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.

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.

Aim: This study aims at clarifying the human leukocyte antigen haplotypes and genotypes conferring susceptibility or resistance to type 1 diabetes in the Japanese population. Methods: The frequencies of human leukocyte antigen DR-DQ haplotypes and genotypes were compared between 83 type 1 diabetic patients, except for fulminant type 1 diabetes, and control subjects in the Japanese population. The patients were divided by onset age into four groups (ages 5–14, 15–29, 30–49, and 50–71 years); the haplotype frequency was compared between each group. Results: The frequencies of DRB1*0405-DQB1*0401 (DR4), DRB1*0802-DQB1*0302 (DR8), DRB1*0901-DQB1*0303 (DR9), and DRB1*1302-DQB1* 0604 (DR13) haplotypes were significantly higher in the patients than in the control subjects. The frequencies of DRB1* 1501-DQB1*0602 and DRB1*1502-DQB1*0601 haplotypes were significantly lower in the patients than in the controls. The frequencies of DR4/8, DR4/13, DR9/9, and DR9/13 genotypes were significantly higher in the patients than in the control subjects. The DR13 haplotype was the most frequent haplotype in the age group 30–49 years, whereas the other haplotypes but DR13 were the most frequent in the other age groups. Conclusion: DR4, DR8, DR9, and DR13 haplotypes confer susceptibility to type 1 diabetes in Japanese patients.

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Although the effects of the various secretagogues on corticotropin (ACTH) secretion have been well studied, their effects on the POMC gene expression have not been thoroughly characterized. In this study, we established a new model system using the AtT20 mouse corticotroph tumor cell line transfected stably with a plasmid containing 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene. The responsiveness to exogenous CRH improved markedly when the cells were cultured with low serum medium (1% FBS) compared with serum rich medium (10%). Using this culture condition, we examined the effects of not only CRH but also other secretagogues such as catecholamines, vasopressin, and angiotensin II, upon the transcriptional activity of the POMC gene. CRH stimulated POMC promoter activity (3.5-fold increase) as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3-5 h after the start of incubation. Catecholamines, especially epinephrine (10 nM and above), also stimulated all parameters, although less potently than CRH, and the effect was mimicked by the beta-, but not alpha-adrenergic, agonist, suggesting the involvement of the beta-adrenergic receptor. The combined effects of epinephrine and CRH were greater in all parameters than those of CRH alone, and the effects of both hormones were completely blocked by H89, an inhibitor of protein kinase A. Vasopressin and angiotensin II showed minimal effects on POMC expression. Our results suggest that 1) catecholamines, as well as CRH, positively regulate the POMC gene at physiological concentrations; 2) the cAMP-PKA system is the common intracellular signaling pathway for CRH and catecholamines; and 3) vasopressin and angiotensin II also have weak but significant stimulatory effects on POMC promoter activity.