REGULATION OF THE PREPROSOMATOSTATIN GENE BY CYCLIC-AMP IN CEREBROCORTICAL NEURONS

The gene coding for preprosomatostatin (ppSom), the molecular precurso r of somatostatin (Som), is regulated at the level of transcription by calcium ions and cyclic-AMP [F. Baldino, S. Fitzpatrick-McElligott, T . O'Kane, I. Oozes, Hormonal regulation of somatostatin, Synapse 2 (19 88) 317-325; M.R. Montminy, M.J. Low, L. Tapia-Arancibia, Cyclic AMP r egulates somatostatin mRNA accumulation in primary diencephalic cultur es and in transfected fibroblast cells, J. Neurosci. 6 (1986) 1171-117 6.], or by agents which increase intracellular levels of cAMP directly , such as forskolin [M.R. Montminy, M.J. Low, L. Tapia-Arancibia, Cycl ic AMP regulates somatostatin mRNA accumulation in primary diencephali c cultures and in transfected fibroblast cells, J. Neurosci. 6 (1986) 1171-1176.]. Transcriptional induction of the ppSom gene as examined i n PC12 cells, transfected fibroblasts and primary diencephalic culture s, requires the highly conserved cAMP response element(CRE), which con fers gene responsiveness to cAMP [M. Comb, N. Mermod, S.E. Hyman, Prot eins bound at adjacent DNA elements act synergistically to regulate hu man proenkephalin cAMP inducible transcription, EMBO J. 7 (1988) 3793- 3805; T. Tsukada, J.S. Fink, G. Mandel, Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regul ation by cyclic AMP, J. Biol. Chem. 262 (1987) 8743-8747.]. The ppSom gene is subject to stringent regulation during cerebrocortical develop ment in vivo; however, little information is available regarding ppSom gene regulation by neurotransmitters or second-messengers in cortical neurons. We used primary cerebrocortical cell cultures from fetal mic e to examine the dose-response and time-course of ppSom gene expressio n in response to the cyclic-AMP analogs, dibutyrl-cAMP (dbcAMP), and 8 -bromo-cAMP (8-BrcAMP). We report a dose-response for both analogs in the range of 0.1-10 mM. Dose-response studies using agents which direc tly stimulate intracellular cAMP synthesis (forskolin) or inhibit its breakdown (3-isobutyl 1-methyl xanthine) were also performed. We obser ved an apparent synergistic effect on ppSom expression when used in co mbination. An increase in ppSom mRNA levels was observed by 4 h, with a maximal response at 12-24 h. No change in ppSom mRNA levels was obse rved in response to phorbol myristate acetate (PMA). Our findings conf irm the specificity of ppSom gene regulation by cAMP and Ca2+ ions, an d demonstrate the utility of using primary cerebrocortical cultures fo r the study of somatostatin gene expression by neurotransmitters and s econd-messengers as a model of human neurologic disorders. (C) 1998 El sevier Science B.V. All rights reserved.