The Effect of Depolarization on Expression of the Human Proenkephalin Gene Is Synergistic-With CAMP and Dependent Upon a CAMP-Inducible Enhancer

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 lesionshave 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 thesediscrepant 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-responsiveDNA element has been identified (Shenget al., 1988; Bartel et al., 1989). We therefore examined The regulation of neural gene expressionby 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 becauseboth 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 effectswere an artifact of the integration site. Two different cell types were usedin 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, KC1and 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 the effects of extra- &i/ml 14C-chloramphenicol (54 mCi/mmol; Amersham), 0.5 mM bu- cellular calcium on levels of expressionin responseto KCl. By tyryl coenzyme A (Pharmacia), and 2.5% glycerol in 0.25 M Tris (pH, altering the electrochemical driving force on calcium, alteration 7.5) in a total reaction volume of 50 ~1 for 2 hr at 37°C. Reactions were of extracellular calcium concentrate should affect the amount terminated with a 2: 1 mixture of pristane : xylenes (obtained from Al- drich and Baker, respectively). The butyrylated chloramphenicol ex- of calcium that enters cells with depolarization. As shown in tracted into the organic (upper) phase was counted in scintillation fluid Figure 2A, the magnitude of the KC1 effect is proportional to (modified from Seed and Sheen, 1988). Protein assays were initially the extracellular calcium concentration over a wide range of performed by the method of Lowry et al. (195 1) to normalize results, concentrations. Similar results were seen with PC12-F3 cells but the protein concentrations did not vary significantly from well to (not shown). Becausethe media was not entirely depleted of well. CAMPassuys. CAMP assays were carried out using a kit assay (Amer- calcium (to retain cell viability), KC1 retained someactivity even sham) based on the competition between unlabeled CAMP (from sam- when no CaCl, was added. ples) and a fixed quantity of tritium-labeled CAMP for binding to a Blockade of calcium entry with verapamil had a marked in- protein that has a high specificity and affinity for CAMP (Brown et al., hibitory effect on depolarization-induced activation of the fu- 1972). Sixteen hours before adding regulators, 1 x lo6 PC12-F3 cells sion gene. At the concentration of verapamil shown in Figure or 5 x lo5 C6-D2 cells were plated into a 6-cm dish. After treatment with regulators for selected times, cells were washed twice with ice-cold 2B (15 PM), there was no statistically significant difference in PBS and harvested in 1.1 ml ice-cold PBS, of which 1 ml was for CAMP forskolin-induced gene expression. Although more complete determination and 100 ~1 for protein determination by the method of blockade of the effects of KC1 could be achieved with higher Lowry et al. (195 1). The cells to be assayed for CAMP were recovered concentrations of verapamil, such concentrations also affected by a I-min spin, then sonicated in 150 ~1 ethanol. The samples were allowed to stand at room temperature for 5 min to coagulate protein, forskolin-induced expression(not shown), suggestingthe emer- and following a 5-min spin, the supematant was transferred to a new genceof nonspecificeffects at high concentrations of verapamil. microfuge tube and dried under vacuum. The residue was dissolved in To characterize more fully the synergistic interaction of cal- 200 ~1 50 mM Tris-Cl buffer (pH, 7.5) containing 4 mM EDTA. After cium with CAMP, we also examined the effects of fixed concen- a 3-min spin to remove insoluble residues, 50 ~1 of the supematant was incubated with 50 ~1 cyclic 3H-AMP (0.5 &i/ml, 18 pmol/ml) and 50 trations of A23 187 (50 nM) and extracellular calcium (1.8 mM) ~1 binding protein solution. After incubation at 4°C for 8 hr and following on expressionproduced by a wide range of forskolin concen- the addition of 100 ~1 charcoal suspension, the tubes were centrifuged trations in PC12-F3 cells. Figure 3 shows log-dose-response for 3 min, and 200 ~1 of the supematant was counted in scintillation curves for forskolin with and without A23187. The phospho- fluid. All values reported were measured within the linear range of the diesteraseinhibitor 3-isobutyl- 1-methylxanthine (IMX) (0.5 mM) assay. was added to achieve maximal levels of CAMP. Calcium entry had a significant synergistic effect on expressionof CAT activity Results at all effective concentrations of forskolin. Calcium produced Proenkephalin gene expressionis synergistically activated by striking increasesin expressioneven at maximally effective con- membranedepolarization or A23187 with forskolin centrations of the CAMP agonists.Higher concentrations of for- Stable clonal lines expressingpENKAT-12 and the dominant skolin produced a decline in expression,presumably as a result selectable marker neomycin resistance were produced in rat of toxicity to the cells (not shown). The Journal of Neuroscience, August 1990, 70(S) 2827

J p 30 - 5 20- 2 E s - 2 20 z 9 B 9 10 - 10 - 9 0.7x 0.8X k Ctrl F FK FA K A I I 0-l ctri K F la= KF KF la= KF I 0 0 0 0 1 5 10 20 (pc.OW [CaCl2] (mM) B 20 1 i 13x 15 -

ki 10 - B 50 n No Inhibitor s 1 q Verapamil(15 pM) 9 40 9

1X 0.9x 27x

K A I I I I I

pc.005

REGULATORS

Figure I. Induction of CAT expression in response to regulators. A, ctt1 F FK K PC1 2-F3 cells. B, C6-D2 cells. The final concentration of forskolin (F) was 10 PM; KC1 (K), 40 mM; and A23187 (A), 500 nM; whether used REGULATORS singly or in combination. Fold induction represents induced CAT activity divided by control CAT activity after a 6-hr incubation with Figure 2. A, The effects of increasing extracellular calcium concentra- regulators. The extracellular (Ca2+) was 1.8 mM. For both PC 12-F3 cells tion on KCl-induced expression of the pENKAT-12 in C6-D2 cells. and C6-D2 cells (A and B, respectively), the effects of forskolin in com- Thirty min prior to adding regulators, standard media (1.8 mM Ca*+) bination with KC1 or A23 187 were sianificantlv different from the effects was replaced with low calcium media (made with calcium-free DMEM of forskolin alone (p < 0.005 by Student’s t test; n = 6 independent [Gibco] and 10% serum). Concentrations of CaCl, added to the media experiments; average and SEM shown). are shown at the bottomof each bar in mM. K, 40 mM KCl; F, 10 FM forskolin. Fold induction over control (ctrl; arbitrarily set at 1) is shown above each bar. B, Effect of the calcium channel blocker verapamil on induced expression of pENKAT- 12 in PC 12-F3 cells. Cells were treated Effects of depolarization and A23187 are not mediated by for 6 hr with forskolin (0, KC1 (K), or the combination in the presence or absence of verapamil (added 30 min prior to other compounds). CAMP, but may be calmodulin-dependent Final concentrations: verapamil, 15 WM; forskolin, 10 PM; KCl, 40 mM. Becauseboth KCl-induced depolarization and A23 187 require Shown are the average and SE of 4 separate determinations. coactivation ofcAMP to induce proenkephalin geneexpression in C6-glioma and PC12 cells, we investigated the mechanisms by which the calcium and CAMP signal transduction pathways to induce gene expression(Fig. 1, A, B). BecauseA23 187 acts might interact to produce the observed synergy. An increasein independently of voltage-sensitive calcium channels, even if CAMP levels hasbeen shown to be required for voltage-sensitive CAMP-dependent phosphorylation of channelsaffects the mag- calcium channels to open in responseto depolarization in the nitude of depolarization-induced gene expression, there must neuroblastomaX glioma line NG 108-15 (Freedman and Miller, be additional sitesof action for CAMP. 1984)and in GH3 pituitary cells (Armstrong and Eckert, 1987). Crosstalk between the CAMP and calcium-dependent signal Becausecalcium entry (and, by inference, opening of calcium transduction pathways might also occur at the level of second channels)appears to be critical for the activation of geneexpres- messengers,protein kinases,transcription factors, or DNA reg- sion by depolarization, this membrane effect of CAMP might ulatory elements. In order to rule out the possibility that de- explain the synergy of forskolin and KCI. However, not only polarization and A23187 were simply acting by increasing in- KCl, but also the ionophore A23 187 required CAMP in order tracellular levels of CAMP, we measured CAMP levels by 2828 Nguyen et al. * Dipolarization-Induced Expression of the Proenkephalin Gene

100000 - Forskolin+lMX+A23187 possiblenonspecific action of both trifluoperazine and W7 even - Forskolin+IMX at low concentrations in inhibition of voltage-sensitive calcium 80000 channels(Harper, 1988). We therefore testedthe effectsof these 1 compounds on both KCl-induced expression,which is dependent on calcium channels,and on A23 187-induced expression, which is not (Fig. 5, A, B). The data from useof theseinhibitors is consistent with a role for calmodulin in calcium-mediated activation of gene expression. We did not test the effects of inhibitors of CAMP-dependent protein kinase in the present studiesbecause it has already been establishedthat expression of pENKAT- 12 is absolutely dependent on the CAMP pathway (Grove et al., 1987; S. Hyman and M. Comb, unpublished observations). Taken together, the data suggesta model in which -11 -10 -9 -8 -7 -6 -5 -4 CAMP plays a permissive role for the actions of calcium on LOG [FORSKOLIN] proenkephalin gene expression,and that the effects of calcium are independently mediated by a calmodulin-dependent path- Figure 3. Synergyof the calciumionophore A23 187with CAMP in- way and therefore possibly by a calcium/calmodulin-dependent ducersforskolin and IMX in activatingexpression of the proenkephalin protein kinase. fusiongene in PC12-F3 cells. The abscissa shows the log-dose of forskolin.The ordinate showsnet CAT activity (total CAT activity from CAMP- and calcium-inducible expressionof the proenkephalin samplesminus a blankprepared from untransfectedC6 cells). IMX (0.5 mM) waspresent in all samples.The curves showthe responseof cells gene convergeon a single DNA regulatory element treatedwith forskolinand IMX in the absence(white squares with black pENKAT-12, which is expressedin the PC12-F3 and C6-D2 dots) and presence(black diamonds) of 50 nM A23 187. cell lines, has previously been shown to be inducible by CAMP and phorbol esters(Comb et al., 1986). We have now demon- strated that it is inducible by calcium, as well. pENKAT-12 radioimmunoassay.A time courseof CAMP generation was first contains only 193 basesof human proenkephalin 5’ flanking determined; forskolin (final concentration, 10 PM) was added at sequence. To determine whether additional 5’ flanking se- time 0 to the media of C6-D2 cells and left there for the duration quencescould also contribute to calcium responsivenessof the of the experiment. Intracellular CAMP levels were found to rise wild-type proenkephalin gene, we constructed a derivative of rapidly in responseto forskolin (Fig. 4.4) and were maximal at pENKAT- 12 containing 3 Kb of proenkephalin 5’ flanking se- 30 min, which correspondsto the time at which steady-state quence (pENKAT-3K). Stable C6-glioma cell lines expressing levels of correctly initiated mRNA from the proenkephalin pro- this plasmid were made and tested for responseto CAMP, de- moter are rising most rapidly (not shown). The CAMP levels polarization, A23 187, and the combination. No additional con- then declined, but remained elevated above baseline. We then tribution to calcium responsivenesswas seen(data not shown). measuredthe effects of KCl-induced depolarization on CAMP We conclude that the physiologically important sequencescon- levels. In C6-D2 cells, KC1 had no significant independenteffect ferring calcium responsivenesson the proenkephalin geneprob- on CAMP levels, nor a significant synergisticeffect with forskolin ably reside within pENKAT- 12. at 30 min (Fig. 4B) or 6 hr (not shown). A 30-min time point pENKAT- 12 contains a CAMP- and phorbol ester-inducible was also measuredfor PC12-F3 cells (Fig. 4C); KC1 and A23 187 enhancer (Comb et al., 1986). This enhancer is comprised of 3 had no significant independent or synergistic effects on CAMP. synergistically acting DNA elements: ENKCRE- 1, ENKCRE- Therefore, the effect of depolarization and calcium on proen- 2, and AP-2 (Fig. 6A; Comb et al., 1988; Hyman et al., 1989). kephalin gene expression cannot be explained by synergistic The middle element, ENKCRE-2, contains the sequence increasesin levels of CAMP. CGTCA, which is the core consensussequence for CAMP-reg- To test the possibility that signal transduction to the nucleus ulatory elements(CREs) (Hyman et al., 1988). This ENKCRE-2 converged at or beyond the level of the protein kinases,we used element is absolutely required for basal and CAMP-inducible the calmodulin antagoniststrifluoperazine and W7 (Hidaka and transcription of the proenkephalin gene (Comb et al., 1988). In Tanaka, 1987). Inhibition of calmodulin should block the ac- order to examine the role of this element in activation of the tivation of calcium/calmodulin-dependent protein kinasesbut geneby depolarization and calcium entry, we constructed plas- not CAMP-dependent protein kinase. Although trifluoperazine mids in which the entire enhancer was removed and then re- and W7 are not entirely specific, even at low concentrations, no placed by synthetic oligonucleotidescontaining the ENKCRE-2 more specificcompounds were available. We tested both com- sequence.The enhancerlessparent plasmid pENKAT-A72, which pounds in order to rule out the possibility that inhibition was lacks all 3 elements(ENKCRE- 1, ENKCRE-2, and AP-2) gives due to an adventitious effect of one of the compounds.In PC12- no correctly initiated transcripts when stably expressedin C6 F3 cells, trifluoperazine (20 PM) had no effect on activation of cells. It producesno CAT activity in responseto forskolin or the proenkephalin fusion gene by forskolin but inhibited most the combination of forskolin with KC1 or A23 187 when tran- of the effectsof KC1 and A23 187 (Fig. 54). At the concentration siently transfected into multiple cell types including C6-glioma used(20 PM), W7 had no effect on forskolin-induced expression and PC12 cells (not shown). Another plasmid, pENKAT-A84, and partially inhibited the synergistic effectsof KC1and A23 187 which contains the AP-2 element, but not ENKCRE- 1 or ENK- (Fig. 5B). More complete inhibition of the effects of KC1 and CRE-2, is also unresponsiveto forskolin (Comb et al., 1986) or A23 187 was achieved with higher concentrations of W7, but to forskolin combined with KC1 or A23 187 (not shown). thesedata were not interpretable because,at higher concentra- In the absenceof the ENKCRE-1 element, a single copy of tions, W7 becomesa nonspecificprotein kinase inhibitor. One ENKCRE-2 is inadequate to produce a significant transcrip- The Journal of Neuroscience, August 1990, IO(8) 2829

40 A 50 A n No Inhibitor 1 Trifluoperazine (20 FM) 1 1

0 15 30 60 160 360 Ctrl F FK FA TIME (min) REGULATORS

50 n No Inhibitor W7 (20 FM) 40

Ctrl F FK K 20 REGULATORS

0 Ctrl F FK FA

REGULATORS Figure 5. Effect of trifluoperazine and W7 on induced expression of pENKAT-12 in PC1 2-F3 cells. Trifluoperazine (20 PM; A) and W7 (20 PM; B) were added 30 min prior to other regulators. Final concentrations of regulators: forskolin (F), 10 PM; KC1 (IQ, 40 mrq A23187 (A), 500 nM. Neither trifluoperazine nor W7 had an effect on expression induced by forskolin alone, but both significantly inhibited the synergistic response to KC1 and A23 187 (the effects of both inhibitors on both KC1 C F FK FA K A and A23 187 were significant bv Student’s t test: TV< 0.005: 4 indenen- dent determinations; average and SEM shown): _ - REGULATORS Figure 4. A, Time course of CAMP induction by forskolin in C6-D2 cells. Forskolin (10 PM) was added at time 0; cells were harvested for tional response(Comb et al., 1988); we therefore constructed assay at the times shown. The average and SEM are shown; 3 indepen- plasmidscontaining multiple copies of an oligonucleotide with dent experiments were performed. B, CAMP levels in C6-D2 cells mea- the ENKCRE-2 sequence.The plasmid pENKCRE2-2 (Fig. 6B), sured 30 min after the addition of regulators (average and SEM shown; which contains 5 copies of the ENKCRE-2 oligonucleotide, was 4 experiments). C, CAMP levels in PC 12-F3 cells measured 30 min after the addition of regulators (average and SEM shown; 6 experiments). testedin detail. Five independent linesof C6-glioma cells stably Final concentrations of regulators: forskolin (0, 10 PM; KC1 (K), 40 expressingpENKCRE2-2 were testedfor responseto CAMP and mM; A23 187 (A), 500 nM. depolarization in order to rule out the possibility that the observed responses were dependent on integration site. Figure 7 showsthe responsesof the cell line C6-Cre2.2; this pattern of responsewas typical of the 4 other stable lines tested. Forskolin producesa robust induction; KC1 producesno independent re- 2830 Nguyen et al. f Dipolarization-Induced Expression of the Proenkephalin Gene

-110 -100 -90 -80 -70 I I I GCGGGGC~GGct3TAGGGCCT&TCAGCTGCaGCGCGGCGGCGATT CGCCCCG AC;GiGCAT CCCGG ‘k&&G+ CGAC:GTC&G&SG CCGCTAA

ENKCRE-1 ENKCRE-2 AP-2

B ENKCRE-2 OLIGOS gatcggcTGCGTCAggg (5X)

-72 DELETION \ I

Figure 6. A, The sequence of the human proenkephalin enhancer. The individual elements that comprise the enhancer (shaded) were identified by extensive mutational analysis (Comb et al.. 1988: Hvman et al.. 1989). B. pBNKCRE2-2. This plasmid was’con: Amp strutted in 2 steps from pENKAT-A84 pENKCRE2-2 (Comb et al., 1986) a derivative of pENKAT- 12 in which the ENKCRE- 1 5.4 Kb and ENKCRE-2 elements were removed using a Pst I site at nucleotide -84. pENKAT-A84 was digested with Bssh II to remove the AP-2 element, and the AP-2 element was replaced with a Bal II linker that does not bind AP-2 (Himan et al., 1989). The resulting plasmid, pENKAT-A72, was digested with Bgl II, and oligonucleotides containing the ENKCRE-2 sequence with Barn HI cohesive termini were cloned into this site as concatamers.

sponse,but in combination with forskolin produces approxi- proteins with which it interacts is therefore sufficient for the mately a 2-fold increase over forskolin alone. Increasing the activation of the proenkephalin gene by both CAMP and cal- concentration of calcium from 1.8 mM (standard medium) to cium. 20 mM producesa small increasein the forskolin responsebut a significant increase in the responseof cells also treated with Discussion KCl, consistent with what had been observed for pENKAT- 12. Intracellular signal transduction pathways Indeed, the pattern of responseto regulators observed in C6- The data presentedhere representan investigation into mech- Cre2.2 cells is quite similar to that seenfor C6-D2 cells (Fig. 7; anisms responsiblefor regulation of gene expressionby mem- cf., Fig. 1B). The ability of multiple ENKCRE-2 elements to brane depolarization. We have shown that depolarization-in- activate transcription in the absenceof an ENKCRE- 1 element duced expression of pENKAT-12 increases with increasing suggeststhat multiple copiesof one protein (or protein complex) concentration of extracellular calcium and is inhibited by the bound in tandem can replace the protein/protein interaction calcium channelblocker verapamil, consistentwith calcium being between factors bound at the ENKCRE- 1 and ENKCRE-2 ele- the effector of the transcriptional responseto depolarization. ments in the wild-type proenkephalin enhancer. Multiple copies This interpretation is consistentwith prior reports that calcium of the ENKCRE-2 element are sufficient to confer responsive- channelantagonists D,,,, verapamil and Co2+(Kley et al., 1986) ness to both CAMP and depolarization on an inactive parent or low calcium media (Wascheket al., 1987) inhibit veratridine- plasmid (pENKAT-A72). The activity of this element and the or KCl-induced activation of proenkephalin geneexpression in The Journal of Neuroscience, August 1990, IO@) 2831 bovine adrenal chromaffin cells. These data suggest that one of the biological roles for voltage-sensitive calcium channels lo- n Standard Medium cated on neuronal cell bodies may be in transduction of depo- High-Calcium Medium larizing signals to the nucleus to regulate gene expression. In PC 12 and C6-glioma cells, stimulation of the transfected 62x proenkephalin gene by KC1 or A23 187 was not only synergistic with agents that increase levels of CAMP, but also absolutely dependent on them (Fig. 1, A, B). In contrast, in bovine adrenal chromaffin cells in primary culture, while activators of CAMP are robustly synergistic with depolarization in activating proenkephalin gene expression (S. Lewis and S. Hyman, unpublished observations), they are not absolutely required (Kley et al., 1986; Waschek et al., 1987; Kley, 1988). This difference between the transformed PC1 2 and C6-glioma cell lines and the primary cells was not an artifact resulting from use of a transfected fusion gene because the endogenous proenkephalin gene in C6-glioma cells is regulated similarly to the transfected gene (data not shown; PC 12 cells do not express an endogenous proenkephalin gene). ctr1 F FK K The reason for the absolute requirement for CAMP in the trans- REGULATOR formed cell lines but not in primary chromaffin cells is unclear, but it may involve cell-type specific differences in the activity Figure 7. C6-Cre2.2 cells were treated for 6 hr with forskolin 0, 10 of different components of the CAMP signal transduction path- PM; KC1 (K), 40 mM; or the combination at 2 concentrations of extra- way. cellular calcium: standard (1.8 mM) and high (20 mM). Forskolin independently induces expression of this proenkephalin fusion gene. KC1 Neither KCl-induced depolarization nor A23 187 had an effect produces no independent response, but when added to forskolin, it gives on CAMP levels; therefore, their effects on proenkephalin gene approximately a 2-fold induction over forskolin alone. The combination expression cannot be ascribed to superinduction of CAMP. of KC1 and forskolin produces a significantly greater induction in high Moreover, the effects of KC1 and A23 187 were inhibited by W7 than in normal calcium media (p < 0.005 by Student’s t test; 6 determinations; average and SEM shown), while forskolin alone does not. and trifluoperazine, while the effects of forskolin were not. These data suggest that calcium, but not CAMP, might be activating gene expression through a calmodulin-dependent pathway. In- terpretation of these data is limited by the nonspecificity of interactions of excitatory neurotransmissionand second-mes- calmodulin antagonists currently available. A possible model is sengerpathways in models of neural plasticity suchas long-term that CAMP, and therefore, presumably, CAMP-dependent pro- associative learning. tein kinase, has a permissive action in activation of proenkepha- One model of transsynaptic activation of gene expressionin lin gene expression, without which the calcium pathway cannot which synergy may have particular relevance is the seizure mod- act. el. Seizures have been shown to induce very high levels of expressionof proenkephalin and a variety of other genes(Mor- Synergy of calcium and CAMP gan et al., 1987; White and Gall, 1987; Saffen et al., 1988). Neurons express a wide variety of neurotransmitter receptors Seizuresrepresent the synchronous firing of many neuronsand on their cell surface membranes, permitting them to respond to are therefore likely. They releasemultiple transmitters, includ- multiple extracellular signals. This diversity of extracellular sig- ing excitatory neurotransmitterssuch asglutamate, which would nals may be transduced into different intracellular signals, in- depolarize target neurons, and other neurotransmitters, such as cluding changes in membrane polarization or activation or deac- peptides, which could activate the CAMP or protein kinase C tivation of second-messenger systems. The integration of multiple pathways. The high levels of proenkephalin mRNA induction intracellular signals is an important mechanism regulating cel- reported in response to seizures (White and Gall, 1987) are lular response: just as neurons summate excitatory and inhib- consistentwith the synergisticmechanisms we have investigated itory postsynaptic potentials as determinants of reaching thresh- here. The hypothesis, that synergistic activation of multiple in- old for an action potential, it is likely that they summate the tracellular messengersmay control the level of gene expression actions of multiple-signal transduction pathways as determi- in seizure and electrical stimulation models of gene expression, nants of other cellular functions. For example, spatial and tem- may be testableby the administration of selectivepharmacolog- poral integration of multiple signals appears to be involved in ic blockers. such phenomena as neuromodulation and long-term potentia- tion. The data presented here demonstrate that cells can inte- Convergenceof calcium and CAMP responseson a common grate multiple signals to regulate gene expression, as well. We DNA element have previously reported that phorbol esters act synergistically We have shown that a concatamer of ENKCRE-2 elements with CAMP to activate proenkephalin gene expression (Comb confers responseto both CAMP and calcium on a transcrip- et al., 1986; Hyman et al., 1989). Synergy of phorbol esters with tionally inactive parent plasmid. These data suggesta model in CAMP has also been reported for the chorionic gonadotropin which a protein that interacts directly or indirectly with the genes (Andersen et al., 1988). Here, we show an interaction ENKCRE-2 element can be activated by both protein kinase A between CAMP and calcium, as well; the magnitude of this and by a calcium-activated protein kinase, perhapsa calcium/ interaction suggests that it is physiologically important (Fig. 3). calmodulin-dependent protein kinase. Such a model would ex- This finding has potential implications for conceptualizing the plain the synergy of CAMP with calcium. The data also suggest, 2832 Nguyen et al. - Dipolarization-Induced Expression of the Proenkephalin Gene

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