The Journal of Neuroscience, August 1990, IO(E): 2825-2833 The Effect of Depolarization on Expression of the Human Proenkephalin Gene Is Synergistic-with CAMP and Dependent upon a CAMP-Inducible Enhancer T. Van Nguyen,1v2-4 Linda Kobierski,‘v6 Michael Comb,iJ,5,6 and Steven E. Hyman1,2,4+6 ‘Molecular Neurobiology Laboratory and Departments of 2Psychiatry and 3Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114, and Departments of 4Psychiatry and 5Neurology, and the ‘jProgram in Neurosciences, Harvard Medical School, Boston, Massachusetts 02115 Membrane depolarization is a critical component of neural maffin cells, proenkephalin mRNA levels have been shown to signaling; in recent years there also has been a great deal increasein responseto nicotinic stimulation (Eiden et al., 1984), of evidence that membrane depolarization can regulate neural K+- or veratridine-induced depolarization (Kley et al., 1986, gene expression. Therefore, excitatory neurotransmission 1987; Waschek et al., 1987), and calcium ionophores (Kley, may be an important mechanism of neural plasticity. We 1988). High-frequency electrical stimulation increasesproen- have investigated the intracellular pathways and DNA reg- kephalin mRNA levels and decreasesprodynorphin mRNA ulatory elements through which membrane depolarization levels in the rat hippocampus (Morris et al., 1988). Seizures activates expression of the neural gene encoding human induced by small dentate gyrus hilus lesions have been shown proenkephalin. In PC1 2 and CG-glioma cells, depolarization- to produce large increasesin proenkephalin mRNA in the en- induced expression of a transfected proenkephalin fusion torhinal cortex (White and Gall, 1987) and to increaselevels of gene was proportional to extracellular calcium concentration enkephalin immunoreactivity and decreaselevels of dynorphin and was inhibited by verapamil. Activation of the gene by immunoreactivity in the mouse hippocampal mossy fiber sys- KCI-induced depolarization or the calcium ionophore A231 87 tem (Gall, 1988). The proenkephalin gene is also regulated by was dependent upon and synergistic with CAMP in PC1 2 and neural activity in rat adrenals. It has been reported that rat CG-glioma cells, but neither depolarization nor treatment with adrenal proenkephalin mRNA levels are increasedby splanch- A23187 affected CAMP levels. Trifluoperazine and W7 in- nit nerve activation (Kanamatsu et al., 1986; Fischer-Colbrie hibited depolarization-induced gene expression but did not et al., 1988). However, it has been reported that, after dener- affect expression induced by the adenylyl cyclase activator vation (Kilpatrick et al., 1984) or explantation of rat adrenals, forskolin. At the level of the DNA, depolarization-induced proenkephalin mRNA levels also increase,and that in the ex- activation is conferred on the proenkephalin gene by a pre- plants, this increaseis suppressedby K+ or veratridine (La Gam- viously characterized CAMP-inducible enhancer. Multiple ma et al., 1985, 1988). The reasonsfor these discrepant results copies of a single component element of that enhancer, in rat adrenals are unclear but may reflect the involvement of containing the CGTCA sequence motif characteristic of CAMP undetected polysynaptic pathways. regulatory elements, can reconstitute the entire repertoire Although a great deal is known about the molecular mecha- of responses to both CAMP and depolarization. These data nisms by which CAMP regulatesneural gene expression(Comb suggest a model in which membrane depolarization acti- et al., 1986; Montminy et al., 1986; Lewis et al., 1987; Hyman vates gene expression through a calcium-dependent path- et al., 1988), relatively little is known about the mechanismsby way, potentially involving calmodulin, and in which the tran- which membrane depolarization produces transcriptional ef- scriptional responses to both CAMP and calcium are fects. The best studied gene to date is proto-oncogene c-fos, for transduced by the same DNA element. which a calcium-responsive DNA element has been identified (Sheng et al., 1988; Bartel et al., 1989). We therefore examined The regulation of neural gene expression by synaptic activity the mechanismsinvolved in depolarization-induced expression and membrane depolarization is well documented (Black et al., of the human proenkephalin gene. Experiments were performed 1985; Greenberg et al., 1986; Kley et al., 1986; Morgan and using rat pheochromocytoma cells (PC12) and rat C6-glioma Curran, 1986). One of the best-studied neural genesin models cells stably transfected with the proenkephalin fusion plasmid of regulation by synaptic activity and depolarization is the pENKAT-12 (Comb et al., 1986) and derivatives. pENKAT- proenkephalin gene.In primary cultures of bovine adrenal chro- 12 contains human proenkephalin genomic sequencesspanning nucleotides - 193 to + 70 with respectto the transcription start site, fusedto the bacterial chloramphenicolacyltransferase (CAT) Received Dec. 29, 1989; revised Apr. 13, 1990; accepted Apr. 17, 1990. transcription unit, and approximately 1 kilobase (Kb) of proen- This work was supported by NIH Grants MH44160 01 and DK01410-04 to S.E.H. and by a grant from the Juvenile Diabetes Foundation. We thank Joe kephalin 3’ flanking sequence. CAT activity derived from Pearlberg, who developed the C6-D2 and PC12-F3 lines. pENKAT-12 has previously been shown to correspond to cor- Correspondence should be addressed to Steven E. Hyman, Molecular Neuro- biology Laboratory, Massachusetts General Hospital, CNY 6, 149, 13th Street, rectly initiated transcription from the human proenkephalin Charlestown, MA 02129. promoter (Comb et al., 1986). C6-glioma and PC12 cells were Copyright 0 1990 Society for Neuroscience 0270-6474/90/082825-09$03.00/O used because both cell lines have voltage sensitive calcium 2626 Nguyen et al. * Dipolarization-Induced Expression of the Proenkephalin Gene channels (Miller, 1987) and depolarize in response to high con- pheochromocytoma PC 12 cells (the PC 12-F3 line) and rat C6- centrations of extracellular K+. Here, we show that depolari- glioma cells (the C6-D2 line). Stable clonal lines were used in zation-induced activation of proenkephalin gene expression is order to perform multiple readily comparableexperiments. The mediated by calcium and possibly calmodulin and is synergistic fold-induction of the proenkephalin gene in responseto regu- with CAMP. In addition, we show that regulation of gene expres- lators in these clonal lines was similar to that observed with sion by calcium and CAMP can converge on a single DNA transient transfection of pENKAT- 12, ruling out the possibility element. that the observed effects were an artifact of the integration site. Two different cell types were used in order to ensure that the Materials and Methods results might be generalizable. Figure 1 shows the effects of Cell culture.PC1 2 cells were grown in Dulbecco’s modification of Eagle’s forskolin, KCl, and the calcium ionophore A23 187 on expres- medium (DMEM), supplemented with 10% fetal bovine serum and 5% sion of CAT activity under the control of the human proen- horse serum; C6-glioma cells were grown in DMEM supplemented with kephalin promoter in PC 12-F3 cells (Fig. 1A) and C6-D2 cells 5% fetal bovine serum and 5% newborn calf serum (Sigma). The PC12- (Fig. 1B). As expected, forskolin, an activator of adenylyl cy- F3 and C6-D2 lines were produced by cotransfecting 3.5 x lo6 PC12 cells or 8 x lo5 C6-glioma cells in a IO-cm dish with 20 rg pENRAT- clase, produced approximately a 6-fold induction of CAT ac- 12 and 5 wg pRSVNeo (Gorman et al., 1983), a plasmid that confers tivity over the controls in both cell lines. In contrast, treatment resistance to the antibiotic G418. Transfections were performed by of cells with depolarizing concentrations of KC1 or with the CaPO, precipitation of DNA as previously described (Howard, 1983). calcium ionophore A23 187 produced no significant induction. Forty-eight hr after transfection, the media was supplemented with 500 However, when combined with forskolin, KC1 and A23 187 pro- .&ml G418. Under these conditions, 25-50% of the G418-resistant colonies expressed the unselected pENRAT- 12 plasmid. Individual col- duced levels of expression significantly higher than those in- onies were isolated and grown in microtiter wells. Media was assayed duced by forskolin alone. In PC 12-F3 cells, depolarization pro- for CAT activity, and single colonies that showed a response to CAMP duced more than a S-fold increaseand calcium ionophore nearly were expanded. a 4-fold increaseover forskolin alone (Fig. lA), and in C6-D2 CAT assays. For CAT assays, 2 x lo5 PC12-F3 cells or 1 x lo5 C6- D2 or C6-Cre2.2 cells were plated into each well of a 24-well plate 16 cells, depolarization and calcium ionophore produced approx- hr before adding regulators. After a 6-hr treatment with regulators, the imately a 2-fold increaseover forskolin alone (Fig. 1B). cells were washed with ice-cold phosphate-buffered saline (PBS), over- Membrane depolarization causesvoltage-sensitive calcium layed with 100 ~1 lysis buffer containing 0.25 M Tris (pH, 7.5) and 0.5% channels on cell membranesto open. To investigate the role of (v/v) Triton, and titurated up and down. The lysate was transferred to a calcium entry in depolarization-induced activation of proen- microfuge tube and pelleted for 10 min. The supematant was assayed for CAT activity by incubating 20% of the extract with a cocktail of 2.5 kephalin gene expression, we determined