Novel functions of PIAS1 and PIAS4 as intracellular feedback repressors of growth hormone signaling in grass carp

Abstract

Growth Hormone (GH), a pituitary hormone essential for body growth and metabolism, is known to exert its biological actions predominantly by activating the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling pathway coupled to growth hormone receptor (GHR). Protein inhibitor of activated STAT (PIAS) is a class of intracellular regulator that inhibits the transcriptional activity of activated STATs, which constitutes an important component for signal termination for cytokine receptors. Previous studies on PIAS have been focused on mammalian models, and very little is known regarding PIAS function and regulation in lower vertebrates, especially in fish and amphibians. To establish the functional link between PIAS and GH signaling in fish model, grass carp was used as a model for modern-day teleosts to examine the role of PIAS as a feedback repressor of GH signaling. Using RT-PCR, the grass carp PIAS1 and PIAS4 cDNAs have been cloned, and results of sequence analysis and protein modeling revealed that the two forms of grass carp PIAS were highly comparable to their respective homologs reported in other vertebrate classes. In grass carp, PIAS1 and PIAS4 have been confirmed to be single-copy genes, and their transcripts were found to be widely expressed at the tissue level including the liver, which is the major target for GH actions. In grass carp hepatocyte cultures, GH treatment increased PIAS1 and PIAS4 mRNA expression in a dose-dependent manner and these stimulatory effects could be negated or attenuated by pharmacological inhibitors for JAK2, STAT5, MEK1/2, p38 MAPK, PI3K and Akt. In CHO cells with stable expression of grass carp GHR, GH treatment promoted the association between PIAS1 and STAT5. Meanwhile, PIAS1 and PIAS4 overexpression dose-dependently inhibited both basal and GH-activated STAT1, STAT3, and STAT5 reporter activity as well as IGF-I promoter activities. Furthermore, PIAS1 and PIAS4 repressed the STAT reporter activities were induced by overexpression of the respective STATs. Interestingly, PIAS1 downregulation of STAT5 activities were partially reverted by blocking the small ubiquitin-like modifier (SUMO) machinery and histone deacetylase (HDAC) activity. Altogether, these results are the first to reveal that (i) GH stimulated PIAS gene expression at the hepatic level by activating the JAK/STAT, MAPK and PI3K/Akt pathways and (ii) PIAS expression could inhibit GH-induced IGF-I gene transcription mediated by JAK/STAT signaling. These findings suggest that PIAS1 and PIAS4 may serve as novel intracellular feedback repressors of GH signaling in the carp liver.