FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1548282121> ?p ?o ?g. }

• W1548282121 endingPage "13829" @default.
• W1548282121 startingPage "13822" @default.
• W1548282121 abstract "Insulin induces apolipoprotein A-I,apoA-I gene transcription via a membrane receptor with intrinsic tyrosine kinase activity. This finding prompted us to ask whether the gene is stimulated by epidermal growth factor (EGF), EGF a peptide hormone that binds to another member of the receptor superfamily with tyrosine kinase activity. Our data showed that like insulin, EGF increased abundance of apoA-I protein and transcription of the gene in human hepatoma, Hep G2 cells. The effects of both hormones appeared direct because their induction of apoA-I gene transcription was not affected by the protein synthesis inhibitor, cycloheximide. Although both insulin and EGF stimulate apoA-I expression, each hormone binds to a distinct membrane receptor thus suggesting differential intracellular signaling. Therefore, we used a panel of inhibitors to define the pathway(s) that mediate the actions of these hormones. Whereas, the actions of EGF required only the Ras-mitogen-activated protein, MAP kinase, those of insulin were mediated by equal participation of both the Ras-MAP kinase and protein kinase C, PKC cascades. Despite differences in signaling pathways triggered by each hormone receptor, the activation ofapoA-I transcription required the participation of a single transcription factor, Sp1. Furthermore, EGF induction of transcription was attenuated by mutating the MAP kinase site at amino acid, Thr266 rendering Sp1 phosphorylation deficient. In summary, EGF stimulation of apoA-I expression is mediated solely by the Ras-MAP kinase cascade and enhanced activity of this pathway requires Sp1 with an intact phosphorylation site at Thr266. However, insulin induction of this gene is different and requires both Ras-MAP kinase and PKC pathways but their actions are also mediated by Sp1. Insulin induces apolipoprotein A-I,apoA-I gene transcription via a membrane receptor with intrinsic tyrosine kinase activity. This finding prompted us to ask whether the gene is stimulated by epidermal growth factor (EGF), EGF a peptide hormone that binds to another member of the receptor superfamily with tyrosine kinase activity. Our data showed that like insulin, EGF increased abundance of apoA-I protein and transcription of the gene in human hepatoma, Hep G2 cells. The effects of both hormones appeared direct because their induction of apoA-I gene transcription was not affected by the protein synthesis inhibitor, cycloheximide. Although both insulin and EGF stimulate apoA-I expression, each hormone binds to a distinct membrane receptor thus suggesting differential intracellular signaling. Therefore, we used a panel of inhibitors to define the pathway(s) that mediate the actions of these hormones. Whereas, the actions of EGF required only the Ras-mitogen-activated protein, MAP kinase, those of insulin were mediated by equal participation of both the Ras-MAP kinase and protein kinase C, PKC cascades. Despite differences in signaling pathways triggered by each hormone receptor, the activation ofapoA-I transcription required the participation of a single transcription factor, Sp1. Furthermore, EGF induction of transcription was attenuated by mutating the MAP kinase site at amino acid, Thr266 rendering Sp1 phosphorylation deficient. In summary, EGF stimulation of apoA-I expression is mediated solely by the Ras-MAP kinase cascade and enhanced activity of this pathway requires Sp1 with an intact phosphorylation site at Thr266. However, insulin induction of this gene is different and requires both Ras-MAP kinase and PKC pathways but their actions are also mediated by Sp1. Apolipoprotein A-I (apoA-I) 1The abbreviations used are: apoA-Iapolipoprotein A-IHDLhigh density lipoproteinIRCEinsulin responsive core elementMAPmitogen-activated proteinPI 3-kinasephosphatidylinositol 3-kinaseRT-PCRreverse transcriptase-polymerase chain reactionEGFepidermal growth factorCATchloramphenicol acetyltransferasePKCprotein kinase CPDBuphorbol 12,13-dibutyrate is a major protein component of the serum high-density lipoprotein (HDL) particles (1Andersson L.O. Curr. Opin. Lipidol. 1997; 8: 225-228Crossref PubMed Scopus (29) Google Scholar, 2Brouillette C.G. Anantharamaiah G.M. Biochim. Biophys. Acta. 1995; 1256: 103-129Crossref PubMed Scopus (167) Google Scholar). The anti-atherogenic properties of apoA-I alone or as part of HDL underlie their inverse correlation with the incidence of ischemic cardiovascular disease, the number 1 cause of premature death in modern societies (3Barter P.J. Rye K.A. Atherosclerosis. 1996; 121: 1-12Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 4De Backer G. De Bacquer D. Kornitzer M. Atherosclerosis. 1998; 137 (suppl.): S1-S6Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The cardioprotective actions of apoA-I or HDL arises from their participation in a normal physiologic process, so called “reverse cholesterol transport” (5Barter P.J. Rye K.A. Curr. Opin. Lipidol. 1996; 7: 82-87Crossref PubMed Scopus (171) Google Scholar, 6Luoma P.V. Pharmacol. Toxicol. 1997; 81: 57-64Crossref PubMed Scopus (42) Google Scholar). ApoA-I acts as a cofactor to facilitate an interaction between HDL particles and the cell membrane. This interaction enables the efflux of intracellular cholesterol to HDL particles, which in turn shuttles the sterol to the liver for further metabolism and excretion (5Barter P.J. Rye K.A. Curr. Opin. Lipidol. 1996; 7: 82-87Crossref PubMed Scopus (171) Google Scholar, 7Miller N.E. La Ville A. Crook D. Nature. 1985; 314: 109-111Crossref PubMed Scopus (158) Google Scholar). Enhanced reverse cholesterol transport lowers total body cholesterol, as demonstrated clearly following the infusion of apoA-I protein into humans (8Nanjee M.N. Crouse J.R. King J.M. Hovorka R. Rees S.E. Carson E.R. Morgenthaler J.J. Lerch P. Miller N.E. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1203-1214Crossref PubMed Scopus (91) Google Scholar). The reduction in cholesterol lowers the risk of arteriosclerosis (9Eisenberg D.A. Am. J. Med. 1998; 104: 2S-5SAbstract Full Text Full Text PDF PubMed Google Scholar), a major cause of ischemic cardiovascular disease. In support of this idea, transgenic mice that overexpress human apoA-I protein had significant reductions of atherosclerotic lesions in vessel walls (10Lawn R.M. Wade D.P. Hammer R.E. Chiesa G. Verstuyft J.G. Rubin E.M. Nature. 1992; 360: 670-672Crossref PubMed Scopus (249) Google Scholar). Therefore, understanding the mechanisms that enhance apoA-Iexpression will lead us to better ways to enhance its expression and thus lower the risk of ischemic cardiovascular disease (11Taylor A.H. Nakamura T. Wong N.C. Proc. West Pharmacol. Soc. 1997; 40: 127-130PubMed Google Scholar). apolipoprotein A-I high density lipoprotein insulin responsive core element mitogen-activated protein phosphatidylinositol 3-kinase reverse transcriptase-polymerase chain reaction epidermal growth factor chloramphenicol acetyltransferase protein kinase C phorbol 12,13-dibutyrate We showed recently that insulin induces rat apoA-I gene transcription and this induction is mediated by an insulin responsive core element (IRCE) a motif recognized by Sp1 (12Murao K. Wada Y. Nakamura T. Taylor A.H. Mooradian A.D. Wong N.C. J. Biol. Chem. 1998; 273: 18959-18965Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 13Zheng X.L. Matsubara S. Diao C. Hollenberg M.D. Wong N.C. J. Biol. Chem. 2000; 275: 31747-31754Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Insulin action is initiated by its binding to a membrane receptor with intrinsic tyrosine kinase activity, prompting us to wonder whether this mechanism is unique to the peptide hormone. The actions of insulin can be categorized into immediate responses: such as glucose transport or delayed responses; including cell differentiation and proliferation (14Kahn C.R. Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar, 15Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar). Several intracellular pathways are activated by insulin action and may include the Ras-MAP kinase, PI 3-kinase, and phospholipase Cγ cascades (14Kahn C.R. Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar). These pathways are not exclusive to the insulin receptor and may be used by other tyrosine kinase receptors, including that for EGF (16Carpenter G. Bioessays. 2000; 22: 697-707Crossref PubMed Scopus (305) Google Scholar). Therefore, we tested the ability of another peptide hormone, epidermal growth factor, EGF onapoA-I expression. If apoA-I is inducible by EGF, it offers a new avenue to augment expression of the gene. But equally important is that this model provides an opportunity to compare or contrast the signaling mechanism(s) activated by EGF and insulin. The results summarized here show that like insulin, EGF also enhances apoA-I expression. However, whereas the actions of EGF are mediated solely by the actions of a single pathway, that of insulin requires the participation of at least two cascades. Construction of the reporter, pAI.474-CAT was described previously (17Romney J.S. Chan J. Carr F.E. Mooradian A.D. Wong N.C. Mol. Endocrinol. 1992; 6: 943-950PubMed Google Scholar). The deletion constructs; pAI.425-, pAI.375-, pAI.325-, and pAI.235-CAT containing ratapoA-I DNA spanning −425, −375, −325, and −235 to −7 were synthesized using the parent pAI.474-CAT as a template in separate PCR (17Romney J.S. Chan J. Carr F.E. Mooradian A.D. Wong N.C. Mol. Endocrinol. 1992; 6: 943-950PubMed Google Scholar). Transverse mutation of the IRCE (−411 to −404) from GAGGCGGG to TCTTATTT was accomplished using a mutant primer in a PCR (12Murao K. Wada Y. Nakamura T. Taylor A.H. Mooradian A.D. Wong N.C. J. Biol. Chem. 1998; 273: 18959-18965Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The RasAsn-17-retroviral vector and Sp1 expression plasmid were gifts from Drs. J. Stone (University of Alberta, Edmonton, Alberta, Canada) and Dr. R. Tjian (University of California, Berkeley, CA), respectively (19Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar). Human hepatoma Hep G2 cells were transiently transfected with plasmid DNA of interest using LipofectAMINE (Life Technologies, Inc.) as per the instructions recommended by the manufacturer. The efficiency of DNA uptake was monitored by co-transfecting 1 μg of the plasmid, RSV-β-galactosidase (20Herbomel P. Bourachot B. Yaniv M. Cell. 1984; 39: 653-662Abstract Full Text PDF PubMed Scopus (556) Google Scholar). Stably transfected Hep G2 cells were created by co-transfecting pAI.474-CAT (17Romney J.S. Chan J. Carr F.E. Mooradian A.D. Wong N.C. Mol. Endocrinol. 1992; 6: 943-950PubMed Google Scholar) and the plasmid, pRc/CMV2 (Invitrogen) that carried neomycin resistance as described (13Zheng X.L. Matsubara S. Diao C. Hollenberg M.D. Wong N.C. J. Biol. Chem. 2000; 275: 31747-31754Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Hep G2 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% bovine calf serum (Life Technologies, Inc.) and penicillin/streptomycin at 37 °C. Hep G2 cells serve as an accepted model for the studying apoA-I (12Murao K. Wada Y. Nakamura T. Taylor A.H. Mooradian A.D. Wong N.C. J. Biol. Chem. 1998; 273: 18959-18965Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 13Zheng X.L. Matsubara S. Diao C. Hollenberg M.D. Wong N.C. J. Biol. Chem. 2000; 275: 31747-31754Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Sakai T. Jin F. Kamanna V.S. Kashyap M.L. Atherosclerosis. 2000; 149: 43-49Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Cells were cultured overnight in serum-free medium prior to the addition of the agent(s) of interest, followed by measuring CAT activity (12Murao K. Wada Y. Nakamura T. Taylor A.H. Mooradian A.D. Wong N.C. J. Biol. Chem. 1998; 273: 18959-18965Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar,23Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). Whole cell extract from control or cells treated with various factors were harvested and lysed in buffer containing orthovanadate 2 mm, Triton X-100 1%, SDS 0.1%, leupeptin and apoprotin 5 μg/ml each, benzamidine and bacitracin 1 mg/ml each, dithiothreitrol 600 mm, Tris 20 mm (pH 7.4), NaCl 300 mm, EDTA 5 mm, NaF 50 mm, sodium pyrophosphate 40 mm, KH2PO450 mm, and Na molybdate 10 mm. An aliquot of equal numbers of control or treated hepatoma cells or culture medium (10 μg of total protein) containing secreted apoA-I protein was separated by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The blot was probed using a monoclonal antibody (Calbiochem) as described (13Zheng X.L. Matsubara S. Diao C. Hollenberg M.D. Wong N.C. J. Biol. Chem. 2000; 275: 31747-31754Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To investigate the activation state of p42/44 MAP kinase in cell lysates of Hep G2 cells in response to various treatments, we employed a phospho-specific antibody probe directed against the activated form of ERK-1/2 (New England Biolabs, Inc.) (24Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (842) Google Scholar). Protein samples of total cell lysates were analyzed for activated p42/44 MAP kinase using Western blot techniques (21Zhong Z.D. Hammani K. Bae W.S. DeClerck Y.A. J. Biol. Chem. 2000; 275: 18602-18610Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Total RNA from cells was extracted from cells using TRITM-reagent (Molecular Research Center, Cincinnati, OH) (25Wu J.Y. Wu Y. Reaves S.K. Wang Y.R. Lei P.P. Lei K.Y. Am. J. Physiol. 1999; 277: C537-C544Crossref PubMed Google Scholar). The RNA was reverse-transcribed with a first strand cDNA synthesis kit using pd(N)6primer (Amersham Pharmacia Biotech) according to manufacturer's protocol. 3 μl of this solution was amplify using PCR primed with a forward primer 5′-CCTGATGAATGCTCATCCG-3′ and reverse primer 5′-AAGCATTCTGCCGACATGG-3′ homologous to the CAT gene. The RT-PCR signal from CAT mRNA transcripts was normalized with the signal obtained from β-actin using the primer pair (forward: 5′-CGTGGGCCGCCCTAGGCACCA-3′; reverse: 5′-TTGGCCTTAGGGTTCAGGGGG-3′) as described previously (26Zheng X.L. Gui Y. Sharkey K.A. Hollenberg M.D. J. Pharmacol. Exp. Ther. 1999; 289: 632-640PubMed Google Scholar). The pBabe-Puro retroviral expression vector (27Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1903) Google Scholar) with and without RasAsn-17 insert was transfected into AmphoPckTM-293 cell (CLONTECH), using LipofectAMINE as per the manufacturer's instructions. The supernatants containing the virus were used to infect stable Hep G2 cells that harbored the pAI.474-CAT reporter gene in the presence of 8 μg/ml Polybrene. After elimination of uninfected cells by adding 2.5 μg/ml puromycin to culture medium, the cells were treated with 17 nm EGF, 100 microunits/ml insulin or 5 μmbpV(phen), prior to assaying for CAT activity. Sp1 mutants were created using the QuickChangTM site-directed mutagenesis kit (Stratagene, Heidelberg, Germany) according to manufacturer's instruction (28Nelson M. Zhang Y. Van Etten J.L. EXS. 1993; 64: 186-211PubMed Google Scholar, 29Vandeyar M.A. Weiner M.P. Hutton C.J. Batt C.A. Gene ( Amst.). 1988; 65: 129-133Crossref PubMed Scopus (226) Google Scholar). In brief, CMV-Sp1 plasmid DNA was denatured and annealed with oligonucleotide primers containing the desired mutation. Using the nonstrand-displacing action of PfuTurbo DNA polymerase, the enzyme extended and incorporated the mutant primers resulting in nicked circular DNA. The methylated, nonmutated parental DNA templates were subsequently digested with DpnI. The circular, nicked double-stranded DNA was transformed into XL1-Blue supercompetent cells. After transformation, the XL1-Blue supercompetent cells repaired the nicks in the mutated plasmid. The mutations were verified using nucleotide sequencing before use in transfection of Hep G2 cells. We recently described the creation of a stable Hep G2 cell line harboring the reporter, pAI.474-CAT. This plasmid is comprised of the −474 to −7 fragment of the rat apoA-I promoter fused to the reporter gene, CAT (23Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). In these cells, CAT activity (Fig.1 A) increased following treatment with 17 nm EGF. Induction appeared to be rapid with detectable increases at 3 h following exposure to the hormone. The extrapolation of the data points to time 0 suggested an almost instantaneous induction by EGF. Similarly, 100 milliunits/ml insulin also stimulated CAT activity with comparable kinetics. The only difference was that following 48 h of exposure to EGF, CAT activity in the cells increased 7-fold versus a 3–3.5-fold caused by insulin. Like insulin, 5 μm mimetic, bpV(phen) also enhanced apoA-I transcription 4–5-fold (Fig.2).Figure 2Inhibiting EGF receptor kinase blocks induction of apoA-I. Panel A , shows an autoradiograph of CAT activity in stable cells treated with either 17 nm EGF, 100 microunits/ml insulin, or 5 μmbpV(phen) for 24 h in the presence (lanes 4, 6, and8) or absence (lanes 3, 5, and 7) of 1 μm PD153035. Panel B, shows a graph of the relative CAT activities in cells treated with conditions noted at thebottom of each bar (mean ± S.E.,n = 4). Asterisk (*) denotes a significant difference with p < 0.01 between the groups with and without PD153035 treatment as determined by ANOVA.View Large Image Figure ViewerDownload (PPT) In search of a potential mechanism underlying EGF induction ofapoA-I promoter activity, the cells were pretreated for 30 min with 10 μm cycloheximide, a protein synthesis inhibitor, or 1 μm actinomycin D, a transcription inhibitor, prior to 24 h of exposure to EGF or insulin. Total RNA was extracted from these cells and assayed for abundance of CAT mRNA using RT-PCR. Results showed that whereas, cycloheximide did not inhibit EGF or insulin induction of CAT mRNA expression (Fig.1 B), as expected actinomycin D blocked transcription of theCAT gene. These findings suggest that both hormones had a direct effect on apoA-I promoter activity and did not require de novo synthesis of other proteins. Whether EGF or insulin induction of apoA-I promoter correlated with increases in apoA-I protein is not known. Therefore, Western blot analysis was used to measure the abundance of the protein in both whole cell lysate and culture medium from cells treated with either EGF or insulin or 5 μm bpV(phen), a insulin mimetic, for 24 h. All three agents increased apoA-I protein in both cell lysate and culture medium (Fig. 1 C). Together the above results show that EGF and insulin share similar activities in the direct induction of apoA-I promoter activity leading to increased abundance of the protein. The action(s) of EGF is initiated by its binding to a specific membrane receptor with intrinsic tyrosine kinase activity. To determine whether EGF induction of apoA-I is mediated by its receptor, stably transfected Hep G2 cells were exposed to 1 μm PD153035, a specific inhibitor for the EGF receptor (30Fry D.W. Kraker A.J. McMichael A. Ambroso L.A. Nelson J.M. Leopold W.R. Connors R.W. Bridges A.J. Science. 1994; 265: 1093-1095Crossref PubMed Scopus (815) Google Scholar), prior to the addition of hormone. The results (Fig. 2) showed that treatment with PD153035 completely blocked EGF induction ofapoA-I activity. In contrast, neither insulin nor bpV(phen) induction of CAT activity in the same cells was affected by PD153035 (Fig. 2). These findings show that the activation of EGF receptor tyrosine kinase is required for apoA-I induction by the hormone. Furthermore, blockade of EGF receptor activity had no effect on the actions of insulin or bpV(phen). Ligand binding to a receptor with intrinsic tyrosine kinase activity triggers this function leading to receptor autophosphorylation. These events initiate signal transduction and cellular responses. Activated tyrosine kinase receptor can initiate several intracellular pathways including; Ras-MAP kinase cascade, PI 3-kinase, and PLCγ pathways (16Carpenter G. Bioessays. 2000; 22: 697-707Crossref PubMed Scopus (305) Google Scholar, 31Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). However, the cellular responses and their signaling mechanisms are largely dependent on the cell type examined. Furthermore, the actions of a single hormone may vary from one cell type to another. Therefore, we wanted to examine the intracellular signaling pathway by which EGF and insulin inducedapoA-I gene expression in Hep G2 cells. To identify the signaling pathways underlying the actions of EGF and insulin, we used a panel of specific inhibitors known to block selected pathways. The results show that inhibitors of PI 3-kinase, 100 nm wortmannin or 10 μm LY294002 (32Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar), did not affect the actions of EGF, insulin, or bpV(phen) (Fig.3). Whereas, the MEK1 inhibitor PD98059 (1 μm) completely blocked EGF induction ofapoA-I expression (Fig. 3), it only inhibited 50% of insulin action on apoA-I activity (Fig. 3). Although the PKC inhibitor GF109203X (2 μm) (33Eichholtz T. de Bont D.B. de Widt J. Liskamp R.M. Ploegh H.L. J. Biol. Chem. 1993; 268: 1982-1986Abstract Full Text PDF PubMed Google Scholar, 34Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar) did not affect EGF response, it did inhibit insulin induction of apoA-I gene by 50% (Fig. 3). More importantly, the combination of PD98059 and GFX together completely blocked insulin induction of apoA-I(Fig. 3). Like insulin, apoA-I induction by bpV(phen) was also blocked in the presence of both inhibitors (data not shown). That MAP kinase was activated during EGF, insulin, or bpV(phen) induction of the gene was assessed by treating the cells with or without the MEK1 inhibitor, PD98059. Cell lysate was assayed for p42/44 kinase using Western blot analysis (Fig. 3 B, inset). The treatment of cells with PD98059 inhibited the phosphorylation of p42/44 MAP kinase (Fig. 3 B, inset). The addition of these data to that above show the following: (i) the PI 3-kinase pathway does not participate in either EGF or insulin induction of apoA-Iexpression; (ii) the MAP kinase cascade is the sole mediator of EGF stimulation of apoA-I; and (iii) insulin or bpV(phen) action requires two independent pathways mediated by PKC and MAP kinase to stimulate the apoA-I gene. Since insulin or bpV(phen) induction of apoA-I is blocked by the PKC inhibitor GFX, this implies that the converse where PKC is activated should enhance apoA-I gene activity. Therefore, we tested whether the PKC activator PDBu stimulated apoA-I expression (Fig. 4) and if this induction is sensitive to the MEK inhibitor PD98059 (13Zheng X.L. Matsubara S. Diao C. Hollenberg M.D. Wong N.C. J. Biol. Chem. 2000; 275: 31747-31754Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Results show treatment of cells with 25 nm PDBu increased apoA-I promoter activity and that was blocked by GFX. However, this induction was not affected by pretreatment of cells with PD98059. These findings suggest that PKC activation does not crossover to the MAP kinase cascade above the level of MEK as described in other cell systems (35Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1161) Google Scholar, 36Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (402) Google Scholar). To determine whether Ras participates in the EGF and insulin induction ofapoA-I, we infected the stable Hep G2 cells with RasAsn-17 retrovirus to express dominant negative RasAsn-17. This mutant interrupts the Ras-dependent signaling pathway (37Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 38Herskowitz I. Nature. 1987; 329: 219-222Crossref PubMed Scopus (869) Google Scholar). Results (Fig.5) showed that expression of RasAsn-17 blocked EGF induction of the apoA-Igene. As expected, the expression of RasAsn-17 partially but significantly attenuated insulin induction of apoA-Ipromoter. Exposure of cells to GFX prior to RasAsn-17retrovirus infection blocked both insulin (Fig. 5) or bpV(phen) (data not shown) induction of the gene. Additionally, the insertion of the dominant negative RasAsn-17 in cells did not affect the stimulation of apoA-I gene induced by PDBu, a PKC activator (data not shown). These findings show that EGF stimulatesapoA-I induction via a Ras-MAP kinase pathway and further solidifying the finding that insulin induction of the gene is mediated independently via both the Ras-MAP kinase and the PKC pathways. Next we searched for the cis-acting element(s) in the apoA-I promoter that mediated the actions of EGF. Thus serial deletion constructs ofapoA-I promoter were transfected into Hep G2 cells and then treated with EGF, insulin, or bpV(phen). Results (Fig.6 A) showed that EGF stimulation of the promoter like insulin or bpV(phen) was abolished following deletion of the −425 to −376 fragment of the promoter. Our previous studies showed that insulin induction of apoA-Igene required a motif called the IRCE −411 to −404. Deletion analysis suggested that the same motif may also be required for response to EGF. Therefore, we tested the activity of a reporter construct containing a mutant of the IRCE. Results (Fig. 6 B) showed that the reporter containing the mutant IRCE was not inducible by EGF, insulin, or bpV(phen). These finding show that the actions of all three agents require the presence of an intact IRCE. The IRCE motif is GC-rich and binds to a transcription factor, Sp1. Therefore, we speculate that Sp1 might participate in EGF, insulin, or bpV(phen) induction of the promoter. To investigate this hypothesis, we used submaximal concentrations of EGF (8.5 nm), insulin (50 microunits/ml), and bpV(phen) (2.5 μm) to stimulate cells that do or do not overexpress of Sp1. Results (Fig. 7) showed that Sp1 expression alone increased apoA-I gene expression 2-fold and submaximal doses of EGF, insulin, and bpV(phen) caused a 2.1-, 1.8-, and 1.7-fold, respectively, in apoA-I transcription. In the presence of both Sp1 and EGF, insulin, or bpV(phen) we observed a marked induction of promoter activity of 7.4-, 6.5-, and 6.4-fold, respectively. The combined actions of Sp1 and hormone were not simply additive but synergistic. Next we postulated that EGF stimulation ofapoA-I was mediated by the MAP kinase pathway involves phosphorylation of Sp1. To test this idea, we inspected Sp1 protein for potential sites of MAP kinase phosphorylation according to published algorithms (39Kreegipuu A. Blom N. Brunak S. Nucleic Acids Res. 1999; 27: 237-239Crossref PubMed Scopus (245) Google Scholar). Six potential sites for MAP kinase were identified (Fig. 8) (40Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 41Merchant J.L. Du M. Todisco A. Biochem. Biophys. Res. Commun. 1999; 254: 454-461Crossref PubMed Scopus (179) Google Scholar). The prediction scores ranged from 0 to 1.000 with values that exceeded 0.5 reflecting potential phosphorylation sites. Three of the sites with the highest scores were Thr266 (0.767), Thr414 (0.664), and Thr650 (0.726) (41Merchant J.L. Du M. Todisco A. Biochem. Biophys. Res. Commun. 1999; 254: 454-461Crossref PubMed Scopus (179) Google Scholar). Thr266 was especially interesting because it was located toward the C-terminal region of the second glutamine-rich domain, one of the known trans-activation domains of Sp1 (19Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar). Since the amino acids surrounding Thr266appeared most homologous against a consensus MAP kinase site, we mutated the threonine residue by replacing it with an alanine to create Sp1-T266A. The use of Sp1-T266A in transfection studies revealed the following results. Although transfection of Sp1-T266A alone into stable Hep G2 cells augmented activity of pAI.474-CAT, the activity was not significantly different from wild-type Sp1. When the transfected cells were exposed to EGF, apoA-I induction was attenuated by 54% in cells containing the mutant Sp1 compared with the wild-type. However, PDBu induction of apoA-I expression mediated by PKC was the same in the presence of the mutant or wild-type Sp1. These data suggest that amino acid Thr266 in Sp1 is required for full EGF induction of apoA-I. ApoA-I is an essential component of HDL. The protein alone or in the form of HDL mediate a normal physiologic process called reverse cholesterol transport, which lowers total body cholesterol (5Barter P.J. Rye K.A. Curr. Opin. Lipidol. 1996; 7: 82-87Crossref PubMed Scopus (171) Google Scholar, 7Miller N.E. La Ville A. Crook D. Nature. 1985; 314: 109-111Crossref PubMed Scopus (158) Google Scholar) thereby reducing the risk of IHD. This function of apoA-I makes it an important target for therapies to enhance expression of the protein. To" @default.
• W1548282121 created "2016-06-24" @default.
• W1548282121 creator A5032080740 @default.
• W1548282121 creator A5034300226 @default.
• W1548282121 creator A5037959449 @default.
• W1548282121 creator A5045627202 @default.
• W1548282121 creator A5052769119 @default.
• W1548282121 date "2001-04-01" @default.
• W1548282121 modified "2023-10-07" @default.
• W1548282121 title "Epidermal Growth Factor Induction of Apolipoprotein A-I Is Mediated by the Ras-MAP Kinase Cascade and Sp1" @default.
• W1548282121 cites W1479869774 @default.
• W1548282121 cites W1529396499 @default.
• W1548282121 cites W1546982941 @default.
• W1548282121 cites W1573821584 @default.
• W1548282121 cites W1607258414 @default.
• W1548282121 cites W1755902553 @default.
• W1548282121 cites W1968129408 @default.
• W1548282121 cites W1969050011 @default.
• W1548282121 cites W1969785844 @default.
• W1548282121 cites W1971915141 @default.
• W1548282121 cites W1979027806 @default.
• W1548282121 cites W1979040872 @default.
• W1548282121 cites W1981223977 @default.
• W1548282121 cites W1981237356 @default.
• W1548282121 cites W1981920188 @default.
• W1548282121 cites W1986868728 @default.
• W1548282121 cites W1994172249 @default.
• W1548282121 cites W1997505548 @default.
• W1548282121 cites W1999200731 @default.
• W1548282121 cites W2001895687 @default.
• W1548282121 cites W2005427150 @default.
• W1548282121 cites W2006590877 @default.
• W1548282121 cites W2006659499 @default.
• W1548282121 cites W2012441637 @default.
• W1548282121 cites W2013525819 @default.
• W1548282121 cites W2018304885 @default.
• W1548282121 cites W2018746110 @default.
• W1548282121 cites W2023620533 @default.
• W1548282121 cites W2024470778 @default.
• W1548282121 cites W2027378911 @default.
• W1548282121 cites W2029551349 @default.
• W1548282121 cites W2030246330 @default.
• W1548282121 cites W2032984787 @default.
• W1548282121 cites W2035414423 @default.
• W1548282121 cites W2037653294 @default.
• W1548282121 cites W2039383779 @default.
• W1548282121 cites W2044349160 @default.
• W1548282121 cites W2056213387 @default.
• W1548282121 cites W2063767041 @default.
• W1548282121 cites W2069637592 @default.
• W1548282121 cites W2078309101 @default.
• W1548282121 cites W2080094531 @default.
• W1548282121 cites W2082022155 @default.
• W1548282121 cites W2088622701 @default.
• W1548282121 cites W2092332672 @default.
• W1548282121 cites W2094703210 @default.
• W1548282121 cites W2095152292 @default.
• W1548282121 cites W2135027079 @default.
• W1548282121 cites W2210815227 @default.
• W1548282121 cites W2306336406 @default.
• W1548282121 doi "https://doi.org/10.1074/jbc.m011031200" @default.
• W1548282121 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11278817" @default.
• W1548282121 hasPublicationYear "2001" @default.
• W1548282121 type Work @default.
• W1548282121 sameAs 1548282121 @default.
• W1548282121 citedByCount "64" @default.
• W1548282121 countsByYear W15482821212012 @default.
• W1548282121 countsByYear W15482821212013 @default.
• W1548282121 countsByYear W15482821212015 @default.
• W1548282121 countsByYear W15482821212017 @default.
• W1548282121 countsByYear W15482821212018 @default.
• W1548282121 countsByYear W15482821212019 @default.
• W1548282121 countsByYear W15482821212020 @default.
• W1548282121 countsByYear W15482821212021 @default.
• W1548282121 countsByYear W15482821212022 @default.
• W1548282121 crossrefType "journal-article" @default.
• W1548282121 hasAuthorship W1548282121A5032080740 @default.
• W1548282121 hasAuthorship W1548282121A5034300226 @default.
• W1548282121 hasAuthorship W1548282121A5037959449 @default.
• W1548282121 hasAuthorship W1548282121A5045627202 @default.
• W1548282121 hasAuthorship W1548282121A5052769119 @default.
• W1548282121 hasBestOaLocation W15482821211 @default.
• W1548282121 hasConcept C184235292 @default.
• W1548282121 hasConcept C185592680 @default.
• W1548282121 hasConcept C2776362946 @default.
• W1548282121 hasConcept C34146451 @default.
• W1548282121 hasConcept C43617362 @default.
• W1548282121 hasConcept C502942594 @default.
• W1548282121 hasConcept C54355233 @default.
• W1548282121 hasConcept C81885089 @default.
• W1548282121 hasConcept C86803240 @default.
• W1548282121 hasConcept C95444343 @default.
• W1548282121 hasConceptScore W1548282121C184235292 @default.
• W1548282121 hasConceptScore W1548282121C185592680 @default.
• W1548282121 hasConceptScore W1548282121C2776362946 @default.
• W1548282121 hasConceptScore W1548282121C34146451 @default.
• W1548282121 hasConceptScore W1548282121C43617362 @default.
• W1548282121 hasConceptScore W1548282121C502942594 @default.

• next