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• W1992480558 abstract "Parathyroid hormone (PTH), a major regulator of bone metabolism, activates the PTHR1 receptor on the osteoblast plasma membrane to initiate signaling and induce transcription of primary response genes. Subsequently, primary genes with transcriptional activity regulate expression of downstream PTH targets. We have identified the adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4bp4) as a PTH-induced primary gene in osteoblasts. E4BP4 is a basic leucine zipper (bZIP) transcription factor that represses or activates transcription in non-osteoblastic cells. We report here that PTH rapidly and transiently induced E4bp4 mRNA in osteoblastic cells and that this induction did not require protein synthesis. PTH also induced E4BP4 protein synthesis and E4BP4 binding to a consensus but not to a mutant E4BP4 response element (EBPRE). E4BP4 overexpression inhibited an EBPRE-containing promoter-reporter construct, whereas PTH treatment attenuated activity of the same construct in primary mouse osteoblasts. Finally, E4BP4 overexpression inhibited PTH-induced activity of a cyclooxygenase-2 promoter-reporter construct. Our data suggest a role for E4BP4 in attenuation of PTH target gene transcription in osteoblasts. Parathyroid hormone (PTH), a major regulator of bone metabolism, activates the PTHR1 receptor on the osteoblast plasma membrane to initiate signaling and induce transcription of primary response genes. Subsequently, primary genes with transcriptional activity regulate expression of downstream PTH targets. We have identified the adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4bp4) as a PTH-induced primary gene in osteoblasts. E4BP4 is a basic leucine zipper (bZIP) transcription factor that represses or activates transcription in non-osteoblastic cells. We report here that PTH rapidly and transiently induced E4bp4 mRNA in osteoblastic cells and that this induction did not require protein synthesis. PTH also induced E4BP4 protein synthesis and E4BP4 binding to a consensus but not to a mutant E4BP4 response element (EBPRE). E4BP4 overexpression inhibited an EBPRE-containing promoter-reporter construct, whereas PTH treatment attenuated activity of the same construct in primary mouse osteoblasts. Finally, E4BP4 overexpression inhibited PTH-induced activity of a cyclooxygenase-2 promoter-reporter construct. Our data suggest a role for E4BP4 in attenuation of PTH target gene transcription in osteoblasts. Parathyroid hormone (PTH 1The abbreviations used are: PTH, parathyroid hormone; RDA, representational difference analysis; bZIP, basic leucine zipper; IL-3, interleukin-3; E4BP4, E4 promoter-binding protein/nuclear factor regulated by IL-3; EBPRE, E4BP4 response element; MOB, mouse osteoblast; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; CHX, cycloheximide; ROS, rat osteosarcoma; ActD, actinomycin D; cox-2, cyclooxygenase-2; ATF, activating transcription factor; TBP, TATA binding protein.1The abbreviations used are: PTH, parathyroid hormone; RDA, representational difference analysis; bZIP, basic leucine zipper; IL-3, interleukin-3; E4BP4, E4 promoter-binding protein/nuclear factor regulated by IL-3; EBPRE, E4BP4 response element; MOB, mouse osteoblast; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; CHX, cycloheximide; ROS, rat osteosarcoma; ActD, actinomycin D; cox-2, cyclooxygenase-2; ATF, activating transcription factor; TBP, TATA binding protein.), a major regulator of bone metabolism, affects both osteoblastic and osteoclastic formation and activity (1Rubin M.R. Cosman F. Lindsay R. Bilezikian J.P. Osteoporos. Int. 2002; 13: 267-277Crossref PubMed Scopus (114) Google Scholar, 2Jerome C.P. Gubler H.P. Calcif. Tissue Int. 1991; 49: 398-402Crossref PubMed Scopus (7) Google Scholar, 3Jerome C.P. Colwell A. Eastell R. Russell R.G.G. Trechsel U. Bone Miner. 1992; 19: 117-125Abstract Full Text PDF PubMed Scopus (16) Google Scholar). However, the target cell of PTH in bone is the osteoblast, which expresses PTHR1, a G protein-coupled heptahelical receptor that also binds PTH-related peptide (4Strewler G.J. J. Clin. Invest. 2001; 107: 271-272Crossref PubMed Scopus (15) Google Scholar, 5Juppner H. Abou-Samra A. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski L.F.J. Hock J. Potts J.T.J. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1025Crossref PubMed Scopus (1140) Google Scholar, 6Abou-Samra A.B. Jupner H. Force T. Freeman M.W. Kong X.F. Schipani A. Urena P. Richards J. Bonvetre J.V. Potts J.T.j. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (995) Google Scholar, 7Schipani E. Karga H. Karaplis A.C. Potts J.T.J. Kronenberg H.M. Segre G.V. Abou-Samra A.B. Juppner H. Endocrinology. 1993; 132: 2157-2165Crossref PubMed Scopus (153) Google Scholar). Ligand-bound PTHR1 initiates signaling cascades that phosphorylate and activate transcription factors that in turn induce transcription of several primary response genes (8Fujimori A. Cheng S.L. Avioli L.V. Civitelli R. Endocrinology. 1992; 130: 29-36Crossref PubMed Google Scholar, 9Partridge N.C. Bloch S.R. Pearman A.T. J. Cell. Biochem. 1994; 55: 321-327Crossref PubMed Scopus (114) Google Scholar, 10Swarthout J.T. D'Alonzo R.C. Selvamurugan N. Partridge N.C. Gene (Amst.). 2002; 282: 1-17Crossref PubMed Scopus (293) Google Scholar). Primary gene products perform many functions and ultimately lead to changes in osteoblastic phenotype. To map early changes in regulation of osteoblastic primary gene expression, we performed representational difference analysis (RDA) of mRNA from control and PTH-treated osteoblastic cells (11Tetradis S. Bezouglaia O. Tsingotjidou A. Endocrinology. 2001; 142: 663-670Crossref PubMed Scopus (64) Google Scholar). Here we report that PTH rapidly induced expression of the bZIP transcription factor adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4BP4). E4bp4 was isolated by screening a placental cDNA λgt11 expression library for binding to the ATF site of the adenovirus E4 promoter (12Cowell I.G. Skinner A. Hurst H.C. Mol. Cell. Biol. 1992; 12: 3070-3077Crossref PubMed Scopus (155) Google Scholar) or to the CRE/ATF-like sequence of the upstream regulatory region of the human IL-1β gene (13Chen W.J. Lewis K.S. Chandra G. Cogswell J.P. Stinnett S.W. Kadwell S.H. Gray J.G. Biochim. Biophys. Acta. 1995; 1264: 388-396Crossref PubMed Scopus (21) Google Scholar). Later, through screening of an expression λgt11 cDNA library prepared from phytohemagglutinin-stimulated human T-cell mRNA for binding to regulatory sequences of the IL3 promoter, a nuclear factor named NF-IL3 was identified and found to be identical to E4bp4 (14Zhang W. Zhang J. Kornuc M. Kwan K. Frank R. Nimer S.D. Mol. Cell. Biol. 1995; 15: 6055-6063Crossref PubMed Scopus (104) Google Scholar). The 2-kb cDNA contains an open reading frame of 1386 bases and encodes a 462-amino acid (51.4 kDa) protein (12Cowell I.G. Skinner A. Hurst H.C. Mol. Cell. Biol. 1992; 12: 3070-3077Crossref PubMed Scopus (155) Google Scholar). E4BP4 is a bZIP transcription factor and acts both as a transcriptional repressor and transcriptional activator. E4BP4 has been hypothesized to be involved in cellular functions such as cell survival versus apoptosis, the anti-inflammatory response, and circadian rhythm regulation (15Cowell I.G. Bioessays. 2002; 24: 1023-1029Crossref PubMed Scopus (103) Google Scholar). However, no involvement of E4BP4 expression in the function of bone cells has been reported. In the present study, we demonstrate that PTH rapidly and transiently induced E4bp4 mRNA in primary mouse osteoblasts and that this induction did not require new protein synthesis. PTH also induced E4BP4 protein synthesis. Furthermore, PTH induced nuclear protein binding to a consensus E4BP4 response element (EBPRE). A specific E4BP4 antibody supershifted the majority of this binding action. E4BP4 overexpression inhibited promoter activity of a reporter luciferase construct containing EBPRE consensus sites in primary osteoblasts. PTH treatment attenuated the promoter activity of the same construct. Finally, E4BP4 overexpression inhibited PTH-induced activity of a cyclooxygenase-2 promoter-reporter construct. Our data suggest that E4BP4 induction might mediate PTH inhibition of target gene transcription. Cell Culture—Primary mouse osteoblasts (MOB) were obtained from the calvariae of neonatal mice as previously described (11Tetradis S. Bezouglaia O. Tsingotjidou A. Endocrinology. 2001; 142: 663-670Crossref PubMed Scopus (64) Google Scholar). MC3T3-E1 cells were plated at 5,000 cells/cm2, and MOB cells were plated at 15,000 cells/cm2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 50 μg/ml streptomycin. ROS 17/2.8 cells were plated at 5,000 cells/cm2 and were grown in F-12 medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum, 100 units/ml penicillin, and 50 μg/ml streptomycin. Cells were grown in a humidified incubator at 37 °C and 5% CO2. Cells were grown in non-differentiating media (no ascorbic acid or β-glycerophosphate) and were treated with agents at the time of confluence. Mouse Calvariae Organ Culture—Hemicalvariae from 6- to 8-day-old CD-1 mice were dissected and cultured individually in 6-well dishes (Costar, Cambridge, MA) containing 2 ml of BGJb medium (Invitrogen) with 1 mg/ml bovine serum albumin, 50 μg/ml ascorbic acid, 100 units/ml penicillin, and 50 μg/ml streptomycin. All animals used in these studies were sacrificed according to protocol approved by the UCLA Institutional Animal Care and Use Committee (ARC number 1998-175-12). RNA Extraction and Northern Blot Assays—Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Twelve to fifteen micrograms of total RNA was separated by electrophoresis on a 1% agarose/3.7% formaldehyde gel and transferred to a Gene Screen Plus hybridization filter (PerkinElmer Life Sciences, Boston, MA). Filters were prehybridized at 68 °C for 15 min, hybridized at 68 °C for 1 h using the QuickHyb hybridization solution (Stratagene, La Jolla, CA) with a 32P-labeled probe (see below). Filters were washed and were used to expose radiographic film (Kodak, Rochester, NY) at –80 °C. E4bp4 mRNA intensity was measured and corrected for GAPDH expression using a PhosphorImager screen (Amersham Biosciences). Reverse Transcriptase-PCR—To generate a full-length E4bp4 probe, mRNA from MC3T3-E1 cells treated with 10 nm PTH for 2 h, was reverse-transcribed using the Moloney murine leukemia virus reverse transcriptase (Stratagene). cDNA was amplified by PCR using oligonucleotide primers that span the full-length open reading frame of E4bp4 (sense primer, 5′-CCG CCA TGC AGC TGA GAA AAA TGC AG-3′; antisense primer, 5′-TTA TTA CCT GGA GTC CGA AGC CG-3′ (16Tetradis S. Nervina J.M. Nemoto K. Kream B.E. J. Bone Miner. Res. 1998; 13: 1846-1851Crossref PubMed Scopus (28) Google Scholar)). The resulting amplification product was purified and subcloned into the pcDNA 3.1 expression vector downstream of the cytomegalovirus promoter (Invitrogen, San Diego, CA). To generate E4bp4 cDNA probe for Northern analysis, the E4bp4 cDNA insert was excised, gel-purified, and labeled with [32P]dCTP by random primer labeling technique. Total and Nuclear Protein Extraction—MOB cells were treated accordingly and then washed twice with ice-cold 1× phosphate-buffered saline. For total protein extracts, cells were resuspended for 30 min in 1 ml of triple detergent lysis buffer containing 50 mm Tris-Cl, 150 mm NaCl, 0.02% sodium azide, 0.1% SDS, 100 μg/ml PMSF, 1 μg/ml aprotinin, 1% Nonidet P-40, 0.5% deoxycholate, and 1× protease inhibitors. Lysates were spun at 15,000 × g for 15 min at 4 °C, and supernatants were used for Western blot assays. Nuclear proteins were prepared as previously described (17Tomaras G.D. Foster D.A. Burrer C.M. Taffet S.M. J. Leukoc. Biol. 1999; 66: 183-193Crossref PubMed Scopus (23) Google Scholar). Cells were resuspended in 400 μl of buffer A (10 mm HEPES (pH 7.9), 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA) supplemented with freshly added 1 mm DTT, 0.5 mm PMSF, and 1× protease inhibitor mixture (Roche Applied Science, Indianapolis, IN) for 15 min on ice. Then 25 μl of 10% Nonidet P-40 was added, and the cells were vortexed vigorously for 10 s and centrifuged at maximum speed for 30 s in a microcentrifuge at 4 °C. Pellets were resuspended in buffer C (20 mm HEPES (pH 7.9), 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA) supplemented with freshly added 1 mm DTT, 0.5 mm PMSF, and 1× protease inhibitor. After incubating for 30 min at 4 °C on a rocking platform, nuclear lysates were centrifuged at maximum speed for 10 min in a microcentrifuge at 4 °C, and the supernatants containing the nuclear proteins were collected. Protein concentrations were determined by the Lowry method. Western Blot Analysis—Fifty micrograms total or nuclear proteins were combined with equal volume of 2× loading buffer, boiled for 5 min, separated by SDS-PAGE (5% stacking at 20 mA and 10% resolving gel at 40 mA), and electrotransferred onto a nitrocellulose membrane overnight at 22 V and 4 °C. Membranes were stained with 0.1% Ponceau S (w/v) in 5% acetic acid (v/v) to determine equal loading. Then membranes were blocked for 1 h with 5% nonfat milk in TBS-T (0.5% Tween), incubated with a polyclonal E4BP4 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, catalog number SC-9549) at 1:100 dilution in 5% nonfat milk/TBS-T for 1–2 h at room temperature, washed with TBS-T, and incubated with anti-goat IgG-horseradish peroxidase conjugated secondary antibody (Santa Cruz Biotechnologies) in 5% nonfat milk/TBS-T for 1 h. Following incubation, membranes were washed with TBS-T, and proteins were visualized using ECL reagent (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions and autoradiography. Electrophoretic Mobility Shift Assay—Oligonucleotides (Invitrogen) carrying an E4BP4 consensus response element (EBPRE, 5′-GGGGATCCGTTATGTAACGGATCC-3′) or EBPRE with single nucleotide mutations (EBPRE-m1, 5′-GGGGATCCGTTATtTAACGGATCC-3′; EBPRE-m2, 5′-GGGGATCCGTaATGTAACGGATCC-3′) were used (12Cowell I.G. Skinner A. Hurst H.C. Mol. Cell. Biol. 1992; 12: 3070-3077Crossref PubMed Scopus (155) Google Scholar). In vitro protein synthesis was performed using the TnT Quickcouple Transcription/Translation kit (Promega Corp., Madison, WI) following the manufacturer's protocol. Nuclear or in vitro-synthesized proteins were incubated with probes in the presence of EMSA buffer (25 mm HEPES, 0.1 mg/ml bovine serum albumin, 10% glycerol, 0.1 mg/ml poly(dI·dC), 1 mm DTT, 0.5 mm PMSF) at room temperature for 30 min. Reaction mixtures were resolved by non-denaturing 4% PAGE in 0.5× TBE at 135 V for 1.5 h. The gel was fixed with 10% methanol, 10% acetic acid for 10 min, dried onto Whatman 3MM paper, and used to expose x-ray film. For supershift experiments, proteins were incubated with 1.5 μl of antibody for 20 min at room temperature prior to loading onto gel. Transfection—Three tandem wild type or mutant EBPREs, described earlier, were subcloned into the pTK-luciferase vector (pTK-LUC, ATCC, Manassas, VA) upstream of the basal thymidine kinase promoter to generate pTK-EBPRE, pTK-EBPRE-m1, and pTK-EBPRE-m2 luciferase reporter constructs. MOB cells were plated in 24-well plates at 50–60% confluence and were co-transfected with 75 ng/well pcDNA3.1-E4BP4 expression vector or with an equimolar amount of pcDNA3.1 empty vector and 75 ng/well pTK-EBPRE, pTK-EBPRE-m1, or pTK-EBPRE-m2 reporter construct utilizing LipofectAMINE Plus and the manufacturer's protocol (Invitrogen). 2–4 days later luciferase assay was performed with the protocol provided by the supplier (Promega). Protein concentrations were determined by Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) using microplate reader (Multiscan, Fisher, Pittsburgh, PA). Statistics—Statistical differences between two groups were detected with Student's t test. Comparisons among groups were performed by analysis of variance, and statistical differences were detected with a Bonferroni post hoc test. All experiments were performed at least three times. PTH Induced E4bp4 Gene Expression in the Absence of Protein Synthesis in MC3T3-E1 Osteoblastic Cells—Using RDA to identify osteoblastic primary response genes induced by PTH (11Tetradis S. Bezouglaia O. Tsingotjidou A. Endocrinology. 2001; 142: 663-670Crossref PubMed Scopus (64) Google Scholar), we cloned a DNA fragment that corresponded to E4bp4 (data not shown). To confirm that E4bp4 was a PTH-induced primary response gene, MC3T3-E1 cells were treated with 10 μg/μl CHX for 30 min followed by 10 nm PTH for 90 min. Total RNA was extracted, and Northern blot analysis was performed using the RDA-isolated cDNA fragment as a probe. E4bp4 was constitutively expressed in CHX-treated cells and was markedly induced by PTH treatment (Fig. 1A). PTH Induced E4bp4 mRNA Levels in MC3T3-E1 and ROS 17/2.8 Osteoblastic Cells in a Time-dependent Manner—Confluent MC3T3-E1 and ROS 17/2.8 cells were treated with 10 nm PTH for 1–6 h. RT-PCR of total RNA utilizing E4bp4-specific primers demonstrated that PTH induced E4BP4 mRNA in both osteoblastic cell lines with a peak at 2 h (Fig. 1B). DNA sequencing confirmed that the PCR product had the previously published E4bp4 sequence (accession number NM_017373, National Institutes of Health data base). PTH Induced E4bp4 Expression in Primary Mouse Osteoblasts and Calvariae—MC3T3-E1 and ROS 17/2.8 cells are immortalized cell lines that retain a pre-osteoblastic/osteoblastic phenotype. To test if PTH induces E4bp4 gene expression in primary osteoblastic cells and normal bone, MOB cells or calvariae organ cultures were treated with 10 nm PTH for various times, total RNA was extracted, and Northern blot analyses were performed. PTH-induced E4bp4 mRNA expression in both MOB cells and cultured calvariae peaked at 2 h and declined thereafter (Fig. 2). PTH Induction of E4bp4 mRNA Does Not Require New Protein Synthesis in MOB Cells and Is Regulated at the Transcriptional Level—To verify that E4bp4 is a PTH-induced primary response gene in MOB cells and to investigate if E4bp4 gene transcription is required for the PTH effects, MOB cells were pretreated with 3 μg/ml CHX or with 5 μg/ml actinomycin D (ActD) for 30 min and then with 10 nm PTH for 0–6 h (Fig. 3). Pretreatment with CHX enhanced PTH-induced E4bp4 mRNA levels that were sustained for the duration of the experiment. Pretreatment with ActD completely inhibited PTH-induced E4bp4 mRNA expression (Fig. 3). PTH Induced E4BP4 Protein Synthesis in MOB Cells—To determine if PTH induced E4BP4 protein synthesis, MOB cells were treated with 10 nm PTH for 0–6 h, and total or nuclear proteins were extracted. Western immunoblot analysis indicated that PTH induced E4BP4 protein expression in MOB cells that peaked between 2 and 4 h and declined thereafter (Fig. 4). Similar results were obtained with both total and nuclear proteins, although nuclear proteins demonstrated a delayed peak and decline (Fig. 4, compare A and B). Recombinant E4BP4 protein was used as a control (lane R, Fig. 4A). PTH Induced Nuclear Protein Binding to a Consensus E4BP4 Response Element (EBPRE)—EMSAs were performed to test the ability of in vitro synthesized E4BP4 protein to bind consensus or either of two mutant EBPREs. E4BP4 bound with high, moderate, and low affinity to wild type EBPRE, to EBPRE-m1, and to EBPRE-m2, respectively (Fig. 5A). In vitro synthesized luciferase control showed no binding to the DNA probes. To investigate potential PTH-induced binding to the consensus or mutant EBPREs, EMSAs utilizing nuclear proteins from PTH-treated MOB cells and the EBPRE probes were performed. Untreated cells showed a low binding of nuclear proteins to the consensus EBPRE. Treatment with 10 nm PTH increased this binding that peaked at 4 h and declined by 6 h. Similar to in vitro synthesized E4BP4, the binding of the nuclear extracts was reduced or almost abolished when EBPRE-m1 or EBPRE-m2 probes were used (Fig. 5B). The PTH-induced binding to the EBPRE probe was completely, partially, or minimally inhibited by 25× and 50× excesses of EBPRE, EBPRE-m1, or EBPRE-m2 cold competitor suggesting specific binding of the nuclear proteins to the wild type probe (Fig. 5C). E4BP4 Protein Is Part of the PTH-induced Binding to a Consensus EBPRE—Basal E4BP4 mRNA and protein levels were very low, whereas PTH treatment significantly induced E4bp4 gene expression. To test if the PTH-induced binding to the consensus EBPRE was due to an existing or to a newly synthesized protein, such as E4BP4, MOB cells were pretreated with 3 μg/ml CHX for 30 min and then treated with 10 nm PTH for 0–6 h. Nuclear extracts were utilized for EMSAs. In the absence of CHX, PTH induced binding to the EBPRE. However, CHX abolished this PTH effect suggesting that the PTH-induced binding was due to newly synthesized proteins (Fig. 6A). To confirm that E4BP4 was part of the PTH-induced complexes, we utilized a polyclonal antibody against E4BP4 protein. First we determined that the antibody was able to supershift in vitro synthesized E4BP4 protein. E4BP4 or luciferase proteins were incubated with EBPRE probes and then with E4BP4 antibody or a nonspecific antibody (Nurr1 antibody at the same concentration, Santa Cruz Biotechnologies, catalog number SC-990). The E4BP4 antibody supershifted the entire E4BP4·EBPRE binding complex, whereas the nonspecific antibody had no effect (Fig. 6B). Similarly, the E4BP4 antibody supershifted part of the PTH-induced nuclear extract·EBPRE complex, whereas the nonspecific antibody had no effect. Interestingly, not all of the PTH-induced complexes were supershifted, in contrast to the in vitro synthesized E4BP4 protein (Fig. 6C). Because the consensus EBPRE is very similar to the consensus C/EBPβ binding site (18Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar), we tested the possibility that C/EBPβ is part of the PTH-inducible binding to the EBPRE. Indeed, a C/EBPβ antibody supershifted part of the PTH-induced nuclear extract·EBPRE complex (Fig. 6D). E4BP4 Inhibited Promoter Activity in Osteoblasts—To study the regulatory role of E4BP4 on osteoblastic gene expression, MOB cells were co-transfected with pcDNA3.1-E4BP4 or empty vector and with pTK-Luc, pTK-EBPRE, pTK-EBPRE-m1, or pTK-EBPRE-m2 reporter constructs. E4BP4 overexpression had no effect on the basal thymidine kinase promoter activity. However, E4BP4 strongly inhibited pTK-EBPRE activity by 92% (Fig. 7A). When mutant EBPREs were inserted upstream of the thymidine kinase promoter, E4BP4 overexpression inhibited promoter activity to a lesser degree (pTK-EBPRE-m1 by 59% and pTK-EBPRE-m2 by 39%), in parallel to the ability of E4BP4 to bind these mutant sites, as described above. Because E4BP4 overexpression inhibited luciferase activity of the pTK-EBPRE reporter construct, we tested the ability of PTH treatment to affect this construct. MOB cells were transfected with pTK-Luc, pTK-EBPRE, pTK-EBPRE-m1, or pTK-EBPRE-m2 reporter constructs and then treated with PTH for 12 h. PTH treatment caused a statistically significant attenuation of the promoter activity by 25%, compared with the untreated control (Fig. 7B). In contrast, PTH treatment had no statistical effect on the pTK-EBPRE-m1 or pTK-EBPRE-m2 constructs (Fig. 7B). Finally, we tested the ability of E4BP4 to affect activity of endogenous promoters. Computer analysis revealed four EBPRE-like elements (seven of eight nucleotides) within the proximal 963 bp of the cyclooxygenase-2 (cox-2) promoter (the kind gift of Dr. Harvey Herschman, UCLA). MOB cells were co-transfected with a luciferase reporter that contained 1 kb of the cox-2 promoter and with pcDNA3.1-E4BP4 or empty vector. Four days later, cells were treated with vehicle or 10 nm PTH for 3 h, and luciferase activity was measured. As previously described (19Tetradis S. Pilbeam C.C. Liu Y. Herschman H.R. Kream B.E. Endocrinology. 1997; 138: 3594-3600Crossref PubMed Scopus (46) Google Scholar), PTH induced cox-2 promoter activity (Fig. 7C). E4BP4 overexpression inhibited basal luciferase activity, although not at statistically significant levels. More importantly, E4BP4 overexpression significantly inhibited PTH-induced cox-2 promoter activity. PTH has important effects on bone metabolism (1Rubin M.R. Cosman F. Lindsay R. Bilezikian J.P. Osteoporos. Int. 2002; 13: 267-277Crossref PubMed Scopus (114) Google Scholar). Osteoblasts are the direct PTH target in bone, yet the molecular mechanisms that mediate the effects of PTH on osteoblastic gene expression remain largely elusive (9Partridge N.C. Bloch S.R. Pearman A.T. J. Cell. Biochem. 1994; 55: 321-327Crossref PubMed Scopus (114) Google Scholar). PTH-triggered osteoblast activity begins with the rapid and transient induction of primary response gene expression (9Partridge N.C. Bloch S.R. Pearman A.T. J. Cell. Biochem. 1994; 55: 321-327Crossref PubMed Scopus (114) Google Scholar, 10Swarthout J.T. D'Alonzo R.C. Selvamurugan N. Partridge N.C. Gene (Amst.). 2002; 282: 1-17Crossref PubMed Scopus (293) Google Scholar). Subsequently, primary gene products mediate downstream PTH effects (19Tetradis S. Pilbeam C.C. Liu Y. Herschman H.R. Kream B.E. Endocrinology. 1997; 138: 3594-3600Crossref PubMed Scopus (46) Google Scholar, 20Kream B.E. LaFrancis D. Petersen D.N. Woody C. Clark S. Rowe D.W. Lichtler A. Mol. Endocrinol. 1993; 7: 399-408Crossref PubMed Scopus (39) Google Scholar, 21Scott D.K. Brakenhoff K.D. Clohisy J.C. Quinn C.O. Partridge N.C. Mol. Endocrinol. 1992; 6: 2153-2159Crossref PubMed Scopus (72) Google Scholar, 22Onyia J.E. Bidwell J. Herring J. Hulman J. Hock J.M. Bone. 1995; 17: 479-484Crossref PubMed Scopus (153) Google Scholar). Among the primary genes induced by PTH are several transcription factors, including the AP-1 transcription factors c-fos and c-jun (23Clohisy J.C. Scott D.K. Brakenhoff K.D. Quinn C.O. Partridge N.C. Mol. Endocrinol. 1992; 6: 1834-1842Crossref PubMed Scopus (77) Google Scholar), the nuclear orphan receptors Nurr1 (11Tetradis S. Bezouglaia O. Tsingotjidou A. Endocrinology. 2001; 142: 663-670Crossref PubMed Scopus (64) Google Scholar) and Nur77 (24Tetradis S. Bezouglaia O. Tsingotjidou A. Vila A. Biochem. Biophys. Res. Commun. 2001; 281: 913-916Crossref PubMed Scopus (34) Google Scholar) and the transcriptional repressor ICER (16Tetradis S. Nervina J.M. Nemoto K. Kream B.E. J. Bone Miner. Res. 1998; 13: 1846-1851Crossref PubMed Scopus (28) Google Scholar). Here, we show that PTH induced E4BP4, which appears to be a transcriptional repressor in osteoblasts. To the best of our knowledge, this is the first report of osseous localization and PTH regulation of E4bp4 gene expression. E4BP4 is constitutively present in a variety of tissues, including spleen, prostate, testis, ovary, heart, brain, lung, liver, and skeletal muscle (25Lai C.K. Ting L.P. J. Virol. 1999; 73: 3197-3209Crossref PubMed Google Scholar, 26Hulme D.J. Blair I.P. Dawkins J.L. Nicholson G.A. Hum. Genet. 2000; 106: 594-596PubMed Google Scholar). In addition, E4BP4 expression is induced by phytohemagglutinin, thapsigargin, and phorbol ester in T-cells (14Zhang W. Zhang J. Kornuc M. Kwan K. Frank R. Nimer S.D. Mol. Cell. Biol. 1995; 15: 6055-6063Crossref PubMed Scopus (104) Google Scholar, 27Nishimura Y. Tanaka T. J. Biol. Chem. 2001; 276: 19921-19928Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), by glucocorticoids in mouse fibroblasts (28Wallace A.D. Wheeler T.T. Young D.A. Biochem. Biophys. Res. Commun. 1997; 232: 403-406Crossref PubMed Scopus (39) Google Scholar), by IL-3 and IL-4 in pro-B lymphocytes (29Ikushima S. Inukai T. Inaba T. Nimer S.D. Cleveland J.L. Look A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2609-2614Crossref PubMed Scopus (119) Google Scholar, 30Chu C.C. Paul W.E. Mol. Immunol. 1998; 35: 487-502Crossref PubMed Scopus (35) Google Scholar, 31Yu Y.L. Chiang Y.J. Yen J.J. J. Biol. Chem. 2002; 277: 27144-27153Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), by oncogenic Ras mutants or by granulocyte-macrophage/colony-stimulating factor treatment in pro-B lymphocytes (32Kuribara R. Kinoshita T. Miyajima A. Shinjyo T. Yoshihara T. Inukai T. Ozawa K. Look A.T. Inaba T. Mol. Cell. Biol. 1999; 19: 2754-2762Crossref PubMed Scopus (73) Google Scholar), and by overexpression of the tumor suppressor phosphatase and tensin homologue in cancer cells (33Unoki M. Nakamura Y. Oncogene. 2001; 20: 4457-4465Crossref PubMed Scopus (308) Google Scholar). Similar to other tissues, osteoblastic cell lines and calvariae demonstrated low basal expression of E4bp4 mRNA levels. PTH treatment rapidly and transiently induced E4bp4 gene expression that peaked around 2 h and declined thereafter. In MC3T3-E1 and MOB cells, PTH-induced E4bp4 mRNA levels did not require protein synthesis suggesting that E4bp4 is a primary response gene in PTH-treated osteoblasts. In fact, CHX pretreatment led to sustained E4bp4 mRNA levels following PTH treatment. This is in agreement with the glucocorticoid induction of E4bp4 in mouse fibroblasts, where CHX pretreatment enhances the induction of E4bp4 mRNA levels (28Wallace A.D. Wheeler T.T. Young D.A. Biochem. Biophys. Res. Commun. 1997; 232: 403-406Crossref PubMed Scopus (39) Google Scholar). Interestingly, our findings contrast the observation that CHX abolishes IL-3-induced E4bp4 expression" @default.
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• W1992480558 cites W1965963242 @default.
• W1992480558 cites W1967334341 @default.
• W1992480558 cites W1968093456 @default.
• W1992480558 cites W1991548299 @default.
• W1992480558 cites W1993733276 @default.
• W1992480558 cites W1997688114 @default.
• W1992480558 cites W2003247523 @default.
• W1992480558 cites W2015955893 @default.
• W1992480558 cites W2021865186 @default.
• W1992480558 cites W2022959303 @default.
• W1992480558 cites W2036042475 @default.
• W1992480558 cites W2037291952 @default.
• W1992480558 cites W2041272447 @default.
• W1992480558 cites W2044399280 @default.
• W1992480558 cites W2046819829 @default.
• W1992480558 cites W2050164560 @default.
• W1992480558 cites W2057772566 @default.
• W1992480558 cites W2061343800 @default.
• W1992480558 cites W2064879068 @default.
• W1992480558 cites W2064905730 @default.
• W1992480558 cites W2066976141 @default.
• W1992480558 cites W2071990427 @default.
• W1992480558 cites W2072572444 @default.
• W1992480558 cites W2083651710 @default.
• W1992480558 cites W2086306018 @default.
• W1992480558 cites W2088870579 @default.
• W1992480558 cites W2090687056 @default.
• W1992480558 cites W2093595636 @default.
• W1992480558 cites W2104082433 @default.
• W1992480558 cites W2104454968 @default.
• W1992480558 cites W2113336371 @default.
• W1992480558 cites W2126122040 @default.
• W1992480558 cites W2126902993 @default.
• W1992480558 cites W2140011725 @default.
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• W1992480558 cites W2151887581 @default.
• W1992480558 cites W2156667942 @default.
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