the gene products of TAC3a and TAC3b were found to potentiate the stimulatory effect of IGF-I/-II on SLα gene expression. These stimulatory effects could be markedly enhanced by SLα co-treatment. These synergistic actions were NK3R dependent and could be correlated with up-regulation of NK3R gene expression induced by IGF-I/-II and SLα treatment. Apparently, IGF and SLα could both enhance NK3R expression in carp pituitary cells via activation of MAPK, PI3K/Akt and/or JAK2/STAT5 pathways. Our findings provide new insights on the biological functions and regulation of TAC3/NK3R system in the pituitary of fish model.