Cytokine Regulation of the Rat Proopiomelanocortin Gene Expression in AtT-20 Cells

Abstract

Although cytokines are known to be involved in the regulation of ACTH secretion, their effects, along with the molecular mechanisms, on POMC gene expression are not thoroughly characterized. In this study we examined the effects of representative cytokines on transcription of the POMC gene in corticotrophs in vitro using AtT20PL, a clone of the AtT20 cell line stably transfected with approximately 0.7 kilobase of the rat POMC 5'-promoter-luciferase fusion gene. In each experiment, cells were incubated with the cytokine tested, and the changes in POMC 5'-promoter activity were determined by a luciferase assay. The results showed that interleukin-1beta (IL-1beta) stimulated promoter activity in a biphasic manner [weak short term effects (2-3 h) followed by potent long term effects (>12-16 h)]. Tumor necrosis factor-alpha had similar effects, but much less potency. IL-6 showed a profound stimulatory, but only a long term (>20 h), effect. IL-2 did not influence POMC expression. In contrast, interferon-alpha (IFN alpha) and IFN-gamma showed acute stimulatory effects (approximately 4 h) followed by marked inhibitory effects (>8 h). Although the acute effects of IL-1beta, IL-6, and tumor necrosis factor-alpha alone were minimal, they significantly potentiated the stimulatory effect of CRH on POMC expression. Finally, pretreatment of the cells with a broad spectrum tyrosine kinase inhibitor, genistein, abolished or significantly diminished the effects of all cytokines except IFNs. Our results suggest that 1) each cytokine tested has a distinct effect on POMC gene expression; 2) there are positive cross-talk effects between CRH and cytokines at the corticotroph level; and 3) tyrosine phosphorylation cascades are involved in the intracellular signaling mechanisms of some cytokines.

In vivo Activation of the Interleukin-6 Receptor/gp130 Signaling Pathway in Pituitary Corticotropes of Lipopolysaccharide-Treated Rats

“…Hypothetically, the intrapituitary proinflammatory cytokines IL-6 or IL-1, which are rapidly produced after a peripheral LPS challenge by folliculostellate cells [28], could regulate the IL-6R component in corticotropes. Such a necessary upregulation of IL-6R complex in the pituitary is in accordance with the long-term effect of IL-6 on ACTH secretion measured in vitro [7, 15, 16, 17]. …”

“…We demonstrated that SOCS-3 mRNA is rapidly induced (within 2 h) in corticotropes during peripheral inflammation. It has been reported that in vivo, LPS-induced SOCS-3 mRNA in mice was concordant with a rapid ACTH release (1–4 h) [30], whereas the effect of IL-6 on ACTH release in vitro generally occurs only after several hours of incubation [7, 15, 16, 17]. The rapid effect of IL-6 on ACTH release could be linked to IL-6 control of exocytosis, but no data are currently available.…”

“…The rapid effect of IL-6 on ACTH release could be linked to IL-6 control of exocytosis, but no data are currently available. On the other hand, IL-6 is probably able to regulate the transcriptional activity of the POMC gene, since the POMC promoter contains STAT-3 nonconsensus boxes [34, 35]and IL-6 treatment of AtT20 cells induces SOCS- 3 [30]and POMC expression [17]. IL-6 could also control ACTH production through the regulation of prohormone convertase 1, which processes POMC into bioactive ACTH.…”

“…In vivo synergistic effects of CRH and IL-6 on the release of ACTH have been described in LPS-challenged rats [8]. Interestingly, the coactivation of POMC promoter by a CRH-dependent pathway and STAT3/SOCS-3 pathway by leukemia inhibitory factor in AtT20 cells potentiated POMC transcription and ACTH secretion [17, 34, 35]. More experiments are needed to demonstrate that IL-6 could also synergize with CRH to control the production/release of POMC/ACTH by corticotropes during an inflammatory event.…”

“…Collectively, these data suggest that IL-6 exerts its effects on ACTH release indirectly via a neural pathway involving CRH neurons. Nevertheless, in vitro administration of IL-6 triggers the production/release of ACTH in human pituitary cultures [15], rat pituitary explants [7]and the AtT20 corticotrope cell line [16, 17]. In vivo, IL-6 administration in CRH-deficient mice stimulates ACTH release, providing evidence that IL-6 can bypass hypothalamic CRH to influence HPA activity [18].…”