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Nuclear Factor of Activated T Cells (NFAT) Is Involved in the Depolarization-Induced Activation of Growth Hormone-Releasing Hormone Gene Transcription in Vitro

Departments of Medicine (M.A., M.Y., N.M.-N., H.A., Y.O.) and Clinical Pathophysiology (Y.I., M.I.), Nagoya University Graduate School of Medicine and Hospital, Nagoya 466-8550, Japan; and Department of Nephrology and Endocrinology (K.T.), Faculty of Medicine, University of Tokyo, Tokyo 112-8655, Japan

Address all correspondence and requests for reprints to: Yasumasa Iwasaki, M.D., Ph.D., Department of Endocrinology, Metabolism, and Nephrology, Kochi University, Kochi Medical School, Kohasu, Oko-cho, Nankoku 783-8505, Japan. E-mail: iwasaki{at}med.kochi-u.ac.jp.

GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5'-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (–417/–403, –402/–387, –317/–301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.

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