Protein Kinase Inhibitor H7 Blocks the Induction of Immediate-Early Genes zif268 and c-fosby a Mechanism Unrelated to Inhibition of Protein Kinase C but Possibly Related to Inhibition of Phosphorylation of RNA Polymerase II
1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês
10.1074/jbc.274.15.10430
ISSN1083-351X
AutoresEiko Kumahara, Tatsuhiko Ebihara, David Saffen,
Tópico(s)Cytokine Signaling Pathways and Interactions
Resumo1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7) has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes,zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca2+ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase (MAPK), an upstream activator of zif268 and c-fos gene expression. H7 also does not inhibit preactivated MAPK in vitro. Taken together, these results suggest that H7 blocks gene expression by acting at a site downstream from MAPK. H7 has previously been shown to block transcription in vitro by blocking the phosphorylation of the carboxyl-terminal domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R., Egly, J.-M., and Bentley, D. L.(1995) J. Biol. Chem. 270, 23922–23925). In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7) has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes,zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca2+ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase (MAPK), an upstream activator of zif268 and c-fos gene expression. H7 also does not inhibit preactivated MAPK in vitro. Taken together, these results suggest that H7 blocks gene expression by acting at a site downstream from MAPK. H7 has previously been shown to block transcription in vitro by blocking the phosphorylation of the carboxyl-terminal domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R., Egly, J.-M., and Bentley, D. L.(1995) J. Biol. Chem. 270, 23922–23925). In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation. nerve growth factor phorbol 12-myristate, 13-acetate protein kinase C protein kinase A mitogen-activated protein kinase serum response element cAMP response element carboxyl-terminal domain (of RNA polymerase II) transcription factor IIH [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide N-[2-(p-bromocinnamino)ethyl]-5-isoquinolinesulfonamide 5, 6-dicholoro-1-β-d-ribofuranosylbenzidazole 4-morpholinepropanesulfonic acid [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide] chloramphenicol acetyltransferase Dulbecco's modified Eagle's medium MAP kinase/ERK-activating kinase phenylmethanesulfonyl fluoride The immediate-early genes zif268 (also termedNGFI-A, egr-1, krox24, TIS8; reviewed in Ref. 1Gashler A. Sukhatme V.P. Prog. Nucleic Acid Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (559) Google Scholar) and c-fos (2$$Google Scholar) encode transcription factors that have been proposed to function as "third messengers" in intracellular signal transduction cascades that convert information conveyed by extracellular stimuli into genomic responses that underlie growth, differentiation, and long term changes in the behavior of cells (3Beckmann A.M. Wilce P.A. Neurochem. Int. 1997; 31: 477-510Crossref PubMed Scopus (280) Google Scholar, 4Herrera D.G. Robertson H.A. Prog. Neurobiol. (New York). 1996; 50: 83-107Crossref PubMed Scopus (573) Google Scholar). We have previously shown that NGF1 (1Gashler A. Sukhatme V.P. Prog. Nucleic Acid Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (559) Google Scholar) and the carbachol (carbamylcholine) cause the rapid induction of zif268mRNA in PC12D cells (5Ebihara T. Saffen D. J. Neurochem. 1997; 68: 1001-1010Crossref PubMed Scopus (32) Google Scholar). Induction of zif268mRNA by NGF is mediated by the high affinity NGF receptor, TrkA, which activates the Ras/MAPK cascade (6Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (908) Google Scholar). Induction by carbachol is mediated by the m1 subtype of muscarinic acetylcholine receptor, which activates phospholipase C to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (5Ebihara T. Saffen D. J. Neurochem. 1997; 68: 1001-1010Crossref PubMed Scopus (32) Google Scholar). Increased intracellular levels of inositol 1,4,5-trisphosphate trigger the release of Ca2+ from internal stores, which in turn opens "capacitative influx" Ca2+ channels in the cell membrane, resulting in a sustained influx of extracellular Ca2+. 2F.-F. Guo, T. Ebihara, and D. Saffen, D., manuscript in preparation. Increased levels of diacylglycerol activate PKC. Both the sustained increase in intracellular Ca2+ and the activation of PKC contribute to the induction of zif268 mRNA (5Ebihara T. Saffen D. J. Neurochem. 1997; 68: 1001-1010Crossref PubMed Scopus (32) Google Scholar), at least in part by activating the MAPK cascade (81Kumahara E. Ebihara T. Saffen D. J. Biochem. (Tokyo). 1999; (in press): 125Google Scholar). Activation of the MAPK cascade is therefore a common element in the intracellular signaling events leading to gene expression that are initiated by NGF and carbachol in PC12D cells. In the course of investigating the involvement of PKC in the induction of zif268 mRNA by NGF and carbachol, we compared the effects of pretreating PC12D cells with the protein kinase inhibitor H7 (7Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2329) Google Scholar) with pretreatment of the cells with the related compound HA1004 (8Asano T. Hidaka H. J. Pharmacol. Exp. Ther. 1984; 231: 141-145PubMed Google Scholar). Both H7 and HA1004 are broad spectrum protein kinase inhibitors, but H7 inhibits PKC more potently than HA1004 (K i values = 6 and 40 μm for H7 and HA1004, respectively) in in vitro assays (7Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2329) Google Scholar). Based upon this difference, many investigators have used these inhibitors in combination to evaluate the role of PKC in various cellular processes, including the induction of gene expression. In many of these studies, inhibition by H7 in the absence of inhibition by HA1004 was taken as evidence for a role for PKC in the process under investigation. The data presented in this paper, however, shows that inhibition of gene expression by H7 does not necessarily imply that PKC is involved. Rather, we found that although H7 potently inhibits the induction ofzif268 and c-fos mRNAs following activation of PKC with phorbol ester, it fails to prevent activation of MAPK by phorbol ester. This shows that H7 can block the induction of gene expression without blocking PKC. Examination of the literature indicates that H7 blocks the induction of a broad spectrum of rapidly inducible genes by a variety of stimuli, including stimuli not previously associated with the activation of PKC. These observations suggest that H7 may block a site, different from PKC, that is universally required for the induction of rapidly inducible genes. A previous report that H7 blocks transcriptionin vitro by inhibiting the phosphorylation of RNA polymerase II by a TFIIH-associated kinase (9Yankulov K. Yamashita K. Roy R. Egly J.-M. Bentley D.L. J. Biol. Chem. 1995; 270: 23922-23925Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), led us to examine the effect of H7 on phosphorylation of RNA polymerase II in vivo. In the present study we show that pretreatment of PC12D cells with H7 significantly reduces levels of phosphorylated RNA polymerase IIin vivo, suggesting that H7 blocks gene expression by directly inhibiting transcription. H7 was purchased from Seikagaku Kogyo and Calbiochem. This H7 is the authentic compound originally described by Hidaka et al. (7Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2329) Google Scholar) and not the less potent iso-H7, which has sometimes been sold under the H7 label (10Quick J. Ware J.A. Driedger P.E. Biochem. Biophys. Res. Commun. 1992; 187: 657-663Crossref PubMed Scopus (57) Google Scholar). HA1004, H8, and H89 were from Seikagaku Kogyo. NGF, carbachol, ATP, chloramphenicol, Ca2+ ionophore A23187, thapsigargin, forskolin, dideoxyforskolin, and dimethyl sulfoxide were purchased from Wako Chemical Industries. K252a and staurosporin were from Kyowa Medex Co., Inc. GF109203x, PD098059, and 5,6-dicholoro-1-β-d-ribofuranosylbenzidazole (DRB) were purchased from Calbiochem. PMA, 4-α-PMA, acetyl-CoA, andS-acetyl-coenzyme A synthetase were from Sigma. [γ-32P]ATP and [α-32P]dCTP were obtained from Amersham Pharmacia Biotech and [3H]sodium acetate was from NEN Life Science Products. Anti-Erk-1 and anti-Erk-2 antibodies were obtained from Santa Cruz Biotechnology. Restriction enzymes and other reagents for modification of DNA were obtained from Toyobo Corp., Takara Shuzo Co., and New England Biolabs. Murinezif268 cDNA (ATCC number 63027), murine c-fos genomic DNA (ATCC number 41041), and the expression vector pBLCAT2 were obtained from the American Tissue Culture Collection. Human cyclophilin cDNA was a gift from Toshio Watanabe (Tohoku University), and pEGF-BOS (11Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar) was a gift from Shigeki Nagata (Osaka Bioscience Institute). PC12D cells (12Katoh-Semba R. Kitajima S. Yamazaki Y. Sano M. J. Neurosci. Res. 1987; 17: 36-44Crossref PubMed Scopus (100) Google Scholar), a rapidly differentiating subline of rat pheochromocytoma-derived PC12 cells (13Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar), were a gift from Mamoru Sano (Dept. of Biology, Faculty of Medicine, Kyoto Prefectural University of Medicine). PC12D cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Nissui) supplemented with 5% fetal bovine serum, 5% horse serum, 0.16% sodium bicarbonate, 3.6 mm glutamine, 10 units/ml penicillin, 45 ng/ml streptomycin at 37 °C under 5% CO2. Non-differentiated PC12D cells were used in all of the experiments. Unless noted, drugs were added directly to the culture medium and were present until the time at which the cells were harvested. The corresponding vehicle (water, Me2SO, or ethanol) was added to control cells. MAPK assays were performed as described by Cook and McCormick (14Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar) with some modifications. Briefly, PC12D cells grown to 80–90% confluency in 3.5 cm in uncoated plastic culture dishes (Corning or Iwaki Glass Co.) were stimulated with various agents for 10 min and then lysed by addition of 200 μl of lysis buffer containing 20 mm Tris-Cl (pH 8.0), 137 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 50 mm NaF, 1 mmNa3VO4, 1 mm phenylmethanesulfonyl fluoride (PMSF), 20 μm leupeptin, 10 μg/ml aprotinin. After brief centrifugation to remove cellular debris, 0.1 μg of anti-Erk-1 and 0.1 μg of anti-Erk-2 antibodies were added to the supernatant fractions, and these were incubated for 1 h at 4 °C with rotation to provide gentle mixing. Protein A-agarose (10 μl of resin suspension, Santa Cruz Biotechnology catalog number sc-2001) was subsequently added to each sample and the incubation continued with rotation at 4 °C for 1 h. The resin in each sample was then collected by centrifugation (2500 rpm) and washed twice with 200 μl of lysis buffer and once with 200 μl of 2× reaction buffer. 1× reaction buffer contained 25 mm MOPS (pH 7.2), 25 mm sodium β-glycerophosphate, 15 mmMgCl2, 1 mm EGTA, 0.1 mm NaF, 4 mm DTT, 1 mm Na3VO4. 22 μl of reaction mix containing 25 μm ATP, 1 μCi of [γ-32P]ATP, 15 μm myelin basic protein in 1× reaction buffer was added to the resin, and the mixture was incubated at 30 °C for 30 min. Reaction mixes were directly spotted on Whatman phosphocellulose filters, and filters were washed 6 times in 1% phosphoric acid for 5 min each wash. Radioactivity retained on the filters was quantified by liquid scintillation counting. During the course of this study, we determined that, unlike the anti-Erk-1 antibodies (Santa Cruz C-16), the anti-ErK-2 antibodies (Santa Cruz Biotechnology catalog number C-14) were not very effective in immunoprecipitating Erk-2 from cell lysates. 3E. Kumahara and D. Saffen, unpublished observations. MAPK activities reported in this paper therefore reflect primarily Erk-1 activities. Qualitative assays of MAPK activation determined by measuring the activation-correlated shift-up in electrophoretic mobilities (15Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (631) Google Scholar) showed, however, that Erk-1 and Erk-2 responded in the same manner to all treatments examined.3 RNA was isolated from PC12D cells grown to 80–90% confluency in 6-cm uncoated plastic dishes, and Northern analysis was carried out as described previously (5Ebihara T. Saffen D. J. Neurochem. 1997; 68: 1001-1010Crossref PubMed Scopus (32) Google Scholar). The amount of RNA in each sample was determined by optical spectroscopy, and the integrity of the RNA in each sample was assessed by examining the ethidium bromide-stained RNA in denaturing gel used for Northern blot analysis. Unless noted, 10 μg of total cellular RNA was electrophoresed in each lane. After blotting onto Pall Biodyne type B transfer membranes (0.45-μm pore size), hybridization was carried out simultaneously using DNA probes prepared fromzif268, c-fos (coding regions), and cyclophilin DNA fragments isolated from agarose gels and labeled using the Amersham Pharmacia Biotech Oligolabeling kit and [α-32P]dCTP. The intensities of bands in Northern blots were quantified using a Fuji Bioimaging analyzer BAS2000. An expression vector, pGLzif420, containing a firefly luciferase reporter gene linked to the ratzif268 promoter was constructed using pGL2 (Promega). Briefly, the zif268 promoter region (from −420 to 0 base pairs) containing 6 SRE sites and 2 CRE sites was amplified by polymerase chain reaction using synthetic oligonucleotide primers (forward primer corresponding to nucleic acid residues 121–149 of the rat zif268 promoter in the numbering system of Changelion et al. (Ref. 16Changelian P.S. Feng P. King T.C. Milbrandt J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 377-381Crossref PubMed Scopus (137) Google Scholar; GenBankTM accession number J04154), 5′-AACACCATATAAGGAGCAGGAAGGATCCC-3′; backward primer containing a synthetic EcoRI site followed by nucleic acid residues 941–920 (14Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar) of the rat zif268gene, 5′-(GCGAATTC)TTGCTCAGCAGCATCATCTCCT-3′). This polymerase chain reaction product was blunt-ended, digested withNruI, and the fragment containing thezif268 promoter sequences cloned into the blunt-endedHindIII site of pGL2. An expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the human elongation factor 1α promoter was constructed as follows: pBLCAT2 was digested with BamHI andBglII and then self-ligated. Digestion of the resulting plasmid with SalI and SmaI yielded a 1.5-kilobase pair fragment containing the CAT gene and a polyadenylation signal derived from SV40. This fragment was isolated, blunt-ended, and cloned into the XbaI site (after converting XbaI-cut ends to blunt ends) of pEF-BOS. The resulting vector, pEF-CAT, was used as an internal control in transfection experiments usingzif268-luciferase expression vectors. Transfections were performed using LipofectAMINETM reagent (Life Technologies, Inc.) essentially as recommended by the manufacturer. Cells were seeded in 6-cm plastic culture dishes (Corning or Iwaki Glass) at a density of 4 × 106 cells/dish and cultured for 1 day prior to transfection. 0.92 μg of pEF-CAT DNA, 2.3 μg of luciferase expression vector DNA, 13.8 μl of LipofectAMINETM reagent were added to each dish of cells and incubated for 4 h, prior to adding the medium containing twice the normal concentration of serum. After incubation overnight, the cells in each 6-cm dish were resuspended and distributed into 12 × 1.1-cm wells. The following day, the medium was replaced with normal DMEM, and the cells were cultured for 1 more day. Drugs were added directly to the culture medium, and cells were harvested after 4 h. Luciferase expression was carried out using the Promega Luciferase or Packard LucLite™, and luciferase activities were quantified using a Packard Tri-Carb or Top count scintillation counter as described in the manuals supplied by Promega and Packard. Background luciferase expression was determined using cells transfected with pGL2, which lacks a promoter for luciferase gene expression. Transfection efficiency was determined by cotransfection with pEF-CAT. CAT activities were measured as described by Nordeen et al. (17Nordeen S.K. Green P.P. Fowlkes D.M. DNA (N. Y.). 1987; 6: 173-178Crossref PubMed Scopus (178) Google Scholar), and these values were used to calculate normalized luciferase activities for each sample. PC12D cells were labeled with 32P in vivo as described (18Cadena D.L. Dahmus M.E. J. Biol. Chem. 1987; 262: 12468-12474Abstract Full Text PDF PubMed Google Scholar). Briefly, PC12D cells grown to 80–90% confluency in 10-cm culture plates were washed twice with 10 mm Tris (pH 7.0)-buffered saline (TBS) and overlaid with 5 ml of phosphate-free DMEM (Life Technologies, Inc.) supplemented with 25 mmHEPES, 5% dialyzed horse serum, and 5% dialyzed fetal bovine serum. 50 μCi of [32P]orthophosphate was added to medium, and the cells were incubated at 37 °C for 3 h in the absence of CO2. H7 (Seikagaku Kogyo) or other inhibitors were added 30 min prior stimulation with 5 ng/ml NGF for 15 min. Cells were washed twice with ice-cold phosphate-buffered saline supplemented with 0.2 mm PMSF, scraped into tubes, and centrifuged at 800 ×g for 5 min. Cells were resuspend in 1 ml of buffer containing 10 mm Tris-Cl (pH 7.9), 1 mmCaCl2, 1.5 mm MgCl2, 0.25m sucrose, 0.2 mm PMSF, 0.5% Triton X-100, homogenized with Dounce homogenizer, and centrifuged at 800 ×g for 5 min. Nuclei pellets were washed once with 1 ml of the above buffer lacking Triton X-100. Pellets were resuspended in 100 μl of buffer containing 25 mm Tris-Cl (pH 8.0), 2.5 mm magnesium acetate, 2 mm CaCl2, 0.05 mm EDTA, 0.1 mm DTT, 0.2 mmPMSF, and 12.5% glycerol and treated with 10 μg of DNase and RNase on ice for 30 min. Nuclei were lysed by adding 100 μl of 2% SDS, boiled for 3 min, and centrifuged at 15,600 × g for 3 min to remove debri. Nuclei extracts (15 μl/lane) were resolved by SDS-PAGE (5% polyacrylamide stacking gel containing 125 mmTris-Cl (pH 6.8), 0.1% SDS; 5% polyacrylamide resolving gel (prepared from 29.5% acrylamide + 0.5% bisacrylamide stock solution) containing 375 mm Tris-Cl (pH 8.8), 0.1% SDS; running buffer containing 25 mm Tris, 250 mm glycine, 0.1% SDS). Electrophoresis was carried out at 25 mA per gel until the tracking dye entered the resolving gel, after which the current was increased to 45 mA per gel. Proteins were then electrophoretically blotted (0.5 A, 45 min) onto polyvinylidene difluoride membranes (ImmobilonTM transfer membrane, Millipore) using a Nihon Eido Western blotting apparatus (20 × 20 cm) in buffer containing 100 mm Tris, 192 mm glycine, 10% methanol, and 0.02% SDS. Following transfer, the membranes were blocked by incubation in phosphate-buffered saline containing 5% skim milk and 0.5% Tween 20 overnight at room temperature. Phosphorylated proteins were detected by autoradiography using Fuji RX-U x-ray film. The membranes were then exposed to 0.02 μg/ml antibodies that recognize the carboxyl-terminal domain (CTD) of RNA polymerase II (C-21, catalog number sc-900, Santa Cruz Biotechnology) in phosphate-buffered saline containing 0.5% skim milk and 0.05% Tween 20 for 2 h at room temperature, washed 3 times (10 min each) with buffer, and incubated in buffer containing anti-rabbit IgG antibodies cross-linked with horseradish peroxidase (Jackson ImmunoResearch, catalog number 111-035-003; 5000-fold final dilution) for 2 h at room temperature. After washing 3 times for 20 min/wash and 3 times for 10 min/wash with buffer, immune complexes were visualized by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech). As shown in Fig. 1, levels of both zif268 and c-fos mRNAs are low or undetectable in unstimulated cells but increase rapidly following exposure to NGF or carbachol, reaching high levels after 45 min. The fact that NGF-stimulated increases in zif268and c-fos mRNA are blocked by the inhibitor K252a (19Knusel B. Hefti F. J. Neurochem. 1992; 59: 1987-1996Crossref PubMed Scopus (236) Google Scholar,20Berg M.M. Sternberg D.W. Parada L.F. Chao M.V. J. Biol. Chem. 1992; 267: 13-16Abstract Full Text PDF PubMed Google Scholar) indicates that these inductions require the activation of the tyrosine kinase of the high affinity NGF receptor, TrkA. Likewise, the ability of atropine to block the inductions of zif268and c-fos mRNAs by carbachol indicates the involvement of muscarinic acetylcholine receptors. Pretreatment of the cells for 30 min with 100 μm H7 completely blocks the induction ofzif268 and c-fos mRNAs by NGF and carbachol. By contrast, pretreatment with 100 μm HA1004 has essentially no effect on these inductions. Neither H7 nor HA1004 affect background levels of zif268 and c-fos mRNAs.3 As shown in Fig.2, H7 blocks the induction ofzif268 and c-fos mRNAs in a dose-dependent manner, with complete inhibition observed at concentrations of 50 μm and greater.Figure 2H7 inhibits the induction ofzif268 and c-fos gene expression in a dose-dependent manner. Northern blot analysis of zif268 and c-fos mRNA in PC12D cells pretreated with water (W), HA1004, or H7. PC12D cells were preincubated with the indicated concentrations of HA1004 or H7 for 30 min prior to stimulation with 5 ng/ml NGF (left) or 100 μm carbachol (right) for 45 min. These data are representative of experiments performed 3 times with similar results.View Large Image Figure ViewerDownload (PPT) The complete inhibition of zif268 and c-fos mRNA inductions by H7 is surprising for two reasons. First, although NGF has previously been reported to activate PKC in PC12 cells (21Hama T. Huang K.P. Guroff G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2353-2357Crossref PubMed Scopus (155) Google Scholar, 22Heasley L.E. Johnson G.L. J. Biol. Chem. 1989; 264: 8646-8652Abstract Full Text PDF PubMed Google Scholar), the inductions of zif268and c-fos mRNAs were not expected to require the activation of PKC, since down-regulation of PKC by prolonged exposure to phorbol ester has only a small effect on the induction of these mRNAs by NGF. 4T. Ebihara and D. Saffen, unpublished observations. We have also previously observed (5Ebihara T. Saffen D. J. Neurochem. 1997; 68: 1001-1010Crossref PubMed Scopus (32) Google Scholar) that induction of zif268mRNA by NGF is not affected by the specific PKC inhibitor GF109203x (23Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 24Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar). Second, although we have previously shown that PKC contributes to m1 muscarinic acetylcholine receptor-mediated induction of zif268 mRNA in PC12D cells, this induction is only partially blocked by pretreatment with GF109203x (5Ebihara T. Saffen D. J. Neurochem. 1997; 68: 1001-1010Crossref PubMed Scopus (32) Google Scholar). Thus, the total block of zif268and c-fos gene induction by H7 seems to be too large an effect. To understand better how H7 blocks gene expression, we examined its effect on the induction of zif268 and c-fos mRNAs by additional agents. Phorbol ester is expected to stimulate increases in immediate-early gene mRNA by activating PKC, and therefore this induction would be expected be inhibited by H7. By contrast, activation of immediate-early gene expression by elevated level of intracellular Ca2+ in PC12 cells has not previously been suggested to require PKC. Rather, activation of Ca2+/calmodulin kinases (25Enslen H. Soderling T.R. J. Biol. Chem. 1994; 269: 20872-20877Abstract Full Text PDF PubMed Google Scholar, 26Johnson C.M. Hill C.S. Chawla S. Treisman R. Bading H. J. Neurosci. 1997; 17: 6189-6202Crossref PubMed Google Scholar) and/or activation of MAPK cascade (27Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 28Xia Z. Dudek H. Miranti C.K. Greenberg M.E. J. Neurosci. 1996; 16: 5425-5436Crossref PubMed Google Scholar) is thought to be sufficient. Similarly, forskolin, which increases intracellular levels of cAMP by stimulating adenylate cyclase, is thought to activate gene expression via activation of PKA (29Meinkoth J.L. Alberts A.S. Went W. Fantozzi D. Taylor S.S. Hagiwara M. Montminy M. Feramisco J.R. Mol. Cell Biochem. 1993; 127/128: 179-186Crossref Scopus (124) Google Scholar) and/or the MAPK cascade (30Frodin M. Peraldi P. 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