Nuclear factor of activated T cells (NFAT) is involved in the depolarization-induced activation of growth hormone-releasing hormone gene transcription in vitro.
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.
Department of Endocrinology, Metabolism, and Nephrology, Kochi University, Kochi Medical School, Kohasu, Oko-cho, Nankoku 783-8505, Japan.
Masato
M
Asai
Yasumasa
Y
Iwasaki
Masanori
M
Yoshida
Noriko
N
Mutsuga-Nakayama
Hiroshi
H
Arima
Masafumi
M
Ito
Koji
K
Takano
Yutaka
Y
Oiso
D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D013006:Growth Hormone / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000032:analysis / Q000235:genetics*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D050778:NFATC Transcription Factors; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; C494006:NFATC2 protein, human; C494008:NFATC3 protein, human; C494009:Nfatc3 protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D004533:Egtazic Acid; D016572:Cyclosporine; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D011188:Potassium; D002118:Calcium