Insulin-like Growth Factor-I Induces bcl-2 Promoter through the Transcription Factor cAMP-Response Element-binding Proteinстатья из журнала
Аннотация: Insulin-like growth factor-I (IGF-I) is known to prevent apoptosis induced by diverse stimuli. The present study examined the effect of IGF-I on the promoter activity ofbcl-2, a gene with antiapoptotic function. A luciferase reporter driven by the promoter region of bcl-2 from −1640 to −1287 base pairs upstream of the translation start site containing a cAMP-response element was used in transient transfection assays. Treatment of PC12 cells with IGF-I enhanced the bcl-2promoter activity by 2.3-fold, which was inhibited significantly (p < 0.01) by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Cotransfection of thebcl-2 promoter with MAPK kinase 6 and the β isozyme of p38 MAPK resulted in 2–3-fold increase in the reporter activity. The dominant negative form of MAPKAP-K3, a downstream kinase activated by p38 MAPK, and the dominant negative form of cAMP-response element-binding protein, inhibited the reporter gene activation by IGF-I and p38β MAPK significantly (p < 0.01). IGF-I increased the activity of p38β MAPK introduced into the cells by adenoviral infection. Thus, we have characterized a novel signaling pathway (MAPK kinase 6/p38β MAPK/MAPKAP-K3) that defines a transcriptional mechanism for the induction of the antiapoptotic protein Bcl-2 by IGF-I through the nuclear transcription factor cAMP-response element-binding protein in PC12 cells. Insulin-like growth factor-I (IGF-I) is known to prevent apoptosis induced by diverse stimuli. The present study examined the effect of IGF-I on the promoter activity ofbcl-2, a gene with antiapoptotic function. A luciferase reporter driven by the promoter region of bcl-2 from −1640 to −1287 base pairs upstream of the translation start site containing a cAMP-response element was used in transient transfection assays. Treatment of PC12 cells with IGF-I enhanced the bcl-2promoter activity by 2.3-fold, which was inhibited significantly (p < 0.01) by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Cotransfection of thebcl-2 promoter with MAPK kinase 6 and the β isozyme of p38 MAPK resulted in 2–3-fold increase in the reporter activity. The dominant negative form of MAPKAP-K3, a downstream kinase activated by p38 MAPK, and the dominant negative form of cAMP-response element-binding protein, inhibited the reporter gene activation by IGF-I and p38β MAPK significantly (p < 0.01). IGF-I increased the activity of p38β MAPK introduced into the cells by adenoviral infection. Thus, we have characterized a novel signaling pathway (MAPK kinase 6/p38β MAPK/MAPKAP-K3) that defines a transcriptional mechanism for the induction of the antiapoptotic protein Bcl-2 by IGF-I through the nuclear transcription factor cAMP-response element-binding protein in PC12 cells. insulin-like growth factor cAMP-response element cAMP-response element-binding protein mitogen-activated protein kinase phosphate-buffered saline wild type dominant negative CREB The products of the bcl-2 gene belong to a growing family of proteins that are involved in the regulation of mammalian apoptosis. They include proapoptotic (Bax, Bad, Bid, and Bik) and antiapoptotic (Bcl-2, Bcl-xL, and Brag-1) proteins (1Merry D. Korsmeyer S. Annu. Rev. Neurosci. 1997; 20: 245-267Crossref PubMed Scopus (546) Google Scholar). Complex interplay between these two groups of proteins seems to decide the fate of cells when exposed to apoptotic stimuli. In transgenic mice overexpressing the bcl-2 gene, the loss of neuronal cells by natural cell death as well as experimental ischemia is significantly reduced (2Martinou J.-C. Dubois-Dauphin M. Staple J.K. Rodriguez I. Frankoowski H. Missotten M. Albertini P. Talabot D. Catsicas S. Pietra C. Huarte J. Neuron. 1994; 13: 1017-1030Abstract Full Text PDF PubMed Scopus (1017) Google Scholar). In bcl-2 gene-ablated mice, loss of neurons and apoptosis in thymus and spleen has been observed (3Veis D. Sorenson C. Shutter J. Korsmeyer S. Cell. 1993; 75: 229-240Abstract Full Text PDF PubMed Scopus (1441) Google Scholar). The expression pattern of Bcl-2 during murine embryogenesis by immunohistochemical analysis shows that this protein is restricted to zones of survival (4Novack D. Korsmeyer S. Am. J. Pathol. 1994; 145: 61-73PubMed Google Scholar). Hence, expression of Bcl-2 appears to be a key regulatory step in promoting cell survival. Insulin-like growth factor-I (IGF-I)1 is known to exert antiapoptotic action in several cell types. One of the mechanisms by which IGF-I promotes cell survival is through down-regulation of the proapoptotic protein Bad. IGF-I stimulates the phosphorylation of Bad by activating phosphatidylinositol 3-kinase and Akt, leading to the sequestration of phospho-Bad in cytosol by the protein 14-3-3 (5Kulik G. Weber M. Mol. Cell. Biol. 1998; 18: 6711-6718Crossref PubMed Scopus (230) Google Scholar). In addition to this cytosolic covalent modification, IGF-I-mediated expression of the antiapoptotic protein Bcl-xL could play a role in the promotion of cell survival (6Párrizas M. LeRoith D. Endocrinology. 1997; 138: 1355-1358Crossref PubMed Scopus (0) Google Scholar). This growth factor has been shown to inhibit the down-regulation of Bcl-2 protein induced by hypoxia in cultured rat cortical neurons and by interleukin-3 deprivation in murine myeloid progenitor cells (7Tamatani M. Ogawa S. Tohyama M. Brain Res. Mol. Brain Res. 1998; 58: 27-39Crossref PubMed Scopus (88) Google Scholar, 8Minshall C. Arkins S. Straza J. Conners J. Dantzer R. Freund G. Kelley K. J. Immunol. 1997; 159: 1225-1232PubMed Google Scholar). The mechanism by which IGF-I sustains the expression of bcl-2 has not been studied. IGF-I is likely to increase the bcl-2 expression at the transcriptional level, since bcl-2 promoter is positively regulated by the nuclear transcription factor CREB, and IGF-I can activate this transcription factor (9Pugazhenthi S. Boras T. O'Connor D. Meintzer M.K. Heidenreich K.A. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 2829-2837Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The bcl-2gene consists of three exons with an untranslated first exon. It has a TATA-less GC-rich promoter with positive and negative regulatory elements (10Young R.L. Korsmeyer S.J. Mol. Cell. Biol. 1993; 13: 3686-3697Crossref PubMed Scopus (182) Google Scholar, 11Chen H.-M. Boxer L.M. Mol. Cell. Biol. 1995; 15: 3840-3847Crossref PubMed Google Scholar, 12Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (377) Google Scholar). The presence of a CRE site in a region between −1526 and −1552 upstream of the translation start site has been reported (12Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (377) Google Scholar). Phosphorylation of CREB by PKC in B lymphocytes leads to induction of the bcl-2 gene in a CRE-dependent fashion and protection from apoptosis (12Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (377) Google Scholar). In a recent study, we demonstrated that insulin-like growth factor-I-induced CREB activation involves p38 MAPK-mediated signaling pathway in PC12 cells (9Pugazhenthi S. Boras T. O'Connor D. Meintzer M.K. Heidenreich K.A. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 2829-2837Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Hence, activation of p38 MAPK could stimulate the bcl-2 promoter activity through CREB in these cells. The p38 MAPK belongs to the MAPK superfamily, the other members being extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/N-terminal Jun kinase. Activation of p38 MAPK has been observed during apoptosis mediated by diverse stimuli such as growth factor withdrawal and exposure to UV irradiation (13Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5036) Google Scholar). p38 MAP kinase is important for programmed cell death, since its specific inhibitor SB203580 can prevent apoptosis (14Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). However, studies have demonstrated that p38 MAPK can be activated by growth factors, leading to induction of growth-promoting genes (9Pugazhenthi S. Boras T. O'Connor D. Meintzer M.K. Heidenreich K.A. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 2829-2837Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15Xing J. Kornhauser J.M. Xia Z. Thiele E.A. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 1946-1955Crossref PubMed Google Scholar). Differentiation of PC12 cells into a neuronal cell type and adipogenesis have been shown to require p38 MAPK (16Engelman J.A. Lisanti M.P. Scherer P.E. J. Biol. Chem. 1998; 273: 32111-32120Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 17Morooka T. Nishida E. J. Biol. Chem. 1998; 273: 24285-24288Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). The apparent discrepancy between these observations can probably be explained by the existence of several p38 MAPK isozymes with distinct functions. So far, four isozymes, α, β, γ, and δ, have been identified with several splice variants (18Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 19Li Z. Jiang Y. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1996; 228: 334-340Crossref PubMed Scopus (353) Google Scholar, 20Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 21Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.-Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). In cardiomyocytes, β isozyme was shown to exert hypertrophic action, whereas p38α induces apoptosis (22Wang Y. Huang S. Sah V.P. Ross Jr., J. Brown J.H. Han J. Chien K.R. J. Biol. Chem. 1998; 273: 2161-2168Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar). Identification of isoform specific regulation by trophic versus toxic factors should clarify this confusing scenario. The objectives of the present investigation were (a) to examine the IGF-I mediated activation of bcl-2 promoter in PC12 cells and characterize the signaling pathway involved in this activation and (b) to examine the isozyme specific role of p38 MAPK in the activation of bcl-2 promoter. We demonstrate that IGF-I-induced bcl-2 promoter activity proceeds in part through a novel signaling pathway involving MAPK kinase 6/p38β MAPK/MAPKAP-K3 and requires CREB. Cell culture media and supplies were from Life Technologies, Inc. (Beverly, MA) and Gemini Bio Products, Inc. (Calabasas, CA). SB 203580 was obtained from Calbiochem. PD98059 was purchased from Biomol (Plymouth Meeting, PA). Different promoter regions of the bcl-2 gene (full-length, −3934 to −1287; truncated with CRE site, −1640 to 1287; truncated without CRE −1526 to −1287; and the CRE mutated (−1640 to 1287)) were linked to luciferase reporter as described previously (12Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (377) Google Scholar). The wild type and constitutively active forms of MAPK kinase 3 and MAPK kinase 6 were obtained from B. Derijard (CNRS, Nice, France) and Joel Raingeaud (Institut Curie, Orsay, France) respectively. The isozymes of p38 MAPK in pcDNA3 were provided by Jiahuai Han (San Diego, CA). The dominant negative triple mutant of MAPKAP-3 was obtained from Peter Young (SmithKline Beecham, King of Prussia, PA). The dominant negative CREB (pRSVKCREB) was provided by Dr. Richard Goodman (Oregon Health Sciences University, Portland, OR). The luciferase assay kit was purchased from Analytical Luminescence Laboratory (San Diego, CA). Antibodies specific for CREB, phospho-CREB (Ser-133), p38 MAPK, phospho-p38 MAPK and phospho-ATF-2, and the ATF-2 fusion protein were obtained from New England Biolabs (Beverly, MA). Plasmids for transfection experiments were purified using Qiagen's (Valencia, CA) Maxi kit. Anti-FLAG antibody and other fine chemicals were purchased from Sigma. cDNA encoding full-length FLAG epitope-tagged p38β or MAPK kinase 6 (WT) were subcloned into HindIII and XbaI sites in the plasmid pACCMVpLpA, which includes the left end of the adenovirus chromosome with the E1A gene and the 5′-half of theE1B gene replaced by the cytomegalovirus major immediate early promoter, a multiple cloning site, and intron and polyadenylation sequences from SV40 (23Gomez-Foix A.M. Coats W.S. Baque S. Alam T. Gerad R.D. Newgard C.B. J. Biol. Chem. 1992; 267: 25129-25134Abstract Full Text PDF PubMed Google Scholar). Recombinant adenovirus containing the various kinases were prepared using homologous recombination in HEK-293 cells (24Graham F. Smiley J. Russell W. Nairn R. J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3507) Google Scholar). Plasmids containing the appropriate constructs in pACCMVpLpA were cotransfected into 293 cells by Ca3(PO4)2 precipitation using 5 μg of the recombinant plasmid and approximately 0.2 μg ofBstBI-digested Ad5dl327Bstβ-gal-TP complex or with 1 μg of the recombinant plasmid and 5 μg of pJM17 (containing the chromosome of Ad5dl309 inserted into a bacterial plasmid vector) (25Jordan M. Schallhorn A. Wurm F. Nucleic Acids Res. 1996; 24: 596-601Crossref PubMed Scopus (732) Google Scholar, 26McGrory W. Bautista D. Graham F. Virology. 1988; 163: 614-617Crossref PubMed Scopus (550) Google Scholar, 27Schaack J. Langer S. Guo X. J. Virol. 1995; 69: 3920-3923Crossref PubMed Google Scholar). Cells were grown until the positive cytopathic effect was evident (7–10 days). Medium from these cells was harvested and freeze-thawed to release virus, and serial dilutions were used to infect 293 cells for plaque purification. The cells were overlaid with 1% Noble agar containing medium and serum 18 h after infection and fed with fresh Noble agar/medium/serum after 4 days. On day 7, the cells were stained with neutral red, and 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside was added to Noble agar containing medium with serum. Clear plaques, which include viruses arising from homologous recombination between the recombinant plasmid and the right (large) arm of Ad5dl327Bstβ-gal-TP complex, were picked and grown in 293 cells, and positive recombinants were identified by Western analysis using the FLAG antibody. Virus was propagated and purified by CsCl gradient centrifugation (28Jones N. Shenk T. Cell. 1978; 13: 181-188Abstract Full Text PDF PubMed Scopus (153) Google Scholar). Rat pheochromocytoma (PC12) cells (provided by Dr. Gary Johnson, Denver, CO) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5% heat-inactivated horse serum, 100 μg/ml streptomycin, and 100 microunits/ml penicillin at 37 °C in a humidified atmosphere at 8% CO2. Cells were cultured in 6 × 35-mm wells for transfection studies. Medium was changed every second day. Confluent cell cultures were split 1:4 and used for the experiments 4 days later. The cells were fasted for five h by maintaining in the medium containing 0.1% fetal bovine serum and 0.05% heat-inactivated horse serum before treatment with growth factors and other agents in the experiments for measuring CREB phosphorylation. Stock solutions of the pharmacological inhibitor SB203580 and PD98059 were prepared in Me2SO at a concentration of 1000-fold, so that when it was added to the culture medium, the concentration of Me2SO was 0.1%. Immunoblotting for phospho-CREB, dual phospho-p38 MAPK, and Bcl-2 was carried out as described previously (9Pugazhenthi S. Boras T. O'Connor D. Meintzer M.K. Heidenreich K.A. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 2829-2837Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). PC12 cells cultured in 60-mm dishes were incubated in serum-free medium before each experiment. After treatment with insulin-like growth factor-I for an appropriate duration, the cells were washed twice with ice-cold PBS, and total cell lysates were prepared by scraping the cells with 200 μl of 1× Laemmli sample buffer containing 100 mm dithiothreitol. The proteins were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were blocked with TBST (20 mmTris-HCl, pH 7.9, 8.5% NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk (blotting grade) at room temperature for 1 h. The blots were then treated with the primary antibody for phospho-p38 MAPK/phospho-CREB/Bcl-2 in TBST containing 5% bovine serum albumin at 4 °C overnight. After three washes with blocking buffer, the blots were incubated with anti-rabbit IgG conjugated to alkaline phosphatase for 1 h at room temperature. This was followed by three washes with blocking buffer, two washes with 10 mm Tris-HCl (pH 9.5), 10 mm NaCl, 1 mm MgCl2, and a 5-min incubation with diluted CDP-Star reagent (New England Biolabs, Beverly, MA) and then exposed to x-ray film. The intensity of bands was quantitated by scanning. The PC12 cells were infected with adenoviral p38β linked to FLAG epitope and MAPK kinase 6 (WT) and exposed to IGF-I as described in the legend to Fig. 7. After washing the cells with PBS, 200 μl of ice-cold cell lysis buffer (20 mmTris (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mmsodium orthovanadate, 10 μg/ml leupeptin, 500 nm okadaic acid, and 1 mm phenylmethylsulfonyl fluoride) was added. The cells were scraped, lysed by sonication, and centrifuged for 20 min. The supernatant (300 μg of protein) was mixed with 15 μg of FLAG antibody overnight at 4 °C. Protein A-Sepharose (20 μl) was added and gently rocked for 3 h at 4 °C. After centrifugation, the pellet was washed twice with cell lysis buffer and twice with kinase assay buffer (25 mm Tris (pH 7.5), 5 mmβ-glycerophosphate, 2 mm dithiothreitol, 0.1 mm sodium orthovanadate, 10 mmMgCl2). The pellet was suspended in 30 μl of kinase buffer with 200 μm ATP and 2 μg of ATF-2 fusion protein and incubated for 30 min at 30 °C. The reaction was terminated by the addition of 10 μl of 4× Laemmli sample buffer. These samples were electrophoresed and immunoblotted with antibody to phospho-ATF-2. The intensities of the bands were measured by scanning. Transient transfection was carried out using LipofectAMINE Plus reagent (Life Technologies, Inc.). PC12 cells were cultured to 60–80% confluence for transfection experiments in 6 × 35-mm plates. For each well, 1 μg of plasmids, 3 μl of Plus reagent, and 10 μg of LipofectAMINE reagent were used as per the manufacturer's instructions. The plasmid containing the β-galactosidase gene driven by the SV40 promoter was included to normalize the transfection efficiency. DNA and the LipofectAMINE reagent were diluted separately in 100 μl of serum-free medium without antibiotics, mixed together, and incubated at room temperature for 30 min. The culture plates were washed with PBS, and 800 μl of serum- and antibiotic-free medium was added. The 200 μl of the plasmid LipofectAMINE mixture was then added to each well, and the plates were incubated at 37 °C for 5 h. Then 1.0 ml of high serum medium (20% fetal bovine serum and 10% heat-inactivated horse serum) was added, and the cells were incubated for approximately 24 h before induction with growth factors for luciferase for 24 h. The cells were washed in PBS and lysed with 100 μl of reporter lysis buffer. The cells were lysed by freezing and thawing, and lysate was centrifuged at 14,000 RPM for 30 min. The supernatant was used for the assay of luciferase and β-galactosidase. Luciferase assays were carried out using the enhanced luciferase assay kit (Analytical Luminescence Laboratory, San Diego, CA) on a Monolight 2010 luminometer. The β-galactosidase assay was performed according to the method of Wadzinski et al. (29Wadzinski B. Wheat W. Jaspers S. Peruski L. Lickteig R. Johnson G. Klemm D. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (286) Google Scholar). Statistical analysis was carried out by Student's ttest. Previous studies have shown the regulation ofbcl-2 promoter activity by positive and negative regulatory elements in the 5′ upstream region (10Young R.L. Korsmeyer S.J. Mol. Cell. Biol. 1993; 13: 3686-3697Crossref PubMed Scopus (182) Google Scholar, 11Chen H.-M. Boxer L.M. Mol. Cell. Biol. 1995; 15: 3840-3847Crossref PubMed Google Scholar, 12Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (377) Google Scholar). To explore the importance of CRE in bcl-2 expression, we first characterized these regulatory regions of bcl-2 promoter in PC12 cells. The sequence from −3934 to −1287 was able to drive the expression of a luciferase gene from a promoterless reporter construct (Fig.1). Truncation of the 5′-end from −3934 to −1640 led to a 2.5-fold increase in the promoter activity (Fig. 1). This increase seems to be due to the loss of negative regulatory regions identified by previous studies (10Young R.L. Korsmeyer S.J. Mol. Cell. Biol. 1993; 13: 3686-3697Crossref PubMed Scopus (182) Google Scholar, 11Chen H.-M. Boxer L.M. Mol. Cell. Biol. 1995; 15: 3840-3847Crossref PubMed Google Scholar). This truncated promoter region contains a CRE site between −1611 and −1526. Mutation of the CRE site decreased the luciferase activity by 50%. Additionally, cotransfection of the CRE-containing reporter construct with dominant negative CREB (KCREB) significantly (p < 0.001) decreased the luciferase induction. Progressive deletion from the 5′-end of the CRE site-containing region resulted in a 68% decrease of reporter activity. These experiments clearly demonstrate the positive regulation of basal bcl-2 promoter activity by the nuclear transcription factor CREB. IGF-I is known to up-regulate the expression of the pro-cell survival protein Bcl-xL (30Parrizas M. LeRoith D. Endocrinology. 1997; 138: 1355-1358Crossref PubMed Scopus (198) Google Scholar). We wanted to examine if this growth factor can increase the expression of Bcl-2. When PC12 cells were treated with 50 and 100 ng/ml concentrations of IGF-I, there was a significant (p < 0.001) increase in the expression of Bcl-2 as shown by the immunoblot (Fig.2 A). To understand the mechanism by which IGF-I stimulates the expression of Bcl-2, we examined the effect of this growth factor on the promoter activity ofbcl-2 gene. When PC12 cells were transiently transfected with a luciferase reporter driven by the CRE site containing truncatedbcl-2 promoter, IGF-I (100 ng/ml) was also able to increase its activity in a time-dependent manner (Fig.2 B). Treatment of the PC12 cells with this growth factor for 18 h led to a 2.3-fold increase in the bcl-2 promoter activity over the untreated cells. When the promoter was cotransfected with dominant negative CREB, IGF-I stimulated reporter activity was decreased by 46% (Table I), indicating that this growth factor induces bcl-2 promoter through activation of CREB.Table IEffect of KCREB on IGF- and p38β MAPK-mediated activation of bcl-2 promoterCotransfection and treatmentCotransfection of reporter with KCREBWithout KCREBWith KCREBControl10051.0 ± 3.5ap < 0.001.IGF-I (100 ng/ml)231.7 ± 25.3124.3 ± 11.9ap < 0.001.MKK6 (Glu)206.0 ± 13.6101.0 ± 12.3ap < 0.001.p38β MAPK224.7 ± 18.0118.7 ± 16.8ap < 0.001.p38β MAPK + IGF-I373.3 ± 52.5182.0 ± 30.1ap < 0.001.PC12 cells cultured in 6 × 35-mm wells to 60–75% confluence were transfected with CRE site-containing bcl-2 promoter linked to luciferase reporter along with indicated plasmids in serum- and antibiotic-free medium. After 24 h of transfection, the cells were incubated in the absence and presence of IGF-I (100 ng/ml). Luciferase and β-galactosidase were assayed in the cell lysates. Control reporter activity in the absence of KCREB was taken as 100%. Values are mean ± S.E. of four independent experiments, each done in duplicate. p values relative to reporter activity in the absence of KCREB were obtained by Student's t test.a p < 0.001. Open table in a new tab PC12 cells cultured in 6 × 35-mm wells to 60–75% confluence were transfected with CRE site-containing bcl-2 promoter linked to luciferase reporter along with indicated plasmids in serum- and antibiotic-free medium. After 24 h of transfection, the cells were incubated in the absence and presence of IGF-I (100 ng/ml). Luciferase and β-galactosidase were assayed in the cell lysates. Control reporter activity in the absence of KCREB was taken as 100%. Values are mean ± S.E. of four independent experiments, each done in duplicate. p values relative to reporter activity in the absence of KCREB were obtained by Student's t test. Having shown the role of CREB in driving thebcl-2 promoter and its induction by IGF-I, we then proceeded to examine the signaling pathways known to be stimulated by IGF-I that are involved in phosphorylation of CREB on serine 133. CREB has been shown to be phosphorylated and activated by signaling cascades mediated by protein kinase A, protein kinase C, Ca2+, Ras, and phosphatidylinositol 3-kinase. In a recent study, we demonstrated that IGF-I-stimulated induction of chromogranin A, a neuroendocrine-specific gene responsive to CREB activation, involves the signaling mediated by MAPK kinase 6 and p38 MAPK (9Pugazhenthi S. Boras T. O'Connor D. Meintzer M.K. Heidenreich K.A. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 2829-2837Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Hence, we examined the stimulation ofbcl-2 promoter activity by IGF-I in the presence of pharmacological inhibitors specific for different MAP kinases. The transfected cells were preincubated with PD98059 (40 μm), an inhibitor of MAPK kinase 1/2 (and therefore extracellular signal-regulated kinase 1/2) and SB203580 (10 μm), a specific inhibitor of p38 MAPK (Fig. 3). IGF-I-stimulated reporter activity was decreased (40%;p < 0.01) by SB203580 (Fig. 3). The MAPK kinase inhibitor (PD98059), on the other hand, increased the bcl-2promoter activity by 80% (p < 0.001) and 52% (p < 0.01) in the absence and presence of IGF-I (Fig.3). The bcl-2 gene has been shown to contain negative regulatory elements that respond to the Ets family of proteins, which are activated through the MAPK kinase/extracellular signal-regulated kinase pathway (11Chen H.-M. Boxer L.M. Mol. Cell. Biol. 1995; 15: 3840-3847Crossref PubMed Google Scholar). Further studies are needed to examine this pathway. The results of the experiments with SB203580 demonstrate the involvement of p38 MAPK-mediated signaling pathway in the induction ofbcl-2 by IGF-I. We next examined the role of this pathway in the regulation of bcl-2 expression in more detail. In the next series of experiments in PC12 cells, the bcl-2reporter construct was cotransfected with wild type and dominant active forms of MAPK kinase 6, the upstream kinase known to activate p38 MAPK. The results of these studies indicated that MAPK kinase 6 is an activator of bcl-2 promoter (Fig.4). The wild type and constitutively active (Glu) forms of MAPK kinase 6 stimulated the luciferase activity by 2–3-fold (p < 0.001). MAPK kinase 6 (Glu)-mediatedbcl-2 promoter activity was decreased by 51% when the cells were cotransfected with KCREB (Table I). There were relatively smaller increases of 40 and 78% in promoter activity when cotransfected with wild type and constitutively active forms of MAPK kinase 3, respectively. This is likely to be due to differences observed in the activation pattern of p38 MAP kinases by MAPK kinase 3 and MAPK kinase 6. Among the isoforms of p38 MAPK, α, γ, and δ have been shown to be activated by both MAPK kinase 3 and MAPK kinase 6, whereas the β isoform is preferentially activated by MAPK kinase 6 (18Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 20Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 31Enslen H. Raingeaud J. Davis R.J. J. Biol. Chem. 1998; 273: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). In order to specifically understand the role of p38 MAP kinase isoforms in regulating the bcl-2 promoter activity, we overexpressed them individually in PC12 cells. Among the various isozymes of p38 MAPK, β was found to stimulate bcl-2 promoter activity in a dose-dependent manner (Fig.5 A). Coexpression of the reporter with 50–100 ng of p38β increased the activity by 2.1–2.8-fold. Although other isozymes of p38, α, γ, and δ, did show a small stimulation it was considerably less when compared with p38β. Optimal activation of p38 MAPK occurs when it is cotransfected with its upstream kinase, although previously it has been shown that p38 MAPK introduced alone is also activated by the endogenous pathway (18Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). We further confirmed the role of MAPK kinase 6 and p38β MAPK inbcl-2 induction by using the specific inhibitor SB203580. This pyridinyl imadazole derivative has been shown to have an inhibitory effect in a highly specific manner toward p38 MAPK when compared with other MAPK family kinases such as extracellular signal-regulated kinase and N-terminal Jun kinase (32Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C.
Год издания: 1999
Авторы: Subbiah Pugazhenthi, Elisa Miller, Carol L. Sable, Peter R. Young, Kim A. Heidenreich, Linda M. Boxer, Jane E.B. Reusch
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Cell death mechanisms and regulation, Cancer-related Molecular Pathways, Cytokine Signaling Pathways and Interactions
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Том: 274
Выпуск: 39
Страницы: 27529–27535