Growth Hormone-Induced Luteinizing Hormone Gene Expression in Grass Carp: -  Signal Transduction and Novel Feedback Via HNF3 Expression at Pituitary Level

Presentation Number: SAT 435
Date of Presentation: April 1st, 2017

Jin Bai*, Mang Leng Lei, Mu-Lan He and Anderson O L Wong
University of Hong Kong, Hong Kong, China


In grass carp, an intrapituitary feedback loop for growth hormone (GH) regulation by local release of luteinizing hormone (LH) has been reported. Interestingly, GH secreted locally can also induce LHβ mRNA expression in carp pituitary cells but the mechanisms involved are still unknown. Recently, the full gene of grass carp LHβ has been cloned and multiple binding sites for hepatocyte nuclear factor-3 (HNF3) are located in the proximal region of LHβ promoter, which raises the possibility that HNF3 may act as a regulatory target for LHβ gene expression. To examine the functional role of HNF3 in LHβ gene expression, three isoforms of carp HNF3, namely HNF3α, HNF3β and HNF3γ, have been cloned and found to be widely expressed at tissue level by RT-PCR. Using LC/MS/MS, protein expression of these HNF3 isoforms was also confirmed in the carp pituitary. In carp pituitary cells, GH treatment was shown to elevate LHβ mRNA and LHβ primary transcript levels with parallel rises in transcript expression of HNF3α, HNF3β and HNF3γ, while the opposite was true by immunoneutralization of endogenous GH with GH antiserum. In these studies, a gradual loss of LHβ mRNA responsiveness was noted with prolonged GH treatment/high dose of GH, indicative of intrinsic mechanisms for signal termination on GH effect. In parallel experiments, GH was also effective in triggering rapid phosphorylation of JAK2, STAT1, STAT3, STAT5, MEK1/2, ERK1/2, PI3K and Akt in carp pituitary cells and GH-induced LHβ mRNA and primary transcript expression could be reduced/negated by the inhibitors targeting JAK2/STAT1,3,5, MEK1/2/ERK1/2 and PI3K/Akt pathways. Except for a lack of inhibition by STAT3 inhibitor for HNF3β mRNA, GH-induced HNF3α & 3β transcript expression were also sensitive to pharmacological blockade of the JAK2/STATs, MEK1/2/ERK1/2 and PI3K/Akt cascades, whereas the corresponding responses for HNF3γ mRNA could be abrogated only by the inhibitors for JAK2 and STAT1 but not for other pathways. In αT3 cells with carp GH receptor expression, LHβ promoter activity conferred by a Luc reporter with a 1417 bp carp LHβ promoter could be up-regulated by GH treatment and this stimulatory effect could be inhibited by over-expression of the dominant negative mutants of Ras, Raf and MEK or co-treatment with inhibitors for JAK2/STAT1,3,5, MEK1/2/ERK1/2 and PI3K/Akt pathways. In the same model, over-expression of carp HNF3α, 3β and 3γ were also found to suppress both basal and GH-induced LHβ promoter activity. These results, as a whole, suggest that (i) GH can induce LHβ gene transcription in carp pituitary by paracrine activation of LHβ promoter via the JAK2/STATs, MEK1/2/ERK1/2 and PI3K/Akt pathways, and (ii) parallel expression of HNF3α, 3β and 3γ induced by GH via differential coupling to JAK2/STATs, MEK1/2/ERK1/2 and PI3K/Akt cascades may serve as novel feedback signals to down-regulate GH-induced LHβ gene expression at the pituitary level.


Nothing to Disclose: JB, MLL, MLH, AOLW