Tyrosine Hydroxylase Gene Promoter Activity Is Regulated by Both Cyclic AMP-responsive Element and AP1 Sites following Calcium Influx
1997; Elsevier BV; Volume: 272; Issue: 9 Linguagem: Inglês
10.1074/jbc.272.9.6051
ISSN1083-351X
AutoresKumi Nagamoto‐Combs, Kristen M. Piech, Joseph A. Best, Baoyong Sun, A. William Tank,
Tópico(s)Adenosine and Purinergic Signaling
ResumoMembrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5′-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response. Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5′-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response. INTRODUCTIONBiosynthesis of the catecholamines is tightly regulated by the activity of the rate-limiting enzyme, tyrosine hydroxylase (TH 1The abbreviations used are: THtyrosine hydroxylase[Ca2+]iintracellular calcium concentrationCATchloramphenicol acetyltransferaseCREBcyclic AMP-responsive element binding proteinCREcyclic AMP-responsive element8-CPT-cAMP8-chlorophenylthio-cyclic AMPPBSphosphate-buffered salineASCREBantisense-CREBbpbase pair(s)RSVRous sarcoma virusCREMcyclic AMP-responsive element-modulating proteinCaREcalcium-responsive elementAP1activating protein-1.; EC 1.14.16.2). Experimental manipulations that lead to long-term stimulation of catecholaminergic cells in sympathetic ganglia and adrenal medulla are associated with increases in TH gene expression (1Thoenen H. Nature. 1970; 228: 861-862Crossref PubMed Scopus (206) Google Scholar, 2Kvetnansky R. Gewirtz G. Weise V.K. Kopin I.J. Am. J. Physiol. 1971; 220: 928-931Crossref PubMed Scopus (70) Google Scholar, 3Tank A.W. Lewis E.J. Chikaraishi D.M. Weiner N. J. Neurochem. 1985; 45: 1030-1033Crossref PubMed Scopus (98) Google Scholar, 4Faucon-Biguet N. Buda M. Lamouroux A. Samolyk D. Mallet J. EMBO J. 1986; 5: 287-291Crossref PubMed Scopus (183) Google Scholar, 5Fossom L.H. Carlson C.D. Tank A.W. Mol. Pharmacol. 1991; 40: 193-202PubMed Google Scholar, 6Richard F. Faucon-Biguet N. Labatut R. Rollet D. Mallet J. Buda M. J. Neurosci. Res. 1988; 20: 32-37Crossref PubMed Scopus (119) Google Scholar, 7Mallet J. Faucon-Biguet N. Buda M. Lamouroux A. Samolyk D. Cold Spring Harbor Symp. Quant. Biol. 1983; 48: 305-308Crossref PubMed Google Scholar, 8Fossom L.H. Sterling C.R. Tank A.W. Mol. Pharmacol. 1992; 42: 898-908PubMed Google Scholar). The mechanisms responsible for regulation of the TH gene are complex and have not been fully elucidated. Cultured rat pheochromocytoma cells have been used extensively to study the underlying mechanisms for TH gene regulation. Using this model system, a number of laboratories have shown that cAMP analogs (9Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 486-496PubMed Google Scholar, 10Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 497-503PubMed Google Scholar, 11Lewis E.J. Tank A.W. Weiner N. Chikaraishi D.M. J. Biol. Chem. 1983; 258: 14632-14637Abstract Full Text PDF PubMed Google Scholar), active phorbol esters (12Vyas S. Faucon-Biguet N. Mallet J. EMBO J. 1990; 9: 3707-3712Crossref PubMed Scopus (92) Google Scholar), and glucocorticoids (8Fossom L.H. Sterling C.R. Tank A.W. Mol. Pharmacol. 1992; 42: 898-908PubMed Google Scholar, 9Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 486-496PubMed Google Scholar, 10Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 497-503PubMed Google Scholar, 13Lewis E.J. Harrington C.A. Chikaraishi D.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3550-3554Crossref PubMed Scopus (299) Google Scholar) induce TH mRNA, stimulate TH gene transcription rate, and/or activate TH gene promoter activity. These results are consistent with the hypothesis that protein kinase A, protein kinase C, and glucocorticoid receptors participate in signaling pathways that regulate the TH gene.Treatments that lead to increased intracellular Ca2+ concentration ([Ca2+]i) also increase expression of the TH gene. Studies by Sabban and co-workers (14Kilbourne E.J. Sabban E.L. Mol. Brain. Res. 1990; 8: 121-127Crossref PubMed Scopus (52) Google Scholar, 15Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar) have shown that membrane depolarization or agonist occupation of nicotinic cholinergic receptors leads to the induction of TH mRNA in PC12 cells. Both of these treatments lead to Ca2+ influx through voltage-sensitive Ca2+ channels. Furthermore, chelation of extracellular Ca2+ with EGTA inhibits the depolarization-mediated increase in TH mRNA (14Kilbourne E.J. Sabban E.L. Mol. Brain. Res. 1990; 8: 121-127Crossref PubMed Scopus (52) Google Scholar). These results suggest that the influx of Ca2+ plays an important role in regulating TH gene expression.A number of studies have been conducted in order to understand the mechanisms that mediate Ca2+- dependent regulation of different genes. Ca2+ influx elicited by either membrane depolarization or Ca2+ ionophore treatment activates and/or induces transcription factors, such as cAMP-responsive element binding protein (CREB) and c-Fos, in PC12 cells (16Greenberg M.E. Ziff E.B. Greene L.A. Science. 1986; 234: 80-83Crossref PubMed Scopus (566) Google Scholar, 17Morgan J.I. Curran T. Nature. 1986; 322: 552-555Crossref PubMed Scopus (825) Google Scholar, 18Bartel D.P. Sheng M. Lau L.F. Greenberg M.E. Genes Dev. 1989; 3: 304-313Crossref PubMed Scopus (394) Google Scholar, 19Sheng M. McFadden G. Greenberg M.E. Neuron. 1990; 4: 571-582Abstract Full Text PDF PubMed Scopus (873) Google Scholar, 20Greenberg M.E. Thompson M.A. Sheng M. J. Physiol. (Lond.). 1992; 86: 99-108Google Scholar). CREB and c-Fos interact with the promoter elements CRE and AP1, respectively, to transactivate expression of numerous genes (21Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6682-6686Crossref PubMed Scopus (1054) Google Scholar, 22Chiu R. Boyle W.J. Meek J. Smeal T. Hunter T. Karin M. Cell. 1988; 54: 541-552Abstract Full Text PDF PubMed Scopus (1003) Google Scholar, 23Curran T. Franza Jr., B.R. Cell. 1988; 55: 395-397Abstract Full Text PDF PubMed Scopus (1309) Google Scholar). Since the 5′-flanking region of the TH gene contains CRE and AP1 sites, it is reasonable to hypothesize that Ca2+ exerts its effects on TH gene transcription rate by influencing transcription factors that bind to these promoter elements. Kilbourne et al. (24Kilbourne E.J. Nankova B.B. Lewis E.J. McMahon A. Osaka H. Sabban D.B. Sabban E.L. J. Biol. Chem. 1992; 267: 7563-7569Abstract Full Text PDF PubMed Google Scholar) have shown that a minimal promoter containing the TH CRE is responsive to Ca2+ influx in PC12 cells. Using deletion analysis, they have also reported that promoter elements upstream of the CRE do not apparently participate in the response of the TH gene promoter to Ca2+. Recently, these results have been extended using TH gene constructs with site-directed mutations (25Nankova B. Hiremagalur B. Menezes A. Zeman R. Sabban E. Mol. Brain Res. 1996; 35: 164-172Crossref PubMed Scopus (36) Google Scholar). Results from these studies suggest that the TH AP1 site is not involved in Ca2+-mediated TH gene regulation. In contrast, deletion analysis by Stachowiak et al. (26Stachowiak M.K. Goc A. Hong J.S. Poisner A. Jiang H.K. Stachowiak E.K. Brain Res. Mol. Brain Res. 1994; 22: 309-319Crossref PubMed Scopus (43) Google Scholar) has shown that the TH CRE is not required for activation of the TH gene promoter by Ca2+ influx in bovine adrenal chromaffin cells and that, by inference, promoter elements upstream of the CRE participate in this response. However, it is not clear from their study which promoter elements participate in this regulation.In the present study, we demonstrate that both the CRE and AP1 sites in the proximal 5′-flanking region of the rat TH gene participate in regulating TH gene expression by Ca2+ influx in PC12 cells. The newly identified TH CRE2 site does not apparently play a major role in this response. To further analyze the involvement of the TH AP1 site in Ca2+-mediated TH gene regulation, we have examined nuclear protein interactions at this site. Finally, we have tested the involvement of CREB in Ca2+-mediated TH gene regulation using PC12 cell-derived CREB-deficient cell lines. Our results indicate that CREB is not required for Ca2+- dependent regulation of the TH gene, supporting the hypothesis that multiple promoter elements are involved in regulation of the TH gene promoter in response to Ca2+ influx.DISCUSSIONMembrane depolarization of excitable cells and the consequent influx of Ca2+ leads to modulation of a number of intracellular events, including changes in transcription rates of numerous genes. Mechanisms responsible for this gene regulation have been extensively studied, particularly with respect to the c-fos gene (19Sheng M. McFadden G. Greenberg M.E. Neuron. 1990; 4: 571-582Abstract Full Text PDF PubMed Scopus (873) Google Scholar, 20Greenberg M.E. Thompson M.A. Sheng M. J. Physiol. (Lond.). 1992; 86: 99-108Google Scholar, 32Ginty D.D. Glowacka D. Bader D.S. Hidaka H. Wagner J.A. J. Biol. Chem. 1991; 266: 17454-17458Abstract Full Text PDF PubMed Google Scholar, 33Enslen H. Soderling T.R. J. Biol. Chem. 1994; 269: 20872-20877Abstract Full Text PDF PubMed Google Scholar, 34Misra R.P. Bonni A. Miranti C.K. Rivera V.M. Sheng M. Greenberg M.E. J. Biol. Chem. 1994; 269: 25483-25493Abstract Full Text PDF PubMed Google Scholar, 35Thompson M.A. Ginty D.D. Bonni A. Greenberg M.E. J. Biol. Chem. 1995; 270: 4224-4235Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). These studies have identified a Ca2+-responsive element (CaRE) within the c-fos gene, which is strikingly similar to the consensus CRE. In addition, the increase in intracellular Ca2+ leads to activation of CREB by phosphorylation of its Ser-133 site (20Greenberg M.E. Thompson M.A. Sheng M. J. Physiol. (Lond.). 1992; 86: 99-108Google Scholar, 36Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1274) Google Scholar). Therefore, it has been postulated that Ca2+ influx evoked by membrane depolarization increases c-fos gene expression via the CRE/CaRE following the phosphorylation of CREB. However, the critical importance of the CRE/CaRE and CREB activation in c-fos gene regulation is unclear, because 1) CREB phosphorylation by itself is not sufficient to increase c-fos gene expression in response to Ca2+ influx in protein kinase A-deficient cell lines (35Thompson M.A. Ginty D.D. Bonni A. Greenberg M.E. J. Biol. Chem. 1995; 270: 4224-4235Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and 2) c-fos gene sequences upstream of the CRE/CaRE also participate in the response to Ca2+ (34Misra R.P. Bonni A. Miranti C.K. Rivera V.M. Sheng M. Greenberg M.E. J. Biol. Chem. 1994; 269: 25483-25493Abstract Full Text PDF PubMed Google Scholar, 37Miranti C.K. Ginty D.D. Huang G. Chatila T. Greenberg M.E. Mol. Cell. Biol. 1995; 15: 3672-3684Crossref PubMed Scopus (197) Google Scholar).Depolarizing stimuli also induce TH mRNA and stimulate TH gene promoter activity in PC12 cells (14Kilbourne E.J. Sabban E.L. Mol. Brain. Res. 1990; 8: 121-127Crossref PubMed Scopus (52) Google Scholar, 24Kilbourne E.J. Nankova B.B. Lewis E.J. McMahon A. Osaka H. Sabban D.B. Sabban E.L. J. Biol. Chem. 1992; 267: 7563-7569Abstract Full Text PDF PubMed Google Scholar) and in cultured bovine adrenal medullary cells (26Stachowiak M.K. Goc A. Hong J.S. Poisner A. Jiang H.K. Stachowiak E.K. Brain Res. Mol. Brain Res. 1994; 22: 309-319Crossref PubMed Scopus (43) Google Scholar). Kilbourne et al. (24Kilbourne E.J. Nankova B.B. Lewis E.J. McMahon A. Osaka H. Sabban D.B. Sabban E.L. J. Biol. Chem. 1992; 267: 7563-7569Abstract Full Text PDF PubMed Google Scholar) have used deletion mutation analysis to show that the region from −60 to +27, which contains the consensus CRE core sequence, is as responsive to veratridine as the region from −773 to +27 in PC12 cells. The construct containing TH gene sequences from −41 to +27 was unresponsive. These authors have interpreted these data to support the participation of the TH CRE (at position −45 to −38) in this veratridine-mediated response. In addition, a construct, in which sequences from −209 to −193 (a region referred to as the FSE) are deleted from TH(−272/+27)-CAT, does not lose responsiveness to veratridine in PC12 cells. Since the TH AP1 site is found within this FSE region, it was concluded that this site does not participate in the response. However, other elements, such as AP2 (−220 to −213), E2A/MyoD (−194 to −189), and POU/Oct (−175 to −168), that may participate positively or negatively in regulating the TH gene are located adjacent to this region. The deletion of this FSE domain alters the normal spacing between these elements. In contrast, Stachowiak et al. (26Stachowiak M.K. Goc A. Hong J.S. Poisner A. Jiang H.K. Stachowiak E.K. Brain Res. Mol. Brain Res. 1994; 22: 309-319Crossref PubMed Scopus (43) Google Scholar) have used deletion analysis of the bovine TH gene promoter to show that regions of the promoter upstream of the CRE are required for the response to veratridine in bovine adrenal chromaffin cells. These regions contain numerous candidate elements, including POU/Oct, SP1, and AP1 sites. From these previous deletion analyses, it is difficult to identify specific promoter elements involved in the response to depolarization, due to the deletion of large segments of the TH gene promoter in the TH-CAT constructs. Furthermore, these deletions alter the normal spacing of elements within the TH gene promoter and eliminate potential interactions between proximal and distal elements.In the present study, we have used a rat TH gene promoter construct similar to that used by Kilbourne et al. (24Kilbourne E.J. Nankova B.B. Lewis E.J. McMahon A. Osaka H. Sabban D.B. Sabban E.L. J. Biol. Chem. 1992; 267: 7563-7569Abstract Full Text PDF PubMed Google Scholar); however, we have employed site-directed mutagenesis to knock out the function of specific sites within the promoter without altering the length of the promoter region. Using this approach, our results suggest that at least two promoter elements participate in the response to Ca2+ influx; these two elements are the CRE and AP1. This conclusion is strongly supported by the 70-80% decrease in response to depolarization, when either the CRE or AP1 site is mutagenized within the context of the intact TH gene promoter.The participation of the TH CRE is also supported by the results of the minimal promoter studies. Treatment of PC12 cells with 50 mM KCl leads to a significant increase in the expression of the minimal promoter construct, THCRE-CAT. This response is modest (about 3-fold) compared with that observed when the cells are treated with a cyclic AMP analog (approximately 15-fold). However, this modest response is perhaps due to the limiting amount of Ca2+ influx that occurs during depolarization with 50 mM KCl, because treatment with 10 μM A23187 (which raises [Ca2+]i to levels more than 3 μM) is associated with more than a 10-fold increase in THCRE-CAT expression.Recently, Nankova et al. (25Nankova B. Hiremagalur B. Menezes A. Zeman R. Sabban E. Mol. Brain Res. 1996; 35: 164-172Crossref PubMed Scopus (36) Google Scholar) have reported a similar study investigating Ca2+-responsive elements within the TH gene promoter using TH-CAT constructs with a mutagenized CRE or AP1 site. Their results with the mutagenized CRE construct agree with our data, indicating that the CRE site plays a significant role in Ca2+-mediated regulation of the TH gene. In contrast to our findings, these investigators do not observe a significant decrease in Ca2+-induced TH-CAT expression when the TH AP1 site is mutagenized. One explanation for this discrepancy may be differences in the number of mutagenized nucleotides within the element. In the mutagenized AP1 construct used in our study, only the first two base pairs of the core AP1 element (TGATTCA) are modified (to GAATTCA). This modification was chosen to minimize its influence on factors binding to the dyad symmetry element that overlaps the 3′ region of the core TH AP1 site (38Yoon S.O. Chikaraishi D.M. Neuron. 1992; 9: 55-67Abstract Full Text PDF PubMed Scopus (137) Google Scholar) or other elements found within this region, such as the FSE (39Gizang-Ginsberg E. Ziff E. Genes Dev. 1990; 4: 477-491Crossref PubMed Scopus (224) Google Scholar) or HIE (40Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The competitive displacement gel shift assays indicate that this 2-bp mutation is sufficient to inhibit the binding of Ca2+-inducible AP1 factors to the mutagenized site (Fig. 1). Furthermore, this 2-bp mutation produces a dramatic decrease in basal promoter activity, which is in agreement with previous studies indicating that the AP1 site or adjacent, overlapping sites participate in controlling basal expression of the gene in rat pheochromocytoma cells. In contrast, the entire core TH AP1 sequence is mutagenized in the construct used by Nankova et al. (25Nankova B. Hiremagalur B. Menezes A. Zeman R. Sabban E. Mol. Brain Res. 1996; 35: 164-172Crossref PubMed Scopus (36) Google Scholar) (mutant 2 in Ref. 38Yoon S.O. Chikaraishi D.M. Neuron. 1992; 9: 55-67Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Since a number of regulatory sequences have been mapped to this region of the gene, it is possible that these different mutations alter the response to Ca2+ due to changes in the binding of activating or repressing proteins that interact with these adjacent sites.An alternative explanation is that the discrepancy is due to differences in the route of Ca2+ influx in the two experiments. Membrane depolarization using high K+ permits Ca2+ influx into PC12 cells via L-type voltage-sensitive calcium channels. 2K. Nagamoto-Combs, K. M. Piech, J. A. Best, B. Sun, and A. W. Tank, unpublished observations. Nankova et al. (25Nankova B. Hiremagalur B. Menezes A. Zeman R. Sabban E. Mol. Brain Res. 1996; 35: 164-172Crossref PubMed Scopus (36) Google Scholar) used ionomycin to promote Ca2+ influx. Hence, the route of Ca2+ entry differs between these two studies. It is possible that CRE and AP1 transcription factors are modulated differently depending upon the route of Ca2+ influx. Support for this hypothesis has been demonstrated for the c-fos gene (41Bading H. Ginty D.D. Greenberg M.E. Science. 1993; 260: 181-186Crossref PubMed Scopus (952) Google Scholar).In order to confirm that the TH AP1 site participates in the response of the promoter to Ca2+ influx, we have performed a number of subsequent studies. Even though the minimal promoter construct, THAP1-CAT, does not respond significantly to 50 mM KCl, a slight, but statistically insignificant, response was observed in each experiment. This lack of significant response is apparently due to either the limited influx of Ca2+ elicited by high K+ treatment or to differences in the route of Ca2+ entry, because treatment of the cells with A23187 elicited a 7-8-fold increase in THAP1-CAT expression. This ability of a single copy of TH AP1 to increase CAT expression upon Ca2+ influx provides evidence that the TH AP1 functions as a Ca2+-responsive element at least under certain stimulus conditions. However, the results using A23187 need to be interpreted with caution, because this ionophore may be producing effects that are not specific to Ca2+ influx.The gel shift assays demonstrate that Ca2+ influx elicited by either depolarization or treatment with A23187 increases the formation of a TH AP1 complex. These results also strongly support the participation of the TH AP1 site in the response to Ca2+ influx. However, it is also possible that unidentified adjacent sites bind to PC12 cell nuclear proteins to form the observed complex. Hence, we used antibodies targeting specific transcription factors to identify the Ca2+-inducible nuclear proteins that bind to the TH AP1 site. These studies have identified c-Fos, c-Jun, JunB, and JunD as members of the inducible TH AP1 complex. Hence, the increased formation of this complex upon Ca2+ influx is due to the increased binding of Fos and Jun family transcription factors to this site, supporting the role of the TH AP1 site in this response.The TH CRE2 is a newly discovered site within the TH gene promoter that is homologous to the proenkephalin gene ENKCRE1 and that is responsive to cyclic AMP in PC12 cells (27Best J.A. Chen Y. Piech K.P. Tank A.W. J. Neurochem. 1995; 65: 1934-1943Crossref PubMed Scopus (32) Google Scholar). The present results suggest that this site does not play a significant role in the response to Ca2+. This conclusion is based on the very modest decrease in response to high K+, when the TH CRE2 is mutagenized within the normal context of the TH gene promoter and the lack of response of the THCRE2 in the minimal promoter studies to either 50 mM KCl or A23187.A current prevailing hypothesis is that Ca2+ influx stimulates genes by activating a calcium/calmodulin- dependent protein kinase, which then phosphorylates and activates CREB, leading to the transactivation of genes downstream of CRE sites. However, our results using the CREB-deficient cell lines suggest that CREB is not necessary for Ca2+-mediated regulation of the TH gene promoter. These results do not rule out CREB as a participant in the response to Ca2+ influx in wild type cells but suggest that other transcription factors can compensate when CREB levels are diminished. In this regard Greenberg and co-workers (42Liu F. Thompson M.A. Wagner S. Greenberg M.E. Green M.R. J. Biol. Chem. 1993; 268: 6714-6720Abstract Full Text PDF PubMed Google Scholar) have shown that the c-fos gene CRE is responsive to Ca2+ via phosphorylation of ATF1. It is also possible that transactivating forms of the CREM gene family may participate in this response (see Ref. 43Sassone-Corssi P. EMBO J. 1994; 13: 4717-4728Crossref PubMed Scopus (68) Google Scholar for review). Both of these factors are still present in the ASCREB cell lines.2 Furthermore, when CREB levels are lowered, regulation via the TH AP1 site may predominate. Further work is needed to elucidate these compensatory mechanisms in the CREB-deficient cell lines. INTRODUCTIONBiosynthesis of the catecholamines is tightly regulated by the activity of the rate-limiting enzyme, tyrosine hydroxylase (TH 1The abbreviations used are: THtyrosine hydroxylase[Ca2+]iintracellular calcium concentrationCATchloramphenicol acetyltransferaseCREBcyclic AMP-responsive element binding proteinCREcyclic AMP-responsive element8-CPT-cAMP8-chlorophenylthio-cyclic AMPPBSphosphate-buffered salineASCREBantisense-CREBbpbase pair(s)RSVRous sarcoma virusCREMcyclic AMP-responsive element-modulating proteinCaREcalcium-responsive elementAP1activating protein-1.; EC 1.14.16.2). Experimental manipulations that lead to long-term stimulation of catecholaminergic cells in sympathetic ganglia and adrenal medulla are associated with increases in TH gene expression (1Thoenen H. Nature. 1970; 228: 861-862Crossref PubMed Scopus (206) Google Scholar, 2Kvetnansky R. Gewirtz G. Weise V.K. Kopin I.J. Am. J. Physiol. 1971; 220: 928-931Crossref PubMed Scopus (70) Google Scholar, 3Tank A.W. Lewis E.J. Chikaraishi D.M. Weiner N. J. Neurochem. 1985; 45: 1030-1033Crossref PubMed Scopus (98) Google Scholar, 4Faucon-Biguet N. Buda M. Lamouroux A. Samolyk D. Mallet J. EMBO J. 1986; 5: 287-291Crossref PubMed Scopus (183) Google Scholar, 5Fossom L.H. Carlson C.D. Tank A.W. Mol. Pharmacol. 1991; 40: 193-202PubMed Google Scholar, 6Richard F. Faucon-Biguet N. Labatut R. Rollet D. Mallet J. Buda M. J. Neurosci. Res. 1988; 20: 32-37Crossref PubMed Scopus (119) Google Scholar, 7Mallet J. Faucon-Biguet N. Buda M. Lamouroux A. Samolyk D. Cold Spring Harbor Symp. Quant. Biol. 1983; 48: 305-308Crossref PubMed Google Scholar, 8Fossom L.H. Sterling C.R. Tank A.W. Mol. Pharmacol. 1992; 42: 898-908PubMed Google Scholar). The mechanisms responsible for regulation of the TH gene are complex and have not been fully elucidated. Cultured rat pheochromocytoma cells have been used extensively to study the underlying mechanisms for TH gene regulation. Using this model system, a number of laboratories have shown that cAMP analogs (9Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 486-496PubMed Google Scholar, 10Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 497-503PubMed Google Scholar, 11Lewis E.J. Tank A.W. Weiner N. Chikaraishi D.M. J. Biol. Chem. 1983; 258: 14632-14637Abstract Full Text PDF PubMed Google Scholar), active phorbol esters (12Vyas S. Faucon-Biguet N. Mallet J. EMBO J. 1990; 9: 3707-3712Crossref PubMed Scopus (92) Google Scholar), and glucocorticoids (8Fossom L.H. Sterling C.R. Tank A.W. Mol. Pharmacol. 1992; 42: 898-908PubMed Google Scholar, 9Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 486-496PubMed Google Scholar, 10Tank A.W. Curella P. Ham L. Mol. Pharmacol. 1986; 30: 497-503PubMed Google Scholar, 13Lewis E.J. Harrington C.A. Chikaraishi D.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3550-3554Crossref PubMed Scopus (299) Google Scholar) induce TH mRNA, stimulate TH gene transcription rate, and/or activate TH gene promoter activity. These results are consistent with the hypothesis that protein kinase A, protein kinase C, and glucocorticoid receptors participate in signaling pathways that regulate the TH gene.Treatments that lead to increased intracellular Ca2+ concentration ([Ca2+]i) also increase expression of the TH gene. Studies by Sabban and co-workers (14Kilbourne E.J. Sabban E.L. Mol. Brain. Res. 1990; 8: 121-127Crossref PubMed Scopus (52) Google Scholar, 15Hiremagalur B. Nankova B. Nitahara J. Zeman R. Sabban E.L. J. Biol. Chem. 1993; 268: 23704-23711Abstract Full Text PDF PubMed Google Scholar) have shown that membrane depolarization or agonist occupation of nicotinic cholinergic receptors leads to the induction of TH mRNA in PC12 cells. Both of these treatments lead to Ca2+ influx through voltage-sensitive Ca2+ channels. Furthermore, chelation of extracellular Ca2+ with EGTA inhibits the depolarization-mediated increase in TH mRNA (14Kilbourne E.J. Sabban E.L. Mol. Brain. Res. 1990; 8: 121-127Crossref PubMed Scopus (52) Google Scholar). These results suggest that the influx of Ca2+ plays an important role in regulating TH gene expression.A number of studies have been conducted in order to understand the mechanisms that mediate Ca2+- dependent regulation of different genes. Ca2+ influx elicited by either membrane depolarization or Ca2+ ionophore treatment activates and/or induces transcription factors, such as cAMP-responsive element binding protein (CREB) and c-Fos, in PC12 cells (16Greenberg M.E. Ziff E.B. Greene L.A. Science. 1986; 234: 80-83Crossref PubMed Scopus (566) Google Scholar, 17Morgan J.I. Curran T. Nature. 1986; 322: 552-555Crossref PubMed Scopus (825) Google Scholar, 18Bartel D.P. Sheng M. Lau L.F. Greenberg M.E. 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Since the 5′-flanking region of the TH gene contains CRE and AP1 sites, it is reasonable to hypothesize that Ca2+ exerts its effects on TH gene transcription rate by influencing transcription factors that bind to these promoter elements. Kilbourne et al. (24Kilbourne E.J. Nankova B.B. Lewis E.J. McMahon A. Osaka H. Sabban D.B. Sabban E.L. J. Biol. Chem. 1992; 267: 7563-7569Abstract Full Text PDF PubMed Google Scholar) have shown that a minimal promoter containing the TH CRE is responsive to Ca2+ influx in PC12 cells. Using deletion analysis, they have also reported that promoter elements upstream of the CRE do not apparently participate in the response of the TH gene promoter to Ca2+. Recently, these results have been extended using TH gene constructs with site-directed mutations (25Nankova B. Hiremagalur B. Menezes A. Zeman R. Sabban E. Mol. Brain Res. 1996; 35: 164-172Crossref PubMed Scopus (36) Google Scholar). Results from these studies suggest that the TH AP1 site is not involved in Ca2+-mediated TH gene regulation. In contrast, deletion analysis by Stachowiak et al. (26Stachowiak M.K. Goc A. Hong J.S. Poisner A. Jiang H.K. Stachowiak E.K. Brain Res. Mol. Brain Res. 1994; 22: 309-319Crossref PubMed Scopus (43) Google Scholar) has shown that the TH CRE is not required for activation of the TH gene promoter by Ca2+ influx in bovine adrenal chromaffin cells and that, by inference, promoter elements upstream of the CRE participate in this response. However, it is not clear from their study which promoter elements participate in this regulation.In the present study, we demonstrate that both the CRE and AP1 sites in the proximal 5′-flanking region of the rat TH gene participate in regulating TH gene expression by Ca2+ influx in PC12 cells. The newly identified TH CRE2 site does not apparently play a major role in this response. To further analyze the involvement of the TH AP1 site in Ca2+-mediated TH gene regulation, we have examined nuclear protein interactions at this site. Finally, we have tested the involvement of CREB in Ca2+-mediated TH gene regulation using PC12 cell-derived CREB-deficient cell lines. Our results indicate that CREB is not required for Ca2+- dependent regulation of the TH gene, supporting the hypothesis that multiple promoter elements are involved in regulation of the TH gene promoter in response to Ca2+ influx.
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