b-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAATyenhancer-binding protein d

Stimulation of b-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAATyenhancer-binding proteins (CyEBP), some of which are known to be cAMP-inducible. In C6–2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased CyEBPd-binding activity, (ii) NGF mRNA levels were increased by overexpressing CyEBPd, and (iii) CyEBPd increased the activity of an NGF promoter– reporter construct. Moreover, DNase footprinting and deletion analyses identified a CyEBPd site in the proximal region of the NGF promoter. CyEBPd appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in CyEBPd-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, CyEBPd is a critical component determining the area-specific expression of NGF in response to BAR stimulation. The ability to precisely alter the expression of individual genes in response to neurotransmitter receptor activation is believed to result from the interaction of multiple transcriptional regulatory factors with their corresponding DNA-regulatory elements. Activation of b-adrenergic receptors (BAR) increases nerve growth factor (NGF) synthesis in the rat cerebral cortex but not in other brain areas (1, 2). This finding is of particular interest for brain plasticity considering that NGF is a neurotrophic factor required for the development of cholinergic neurons of the basal forebrain (reviewed in ref. 3), which play an important role in learning and memory processes (4, 5). Given the role of NGF in fostering cholinergic plasticity, BAR agonists, by inducing the synthesis and availability of NGF, may be a potential therapeutic approach for the treatment of neurodegenerative diseases affecting the cholinergic system. Activityand neurotransmitter-dependent regulation of NGF expression have been shown to occur within the central nervous system in an area-specific manner. For instance, excitatory neurotransmitters increase NGF expression mainly in limbic areas (6, 7), glucocorticoid hormones in the hippocampus and cortex (8–10), interleukin-1 in the hippocampus (11), and BAR stimulation in specific layers of the cerebral cortex (2). These observations suggest that the NGF gene may be regulated by multiple cell-specific transcription factors that are activated by diverse stimuli. The molecular mechanisms underlying the area-specific, BAR-mediated induction of NGF synthesis are still poorly understood. Stimulation of BAR activates cAMP-dependent protein kinase A, which causes a rapid and time-dependent increase in early inducible genes such as c-fos (12, 13) and the associated AP-1-binding activity (14, 15). The NGF promoter contains an AP-1 element important for the expression of NGF (16, 17); thus, AP-1 may be involved in the corticalspecific induction of NGF gene in response to BAR agonists. However, other cAMP-inducible transcription factors could contribute to BAR-mediated induction of NGF expression, including members of the CCAATyenhancer-binding protein (CyEBP) family of basic region–leucine zipper DNA-binding proteins (18, 19), such as CyEBPb and CyEBPd (reviewed in ref. 20). The CyEBP proteins display cell-specific patterns of expression in a number of tissues, including the brain (21, 22), and play a role in synaptic plasticity (23). In this study, we have examined the contribution of AP-1 and CyEBPs in the BARmediated increase of NGF expression in the cerebral cortex. We provide evidence that CyEBPd is one of the transcription factors responsible for the selective induction of NGF expression in the cerebral cortex. MATERIALS AND METHODS Cell Cultures. C6–2B glioma and PC12 cells were grown as described (24). CTX-TNA2 rat astrocytes, established from primary type 1 astrocytes from frontal cortex (25), were grown as monolayer in DMEM supplemented with 10% fetal bovine serum (GIBCO). Antibodies. c-fos antiserum (a-c-fos) (26) was kindly provided by M. Iadarola (National Institute on Dental Research, National Institutes of Health, Bethesda, MD). Anti-CyEBPb (a-CyEBPb) antibody was obtained from Santa Cruz Biotechnology. Anti-CyEBPd (a-CyEBPd) antiserum (C. Cantwell and P.F.J., unpublished data) was raised against the Nterminal portion of murine CyEBPd and will be described in detail elsewhere. Nuclear Extracts. Brain areas were dissected on ice and immediately homogenized in lysis buffer (50 mM TriszHCl, pH 7.5y1.5 mM MgCl2y200 mM sucrosey0.1% Triton X-100y2 mM 2-mercaptoethanoly10 mM NaFy0.5 mM phenylmethylsulfonyl f luoride) and incubated on ice for 5 min. Nuclei were The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9510920-6$2.00y0 PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: BAR, b-adrenergic receptors; NGF, nerve growth factor; CyEBP, CCAATyenhancer-binding protein; CLE, clenbuterol; FGF2, basic fibroblast growth factor; EMSA, electrophoretic mobilityshift assay; ISO, l-isoproterenol; PR, l-propranolol. ‡To whom reprint requests should be addressed at: Department of Cell Biology, Georgetown University, 3900 Reservoir Road NW, Washington, DC 20007. e-mail: [email protected].