FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079081366> ?p ?o ?g. }

• W2079081366 endingPage "297" @default.
• W2079081366 startingPage "287" @default.
• W2079081366 abstract "Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through a novel IFN response element called the γ-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626–12632. Here, we describe a new IFN-γ-stimulated pathway that operates C/EBP-β-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-γ to stimulate C/EBP-β-dependent expression. Sustained ERK activation directly correlated with C/EBP-βdependent gene expression in response to IFN-γ. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-γ-stimulated gene induction through the γ-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-β-induced gene expression required STAT1, but not JAK1. A C/EBP-β mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-β to IFN-γ signaling through ERKs. Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through a novel IFN response element called the γ-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626–12632. Here, we describe a new IFN-γ-stimulated pathway that operates C/EBP-β-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-γ to stimulate C/EBP-β-dependent expression. Sustained ERK activation directly correlated with C/EBP-βdependent gene expression in response to IFN-γ. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-γ-stimulated gene induction through the γ-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-β-induced gene expression required STAT1, but not JAK1. A C/EBP-β mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-β to IFN-γ signaling through ERKs. interferons interferon-stimulated gene interferon-stimulated gene factor Janus kinase signal transducer and activator of transcription γ-interferon-activated transcriptional element CCAAT/enhancer-binding protein-β extracellular signal-regulated kinase diphosphorylated extracellular signal-regulated kinase mitogen-activated protein kinase mitogen-activated protein kinase kinase interleukin activator protein-1 response element polyacrylamide gel electrophoresis myelin basic protein mitogen-activated protein kinase/extracellular signal-regulated kinase kinase epidermal growth factor humanγ-interferon receptor Interferons (IFNs)1regulate the antiviral, antitumor, and immune responses in vertebrates by inducing the transcription of a number of IFN-stimulated genes (ISGs). Induction of ISGs occurs primarily due to the activation of the JAK-STAT pathway (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). Type I (IFN-α/β) and type II (IFN-γ) IFNs bind to distinct cell-surface receptors and activate the signals that up-regulate the expression of ISGs (2Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). Upon binding to their receptor, IFN-α/β induce the tyrosine phosphorylation of the cytoplasmic tails of receptor using the JAKs Tyk2 and JAK1. JAKs undergo tyrosyl phosphorylation prior to inducing the receptor phosphorylation. Activated JAKs phosphorylate the STAT2 and STAT1 proteins at critical tyrosines. The STAT2-STAT1 dimer dissociates from the receptor and forms a heteromeric complex with a DNA-binding protein, p48 or IFN regulatory factor-9 or ISGF3γ (3Veals S.A. Schindler C. Leonard D. Fu X.Y. Aebersold R. Darnell Jr., J.E. Levy D.E. Mol. Cell. Biol. 1992; 12: 3315-3324Crossref PubMed Scopus (349) Google Scholar). This complex, known as ISGF3, binds to the IFN-stimulated response element of the ISGs and induces gene expression (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). Ligand-activated IFN-γ receptor recruits the Janus kinases JAK1 and JAK2, which selectively stimulate the phosphorylation of STAT1 (4Bach E.A. Tanner J.W. Marsters S. Ashkenazi A. Aguet M. Shaw A.S. Schreiber R.D. Mol. Cell. Biol. 1996; 16: 3214-3221Crossref PubMed Scopus (126) Google Scholar). STAT1 dimers then rapidly migrate to the nucleus and induce the expression of ISGs that contain a γ-IFN-activated site (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). STAT activation does not require new protein synthesis and is short-lived, lasting no longer than 30 min after ligand/receptor engagement. It does not persist over longer periods of time, despite the presence of IFN in the extracellular environment (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). Such deregulation may occur due to the action of tyrosine phosphatases (5David M. Grimley P.M. Finbloom D.S. Larner A.C. Mol. Cell. Biol. 1993; 13: 7515-7521Crossref PubMed Scopus (102) Google Scholar, 6Haspel R.L. Salditt-Georgieff M. Darnell Jr., J.E. EMBO J. 1996; 15: 6262-6268Crossref PubMed Scopus (272) Google Scholar) or degradation of STATs by proteasome (7Kim T.K. Maniatis T. Science. 1996; 273: 1717-1719Crossref PubMed Scopus (361) Google Scholar). The SOCS-1 (suppressor ofcytokine signaling-1) protein has been shown to be critical for inhibiting IFN-γ responses in vivo(8Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Induction of ISGs by IFN-γ is far more complex than that of IFN-α/β, largely due to the facts that the temporal control of these genes is variable and that, in some cases, blockade of protein synthesis prevents their expression (9Sen G.C. Ransohoff R.M. Adv. Virus Res. 1993; 42: 57-102Crossref PubMed Scopus (313) Google Scholar). These data suggest that IFN-γ may activate different transactivating factors in a JAK-STAT-dependent or -independent manner. Evidence for these pathways is only beginning to accumulate. For example, the repression of the c-myc gene by IFN-γ occurs via both STAT1-dependent and -independent mechanisms (10Ramana C.V. Grammatikakis N. Chernov M. Nguyen H. Goh K.C. Williams B.R. Stark G.R. EMBO J. 2000; 19: 263-272Crossref PubMed Scopus (251) Google Scholar). A number of transcription factors such as IFN regulatory factor-1 (11Briken V. Ruffner H. Schultz U. Schwarz A. Reis L.F. Strehlow I. Decker T. Staeheli P. Mol. Cell. Biol. 1995; 15: 975-982Crossref PubMed Google Scholar), IFN consensus sequence binding protein (12Contursi C. Wang I.M. Gabriele L. Gadina M. O'Shea J. Morse III, H.C. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 91-96Crossref PubMed Scopus (65) Google Scholar), class II transactivator (13Steimle V. Siegrist C.A. Mottet A. Lisowska-Grospierre B. Mach B. Science. 1994; 265: 106-109Crossref PubMed Scopus (710) Google Scholar, 14Chin K.C. Mao C. Skinner C. Riley J.L. Wright K.L. Moreno C.S. Stark G.R. Boss J.M. Ting J.P. Immunity. 1994; 1: 687-697Abstract Full Text PDF PubMed Scopus (130) Google Scholar), and RF-X (15Brickey W.J. Wright K.L. Zhu X.S. Ting J.P. J. Immunol. 1999; 163: 6622-6630PubMed Google Scholar) activate specific sets of ISGs in an IFN-γ-dependent manner. IFN-γ augments IFN-α/β-induced gene expression by up-regulating the gene encoding the p48 subunit of ISGF3 (16Bandyopadhyay S.K. Kalvakolanu D.V. Sen G.C. Mol. Cell. Biol. 1990; 10: 5055-5063Crossref PubMed Scopus (55) Google Scholar, 17Levy D.E. Lew D.J. Decker T. Kessler D.S. Darnell Jr., J.E. EMBO J. 1990; 9: 1105-1111Crossref PubMed Scopus (176) Google Scholar). A central role for p48 in IFN-regulated pathways is highlighted by several observations. Certain oncogenic viruses down-regulate p48 expression to evade the action of IFNs (18Kalvakolanu D.V. Trends Microbiol. 1999; 7: 166-171Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and its activity is inhibited in some human tumor cell lines (19Petricoin E.R. David M. Fang H. Grimley P. Larner A.C. Vande Pol S. Mol. Cell. Biol. 1994; 14: 1477-1486Crossref PubMed Scopus (39) Google Scholar). IFN-γ-induced expression of the p48 gene is rather slow (12–18 h) and is inhibited by the protein synthesis inhibitor cycloheximide (16Bandyopadhyay S.K. Kalvakolanu D.V. Sen G.C. Mol. Cell. Biol. 1990; 10: 5055-5063Crossref PubMed Scopus (55) Google Scholar, 17Levy D.E. Lew D.J. Decker T. Kessler D.S. Darnell Jr., J.E. EMBO J. 1990; 9: 1105-1111Crossref PubMed Scopus (176) Google Scholar, 20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar), indicating the involvement of a novel regulatory element and its cognate factors. Promoter analysis revealed that the p48 gene promoter has no γ-IFN-activated site, but instead contains a unique IFN-γ response element termed GATE (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). Although GATE is partially homologous to the IFN-stimulated response element, factors that bind to the IFN-stimulated response element do not bind to GATE. The activity of GATE-binding factors is modulated by IFN-γ and is inhibited by cycloheximide and staurosporine (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). These observations suggest that IFN-γ regulates not only the synthesis of GATE-binding factors, but also their post-translational modifications. We have recently identified one of these as the transcription factor CCAAT/enhancer-binding protein-β (C/EBP-β) (21Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), a regulator of acute-phase responses and cell differentiation (22Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar, 23Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Although overexpression of C/EBP-β alone elevates basal gene expression, treatment with IFN-γ further augments gene expression through GATE (21Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Our recent study demonstrated for the first time a role for C/EBP-β in IFN-γ-regulated gene expression. Since the JAK-STAT pathway is rapidly activated and deactivated after IFN-γ treatment (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar), it was unclear which kinases could regulate the function of C/EBP-β under these conditions. C/EBP-β binds to the consensus sequence TTNNGNAAT (14Chin K.C. Mao C. Skinner C. Riley J.L. Wright K.L. Moreno C.S. Stark G.R. Boss J.M. Ting J.P. Immunity. 1994; 1: 687-697Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 15Brickey W.J. Wright K.L. Zhu X.S. Ting J.P. J. Immunol. 1999; 163: 6622-6630PubMed Google Scholar). The nucleotide sequence of GATE is as following: 5′-CCCGAGGAGAATTGAAACTTAGGG-3′. Six of the nine nucleotides in this region of GATE are homologous to the consensus C/EBP-β-binding site. Mutation of the AAACTT nucleotides of wild-type GATE resulted in a loss of C/EBP-β binding. In addition, a shorter GATE (GAGGAGGAATTGAAACTT) that encompassed the C/EBP-β-binding site (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar) or the C/EBP-binding site of GATE alone did not support gene induction by IFN-γ (data not shown). Furthermore, the majority (80%) of the IFN-γ-induced response was lost upon mutation of the C/EBP-β-binding site. Thus, a full-length GATE including the C/EBP-β-binding site is critical for IFN-γ induction. In addition, a C/EBP-β mutant lacking the transactivation domain blunted GATE-dependent gene expression (21Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). These data together indicate that C/EBP-β is an important component of GATE-dependent gene expression. In this investigation, we show that IFN-γ stimulates the transcriptional activity of C/EBP-β through activation of ERK1 and ERK2 MAPKs, but independently of c-Raf and c-Ras. Pharmacological and gene-specific dominant-negative inhibitors of MKK1, ERK1, and ERK2 block gene expression. Surprisingly, STAT1, but not JAK1, is required to activate the C/EBP-β-dependent IFN-γ response. Together, our study identifies a new IFN-γ signaling pathway that couples the ERKs (MAPKs) to C/EBP-β-dependent gene regulation. Recombinant murine IFN-γ (Roche Molecular Biochemicals); human IFN-γ (Pestka Biomedical Labs); IL-6 (Genzyme Corp.); staurosporine (Sigma); PD98059, SB202190, and SB202474 (Calbiochem); and U0126 (Promega) were used in this study. Antibodies specific for phospho-ERK1/2 (Sigma) and ERK1, ERK2, JNK1, p38, Raf-1, C/EBP-β, tubulin, and actin (Santa Cruz Biotechnology) were used as recommended by the manufacturers. The RAW murine macrophage cell line (RAW264.7) was grown in RPMI 1640 medium supplemented with 5% fetal bovine serum. The murine ISGF3γ promoter construct P4 has been described previously (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). Human 2fTGH (parental), U3A (STAT1-deficient), and U4A (JAK1-deficient) cell lines (24Muller M. Laxton C. Briscoe J. Schindler C. Improta T. Darnell Jr., J.E. Stark G.R. Kerr I.M. EMBO J. 1993; 12: 4221-4228Crossref PubMed Scopus (373) Google Scholar, 25Muller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (646) Google Scholar) were a gift from George Stark (Cleveland Clinic Lerner Research Institute). They were cultured in Dulbecco's modified Eagle's medium with 5% newborn calf serum and hygromycin (100 μg/ml) to retain the mutant characteristics. The isogenic mouse fibroblasts WTSIM (wild-type), SKIM (STAT1−/−), WTJIM (wild-type), and JKIM (JAK1−/−) have been described elsewhere (26Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1401) Google Scholar,27Rodig S.J. Meraz M.A. White J.M. Lampe P.A. Riley J.K. Arthur C.D. King K.L. Sheehan K.C. Yin L. Pennica D. Johnson Jr., E.M. Schreiber R.D. Cell. 1998; 93: 373-383Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). The SCC mouse fibroblast cell line expressing the wild-type or mutant human IFN-γ receptor has been described (28Kaplan D.H. Greenlund A.C. Tanner J.W. Shaw A.S. Schreiber R.D. J. Biol. Chem. 1996; 271: 9-12Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These cell lines express human chromosome 21, which provides the accessory factor of the receptor, and permit human IFN-γ-induced actions in the mouse background. The murine ISGF3γ (p48) promoter construct P4 has been described in our previous study (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). In this construct, a 74-base pair element of the murine p48 gene promoter encompassing GATE was cloned upstream of the SV40 early promoter. This construct, like the wild-type full-length promoter, consistently responds to IFN-γ and C/EBP-β (21Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) when transfected into multiple cell types. Mutation of the GATE sequence in this construct caused a loss of IFN-γ response and C/EBP-β binding (21Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). C/EBP-β cDNA cloned into a mammalian expression vector was provided by Richard Hanson (Case Western Reserve University, Cleveland, OH). Wild-type and mutant (Mut1 and Mut2) murine C/EBP-β (C/EBP-related protein-2) proteins were provided by Peter Johnson (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) (29Williams S.C. Baer M. Dillner A.J. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar). Wild-type and point mutant (T235A) human C/EBP-β (NF-IL6) proteins cloned into the pCMV vector were a gift from S. Akira (30Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (518) Google Scholar). Human STAT1 cDNA cloned under the control of a constitutive enhancer of the pCXN2 vector has been described (31Weihua X. Kolla V. Kalvakolanu D.V. J. Biol. Chem. 1997; 272: 9742-9748Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Wild-type ERK2 cDNA cloned into the pCMV vector has been described earlier (32Shapiro P.S. Ahn N.G. J. Biol. Chem. 1998; 273: 1788-1793Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Catalytically inactive dominant-negative mutants of ERK1 (K71R) and ERK2 (K52R) cloned into the pCEP4 vector (33Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar) were provided by Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, TX). The wild-type, constitutively active, and kinase-inactive MKK1 cDNAs cloned into pCMV were described previously (32Shapiro P.S. Ahn N.G. J. Biol. Chem. 1998; 273: 1788-1793Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 34Tolwinski N.S. Shapiro P.S. Goueli S. Ahn N.G. J. Biol. Chem. 1999; 274: 6168-6174Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The p38 kinase AGF mutant (35Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar) was provided by Roger Davis (Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA). Kinase-inactive MKK1 has a K97M mutation, and the constitutively active MKK1 mutant has a deletion in the N-terminal region from amino acids 44 to 51 and serines 218 and 222 mutated to glutamate and aspartate, respectively (32Shapiro P.S. Ahn N.G. J. Biol. Chem. 1998; 273: 1788-1793Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 34Tolwinski N.S. Shapiro P.S. Goueli S. Ahn N.G. J. Biol. Chem. 1999; 274: 6168-6174Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Wild-type Ras and dominant-negative Ras (N-17) constructs were described elsewhere (36Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar). Constitutively active c-Raf (Raf-BXB) and dominant-negative c-Raf were described previously (37Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (397) Google Scholar). The authenticity of these mutants has been confirmed using a luciferase reporter gene driven by the AP-1 response element (AP-1RE). The AP-1RE-Luc plasmid was provided by Robert Freund (University of Maryland, Baltimore, MD). In this construct, two copies of the consensus AP-1RE were inserted upstream of the SV40 early promoter in the pGL3 promoter vector (Promega). Northern and Western blot analyses, transfection, β-galactosidase and luciferase assays, and SDS-PAGE analyses were performed as described in our earlier work (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). The total amount of transfected DNA (1.5 μg) was kept constant by adding pBluescript SK DNA where required. In general, 0.4 μg of luciferase and 0.1 μg of C/EBP-β expression vector were used. A β-actin promoter-driven β-galactosidase reporter (0.2 μg) was used as an internal control for normalizing variations in transfection efficiency as described in our previous study (20Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). Transfection assays were repeated at least three times. Luciferase and β-galactosidase assays were essentially similar to those described in common laboratory manuals (38Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1994Google Scholar). After stimulation with the indicated reagents, cell extracts were prepared using a lysis buffer supplemented with protease and phosphatase inhibitors as described (32Shapiro P.S. Ahn N.G. J. Biol. Chem. 1998; 273: 1788-1793Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). ERK2-, JNK1-, p38-, or Raf-1-specific antibodies (0.4 μg) conjugated to protein A-Sepharose (Amersham Pharmacia Biotech) were used to immunoprecipitate the cognate proteins from ∼200 μg of total cell protein at 4 °C for 2 h. Immunoprecipitates were washed extensively with 25 mm HEPES (pH 7.4), 25 mm MgCl2, 1 mm dithiothreitol, and 0.2 mm sodium orthovanadate. ERK2, JNK1, p38, and Raf-1 kinase activities were determined using 2.5 μg of myelin basic protein (MBP), glutathione S-transferase-Jun-(1–79), glutathione S-transferase-activating transcription factor-2, and wild-type MKK1 coupled to kinase-inactive ERK2, respectively. Kinase reactions were stopped with SDS-PAGE loading buffer after incubation at 30 °C for 30 min in the presence of [γ-32P]ATP. Proteins in the kinase reactions were separated by SDS-PAGE and Western-blotted, and substrate phosphorylation was determined by PhosphorImager analysis. For analyzing active ERK1/2, cell lysates were prepared as described above, separated by SDS-PAGE, and immunoblotted using the phospho-ERK1/2-specific antibody. Total ERK was determined in these samples by using antibodies specific for ERK2 or ERK1. Intensity of the activated ERK bands was quantified using a Molecular Dynamics laser densitometer. Data from at least three independent samples are presented in the bar graphs. To understand the role of protein kinases in IFN-γ-stimulated gene expression through GATE, we studied the influence of staurosporine, a protein kinase inhibitor. The RAW macrophage cell line was transfected with the P4 reporter gene, in which a 74-base pair element from the p48 promoter drives the expression of the luciferase gene. Cells were treated with IFN-γ in the presence or absence of staurosporine. Although IFN-γ alone strongly up-regulated GATE-dependent gene expression, it was strongly blocked by staurosporine (Fig.1 A). Since C/EBP-β binds to GATE and induces gene expression (21Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we examined whether staurosporine also blocks C/EBP-β-regulated gene expression. As expected, C/EBP-β strongly induced luciferase gene expression upon cotransfection with the P4 reporter compared with the vector-transfected cells (compare the scales of Fig. 1, Aand B). Treatment of cells with IFN-γ further enhanced C/EBP-induced expression. Staurosporine treatment not only inhibited IFN-γ-augmented gene expression, but also yielded lower luciferase activity than that obtained with C/EBP-β alone. These data suggest that C/EBP-β undergoes phosphorylation in response to IFN-γ treatment. Since staurosporine is a general protein kinase inhibitor and recent reports indicated that MAPKs are activated during IFN treatment (39Goh K.C. Haque S.J. Williams B.R. EMBO J. 1999; 18: 5601-5608Crossref PubMed Scopus (328) Google Scholar,40Uddin S. Majchrzak B. Woodson J. Arunkumar P. Alsayed Y. Pine R. Young P.R. Fish E.N. Platanias L.C. J. Biol. Chem. 1999; 274: 30127-30131Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), we examined the effect of different inhibitors of the MAPK pathways on IFN-γ-induced gene expression through GATE. Three inhibitors, PD98059 (an MKK1-specific inhibitor), SB202474 (a negative control inhibitor for MKK1), and SB202190 (a p38 MAPK-specific inhibitor), were used in this study. RAW cells were transfected with the P4 reporter gene and then treated with a 25 μmconcentration of each inhibitor prepared in Me2SO prior to IFN-γ treatment. PD98059, but not the negative control inhibitor SB202474, strongly blocked gene expression (Fig. 1 C). SB202190 was marginally inhibitory at this dose, but this low-level inhibition is probably not specific since p38 MAPK is not activated under these conditions (see below). PD98059 inhibits activation of MKK1 and suppresses the phosphorylation of ERKs (41Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar). We also studied the effect of a synthetic compound, U0126 (42Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2754) Google Scholar), that specifically blocks the activated MKK1 function (i.e. ERK activation) on IFN-γ-induced gene expression. U0126 inhibited IFN-γ-induced gene expression in a concentration-dependent manner (Fig.1 D). Treatment with Me2SO (vehicle) had no effect on gene induction by IFN-γ. Thus, MKK1 activation appears to be necessary for IFN-γ-induced gene expression. The effect of MKK1 inhibitors on endogenous p48 (ISGF3γ) gene expression was also studied. RAW cells were treated with IFN-γ in the presence or absence of either SB202190 or PD98059 for 16 h. Poly(A)+ RNA was extracted, Northern-blotted, and probed with p48 cDNA (Fig. 2 A). IFN-γ strongly induced the expression of p48 mRNA (compare lanes 1 and 2). However, PD98059, but not SB202190, strongly inhibited IFN-γ-induced gene expression (compare lanes 3 and 4). A 6–8-fold reduction of p48 mRNA expression occurred. Neither inhibitor alone significantly altered p48 mRNA levels (lanes 5 and 6). Although the blot shows a marginal increase in the p48 mRNA signal with the inhibitors alone, this is not a reproducible effect. A reprobing of these blots with 32P-labeled β-actin cDNA revealed the presence of a comparable amount of RNA in all lanes (Fig. 2 B). Thus, a reporter driven by p48 promoter elements and endogenous p48 behave in similar manner in the presence of the MKK inhibitor PD98059. Based on above results, we next examined the effect of MKK1 inhibitors on C/EBP-β-dependent gene expression. Cells were transfected with the P4 reporter and C/EBP-β expression vector and then treated with IFN-γ in the absence or presence of the indicated inhibitors. C/EBP-β itself induced gene expression, and IFN-γ, as expected, stimulated it very strongly (Fig. 1 E). PD98059, but not SB202474, strongly inhibited C/EBP-β-dependent gene expression stimulated by IFN-γ. To ascertain the specificity of such inhibition, a similar experiment was performed using U0126. As expected, U0126 inhibited IFN-γ-stimulated expression in a concentration-dependent manner (Fig. 1 F). To directly demonstrate the role of MKK1 (MEK1), the effect of MKK1 coexpression on GATE-dependent gene expression was determined. Three different MKK1 expression vectors that carry the wild-type, constitutively active, or non-catalytic mutant MKK1 cDNA were employed in these experiments. A control transfection with the pCMV4 vector was also performed. Overexpression of the wild-type MKK1 cDNA alone slightly induced the luciferase gene compared with the vector control (Fig.3 A). Treatment with IFN-γ further stimulated the activity. The constitutively active MKK1 mutant elevated basal expression and augmented IFN-γ-induced expression to significantly higher levels compared with the wild-type MKK1 control. Thus, MKK1 may drive gene expres" @default.
• W2079081366 created "2016-06-24" @default.
• W2079081366 creator A5019299639 @default.
• W2079081366 creator A5020339890 @default.
• W2079081366 creator A5031774412 @default.
• W2079081366 creator A5035965082 @default.
• W2079081366 creator A5051335860 @default.
• W2079081366 creator A5066736391 @default.
• W2079081366 creator A5073626775 @default.
• W2079081366 creator A5086001447 @default.
• W2079081366 date "2001-01-01" @default.
• W2079081366 modified "2023-10-16" @default.
• W2079081366 title "ERK1 and ERK2 Activate CCAAAT/Enhancer-binding Protein-β-dependent Gene Transcription in Response to Interferon-γ" @default.
• W2079081366 cites W1486631729 @default.
• W2079081366 cites W1495187352 @default.
• W2079081366 cites W1516222795 @default.
• W2079081366 cites W1518732132 @default.
• W2079081366 cites W1521367633 @default.
• W2079081366 cites W1532900970 @default.
• W2079081366 cites W1566737177 @default.
• W2079081366 cites W1592356588 @default.
• W2079081366 cites W161652132 @default.
• W2079081366 cites W1702674772 @default.
• W2079081366 cites W179818011 @default.
• W2079081366 cites W1964842789 @default.
• W2079081366 cites W1966474085 @default.
• W2079081366 cites W1971915141 @default.
• W2079081366 cites W1974684256 @default.
• W2079081366 cites W1980687898 @default.
• W2079081366 cites W1983683479 @default.
• W2079081366 cites W1986474736 @default.
• W2079081366 cites W1994828005 @default.
• W2079081366 cites W1996520665 @default.
• W2079081366 cites W1998487776 @default.
• W2079081366 cites W2000583211 @default.
• W2079081366 cites W2001038614 @default.
• W2079081366 cites W2007780460 @default.
• W2079081366 cites W2010489503 @default.
• W2079081366 cites W2013036001 @default.
• W2079081366 cites W2020482185 @default.
• W2079081366 cites W2022139382 @default.
• W2079081366 cites W2022308523 @default.
• W2079081366 cites W2022819740 @default.
• W2079081366 cites W2034779553 @default.
• W2079081366 cites W2043106387 @default.
• W2079081366 cites W2048789360 @default.
• W2079081366 cites W2060320549 @default.
• W2079081366 cites W2068224514 @default.
• W2079081366 cites W2071268780 @default.
• W2079081366 cites W2077349366 @default.
• W2079081366 cites W2079357798 @default.
• W2079081366 cites W2083802827 @default.
• W2079081366 cites W2086262311 @default.
• W2079081366 cites W2093392246 @default.
• W2079081366 cites W2103565638 @default.
• W2079081366 cites W2122506044 @default.
• W2079081366 cites W2128513949 @default.
• W2079081366 cites W2134768631 @default.
• W2079081366 cites W2135177247 @default.
• W2079081366 cites W2146736041 @default.
• W2079081366 cites W2161538693 @default.
• W2079081366 cites W2171569176 @default.
• W2079081366 cites W2252225016 @default.
• W2079081366 cites W2399467729 @default.
• W2079081366 cites W318497777 @default.
• W2079081366 cites W4300659364 @default.
• W2079081366 cites W4313309953 @default.
• W2079081366 cites W98489723 @default.
• W2079081366 doi "https://doi.org/10.1074/jbc.m004885200" @default.
• W2079081366 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10995751" @default.
• W2079081366 hasPublicationYear "2001" @default.
• W2079081366 type Work @default.
• W2079081366 sameAs 2079081366 @default.
• W2079081366 citedByCount "144" @default.
• W2079081366 countsByYear W20790813662012 @default.
• W2079081366 countsByYear W20790813662013 @default.
• W2079081366 countsByYear W20790813662014 @default.
• W2079081366 countsByYear W20790813662015 @default.
• W2079081366 countsByYear W20790813662016 @default.
• W2079081366 countsByYear W20790813662017 @default.
• W2079081366 countsByYear W20790813662018 @default.
• W2079081366 countsByYear W20790813662019 @default.
• W2079081366 countsByYear W20790813662020 @default.
• W2079081366 countsByYear W20790813662021 @default.
• W2079081366 countsByYear W20790813662022 @default.
• W2079081366 crossrefType "journal-article" @default.
• W2079081366 hasAuthorship W2079081366A5019299639 @default.
• W2079081366 hasAuthorship W2079081366A5020339890 @default.
• W2079081366 hasAuthorship W2079081366A5031774412 @default.
• W2079081366 hasAuthorship W2079081366A5035965082 @default.
• W2079081366 hasAuthorship W2079081366A5051335860 @default.
• W2079081366 hasAuthorship W2079081366A5066736391 @default.
• W2079081366 hasAuthorship W2079081366A5073626775 @default.
• W2079081366 hasAuthorship W2079081366A5086001447 @default.
• W2079081366 hasBestOaLocation W20790813661 @default.
• W2079081366 hasConcept C104317684 @default.
• W2079081366 hasConcept C111936080 @default.
• W2079081366 hasConcept C138885662 @default.

• next