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• W2044332034 abstract "CCAAT/enhancer-binding protein β (C/EBPβ) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBPβ gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBPβ genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBPβ promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3′-untranslated region of the C/EBPβ mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt +1614–1621 (5′-TGACGCAA-3′) is responsible for activation of the C/EBPβ gene. Electrophoresis mobility shift analysis revealed that proteins are bound to this element and that the amount of binding is increased following glucose deprivation. This element is homologous to a previously reported mammalian unfolded protein response element that binds XBP-1. Consistent with those data, overexpression of XBP-1 caused an increase in transcription that was mediated by the C/EBPβ mammalian unfolded protein response element. CCAAT/enhancer-binding protein β (C/EBPβ) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBPβ gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBPβ genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBPβ promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3′-untranslated region of the C/EBPβ mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt +1614–1621 (5′-TGACGCAA-3′) is responsible for activation of the C/EBPβ gene. Electrophoresis mobility shift analysis revealed that proteins are bound to this element and that the amount of binding is increased following glucose deprivation. This element is homologous to a previously reported mammalian unfolded protein response element that binds XBP-1. Consistent with those data, overexpression of XBP-1 caused an increase in transcription that was mediated by the C/EBPβ mammalian unfolded protein response element. CCAAT/enhancer-binding protein β (C/EBPβ) 1The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP homology protein; UPR, unfolded protein response; UPRE, UPR element; mUPRE, mammalian UPRE; ERSR, endoplasmic reticulum stress response; ERSE, ERSR element; GRP, glucose-regulated protein; nt, nucleotide(s); ER, endoplasmic reticulum; ASNS, asparagine synthetase; UTR, untranslated region; MEM, minimal essential medium; Tg, thapsigargin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase. is a member of a family of transcription factors that also includes C/EBPα, -δ, -γ, and -ϵ and C/EBP homology protein (CHOP) reviewed in Refs. 1Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 2Takiguchi M. Int. J. Exp. Pathol. 1998; 79: 369-391Crossref PubMed Scopus (98) Google Scholar, 3Ramji D.P. Foka P. Biochem. J. 2002; 365: 561-575Crossref PubMed Google Scholar. The C/EBP members contain a basic leucine zipper (bZIP) domain at their C terminus, which is responsible for selective dimerization with other bZIP family members, as reviewed by Newman and Keating (4Newman J.R. Keating A.E. Science. 2003; 300: 2097-2101Crossref PubMed Scopus (393) Google Scholar). Although C/EBPβ plays a role in a wide range of important cellular processes, such as adipocyte differentiation, carbohydrate metabolism, inflammation, and cellular proliferation, investigation of the transcriptional control of the C/EBPβ gene itself is limited. It has been reported that the cAMP-response element-like sequences, located within the rat C/EBPβ proximal promoter, are required for C/EBPβ expression and IL-6-mediated induction of the gene during the acute phase response (5Niehof M. Kubicka S. Zender L. Manns M.P. Trautwein C. J. Mol. Biol. 2001; 309: 855-868Crossref PubMed Scopus (44) Google Scholar). Disturbance of the protein folding process in the endoplasmic reticulum (ER) activates an ER stress-signaling pathway called the unfolded protein response (UPR), reviewed in Refs. 6Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (679) Google Scholar, 7Kaufman R.J. J. Clin. Invest. 2002; 110: 1389-1398Crossref PubMed Scopus (1105) Google Scholar, 8Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell Dev. Biol. 2002; 18: 575-599Crossref PubMed Scopus (812) Google Scholar. In the yeast Saccharomyces cerevisiae, ER stress activates the kinase/endonuclease Ire1p, which in turn mediates an unconventional splicing of HAC1 mRNA. The processed HAC1 mRNA codes for a bZIP protein, Hac1p, which binds to a UPR element (UPRE) in the appropriate target genes. Two mammalian Irelp forms, IRE1α and IRE1β (9Wang X.-Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (661) Google Scholar, 10Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (751) Google Scholar), have been identified, and XBPI has been described as the mammalian counterpart to yeast Hac1p (11Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (3014) Google Scholar, 12Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (840) Google Scholar, 13Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2161) Google Scholar). However, at least two ER stress-responsive pathways exist in mammals, and proteolytic activation of the transcription factor ATF6 also plays an important role (14Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1562) Google Scholar, 15Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2000; 20: 6755-6767Crossref PubMed Scopus (796) Google Scholar, 16Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar). The precursor form of ATF6 is an ER transmembrane protein that is proteolytically cleaved in response to ER stress, and the N-terminal portion is thereby released as an active transcription factor, which translocates to the nucleus. A bipartite mammalian ER stress response element (ERSE), 5′-CCAATN9CCACG-3′, was identified (17Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 18Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar), which was shown to bind either XBP-1 or ATF6 at the CCACG half-site (17Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). However, more recently it has been demonstrated that XBP-1 activates a second set of ER stress-responsive genes by binding to a genomic element composed of the consensus sequence 5′-TGACGTG(G/A)-3′ (12Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (840) Google Scholar, 19Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar), and this element has been referred to as the mammalian UPRE (11Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (3014) Google Scholar). Asparagine synthetase (ASNS), which catalyzes asparagine biosynthesis, is transcriptionally regulated in response to a variety of cellular stress signals, including either amino acid limitation or ER stress (20Guerrini L. Gong S.S. Mangasarian K. Basilico C. Mol. Cell. Biol. 1993; 13: 3202-3212Crossref PubMed Scopus (88) Google Scholar, 21Barbosa-Tessmann I.P. Chen C. Zhong C. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 1999; 274: 31139-31144Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 22Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar). A cis-element, termed nutrient-sensing response element-1, located within the ASNS proximal promoter at nt –68 to –60 contributes to this induction under the stress conditions mentioned above, and binding of C/EBPβ to the NSRE-1 is increased following activation of these stress pathways. Marten et al. (23Marten N.W. Burke E.J. Hayden J.M. Straus D.S. FASEB J. 1994; 8: 538-544Crossref PubMed Scopus (115) Google Scholar) was the first to document that the mRNA content of C/EBPβ is increased by amino acid deprivation, but regulation of the C/EBPβ gene by ER stress has yet to be examined. The present study was designed to test the hypothesis that transcription from the human C/EBPβ gene is induced in response to ER stress and to examine the mechanism of this induction. C/EBPβ mRNA content was increased following ER stress conditions including glucose deprivation, tunicamycin, or thapsigargin treatment. Given that ER stress did not alter the turnover rate of the C/EBPβ mRNA, it was proposed that this increase was due to increased transcription. Transient transfection of genomic fragments linked to a luciferase reporter gene demonstrated that a 46-bp region, located at a genomic site that corresponds to the 3′-untranslated region (UTR) of the C/EBPβ mRNA is required for the induction following ERSR activation, and that the C/EBPβ promoter plays no major regulatory role. Mutagenesis further indicated that a cis-regulatory element located at nt +1614–1621 (5′-TGACGCAA-3′) is responsible for activation of the C/EBPβ gene by ER stress. This element differs from the consensus mUPRE (see above) by 2 nucleotides (19Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar), but expression of exogenous XPBP1 activated transcription from a C/EBPβ genomic fragment containing this sequence. Cell Culture—Human hepatoma HepG2 cells were cultured in minimal essential medium (MEM), pH 7.4, (Mediatech Inc., Herndon, VA), supplemented to contain 25 mm NaHCO3, 4 mm glutamine, 1× nonessential amino acids, 10 μg/ml streptomycin sulfate, 100 μg/ml penicillin G, 28.4 μg/ml gentamycin, 0.023 μg/ml N-butyl-p-hydroxybenzoate, 0.2% (w/v) bovine serum albumin, and 10% (v/v) fetal bovine serum. Cells were maintained at 37 °C in a 5% CO2, 95% air incubator. Wild type and XBP-1-deficient mouse embryonic fibroblasts, generously supplied by Dr. Laurie H. Glimcher (Harvard School of Public Health) were maintained as described previously (24Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1639) Google Scholar). Northern Blot Analysis—HepG2 cells were cultured to 70–80% confluence in 60-mm dishes and then incubated for the indicated time in complete MEM, glucose-free MEM, complete MEM containing either 300 nm thapsigargin (Tg) or 5 μg/ml tunicamycin. Total cellular RNA was isolated using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA). 32P-Radiolabeled cDNA probe synthesis and Northern analysis was performed as described by Aslanian et al. (25Aslanian A.M. Fletcher B.S. Kilberg M.S. Biochem. J. 2001; 357: 321-328Crossref PubMed Scopus (138) Google Scholar). The cDNA probe for C/EBPβ was nt +1425–1632, which corresponds to a fragment of the 3′-untranslated region of the human C/EBPβ mRNA. The cDNA probe for human C/EBPα was the entire 3′-untranslated region of the human C/EBPα mRNA. The ribosomal protein L7a cDNA probe was the entire coding sequence obtained from Dr. Tatsuo Tanaka (University of Ryukyus, Okinawa, Japan), and the cDNA probe for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the entire coding sequence obtained from Dr. Anupam Agarwal (University of Alabama, Birmingham, AL). Real Time Quantitative RT-PCR—25–200 ng of HepG2 total RNA was used in each reaction to measure the mRNA content for C/EBPβ, Asns, and GAPDH. SYBR green chemistry was used to detect the products of interest (Applied Biosystems Inc., Foster City, CA). The primers for amplification were as follows: C/EBPβ, 5′-AGAACGAGCGGCTGCAGAAGA-3′ and 5′-CAAGTTCCGCAGGGTGCTGA-3′; ASNS, 5′-GCAGCTGAAAGAAGCCCAAGT-3′ and 5′-TGTCTTCCATGCCAATTGCA-3′; GAPDH, 5′-TTGGTATCGTGGAAGGACTC-3′ and 5′-ACAGTCTTCTGGGTGGCAGT-3′. The RT-PCRs were performed and quantified using a DNA Engine Opticon 2 system (MJ Research, Reno, NV). Each RNA sample was measured in duplicate, and three independent RNA samples were collected for each time point. Subcloning of the Human C/EBPβ Gene—A BAC clone (RP11-112L6) containing the human DNA sequence from chromosome 20 was obtained from the Sanger Institute (Cambridge, UK). To obtain a C/EBPβ-containing genomic fragment, the BAC clone was digested with EcoRI and PvuI, and fragments were separated by preparative field inversion gel electrophoresis and then ligated into the EcoRI site of the pBluescript II SK vector (Stratagene, La Jolla, CA). Using the C/EBPβ cDNA probe described above, colony hybridization was used to screen the resulting DH5α colonies, and an 11.5-kb C/EBPβ clone was obtained that contained nt –8451 to +3074. Deletion Analysis—C/EBPβ fragments containing nt –8451/+157 and –1595/+157 were obtained by restriction digestion of the –8451/+3074 clone, whereas the C/EBPβ promoter fragment (nt –325/+157) was prepared by PCR. The C/EBPβ sequences +1554/+1646, +1423/+2213, and +1423/+3541, which are 3′ to the coding sequence, were amplified by PCR using either nt –8451/+3074 or the original BAC clone as template. The promoter fragments were ligated into the SmaI site of the pGL3-basic vector (Promega, Madison, WI), which contains the Firefly luciferase gene as a reporter, whereas the C/EBPβ gene downstream sequences were ligated into the BamHI site. To test the C/EBPβ genomic sequences +1554/+1600 and +1601/+1646 with the SV40 promoter, oligonucleotides were synthesized with BamHI linkers (Invitrogen) and ligated into the BamHI site of the pGL3-promoter vector (Promega, Madison, WI). Mutagenesis—All site-directed mutagenesis was performed using the QuikChange® site-directed mutagenesis kit from Stratagene. For mutagenesis, substitutions were made within the C/EBPβ +1423 to +2213 or the +1601/+1646 sequence, cloned into BamHI site of the firefly luciferase reporter gene, and expressed under the control of either the C/EBPβ promoter –1593/+157 or the SV40 promoter. The specific mutations made are given under “Results.” The C/EBPβ-luciferase constructs were transiently transfected into HepG2 cells, and the firefly luciferase activity was measured as described below. Transient Transfection and Transcription Factor Overexpression— For each transfection, 1 μg of the pGL3 firefly luciferase reporter construct, driven by either the human GRP78 promoter –132/+7 (a generous gift from Dr. Kazutoshi Mori, Kyoto, Japan) or by the C/EBPβ sequence +1601/+1646 under the control of the SV40 promoter, was co-transfected along with 0.5 ng of a reference Renilla luciferase expression plasmid, phRL-SV40 (Promega). When indicated, 0.5 μg of pcDNA3.1 vector only (Invitrogen) or pcDNA3.1 containing the cDNA sequence for the active and nuclear form of ATF6α (amino acids 1–373) or the spliced and active form of XBP-1 (both gifts from Dr. Kazutoshi Mori, Kyoto, Japan) was included. HepG2 cells were transfected at ∼50% confluence in 24-well plates (2 × 105 cells/well) using the SuperFect™ transfection reagent (Qiagen). After transfection and a subsequent 18-h recovery in complete MEM containing 10% fetal bovine serum, cells were then incubated for 12 h in either fresh complete MEM or MEM plus 300 nm Tg, each supplemented with 10% dialyzed fetal bovine serum. The effect of transcription factor overexpression was measured by luciferase activity. Luciferase Reporter Assay—HepG2 cells were transfected with the C/EBPβ-firefly luciferase reporter plasmids described above. The relative transfection efficiency was corrected by co-transfection with Renilla luciferase reporter (phRL-SV40) driven by the SV40 promoter. The firefly and Renilla luciferase activities were assayed by the dual luciferase reporter system according to the manufacturer's directions (Promega). The cells were transfected at ∼50% confluence in 24-well plates (2 × 105 cells/well) using the SuperFect transfection reagent, as described above. The data are expressed as the averages ± S.D. of 3–4 assays, and each experiment was repeated with multiple batches of cells. Immunoblotting—Total cell extracts (30–60 μg/sample) were separated on an SDS-polyacrylamide gel and electrotransferred to a Protran nitrocellulose membrane (Schleicher & Schuell), as previously described (26Pan Y.-X. Chen H. Siu F. Kilberg M.S. J. Biol. Chem. 2003; 278: 38402-38412Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The membrane was stained with Fast Green Stain to check for equal loading between lanes. Primary antibodies (1:100 to 1:1000 dilution) were rabbit polyclonals against either C/EBPβ (catalog no. sc-150) or XBP-1 (catalog no. sc-7160) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The secondary antibody was goat anti-rabbit IgG and was used at a dilution of 1:2000 (XBP-1) or 1:20,000 (C/EBPβ). The bound secondary antibody was detected using an enhanced chemiluminescence kit (Amersham Biosciences) and exposed to Biomax MR film (Eastman Kodak Co.). Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay—HepG2 cells were seeded on 150-mm dishes (15 × 106 cells/dish). After 16 h of culture, the cells were washed twice with PBS and incubated for 18 h in either complete MEM or MEM lacking glucose, both supplemented with 5% dialyzed fetal bovine serum. The nuclear extraction was performed as previously described (27Leung-Pineda V. Kilberg M.S. J. Biol. Chem. 2002; 277: 16585-16591Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Protein concentration was determined using a modified Lowry assay (28Kilberg M.S. Methods Enzymol. 1989; 173: 564-575Crossref PubMed Scopus (47) Google Scholar). Singlestranded oligonucleotides were annealed by adding 4.8 nmol of each oligonucleotide, with 20 μl of 10× annealing buffer (100 nm Tris-HCl, pH 7.5, 1 m sodium chloride, 10 mm EDTA) in a total volume of 200 μl. The oligonucleotide solution was heated to 95 °C for 5 min and then allowed to gradually cool to 4 °C over 2 h. The oligonucleotides used either as electrophoretic mobility shift assay probes or competitors are listed in Fig. 10. The double-stranded oligonucleotides were radiolabeled by extension of overlapping ends with Klenow fragment in the presence of [α-32P]dATP. For each binding reaction, 10 μg of nuclear extract protein was incubated with 40 mm Tris-base, 200 mm NaCl, 2 mm dithiothreitol, 10% (v/v) glycerol, 0.05% (v/v) Nonidet P-40, 3 μg of poly(dI-dC) (Amersham Biosciences), and 0.05 mm EDTA for 20 min on ice. The radiolabeled probe was added at a concentration of 0.004 pmol/reaction (∼20,000 cpm), and where indicated, unlabeled competitor oligonucleotides were added at 0.04 pmol/reaction. The reaction mixture (30-μl final volume), was incubated at room temperature for 20 min. The reactions were subjected to electrophoresis as described previously (27Leung-Pineda V. Kilberg M.S. J. Biol. Chem. 2002; 277: 16585-16591Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).Fig. 10Electrophoretic mobility shift analysis of the human C/EBPβ mUPRE sequence. Nuclear extracts were prepared from HepG2 cells maintained in either complete (MEM) or glucose-deficient (MEM-Glc) MEM medium for 18 h, as described under “Materials and Methods.” The sequence of the 32P-radiolabeled oligonucleotide probes (WT or Mut) and competitor oligonucleotides (WT, Mut, or NS) are shown in the lower panel and correspond to the wild type C/EBPβ sequence or one that has the mUPRE core sequence mutated as shown (Mut). Unlabeled competitor oligonucleotides were prepared corresponding to the wild type C/EBPβ sequence (WT), the C/EBPβ sequence with the mUPRE core mutated (Mut), or an unrelated, nonspecific sequence (NS). The competitor oligonucleotides were added at 100 times the concentration of the labeled probe.View Large Image Figure ViewerDownload (PPT) Induction of C/EBPβ mRNA by Glucose Deprivation—To examine whether or not C/EBPα or C/EBPβ mRNA content was altered in response to glucose deprivation of HepG2 cells, the cells were incubated in glucose-free MEM for 0–12 h, and Northern blot analysis was performed (Fig. 1A). An initial increase in C/EBPβ mRNA content was observed between 2 and 4 h of glucose deprivation and then reached a maximum of about 7 times the control value (5.5 mm glucose) at 8 h (Fig. 1B). In contrast, C/EBPα mRNA content remained relatively steady for 4 h and then declined during the 8–12-h period of glucose deprivation (Fig. 1C). To obtain a quantitative analysis of C/EBPβ regulation by glucose, the mRNA level for C/EBPβ was measured for three independent experiments by real time quantitative RT-PCR (data not shown). The induction of C/EBPβ mRNA measured by Northern blot analysis and quantitative RT-PCR followed a similar pattern, reaching a comparable degree of induction at 8 h. Induction of C/EBPβ mRNA and Protein by ER Stress—To further investigate whether or not the ERSR pathway was responsible for the increased C/EBPβ mRNA content, HepG2 cells were incubated with known ERSR activators such as thapsigargin (Tg), an ER Ca2+-ATPase inhibitor, or tunicamycin, an inhibitor of protein glycosylation (29Pahl H.L. Physiol. Rev. 1999; 79: 683-701Crossref PubMed Scopus (307) Google Scholar). Consistent with their common mode of action, tunicamycin treatment and glucose deprivation increased C/EBPβ mRNA content over a similar time frame, with the initial increase being observed between 2 and 4 h, and reaching a 7- or 8-fold induction at 8 h (Fig. 2). In contrast, thapsigargin treatment also induced C/EBPβ mRNA content, but the maximal induction of 8-fold was achieved by 4 h. To test whether or not C/EBPβ protein content was also increased, immunoblots of control (MEM-incubated cells) or Tg-treated whole cell extracts were subjected to immunoblotting (Fig. 3). ER stress caused an elevation of full-length (liverenriched activating protein) C/EBPβ protein that was consistent with the rise in mRNA level, although the absolute magnitude of the protein increase was less than that for the mRNA. The peak of C/EBPβ protein occurred at 4 h and stayed relatively high for the remainder of the 12-h period investigated. To show equal lane loading, the blot was stained with Fast Green, and as a negative control, the blot was probed with antibody specific for the bZIP transcription factor ATF2, which did not show increased expression (Fig. 3, upper panel). Induction of C/EBPβ mRNA by the ERSR Is Dependent on de Novo Protein Synthesis—To determine whether or not synthesis of an upstream regulatory protein was required for induction of the C/EBPβ gene, HepG2 cells were incubated with thapsigargin in the presence or absence of 0.1 mm cycloheximide (Fig. 4). Cycloheximide completely prevented the increase in C/EBPβ mRNA content following ERSR activation, suggesting that de novo protein synthesis was required at some unidentified step leading to activation of the C/EBPβ gene. Inhibition of protein synthesis may also have a minor effect on the turnover of C/EBPβ mRNA, as indicated by the small, but consistent elevation in mRNA content in cells treated with cycloheximide in the absence of thapsigargin (Fig. 4). Induction of C/EBPβ mRNA Content by the ERSR Is Not Due to mRNA Stabilization—To test for increased mRNA stability as a possible mechanism for the ERSR induction, HepG2 cells were incubated in glucose-free MEM for 8 h to elevate the C/EBPβ mRNA content and were then transferred to either fresh glucose-free MEM or complete MEM, both containing 5 μm actinomycin D (Fig. 5). The results showed that the half-life of C/EBPβ mRNA with or without glucose was ∼1.5 h, indicating that the ERSR-dependent elevation in C/EBPβ mRNA is probably not the result of mRNA stabilization. C/EBPβ Genomic 5′ Upstream Region Does Not Respond to the ERSR—To investigate the role of genomic 5′ upstream sequences in mediating C/EBPβ transcription in response to ER stress, a fragment corresponding to the human C/EBPβ proximal promoter nt –325/+157 was tested initially (Fig. 6). Compared with the 7-fold induction of endogenous mRNA by glucose deprivation or Tg treatment (Figs. 1 and 2), this promoter fragment did not respond to the glucose-limited condition and resulted in less than a 70% increase in Tg-treated cells (Fig. 6B). To test the possibility that the cis-acting elements required for full induction may be located farther upstream, much longer promoter fragments (–1595/+157 and –8451/+157) were examined, but similar results were obtained (Fig. 6B). C/EBPβ Genomic Sequence 3′ to the Protein Coding Sequence Is Essential for the ERSR—As depicted in Fig. 6A, the human C/EBPβ gene is intronless and relative to the transcription start site, the first of multiple translation start sites is at nt +206, and the single translation stop codon is at +1243 (30Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar). Although not fully characterized, an apparent polyadenylation signal (5′-AATAAA-3′) is ∼1.8 kb downstream from the transcription start (GenBank™ number NM_005194). Given that a 5′ upstream fragment of nearly 8.5 kbp did not support induction by ER stress, the C/EBPβ genomic region 3′ to the protein coding sequence was investigated (Fig. 7). Sequentially deleted fragments were ligated downstream of the firefly luciferase reporter gene, driven by a C/EBPβ promoter fragment containing nt –1595 to +157. To approximate the endogenous location, the C/EBPβ 3′ genomic fragments were inserted downstream of the firefly coding sequence within the reporter plasmid. The C/EBPβ sequence from nt +1423 to +3541, containing the sequence corresponding to the mRNA 3′-untranslated region and some additional 3′ genomic sequence, induced transcription by 17-fold when cells were treated with Tg (Fig. 7). The degree of induction was similar when this 2.1-kb genomic sequence was deleted to an 800-bp DNA fragment covering nt +1423–2213. The ER stress-responsive region was narrowed even further by establishing that a 93-bp DNA fragment containing nt +1554–1646 activated transcription (Fig. 7). The 93-bp sequence yielded an induction level of about 8 times the control, less than the longer fragments but nearly identical in magnitude to the maximal increase in endogenous mRNA content following Tg treatment (Fig. 2). The decline in the relative increase was due to an increase in the basal rate rather than a decline in the absolute transcription rate following ER stress. Taken together, the data of Figs. 6 and 7 indicate that the DNA regulatory element necessary to mediate the ERSR activation of the C/EBPβ gene is located 3′ to the protein coding sequence and within nt +1554–1646. C/EBPβ 3′ Genomic Sequence Can Confer ER Stress Responsiveness to an Otherwise Inert Promoter—To test the hypothesis that nt +1554–1646 of the C/EBPβ gene (Fig. 8A) could confer ER stress-regulated transcription to an unrelated promoter, the C/EBPβ promoter fragment used in the previous experiments was replaced with the SV40 promoter (Fig. 8B). The SV40 promoter alone was inert to Tg treatment, but when a single copy of the 93-bp C/EBPβ genomic sequence was present, transcription was induced to 9 times the MEM control. A UPRE-like Binding Site Is Responsible for the Induction of the C/EBPβ Gene by ER Stress—Computer analysis of the 93-bp region revealed no perfect match for either the ERSE (5′-CCAATN9CCACG-3′) (17Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 18Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar) or the nutrient-sensing response unit (5′-TGATGAAACN11GTTACA-3′) (21Barbosa-Tessmann I.P. Chen C. Zhong C. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 1999; 274: 31139-31144Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 22Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar, 31Zhong C. Chen C. Kilberg M.S. Biochem. J. 2003; 372: 603-609Crossref PubMed Scopus (36) Google Scholar), either of which can mediate the ER stress signal. However, a sequence" @default.
• W2044332034 created "2016-06-24" @default.
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• W2044332034 date "2004-07-01" @default.
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• W2044332034 title "Human CCAAT/Enhancer-binding Protein β Gene Expression Is Activated by Endoplasmic Reticulum Stress through an Unfolded Protein Response Element Downstream of the Protein Coding Sequence" @default.
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