Multiple forms of hepatic nuclear factors for IGF-I and LHβ gene regulation in grass carp

Abstract

Hepatic nuclear factors (HNF) are liver-enriched transcription factors and play an essential role in hepatic development and metabolic functions in mammals. However, little is known regarding their functions/regulation in non-mammalian species, especially in lower vertebrates. In our recent study, the 5’ promoters of IGF-I and LHβ genes were isolated in grass carp. A cluster of cis-elements with binding sites for HNF1α, STAT5 and CREB close to a well-conserved HNF4 binding site was located in the carp IGF-I promoter whereas a total of 21 HNF3 binding sites could be mapped in the carp LHβ promoter, implying that different HNF members may be involved in IGF-I and LHβ gene transcription in carp species. In grass carp, hepatic expression of IGF-I and pituitary expression LHβ are known to be under the influence of growth hormone (GH) but HNF involvement in GH regulation of these two target genes is still unclear. In this study, different members of HNF, namely HNF1α, HNF3α, HNF3β, HNF3γ, HNF4α and HNF4β, were cloned in grass carp and confirmed to have the structural characteristics of individual subtypes and functionality in transactivating target promoters with the respective binding sites. Using RT-PCR, the newly cloned HNFs were shown to be widely expressed at tissue level including the liver and pituitary. In carp hepatocytes, glucagon treatment could elevate IGF-I mRNA level with parallel rises in HNF1α and CREB transcripts and these effects were caused by cAMP/PKA and PLC/IP3/PKC pathways functionally coupled with MAPK and PI3K/Akt cascades. IGF-I mRNA expression could also be induced by GH treatment with parallel rises in HNF1α, STAT5, CREB, HNF4α and HNF4β transcripts and these effects were mediated by the JAK2/STAT5, MAPK and PI3K/Akt cascades. With glucagon or GH treatment, HNF1α/CREB phosphorylation, protein expression of HNF1α, CREB and/or STAT5, and protein:protein interaction among these transcription factor/their association with IGF-I promoter were also noted. In transfection studies with cell lines, IGF-I promoter activity of carp origin was shown to be up-regulated by glucagon via the HNF1α and CREB binding sites and by GH via the HNF1α, STAT5 and CREB binding sites within the IGF-I promoter. The stimulatory effect of GH on IGF-I promoter activation could be blocked by HNF4α/4β expression and the effect was dependent of the HNF4 binding site within the IGF-I promoter. In carp pituitary cells, GH induction was effective in elevating LHβ mRNA and primary transcript expression with parallel rises in HNF3α, 3β and 3γ transcripts and these effects were mediated by JAK2/STAT5 pathway with differential coupling of STAT1, STAT3, MEK/ERK, JNK, p38MAPK and PI3K/Akt signaling. In parallel transfection studies, LHβ promoter activation could be induced by GH and expression of the three isoforms of HNF3 not only could reduce basal but also abolish GH-induced LHβ promoter activity. Our findings, as a whole, provide evidence that different members of HNF can serve as the regulatory targets of GH and contribute to regulation of IGF-I and LHβ gene expression in carp species.