Transcriptional Regulation of the Osterix (Osx, Sp7) Promoter by Tumor Necrosis Factor Identifies Disparate Effects of Mitogen-activated Protein Kinase and NFκB Pathways
2006; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m507804200
ISSN1083-351X
AutoresXianghuai Lu, Linda Gilbert, Xiaofei He, Janet Rubin, Mark S. Nanes,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoOsteoblast (OB) differentiation is suppressed by tumor necrosis factor-α (TNF-α), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFκB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFκB nuclear entry. The forced expression of high levels of NFκB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis. Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-α (TNF-α), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFκB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFκB nuclear entry. The forced expression of high levels of NFκB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis. Osteoblasts (OBs) 2The abbreviations used are: OB, osteoblast; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; TNF-α, tumor necrosis factor-α; siRNA, small interfering RNA; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance; BMP, bone morphogenic protein. are derived from pluripotent precursor cells of mesenchymal origin that are capable of differentiation to chondrocytes, myocytes, adipocytes, or fibroblasts (1Lian J.B. Stein G.S. Marcus R. Feldman D. Kelsey J. Osteoporosis. 2nd. 1. Academic Press, San Diego, CA2001: 21-72Crossref Google Scholar). Bone formation in the embryo and remodeling in the adult require that a sufficient number of precursor cells differentiate to functional OBs. New OBs are continuously required for the synthesis of bone matrix and replacement of cells becoming osteocytes or undergoing apoptosis. A coordinated expression of transcription factors determines the commitment of precursor cells toward the OB phenotype under the control of autocrine, paracrine, and hormonal stimuli. Two of these transcription factors, RUNX2 (Cbfa1/AML3/Pebp2αA) and Osx, are required for differentiation to the OB lineage. In mice, RUNX2 gene knock-out causes a lethal mutation with a cartilaginous skeleton. RUNX2 is presumed to function as an organizer on promoters of skeletal-specific genes (2Lian J.B. Javed A. Zaidi S.K. Lengner C. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Crit. Rev. Eukaryotic Gene Expression. 2004; 14: 1-41Crossref PubMed Google Scholar). A phenotype similar to the RUNX2 knock-out is observed with knock-out of Osx. Here the arrest in development occurs slightly later but also results in a cartilaginous skeleton (3Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2835) Google Scholar). In addition, Osx induces OB differentiation of dispersed embryonic cells (4Tai G. Polak J.M. Bishop A.E. Christodoulou I. Buttery L.D. Tissue Eng. 2004; 10: 1456-1466Crossref PubMed Scopus (70) Google Scholar). RUNX2 is expressed in Osx knock-outs, suggesting that Osx functions downstream of RUNX2 in the differentiation pathway. Differentiation of precursor cells to OBs in adult bone is impaired by inflammatory stimuli. In rheumatoid arthritis, estrogen deficiency, and aging, there is an increased expression of cytokines, including tumor necrosis factor-α (TNF-α). In adult bone, inflammatory cytokines blunt the formation rate of new bone in the face of increased resorption, contributing to net bone loss (5Pacifici R. Calcif. Tissue Int. 1999; 65: 345-351Crossref PubMed Scopus (36) Google Scholar, 6Gilbert L. He X. Farmer P. Boden S. Kozlowski M. Rubin J. Nanes M.S. Endocrinology. 2000; 141: 3956-3964Crossref PubMed Scopus (0) Google Scholar, 7Cenci S. Weitzmann M.N. Roggia C. Namba N. Novack D. Woodring J. Pacifici R. J. Clin. Investig. 2000; 106: 1229-1237Crossref PubMed Scopus (560) Google Scholar, 8Roggia C. Gao Y. Cenci S. Weitzmann M.N. Toraldo G. Isaia G. Pacifici R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13960-13965Crossref PubMed Scopus (442) Google Scholar, 9Bingham III, C.O. J. Rheumatol. 2002; 65: 3-9Google Scholar, 10Weitzmann M.N. Roggia C. Toraldo G. Weitzmann L. Pacifici R. J. Clin. Investig. 2002; 110: 1643-1650Crossref PubMed Scopus (227) Google Scholar, 11Nanes M.S. Gene. 2003; 321: 1-15Crossref PubMed Scopus (375) Google Scholar). Cytokines could interfere with the expression of factors required for OB differentiation. We have previously shown that TNF inhibits osteoblast differentiation at the stage of precursor cell commitment to the OB lineage (6Gilbert L. He X. Farmer P. Boden S. Kozlowski M. Rubin J. Nanes M.S. Endocrinology. 2000; 141: 3956-3964Crossref PubMed Scopus (0) Google Scholar). Osteoblast precursors, including fetal calvaria cells, murine marrow stromal cells, and the clonal pre-osteoblastic cell line MC3T3, fail to differentiate in the presence of TNF. These models of osteoblast progenitors uniformly show enhanced sensitivity to TNF blockade of differentiation at an early stage in culture when the key transcription factors RUNX2 and Osx are required. An inhibitory effect of TNF on the RUNX2 promoter that could contribute to decreased expression and differentiation of cells has been described previously (12Gilbert L. He X. Farmer P. Rubin J. Drissi H. van Wijnen A.J. Lian J.B. Stein G.S. Nanes M.S. J. Biol. Chem. 2002; 277: 2695-2701Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). This effect of TNF is isoform-specific, inhibiting the osteoblastic MASNS RUNX2 isoform 50% and the more ubiquitously expressed MRIPV isoform >90%. These results suggest that TNF may have additional targets. Here we present evidence that TNF is a potent inhibitor of Osx expression. In addition, we have evaluated the structure and regulation of the Osx promoter and report transcriptional regulation by TNF at a discrete site via a mitogen-activated protein kinase (MAPK) signal. Reagents—MC3T3-E1 (clone 14) mouse pre-osteoblast cells were provided by Dr. Renny Franceschi (University of Michigan). C3H10T1/2 cells were obtained from the America Type Culture Collection (Manassas, VA). Human TNF-α was purchased from PeproTech (Rocky Hill, NJ). Real-time PCR was done using the Bio-Rad ICycler. SYBR Green was obtained from Bio-Rad. MAPK inhibitors PD98059 and SB203580 were obtained from Calbiochem. SP600125 was purchased from Tocris Cookson (Ellisville, MO). Minimal essential medium was purchased from Invitrogen and fetal bovine serum from Hyclone (Logan, UT). Other reagents were obtained from Sigma. Cell Treatment and RNA Harvest—MC3T3-E1 cells were plated on day 0 at 7.4 × 106 cells/150-mm plate in minimal essential medium + 10% fetal bovine serum (medium). On day 1, medium was replaced with differentiating medium (minimal essential medium + 10% fetal bovine serum + 50 μg/ml l-ascorbate). On day 2, TNF-α was added in the doses indicated for each experiment. The half-life of Osx mRNA was measured in MC3T3-E1 cells plated on day 0 at 4.4 × 105 cells/well in 6-well plates in medium. Actinomycin D (0.5 μg/ml) was added 2 h prior to TNF-α, and RNA was obtained at the time points indicated under "Results." For experiments using cycloheximide, the same protocol was followed as for actinomycin D, except 5 μg/ml cycloheximide was used. RNA was prepared using the RNeasy Mini Kit (Qiagen, Valencia, CA). The addition of MAPK chemical inhibitors was done 2 h prior to TNF treatment. Transient transfection of dominant negative MEK1 or ERK1 was done with the Osx promoter reporter construct followed by TNF 24 h later and cell lysates for luciferase assay after an additional 24 h. The addition of the siRNA-p65 expression plasmid or the siRNA control (Santa Cruz Biotechnology, Santa Cruz, CA) was done 48 h prior to TNF and transfection of the N terminus-deleted IκB 24 h prior to TNF treatment. Real-time RT-PCR—Quantitation of Osx mRNA in total cell RNA samples was carried out using standard RT and PCR procedures, setting up duplicate or triplicate reactions. The primers used were OsxQRTf, 5′-CCT CTC GAC CCG ACT GCA GAT C-3′ and OsxQRTr, 5′-AGC TGC AAG CTC TCT GTA ACC ATG AC-3′. The PCR reaction conditions were as follows: 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 20 s for 40 cycles. Reporter and Deletion Constructs—The Osx promoter sequence selected for further study spans nucleotides –1360 to –10 of the Osx gene locus relative to the position (+1) of the initiation methionine for the Osx open reading frame. The sequence was amplified from mouse genomic F-factor-based bacterial artificial chromosome RP23–118K10 by PCR using the following 5′ and 3′ primers, respectively: 5′-ACA GGT ACC CAC ACA TAC ACG-3′ and 5′-CAA GCA GAG AGG ACG CCA TCC TCG AGC T-3′. The resulting PCR product was digested with KpnI and XhoI and inserted into the pGL3 Basic luciferase reporter vector (Promega, Madison, WI). The serial 5′ deletion mutants of the Osx promoter were constructed by PCR using the same 3′ primer that amplified the F-factor-based bacterial artificial chromosome and the following 5′ primers, respectively: 5′-CCTTT CGGTA CCAGC GCGGC-3′ (–665/+91); 5′-AGATG GTACC CAGCG CCCTC-3′ (–471/+91); 5′-CCAAG GTACC TCTGA CAACT TG-3′ (–304/+91); and 5′-GGTGG TACCC TACAG ACAGA-3′ (–121/+91). The two 3′ deletion mutants used the same 5′ primer as for the F-factor-based bacterial artificial chromosome amplification and the following 3′ primers, respectively: 5′-TGATG AAGCT TCACA GACAA-3′ (–1269/–469) and 5′-CTCTG AAGCT TGGGATCCAC-3′ (–1269/–69). The resulting PCR products were digested by KpnI and HindIII and inserted into the pGL3 Basic luciferase reporter. The two smaller deletion and mutation mutants of the Osx promoter were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations. The Osx promoter –665/+91 deletion mutant was used as the DNA template with the following pair of PCR primers: pair 1, 5′-GATTG TAGGA TTGGA AGCTG GCCTG AGAGA-3′ and 5′-TCTCT CAGGC CAGCT TCCAA TCCTA CAATC-3′ (D520/500); pair 2, 5′-AACAA GAGTG AGCTG GCCTG TTGCC AGTAA TCTTC AAGCC-3′ and 5′-GGCTT GAAGA TTACT GGCAA CAGGC CAGCT CACTC TTGTT-3′ (D488/450); pair 3, 5′-TTGGA TCTGA GGTTT AACAA GAGTG AGCTG-3′ and 5′-CAGCT CACTC TTGTT AAACC TCAGA TCCAA-3′ (mutated 514/510). Direct ligation into the promoter SV40-luciferase (pGL-promoter) was done to create the heterologous reporter with a dimer of the sequence 5′-CTCTG AGTGG GAACA AGAGT GTCTG AGTGG GAACA AGAGT GTCTG AGTGG GAACA AGAGT G-3′. All constructs were confirmed by automated sequencing. Transfection—The C3H10T1/2 cells (n = 3 wells/group) were plated at a density of 7 × 104 cells/well in 12-well tissue culture plates (Corning, NY). After 24 h, the cells were transfected with a mixture of Superfect transfection reagent (Qiagen, Valencia, CA), medium, promoter reporter, and pRL-TK control vector (Promega, Madison, WI). 48 h after transfection, cells were harvested and assayed using firefly luciferase and Renilla luciferase substrates in the Dual Luciferase assay system (Promega, Madison, WI). Luciferase values were normalized to Renilla luciferase data to correct for variation in transfection efficiency. Rapid Amplification of 5′-cDNA Ends—The 5′-end sequence of the alternatively spliced exon was obtained using BD SMART RACE cDNA amplification kit (BD Biosciences) following the manufacturer's instructions. The primer used was 5′-GAG CTT CTT CCT CAA GCA GAG AGG ACG CCA TCC TCG A-3′. The PCR products were cloned into the pCR-TOPO vector using the TOPO TA cloning kit (Invitrogen). Automated sequencing was done to determine the transcription start sites from the 5′-RACE products. Primer Extension—Total RNA was extracted as described above and used for primer extension analysis. Reactions were performed by annealing 1 pmol of [32P]-labeled primer, complementary to the Osx gene untranslated 5′-end (GGACT GGAGC CATAG TGAGC TTC), to 10 μg of total RNA. Following annealing at 58 °C for 20 min, extension was performed with 100 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and deoxyribonucleotide triphosphates (0.5 mm) for 30 min at 42 °C. cDNA products were denatured and analyzed on 6% (w/v) denaturing polyacrylamide gels. The gels were dried and analyzed in a phosphorimaging device. Electrophoretic Mobility Shift Assay (EMSA)—Electrophoretic mobility shifts were done as previously described (12Gilbert L. He X. Farmer P. Rubin J. Drissi H. van Wijnen A.J. Lian J.B. Stein G.S. Nanes M.S. J. Biol. Chem. 2002; 277: 2695-2701Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 13Farmer P.K. He X. Schmitz M.L. Rubin J. Nanes M.S. Am. J. Physiol. 2000; 279: E213-E220Crossref PubMed Google Scholar). Statistical Analysis—ANOVA was used to determine a statistical difference between multiple groups. Multiple comparisons between individual groups were done by the method of Tukey. Comparisons between any one group and a common control were done by the method of Dunnet. Analyses were done using Prism software, (GraphPad, San Diego, CA). TNF Inhibits Osx Steady State mRNA—Osx expression was observed by day 2 of culture in MC3T3 cells. The effect of TNF was studied using doses previously shown to inhibit osteoblast differentiation in MC3T3, bone marrow stromal, and fetal rat calvaria pre-osteoblasts (6Gilbert L. He X. Farmer P. Boden S. Kozlowski M. Rubin J. Nanes M.S. Endocrinology. 2000; 141: 3956-3964Crossref PubMed Scopus (0) Google Scholar, 14Gilbert L.C. Rubin J. Nanes M.S. Am. J. Physiol. 2004; 288: E1011-E1018Google Scholar). Fig. 1A shows that treatment with 10 ng/ml TNF on day 2 of culture inhibited Osx steady state mRNA. TNF inhibited Osx mRNA 50% by 4 h, 85% by 8 h, and 95% by 24 h compared with levels in the control cultures. Fig. 1B shows that the inhibitory effect of TNF was dose-dependent with 50% inhibition of mRNA occurring at 0.75 ng/ml. This IC50 is similar to that reported for TNF inhibition of osteoblast differentiation (6Gilbert L. He X. Farmer P. Boden S. Kozlowski M. Rubin J. Nanes M.S. Endocrinology. 2000; 141: 3956-3964Crossref PubMed Scopus (0) Google Scholar). To determine whether TNF decreased Osx by destabilization of mRNA, MC3T3 cells were treated with actinomycin D 2 h prior to the addition of TNF to stop RNA synthesis. The half-life of the Osx mRNA was then measured over 14 h using real-time RT-PCR. TNF did not decrease the brief (1.5 h) Osx mRNA half-life (Fig. 1C). MC3T3 cells were treated with cycloheximide to determine whether TNF action required new protein synthesis. Fig. 1D shows that cycloheximide alone decreased Osx mRNA, although TNF further inhibited the Osx mRNA level in the presence of cycloheximide treatment. Cloning of the Osx Promoter and Determination of Transcriptional Start Sites—A 1357-bp fragment of the proximal promoter including a portion of 5′-untranslated RNA was amplified from a murine F-factor-based bacterial artificial chromosome containing genomic Osx, sequenced for confirmation, and cloned into the KpnI and XhoI sites of the pGL3 Basic-luciferase reporter. A map of homologous transcription factor binding sites was generated using MatInspector to assess potential regulatory sites (15Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2427) Google Scholar). Fig. 2 shows that the promoter contains putative homologous binding sites for factors known to influence differentiation of pluripotent precursor cells to the osteoblast, chondrocyte, adipocyte, or myoblast lineage. These included MyoD, AML-1, RUNX2, cEBP, Msx, DLX, and NFκB core consensus sequences. The Osx promoter fragment also included 14 contiguous repeats of a Myf5-binding site. The transcriptional start site of the Osx promoter was determined by 5′-RACE. Fig. 3A shows that 5′-RACE yielded two bands of 100 and 300 kb size, suggesting two potential start sites. (also identified in Fig. 2). These sites were confirmed by primer extension (not shown). Amplification using unique 5′ primers and a common 3′ primer yielded two mRNA species designated Osx1 and Osx2 (Fig. 3B). Sequencing revealed that the more abundant Osx1 corresponded to the expected mRNA as reported previously for murine Osx and the highly homologous human Osx (16Milona M.A. Gough J.E. Edgar A.J. BMC Genomics. 2003; 4: 43Crossref PubMed Scopus (59) Google Scholar, 17Gao Y. Jheon A. Nourkeyhani H. Kobayashi H. Ganss B. Gene. 2004; 341: 101-110Crossref PubMed Scopus (58) Google Scholar). The less abundant Osx2 includes alternatively spliced exons 1 and 2. Fig. 3C maps the genomic Osx structure as deduced from these experiments. Deletion Analysis Reveals Independent Promoter Activities Upstream Of Osx1 and Osx2—The basal activity of the Osx promoter was analyzed by making successive deletions from the 3′- or 5′-end. These were inserted upstream of a promoterless pGL3basic luciferase reporter. Fig. 4A shows a diagram of the –1269/+91 promoter with the locations of the Osx1 and Osx2 transcription start sites labeled for reference. Fig. 4B shows the effect of the deletions on promoter activity. The proximal promoter containing the Osx1, but not Osx2, start site had 40% of the activity of the full-length reporter (Fig. 4B, construct C versus A). Interestingly, deletion of the regions proximal to the Osx1 start site retained substantial activity (Fig. 4B, constructs D, E, and F versus A). Activity was lost with deletions upstream of –469 (Fig. 4B, constructs G, J, and K versus A). To determine whether there were independent promoter activities associated with the Osx1 and Osx2 start sites, –669/–469 and –269/+91 fragments were cloned upstream of the promoterless pGLbasic. These reporters retained independent activity that was 40% of the full-length –1269/+91 (Fig. 4B, constructs H and I versus A) and five times that of the pGLbasic control (H versus L). As previously noted, the region around Osx1 was also capable of independent promoter activity (Fig. 4B, construct C versus A). TNF Regulation of the Osx Promoter—The effect of TNF on the Osx promoter was then measured. Time course and dose response effects of TNF on the –1269/+91 Osx promoter were done in MC3T3 cells (Fig. 5, A and B) and also the primitive mesenchymal cell line C3H10T1/2 (Fig. 5, C and D). TNF caused a time- and dose-dependent inhibition of Osx promoter activity with an IC50 between 0.5 and 1.0 ng/ml, consistent with the effects on Osx mRNA and the inhibition of OB differentiation. Two major signal pathways mediating TNF action are NFκB and MAPK (11Nanes M.S. Gene. 2003; 321: 1-15Crossref PubMed Scopus (375) Google Scholar). The effect of NFκB expression on the promoter activity was determined by transfection of C3H10T1/2 cells with NFκB or its individual p50 or p65 subunits. Expression was done with a pef-Myc-nuc vector containing an independent nuclear localization signal and driven by a cytomegalovirus promoter (18Lu X. Farmer P. Rubin J. Nanes M.S. J. Cell. Biochem. 2004; 92: 833-848Crossref PubMed Scopus (75) Google Scholar). Fig. 6 shows the results for this transfection and also the effect of other stimuli for comparison, including RUNX2, Msx2, dexamethasone, 1,25(OH)2D3, or parathyroid hormone. Surprisingly, NFκB caused a potent stimulation of the Osx promoter. This effect was mediated by the p65 subunit of NFκB, which retained 40% of the activity of the intact dimer. The p50 subunit did not significantly increase Osx promoter activity. Msx2 expression increased Osx promoter activity 5.7-fold, but none of the other stimuli were effective. In a separate experiment, treatment with bone morphogenic protein-2 (BMP-2) did not stimulate Osx promoter activity (not shown). Deletions of the promoter were studied to localize the regions conferring the inhibitory effect of TNF. Fig. 7 shows the effect of TNF as fold stimulation relative to the activity of the respective control constructs. Most of the TNF inhibition was localized to a region between –514 to –510, as seen by the effect of deletion of this region (Fig. 7, constructs F and G versus A). This region was downstream of the Osx2 start site and embedded within the region of independent promoter activity shown in Fig. 4B. To further localize the TNF response element, small deletions or a four-base mutation were made within the –665/+91 construct (Fig. 7, constructs D and E). These localized TNF inhibition of the promoter to a region between –514 and –510. Confirmation of the localization was done by inserting three copies of the –520/–500 sequence upstream of the heterologous SV40 promoter (Fig. 7, construct H). TNF inhibited the activity of this promoter but had no activity on pSV40 alone (not shown). Identification of Protein-DNA Binding in the TNF-responsive Region—EMSA was done using overlapping probes that spanned 200 bp around the TNF-responsive region to determine sites of nuclear protein-DNA binding. Nuclear extract obtained from control and TNF-treated C3H10T1/2 cells was used for incubation with the probes. Five sites were found with strong protein-DNA interaction, one of which overlapped the TNF-responsive region (Fig. 8, probe 9, specific). Binding to probes 3 and 5 represented binding to the Myf5 consensus, whereas probe 6 spanned the Osx2 start site. Localization of the TNF response by deletion and mutational analysis led us to focus on a region within probe 9 (–520/–500); however, TNF did not change the pattern of binding at this site, although a small 2-fold increase in binding intensity was confirmed in repeated experiments. Incubation with antibodies to transcription factors was done using probe 9, but no supershifts were observed for p65, p50, RUNX2, RUNX1, JunD, RBP-Jκ, Fra-1, or Fra-2 (not shown). TNF Inhibition of Osx Is Mediated by MAPK Signaling—The TNF-bound p55 receptor activates a MAPK cascade in addition to the NFκB pathway. These pathways diverge downstream of activation of the cytosolic adapter protein TRAF2 in osteoblastic cells (11Nanes M.S. Gene. 2003; 321: 1-15Crossref PubMed Scopus (375) Google Scholar). We evaluated MAPK as a possible mediator of TNF inhibitory action, because NFκB paradoxically stimulated Osx transcription. Cells were pre-incubated for 2 h with PD98059, SP600125, or SB203580, inhibitors of MEK1 (the immediate upstream activator of ERK1/2), cJun-N-terminal kinase (JNK), or p38 kinase, respectively. The effect of these treatments on TNF inhibition of the Osx promoter was measured in the –1269/+91 Osx reporter. Fig. 9A shows that the MEK inhibitor reversed TNF inhibition of Osx transcription, whereas the inhibitors of JNK and p38 had no effect. The MEK inhibitor alone increased Osx promoter activity above the level of control. The effect of the MEK inhibitor was also observed using a reporter limited to the –669/–469 TNF-responsive region. The MEK inhibitor (PD98059) completely abrogated TNF inhibition of this limited promoter and also increased basal activity (Fig. 9B). Fig. 9C shows that the MEK inhibitor also abrogated TNF inhibition of the heterologous SV40-LUC promoter bearing the 3×(–520/–500) sequence, indicating that the TNF element was sufficient to confer MAPK responsiveness. To determine whether the MEK inhibitor blocked TNF inhibition of Osx mRNA, MC3T3 cells were treated with PD98059 for 5 h before the addition of TNF. RNA was harvested 18 h later for measurement of Osx mRNA by real-time RT-PCR. Fig. 9D shows that the MEK inhibitor completely abrogated TNF inhibition of Osx mRNA. PD98059 alone caused a significant increase in steady state Osx mRNA. Similar results were obtained for the –1269/+91 Osx promoter using a dominant negative MEK1 or ERK1. Fig. 10 shows that these dominant negatives partially reversed TNF inhibition of the promoter, confirming the results obtained using PD98059.FIGURE 10Blockade of MAPK with dominant negative MEK1 or ERK1 partially reverses TNF inhibition of the Osx promoter. C3H10T1/2 cells were cultured as described in the legend to Fig. 9. Dominant negative constructs were co-transfected with the –1269/+91 Osx promoter, and 24 h later, the cells were treated with TNF (10 ng/ml) or control. After an additional 24 h, cell lysates were obtained for luciferase activity. Results show arbitrary luciferase units corrected for a constitutively expressed Renilla luciferase control. Bars with different letters (a, b, and c) to the right are significantly different from each other (p < 0.05 by ANOVA).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of the NFκB Regulatory Element—The pattern of NFκB stimulation of the promoter was determined by overexpression of the p65 subunit, as described for Fig. 6. NFκB responsiveness was abolished with deletion of the proximal promoter between –270 and –70 (Fig. 11, construct J versus A or I). A promoter fragment containing the TNF response site, but not the proximal promoter sequence, was not stimulated by NFκB (Fig. 7, construct H). Further deletions localized the NFκB response to a region within 200 bp of the consensus p50 binding site indicated in Fig. 2 (-214/-217). Mutation of four bases representing the core binding motif abolished NFκB stimulation of the –1269/+91 promoter (Fig. 11K). These bases and their flanking sequence conferred NFκB activation in a heterologous SV40 promoter, confirming independent NFκB enhancer activity (Fig. 11L). The potency of the NFκB stimulus to Osx transcription was predominant over the inhibitory effect of TNF in the full-length –1269/+91 construct, because TNF treatment did not inhibit transcription when NFκB was simultaneously overexpressed (not shown, C = 1.0 ± 0.1, p65 = 13.1 ± 0.6, TNF + p65 = 12.4 ± 0.8). This paradox led to a more detailed assessment of the effects of these treatments on binding to the NFκB element. The NFκB Element Is Not Activated by TNF—The nuclear protein binding characteristics of the NFκB-responsive region were investigated by generating probes spanning a 231-bp sequence centered around the p50 binding motif. Fig. 12A shows a map of the promoter and indicates the sequence of probes used for EMSA analysis with recombinant NFκB. Fig. 12B shows that probes 2 and 4 bound a recombinant p50 monomer and a p50/p65 dimer but not the p65 monomer. The sequence bound was concordant with the functional sequence identified by deletion and mutational analysis of the promoter. This sequence is compared with the NFκB consensus in Fig. 12C. Binding of recombinant p50 to this sequence was of very low affinity compared with binding to the NFκB consensus sequence (Fig. 12D). Nuclear protein from TNF-treated C3H10T1/2 cells did not bind this low affinity site, demonstrating that the amount of NFκB stimulated by TNF was insufficient to bind and activate the enhancer (Fig. 12E, lane 5). To confirm that NFκB activation was not modulating TNF inhibition of the Osx promoter, NFκB activation was blocked by siRNA-p65 or expression of a degradation-resistant N terminus-deleted IκB (ΔIκB, (19Chu Z.L. McKinsey T.A. Liu L. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (826) Google Scholar)) in C3H10T1/2 cells. Fig. 13A shows
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