Type II suppressor of cytokine signaling (SOCS) serve as feedback repressors for cytokines and are known to inhibit growth hormone (GH) actions. However, direct evidence for SOCS modulation of GH-induced insulin-like growth factor 1 (Igf1) expression is lacking, and the post-receptor signaling for SOCS expression at the hepatic level is still unclear. To shed light on the comparative aspects of SOCS in GH functions, grass carp was used as a model to study the role of type II SOCS in GH-induced Igf1 expression. Structural identity of type II SOCS, Socs1–3 and cytokine-inducible SH2-containing protein (Cish), was established in grass carp by 5’/3’-RACE, and their expression at both transcript and protein levels were confirmed in the liver by RT-PCR and LC/MS/MS respectively. In carp hepatocytes, GH treatment induced rapid phosphorylation of JAK2, STATs, MAPK, PI3K, and protein kinase B (Akt) with parallel rises in socs1–3 and cish mRNA levels, and these stimulatory effects on type II SOCS were shown to occur before the gradual loss of igf1 gene expression caused by prolonged exposure of GH. Furthermore, GH-induced type II SOCS gene expression could be negated by inhibiting JAK2, STATs, MEK1/2, P38 MAPK, PI3K, and/or Akt respectively. In CHO cells transfected with carp GH receptor, over-expression of these newly cloned type II SOCS not only suppressed JAK2/STAT5 signaling with GH treatment but also inhibited GH-induced grass carp Igf1 promoter activity. These results, taken together, suggest that type II SOCS could be induced by GH in the carp liver via JAK2/STATs, MAPK, and PI3K/Akt cascades and serve as feedback repressors for GH signaling and induction of igf1 gene expression.
Xue Jiang, Jia Xiao, Mulan He, Ani Ma and Anderson O L Wong
Chengyuan Lin, Xue Jiang, Mulan He, Ling Zhao, Tao Huang, Zhaoxiang Bian and Anderson O L Wong
In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone with diverse functions but its role in prolactin (PRL) regulation is highly controversial. To shed light on Prl regulation by PACAP in fish model, grass carp pituitary cells was used as a model to examine the receptor specificity and signal transduction for PACAP modulation of prl gene expression in the carp pituitary. Using RT-PCR, PACAP-selective PAC1 receptor was detected in carp lactotrophs. In carp pituitary cells, nanomolar doses of PACAP, but not VIP, could elevate Prl secretion and protein production with concurrent rise in prl mRNA and these stimulatory effects were blocked by PACAP antagonist but not VIP antagonist. PACAP-induced prl mRNA expression could be mimicked by activating adenylate cyclase (AC), increasing cAMP level by cAMP analog, or increasing intracellular Ca2+ ([Ca2+]i) by Ca2+ ionophore/voltage-sensitive Ca2+ channel (VSCC) activator. PACAP-induced prl gene expression, however, was attenuated/abolished by suppressing cAMP production, inhibiting PKA activity, blocking [Ca2+]i mobilization and VSCC activation, calmodulin (CaM) antagonism, and inactivation of JNK and CaM Kinase II (CaMK-II). Similar sensitivity to CaM, JNK, and CaMK-II blockade was also noted by substituting cAMP analog for PACAP as the stimulant for prl mRNA expression. These results, as a whole, provide evidence for the first time that (i) PACAP activation of PAC1 receptor expressed in carp lactotrophs could induce Prl synthesis and secretion, and (ii) Prl production induced by PACAP was mediated by upregulation of prl gene expression, presumably via functional coupling of cAMP/PKA-, Ca2+/CaM-, and MAPK-dependent cascades.