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The Signal Transduction Pathway Underlying Ion Channel Gene Regulation by Sp1-c-Jun Interactions

2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês

10.1074/jbc.m010735200

1083-351X

Irena N. Melnikova, Paul Gardner,

Neuroscience and Neuropharmacology Research

During neuronal differentiation, an exquisitely controlled program of signal transduction events takes place, leading to the temporally and spatially regulated expression of genes associated with the differentiated phenotype. A critical class of genes involved in this phenomenon is that made up of genes encoding neurotransmitter-gated ion channels that play a central role in signal generation and propagation within the nervous system. We used the well established PC12 cell line to investigate the molecular details underlying the expression of the neuronal nicotinic acetylcholine receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in neuronal nicotinic acetylcholine receptor gene expression. Nerve growth factor initiates several signal transduction cascades. Here, we show that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the nerve growth factor-mediated increase in the transcriptional activity of a neuronal nicotinic acetylcholine receptor gene promoter. In addition, we show that a component of the Ras-dependent mitogen-activated protein kinase pathway, nerve growth factor-inducible c-Jun, exerts its effects on receptor gene promoter activity most likely through protein-protein interactions with Sp1. Finally, we demonstrate that the target for nerve growth factor signaling is an Sp1-binding site within the neuronal nicotinic acetylcholine receptor gene promoter. During neuronal differentiation, an exquisitely controlled program of signal transduction events takes place, leading to the temporally and spatially regulated expression of genes associated with the differentiated phenotype. A critical class of genes involved in this phenomenon is that made up of genes encoding neurotransmitter-gated ion channels that play a central role in signal generation and propagation within the nervous system. We used the well established PC12 cell line to investigate the molecular details underlying the expression of the neuronal nicotinic acetylcholine receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in neuronal nicotinic acetylcholine receptor gene expression. Nerve growth factor initiates several signal transduction cascades. Here, we show that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the nerve growth factor-mediated increase in the transcriptional activity of a neuronal nicotinic acetylcholine receptor gene promoter. In addition, we show that a component of the Ras-dependent mitogen-activated protein kinase pathway, nerve growth factor-inducible c-Jun, exerts its effects on receptor gene promoter activity most likely through protein-protein interactions with Sp1. Finally, we demonstrate that the target for nerve growth factor signaling is an Sp1-binding site within the neuronal nicotinic acetylcholine receptor gene promoter. nerve growth factor ras-dependent mitogen-activated protein kinase phosphoinositide 3-kinase extracellular signal-regulated kinase MAPK/ERK kinase acetylcholine neuronal nicotinic acetylcholine receptor base pair Nerve growth factor (NGF)1 is critical for the survival and differentiation of sensory and sympathetic neurons in the peripheral nervous system (1Kaplan D. Zirrgiebel U. Atwal J. Neuron. 2000; 25: 253-254Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) and of basal forebrain and hippocampal cholinergic neurons in the central nervous system (2Ruberti F. Capsoni S. Comparini A. Di Daniel E. Franzot J. Gonfloni S. Rossi G. Berardi N. Cattaneo A. J. Neurosci. 2000; 20: 2589-2601Crossref PubMed Google Scholar). NGF signals through binding to its receptors, a high affinity tyrosine kinase receptor, TrkA, and a low affinity receptor, p75 (3Friedman W.J. Greene L.A. Exp. Cell Res. 1999; 253: 131-142Crossref PubMed Scopus (312) Google Scholar). Binding of NGF to its receptors initiates several signal transduction cascades, including, among others, ras-dependent mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3-K), and the cAMP-dependent protein kinase pathways (3Friedman W.J. Greene L.A. Exp. Cell Res. 1999; 253: 131-142Crossref PubMed Scopus (312) Google Scholar). Some of the downstream targets of the activated protein kinase pathways are transcription factors such as AP-1, cAMP responsive element-binding protein, and NF-κB. Rat pheochromocytoma PC12 cells have been extensively used to study the molecular mechanisms involved in NGF signaling. PC12 cells are chromaffin-like cells that in response to NGF treatment withdraw from the cell cycle and differentiate into sympathetic-like neurons, a process accompanied by neurite outgrowth, increased electrical excitability, and changes in neurotransmitter synthesis (4Dichter M.A. Tischler A.S. Greene L.A. Nature. 1977; 268: 501-504Crossref PubMed Scopus (288) Google Scholar, 5Greene L.A. Tischler A.S. Adv. Cell. Neurobiol. 1982; 3: 373-414Crossref Google Scholar, 6Halegoua S. Armstrong R.C. Kremer N.E. Curr. Top. Microbiol. Immunol. 1991; 165: 119-170PubMed Google Scholar). Among the signal transduction cascades activated by NGF in PC12 cells, the MAPK and the PI3-K pathways are thought to play a central role, although other pathways are clearly important (7Kalman D. Wong B. Horvai A.E. Cline M.J. O'Lague P.H. Neuron. 1990; 4: 355-366Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 8Fanger G.R. Erhardt P. Cooper G.M. Maue R.A. J. Neurochem. 1993; 61: 1977-1980Crossref PubMed Scopus (27) Google Scholar, 9Obermeier A. Bradshaw R.A. Seedorf K. Choidas A. Schlessinger J. Ullrich A. EMBO J. 1994; 13: 1585-1590Crossref PubMed Scopus (268) Google Scholar, 10Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar). Activated TrkA promotes conversion of the membrane-anchored Ras-GDP into Ras-GTP. Ras then cooperates in activation of a serine/threonine kinase, Raf-1, which phosphorylates the dual specificity MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK), which in turn phosphorylates and activates the ERKs, ERK1 and ERK2. Recently, it was demonstrated that activation of ERK in response to NGF in both primary sensory neurons and in PC12 cells also requires activation of the PI3-K pathway (11York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (761) Google Scholar, 12York R.D. Molliver D.C. Grewal S.S. Stenberg P.E. McCleskey E.W. Stork P.J. Mol. Cell. Biol. 2000; 20: 8069-8083Crossref PubMed Scopus (204) Google Scholar). PI3-K is required for internalization of TrkA and for its signaling to ERK via a Ras family member, Rap 1 (11York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (761) Google Scholar, 12York R.D. 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Activated ERKs translocate to the nucleus, where they phosphorylate and activate the existing transcription factor c-Jun, as well as induce expression of c-Jun (20Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1998; 17: 4404-4413Crossref PubMed Scopus (291) Google Scholar). Interestingly, overexpression of activated c-Jun in PC12 cells induces neurite outgrowth, suggesting that c-Jun plays an important role in promoting PC12 neural differentiation (20Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1998; 17: 4404-4413Crossref PubMed Scopus (291) Google Scholar). Among the neuronal genes whose expression is up-regulated in response to NGF in PC12 cells are the genes encoding several subunits (α3, α5, α7, β2, and β4) of neuronal nicotinic acetylcholine receptors (nAChRs) (21Henderson L.P. Gdovin M.J. Liu C. Gardner P.D. Maue R.A. J. Neurosci. 1994; 14: 1153-1163Crossref PubMed Google Scholar, 22Boyd R.T. Neurosci. Lett. 1996; 208: 73-76Crossref PubMed Scopus (18) Google Scholar, 23Nakayama H. Ueno S. Ikeuch I.T. Hatanaka H. J. Neurochem. 2000; 74: 1346-1354Crossref PubMed Scopus (10) Google Scholar). nAChRs are pentameric ligand-gated ion channels important for synaptic transmission in the nervous system (24Cordero-Erausquin M. Marubio L.M. Klink R. Changeux J.P. Trends Pharmacol. Sci. 2000; 21: 211-217Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Previously, we demonstrated that the NGF-mediated increase in nAChR gene expression is independent of cAMP-dependent protein kinase signaling (21Henderson L.P. Gdovin M.J. Liu C. Gardner P.D. Maue R.A. J. Neurosci. 1994; 14: 1153-1163Crossref PubMed Google Scholar) and that NGF treatment of PC12 cells increases the transcriptional activity of the β4 gene promoter (25Hu M. Whiting-Theobald N.L. Gardner P.D. J. Neurochem. 1994; 62: 392-395Crossref PubMed Scopus (24) Google Scholar). In this study we investigated the involvement of the other NGF-signaling pathways, namely MAPK, PI3-K, protein kinase C, and phospholipase C, in the regulation of nAChR gene expression. We show that in PC12 cells, activation of PI3-K and one of the components of the MAPK pathway, MEK, is important for up-regulation of β4 subunit gene promoter activity in response to NGF. We also show that NGF-inducible c-Jun can transactivate the promoter of the β4 gene through cooperation with Sp1 protein bound to the β4 promoter at a previously identified Sp1-binding site, a CA box. We further demonstrate that this Sp1-binding site is necessary for the responsiveness of the β4 promoter to NGF. SN17 cells (26Hammond D.N. Lee H.J. Tonsgard J.H. Wainer B.H. Brain Res. 1989; 512: 190-200Crossref Scopus (89) Google Scholar) andDrosophila melanogaster Schneider SL2 cells were maintained as described (27Liu Q. Melnikova I.N. Hu M. Gardner P.D. J. Neurosci. 1999; 19: 9747-9755Crossref PubMed Google Scholar, 28Du Q. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1998; 273: 19877-19883Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). PC12 cells (29Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar) were cultured and differentiated with NGF (Upstate Biotechnology, Lake Placid, NY) as previously described (25Hu M. Whiting-Theobald N.L. Gardner P.D. J. Neurochem. 1994; 62: 392-395Crossref PubMed Scopus (24) Google Scholar). Twenty-four h prior to transfections, SN17 cells were plated at a density of 250,000 cells per 35-mm dish. Transfections were performed by a calcium phosphate method using a commercially available kit (Eppendorf-5 Prime, Inc., Boulder, CO). The wild type rat β4-luciferase expression plasmid, pX1B4FH, containing a 226-bpFokI/HindIII fragment spanning nucleotides −89 to +137, relative to the β4 transcription initiation site, and the construct pX1B4FHmut4, containing a mutation in the CA box of the β4 promoter, were described previously (25Hu M. Whiting-Theobald N.L. Gardner P.D. J. Neurochem. 1994; 62: 392-395Crossref PubMed Scopus (24) Google Scholar, 30Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Cells were transfected with 2.5 μg of test DNA (pX1B4FH or pX1B4FHmut4), 2.5 μg of effector DNA (the empty pCMV5 vector (Invitrogen, Carlsbad, CA) or pCMV-jun constructs), and 2.5 μg of a β-galactosidase expression vector, RSV-βgal. In some cases, no effector DNA was included in the transfections. To ensure that the calcium phosphate/DNA precipitates had equal amounts of DNA, appropriate quantities of pBluescript II SK DNA (Stratagene, La Jolla, CA) were added to each sample. PC12 cells were transfected in 60-mm dishes at a density of 106cells/ml using 30 μl of LipofectAMINE (2 mg/ml; Life Technologies, Inc.) and 2.5 μg of each DNA. After a 5-h incubation in the lipid/DNA mix, cells were washed twice with Dulbecco's modified Eagle's medium and fed with growth medium. For experiments involving the pharmacological kinase inhibitors, growth media were supplemented with or without 100 ng/ml NGF and with or without inhibitors, as indicated. All inhibitors were purchased from Calbiochem (San Diego, CA) and were used at the following concentrations: MEK inhibitor PD98059 at 100 μm (31Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar), PI3-K inhibitor LY294002 at 50 μm (12York R.D. Molliver D.C. Grewal S.S. Stenberg P.E. McCleskey E.W. Stork P.J. Mol. Cell. Biol. 2000; 20: 8069-8083Crossref PubMed Scopus (204) Google Scholar, 32Zheng W.-H. Kar S. Quirion R. J. Biol. Chem. 2000; 275: 39152-39158Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), phospholipase C inhibitor U-73122 at 1 μm (33Kolkova K. Novitskaya V. Pedersen N. Berezin V. Bock E. J. Neurosci. 2000; 20: 2238-2246Crossref PubMed Google Scholar), and protein kinase C inhibitors calphostin C (33Kolkova K. Novitskaya V. Pedersen N. Berezin V. Bock E. J. Neurosci. 2000; 20: 2238-2246Crossref PubMed Google Scholar) and bisinodylmalemide (BIM) (34Melikina H.E. Buckley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar) at 400 nm and 1 μm, respectively. SL2 cells were transfected as previously described (28Du Q. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1998; 273: 19877-19883Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), except that 100 ng of the effector DNAs (pActSp1 or pPacJun) were used. The pPacJun plasmid was a kind gift of Dr. J. Noti and is described elsewhere (35Noti J.D. J. Biol. Chem. 1997; 272: 24038-24045Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Forty-eight h following transfection, cells were harvested and assayed for luciferase activity using a commercially available kit (Promega Corp., Madison, WI) and an Autolumat LB953 luminometer (EG&G Berthold, Gaithersburg, MD). All transfections were done a minimum of two times with two different preparations of plasmid DNAs. To correct for differences in transfection efficiencies between dishes, the luciferase activity in each sample was normalized to the β-galactosidase activity in the same sample, which was measured using a commercially available kit (Galacto-Light; Tropix, Inc., Bedford, MA). Immunoprecipitations and Western blotting were performed as previously described (36Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) using 250 μg of nuclear extracts prepared from NGF-treated PC12 cells. Anti-c-Jun rabbit polyclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Nuclear extracts were prepared by the method of Dignam et al. (37Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) as previously described (38Melnikova I.N. Christy B.A. Cell Growth Differ. 1996; 7: 1067-1079PubMed Google Scholar), except that nuclear extracts were dialyzed against RIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1%@ sodium dodecyl sulfate). NGF-dependent up-regulation of neuronal nAChR gene expression in PC12 cells is thought to occur partly at the level of transcription (25Hu M. Whiting-Theobald N.L. Gardner P.D. J. Neurochem. 1994; 62: 392-395Crossref PubMed Scopus (24) Google Scholar). To investigate which of the NGF-activated pathways is involved in the transcriptional regulation of neuronal nAChR gene expression in PC12 cells, we examined the effects of blocking several of these pathways on the transcriptional activity of the β4 gene promoter. We previously identified a 226-bp promoter fragment of the β4 gene that can confer neuron-specific expression to a reporter gene in transient transfection assays (27Liu Q. Melnikova I.N. Hu M. Gardner P.D. J. Neurosci. 1999; 19: 9747-9755Crossref PubMed Google Scholar). We have also demonstrated that treatment of PC12 cells with NGF results in a significant increase in β4 promoter activity and that the 226-bp region is sufficient to mediate this response (25Hu M. Whiting-Theobald N.L. Gardner P.D. J. Neurochem. 1994; 62: 392-395Crossref PubMed Scopus (24) Google Scholar,27Liu Q. Melnikova I.N. Hu M. Gardner P.D. J. Neurosci. 1999; 19: 9747-9755Crossref PubMed Google Scholar). We therefore investigated whether MEK, PI3-K, protein kinase C, or phospholipase C is involved in the regulation of transcription of the β4 gene through elements in the 5′ regulatory region of the gene. The 226-bp β4 fragment fused upstream of the luciferase gene (pX1B4FH) was transiently transfected into PC12 cells. Following transfection cells were treated with NGF for 36 h. A parallel set of transfected cells was left untreated and served as a control. Consistent with our previous results, 36 h of NGF treatment stimulated β4 promoter activity and resulted in induction of reporter gene activity (Fig. 1). When NGF treatment of the transfected cells was carried out in the presence of PD98059, a selective pharmacological agent that blocks activation of MEK, or LY294002, a specific pharmacological inhibitor of PI3-K, stimulation of the β4 promoter was significantly reduced, as judged by reporter gene activity (Fig. 1). In contrast, inhibitors of phospholipase C, U-73122, and protein kinase C, calphostin C, and bisinodylmalemide, had little effect on the activity of the β4 promoter in transfected cells in response to NGF (Fig. 1). These results suggest that activation of the β4 promoter in response to NGF is mediated in part via the MAPK- and PI3-K-dependent pathways in PC12 cells. This is consistent with a model suggesting that these two pathways share overlapping functions in neuronal cells (12York R.D. Molliver D.C. Grewal S.S. Stenberg P.E. McCleskey E.W. Stork P.J. Mol. Cell. Biol. 2000; 20: 8069-8083Crossref PubMed Scopus (204) Google Scholar). In addition, these data indicate that the 226-bp fragment of the β4 promoter contains the necessary elements to mediate the response to ERK activation, a common downstream target of MEK and PI3-K (12York R.D. Molliver D.C. Grewal S.S. Stenberg P.E. McCleskey E.W. Stork P.J. Mol. Cell. Biol. 2000; 20: 8069-8083Crossref PubMed Scopus (204) Google Scholar). It is well established that in PC12 cells, activation of the ERK cascade by NGF induces c-Jun expression and phosphorylation of its gene product (20Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1998; 17: 4404-4413Crossref PubMed Scopus (291) Google Scholar). c-Jun is a transcription factor that has been implicated in regulation of gene expression in response to multiple extracellular stimuli (39Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar). To address the question whether c-Jun is capable of regulating the activity of the β4 promoter, PC12 cells were transiently transfected with the 226-bp β4 promoter-luciferase construct, pX1B4FH, alone or in combination with an expression construct for c-Jun, pCMV-jun, in which the c-Jun gene is under the control of the CMV promoter, or with an empty parental vector, pCMV5. As shown in Fig.2 A (left side), transfections of the empty expression vector pCMV5 had no effect on reporter gene activity. However, when the β4-luciferase construct was cotransfected with c-Jun, a dramatic increase in reporter gene activity was observed (Fig. 2 A). Similar results were observed in the cholinergic cell line SN17 (Fig. 2 B). Together, these data indicate that, indeed, c-Jun is capable of transactivating the β4 promoter and suggest that c-Jun-responsive elements are present within this 226-bp fragment of the β4 promoter. Jun proteins bind to the DNA sequence 5′-TGACTCA-3′, referred to as an AP-1 site, as homodimers or as heterodimers with Fos proteins (39Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar). Interestingly, sequence analysis of the β4 promoter fragment used in these experiments indicates that there are no AP-1-binding sites within this region. It is therefore possible that c-Jun transactivates the β4 promoter through protein-protein interactions with a factor that can bind to β4 regulatory elements. c-Jun has been shown to functionally interact with a number of transcription factors, including members of the Sp family, Sp1 and Sp3 (35Noti J.D. J. Biol. Chem. 1997; 272: 24038-24045Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 40McDonough P.M. Hanford D.S. Sprenkle A.B. Mellon N.R. Glembotski C.C. J. Biol. Chem. 1997; 272: 24046-24053Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 41Kardassis D. Papakosta P. Pardali K. Moustakas A. J. Biol. Chem. 1999; 274: 29572-29581Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 42Falvo J.V. Uglialoro A.M. Brinkman B.M. Merika M. Parekh B.S. Tsai E.Y. King H.C. Morielli A.D. Peralta E.G. Maniatis T. Thanos D. Goldfeld A.E. Mol. Cell. Biol. 2000; 20: 2239-2247Crossref PubMed Scopus (146) Google Scholar). Moreover, c-Jun can directly interact with Sp1 in vitro(41Kardassis D. Papakosta P. Pardali K. Moustakas A. J. Biol. Chem. 1999; 274: 29572-29581Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). This is particularly relevant to the β4 promoter, because we previously reported that both Sp1 and Sp3 can efficiently transactivate this promoter (28Du Q. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1998; 273: 19877-19883Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 30Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 36Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Therefore, it is possible that c-Jun activates the β4 promoter through protein-protein interactions with Sp1 and/or Sp3. To test this hypothesis, we first wanted to address the question of whether c-Jun and Sp1 are physically associated in PC12 cells. To that end, we performed immunoprecipitation experiments followed by Western blot analysis. Anti-Sp1 or anti-c-Jun antiserum was used to immunoprecipitate proteins from nuclear extracts prepared from NGF-treated PC12 cells. The precipitated proteins were separated via SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane that was subsequently incubated with anti-Sp1 or anti-c-Jun antiserum. As shown in Fig.3, preimmune serum did not precipitate either Sp1 or c-Jun in this experiment. However, complexes containing Sp1 and c-Jun were detected by immunoprecipitations using antibodies directed toward one or the other protein (Fig. 3). It should be noted that the levels of Sp1 found in c-Jun immunoprecipitates were somewhat lower when compared with those precipitated with anti-Sp1 antibody (Fig. 3), suggesting that not all cellular Sp1 is engaged in a complex with c-Jun. The same was true with c-Jun as well (Fig. 3). These data provide compelling evidence for direct physical interactions between Sp1 and c-Jun in PC12 cells and support the idea that c-Jun may regulate the β4 promoter through its association with Sp1. Previously, we showed that Sp1 can specifically bind to a CA box in the 226-bp region of the β4 promoter (30Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 36Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and that this interaction is critical for Sp-mediated transactivation of the β4 promoter (36Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). If c-Jun activates the β4 promoter through its association with Sp1, then the Sp1-binding site within this promoter, the CA box, should be important for the ability of c-Jun to regulate the β4 promoter. To test this hypothesis, PC12 cells were cotransfected with a wild type β4 promoter-luciferase expression construct, pX1B4FH, or with a construct in which the CA box is mutated at three nucleotide positions, pX1B4FHmut4, and with an expression construct for c-Jun, pCMV-jun. As mentioned earlier, c-Jun was able to strongly transactivate the wild type β4 promoter, pX1B4FH (Fig.2 A). However, when the β4 promoter-luciferase reporter containing mutations in the CA box, pX1B4FHmut4, was used in this experiment, c-Jun activation of the promoter was marginal (Fig.2 A). Similar results were obtained from transfections performed in SN17 cells (Fig. 2 B). Thus, the CA box appears to be critical for c-Jun-mediated transactivation of the β4 promoter. To further investigate the importance of c-Jun-Sp1 interactions for the transactivation of the β4 promoter, we performed transfection experiments using theDrosophila SL2 cell line. These cells lack endogenous Sp1 activity (43Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar, 44Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 45Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Crossref PubMed Scopus (356) Google Scholar, 46Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (657) Google Scholar), as well as endogenous c-Jun activity (35Noti J.D. J. Biol. Chem. 1997; 272: 24038-24045Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), making them a useful system to study the functional consequences of the protein-protein interactions mentioned above. We previously reported that Sp1 can strongly transactivate the β4 promoter when transfected into SL2 cells (28Du Q. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1998; 273: 19877-19883Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 30Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 36Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To determine whether c-Jun activates the β4 promoter through its interactions with Sp1, SL2 cells were cotransfected with a wild type β4 promoter-luciferase expression construct (pX1B4FH) and pActSp1 or pPacJun or both. To confirm that Sp1 and c-Jun expression was involved in transactivation, the reporter DNA was also transfected with the parental vectors devoid of Sp1 and c-Jun coding sequences, pAct and pPacO, alone. As expected, Sp1 was capable of transactivating the wild type β4 promoter-luciferase construct ∼20-fold (Fig. 4). Consistent with the observation that the β4 promoter fragment used in these experiments does not contain an AP-1-binding site, c-Jun by itself did not have any effect on reporter gene activity (Fig. 4). However, when Sp1 and c-Jun were cotransfected with the β4 promoter-luciferase construct, an ∼120-fold increase in reporter gene activity was observed, indicating a synergistic effect of the two regulatory factors (Fig. 4). These results are consistent with the hypothesis that c-Jun activates the β4 promoter through direct interactions with Sp1. To investigate the importance of the Sp1-binding site for the ability of Sp1 and c-Jun to synergistically activate the β4 promoter, similar transfection experiments were performed using a β4 promoter-luciferase construct in which the CA box is mutated at three nucleotide positions (pX1B4FHmut4). Mutation in this site reduces the ability of Sp1 to transactivate the β4 promoter in SL2 cells (Fig. 4) (36Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstr