Transcriptional Regulation of the Osterix (Osx, Sp7) Promoter by Tumor Necrosis Factor Identifies Disparate Effects of Mitogen-activated Protein Kinase and NFκB Pathways

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. 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 On day TNF-α was in the for each The of Osx mRNA was in cells plated on day 0 at × in in was to TNF-α, and RNA was obtained at the under using the was as for μg/ml was RNA was using the The of MAPK inhibitors was done to TNF of MEK1 or was done with the Osx promoter by TNF later and cell for luciferase an additional The of the expression or the control was done to TNF and of the to TNF Real-time of Osx mRNA in cell RNA was using and PCR or The used were and The PCR were as for for and for for and Deletion Osx promoter for to of the Osx gene to the of the for the Osx The was from mouse by PCR using the and and The PCR was with and and inserted the luciferase The of the Osx promoter were constructed by PCR using the that the and the and The two used the as for the and the and The PCR were by and and inserted the luciferase The two and mutation of the Osx promoter were constructed using the the The Osx promoter was used as the with the of PCR 1, and and and the promoter was done to the heterologous with a of the were by C3H10T1/2 cells were plated at a of × in culture the cells were with a of promoter and control cells were and using luciferase and luciferase in the were to luciferase to for in of of the alternatively spliced was obtained using the The used was The PCR were the using the was done to the transcription start sites from the RNA was as described and used for were by of to the Osx gene to of at for was with of murine reverse and for at were and on The were and in a mobility 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, He X. Rubin J. Nanes M.S. J. 2000; PubMed Google Scholar). was used to a were done by the of and a control were done by the of were done using San Diego, TNF Osx expression was observed by day of culture in The effect of TNF was using previously shown to osteoblast differentiation in MC3T3, bone marrow and fetal calvaria (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, Rubin J. Nanes M.S. J. 2004; Scholar). that with TNF on day of culture inhibited Osx TNF inhibited Osx mRNA 50% by by and by with levels in the control that the inhibitory effect of TNF was with 50% inhibition of mRNA at This is similar to that 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). TNF decreased Osx by of cells were with to the of TNF to RNA synthesis. The of the Osx mRNA was using TNF did not decrease the Osx mRNA cells were with to TNF action required new protein synthesis. that decreased Osx although TNF inhibited the Osx mRNA in the presence of of the Osx Promoter and of of the proximal promoter including a of RNA was from a murine Osx, for and the and sites of the A of transcription factor binding sites was using to regulatory sites K. K. H. PubMed Scopus Google Scholar). that the promoter binding sites for factors to differentiation of pluripotent precursor cells to the or lineage. These and NFκB sequences. The Osx promoter also of a site. The transcriptional start site of the Osx promoter was by that two of and suggesting two start identified in These sites were by using and a two mRNA and Osx2 revealed that the more abundant to the mRNA as previously for murine Osx and the Osx A.J. 2003; PubMed Scopus Google Scholar, Y. A. H. H. B. Gene. 2004; PubMed Scopus Google Scholar). The abundant Osx2 alternatively spliced and the Osx structure as from these Deletion Analysis Promoter and of the Osx promoter was by from the or These were inserted upstream of a luciferase a of the promoter with the of the and Osx2 transcription start sites for the effect of the on promoter activity. The proximal promoter the but not start site of the of the of the proximal to the start site retained and was with upstream of and there were independent promoter with the and Osx2 start and fragments were upstream of the These retained independent that was of the and and that of the control previously the region was also capable of independent promoter TNF of the Osx effect of TNF on the Osx promoter was and response effects of TNF on the Osx promoter were done in cells A and and also the mesenchymal cell line C3H10T1/2 and TNF a and inhibition of Osx promoter with an and with the effects on Osx mRNA and the inhibition of OB differentiation. Two major signal TNF action are NFκB and MAPK M.S. Gene. 2003; 321: 1-15Crossref PubMed Scopus (375) Google Scholar). The effect of NFκB expression on the promoter was by of C3H10T1/2 cells with NFκB or or was done with a an independent nuclear signal and by a promoter X. Farmer P. Rubin J. Nanes M.S. J. Cell. 2004; PubMed Scopus Google Scholar). the results for this and also the effect of for including or NFκB a potent of the Osx promoter. This effect was by the of retained of the of the The did not Osx promoter activity. expression increased Osx promoter but of the were In a with bone morphogenic did not Osx promoter of the promoter were to the the inhibitory effect of TNF. the effect of TNF as to the of the control of the TNF inhibition was to a region to as by the effect of of this region and This region was downstream of the Osx2 start site and the region of independent promoter shown in the TNF response small or a mutation were the and These TNF inhibition of the promoter to a region and of the was done by of the upstream of the heterologous promoter TNF inhibited the of this promoter but on of in the TNF-responsive was done using that the TNF-responsive region to sites of nuclear obtained from control and C3H10T1/2 cells was used for with the sites were with of the TNF-responsive region to and binding to the the Osx2 start site. of the TNF response by and mutational analysis to on a region however, TNF did not change the of binding at this although a small in binding was in with to transcription factors was done using but were observed for or TNF Inhibition of Osx by MAPK a MAPK in to the NFκB pathway. These downstream of of the protein in osteoblastic cells M.S. Gene. 2003; 321: 1-15Crossref PubMed Scopus (375) Google Scholar). We evaluated MAPK as a of TNF inhibitory NFκB Osx were for with or inhibitors of MEK1 upstream of kinase or p38 kinase, The effect of these on TNF inhibition of the Osx promoter was in the Osx that the inhibitor TNF inhibition of Osx the inhibitors of JNK and p38 The inhibitor increased Osx promoter the of The effect of the inhibitor was also observed using a to the TNF-responsive The inhibitor abrogated TNF inhibition of this promoter and also increased that the inhibitor also abrogated TNF inhibition of the heterologous promoter the that the TNF was sufficient to MAPK the inhibitor TNF inhibition of Osx cells were with PD98059 for the of TNF. RNA was later for of Osx mRNA by that the inhibitor abrogated TNF inhibition of Osx PD98059 a in Osx results were obtained for the Osx promoter using a MEK1 or that these TNF inhibition of the the results obtained using of MAPK with MEK1 or TNF inhibition of the Osx promoter. C3H10T1/2 cells were as described in the to were with the Osx and the cells were with TNF or an additional cell were obtained for luciferase activity. show luciferase for a expressed luciferase with and to the are from each by of the NFκB of NFκB of the promoter was by of the as described for NFκB responsiveness was with of the proximal promoter and A or A promoter the TNF response but not the proximal promoter was not by NFκB the NFκB response to a region of the binding site in of the binding NFκB of the promoter These and NFκB in a heterologous independent NFκB The of the NFκB stimulus to Osx transcription was the inhibitory effect of TNF in the TNF did not transcription when NFκB was TNF + This to a more of the effects of these on binding to the NFκB The NFκB by nuclear protein binding of the region were investigated by a the binding a of the promoter and the of used for analysis with that and a and a but not the The was with the functional identified by and mutational analysis of the promoter. This is with the NFκB in of to this was of with binding to the NFκB protein from C3H10T1/2 cells did not this that the of NFκB by TNF was to and the that NFκB was not TNF inhibition of the Osx NFκB was by or expression of a L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google in C3H10T1/2 that the the could TNF inhibition of Osx promoter activity. the of in TNF of an results show that TNF expression of Osx by inhibiting the transcriptional of promoter. The of TNF that inhibits Osx expression is similar to that the differentiation of OB (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, Rubin J. Nanes M.S. J. 2004; Scholar). TNF inhibition of Osx was and and observed in two cell of the early of OB differentiation. We have previously shown (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 that TNF inhibits the expression of transcription factor required for OB differentiation (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). have shown that both RUNX2 and Osx are for OB differentiation. the effect of TNF to of both factors the early in OB differentiation. TNF action on Osx is to there was effect of TNF on Osx mRNA with although inhibitory on was to a inhibitory action of TNF. These suggest that the effects of TNF are direct the of a protein The for protein synthesis has been for the of Osx mRNA by BMP, is prevented by J.M. 2003; PubMed Scopus Google Scholar). with these we did not of Osx promoter by of the Osx promoter regulation of in driving expression of TNF action on the Osx we a upstream of the luciferase and analysis revealed two transcription start of the start site for the more expressed and previously described mRNA A.J. 2003; PubMed Scopus Google Scholar, Y. A. H. H. B. Gene. 2004; PubMed Scopus Google has shown that the Osx mRNA has at two These an abundant to the murine Osx and a alternatively spliced we the expression of the two in cells and that from the two transcription start also show that the Osx promoter at two capable of independent promoter This that the two mRNA could expressed under the regulation of the two although transcriptional requires Osx2 mRNA could function to have a or expressed as an that is to the function of and two protein are to the major of Osx TNF inhibited the of the Osx promoter in a and in both C3H10T1/2 and These results were similar to the and regulation of Osx with a small in TNF action that to the of luciferase Deletion analysis of the promoter the inhibitory effect of TNF to a region proximal to the Osx2 start site. This region and sites for transcription factor gene and the site. TNF did not change the of binding at this a small in binding was at this site could of of the in the the regulatory effect of the from an to a to identify the protein in this that is by TNF. TNF M.S. Gene. 2003; 321: 1-15Crossref PubMed Scopus (375) Google Scholar). We to the roles of two major TNF MAPK and as mediators of TNF inhibition of Osx In a TNF two or of the 1, inhibition of OB differentiation Rubin J. Nanes M.S. J. 2004; Scholar). In a the a that and kinase The kinase isoform an NFκB binding protein that the of the to and of NFκB for nuclear and gene also the MAPK with downstream of or suggest that TNF inhibition of Osx expression is via MAPK, the MEK1 inhibitor PD98059 TNF effects on Osx promoter and Osx mRNA expression. This blockade of TNF action was to the inhibitor of and not observed with inhibitors of p38 or The inhibitor Osx promoter and mRNA suggesting that a inhibitory of MAPK on the Osx promoter The inhibitor PD98059 also abrogated TNF inhibition of a the region or the response upstream of a heterologous promoter. results on of TNF action and of the TNF signal by MAPK are results a for JNK or p38 in TNF regulation of Osx, PD98059 could additional as the for TNF action was also by the of TNF action by MEK1 and to the MAPK by TNF and the nuclear protein of this evidence on the of MAPK in OB differentiation that may by of the of pluripotent to the OB phenotype is by a of factors, including that expression of the key transcription factor RUNX2 or that Osx expression. The sensitivity to may increased or decreased by the MAPK of OB differentiation by has been shown to require p38 kinase Y. B. Endocrinology. 2003; PubMed Scopus Google Scholar, J. C. J. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. C. A. G. J. J. Bone 2003; PubMed Scopus Google Scholar, U. R. R. M. A. Cell 2003; PubMed Scopus Google Scholar). of is with with this In of this C. A. N. K. K. H. K. J. Bone 2002; PubMed Scopus Google that of of including expression of and in cells and of in more pluripotent and Endocrinology. PubMed Scopus Google that inhibition of using PD98059 of OB gene expression in marrow stromal cells, a inhibited this In with on blockade has been shown to inhibition of OB differentiation by factors, including and K. Y. M. S. S. M. K. Y. J. Bone 2003; PubMed Scopus Google Scholar). These results suggest that inflammatory via OB via p38 OB differentiation. are to an inhibitory effect of Cell with matrix is signal that and an for RUNX2 of gene promoters G. D. P. K. G. Franceschi J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G. R. D. Franceschi J. Bone 2002; PubMed Scopus Google Scholar, G. D. R. Franceschi J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. Cell. 2003; PubMed Scopus Google Scholar). of pre-osteoblast by factors was shown to M. L. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). effects may on the stimulus also signal or on the stage of cell differentiation C. A. N. K. K. H. K. J. Bone 2002; PubMed Scopus Google Scholar). although the MAPK inhibitors used in the present ERK, and JNK, there may to that could inhibited by PD98059 that have to evaluated J. M. S. C. Z. J. 2001; PubMed Google Scholar). Because TNF also of we evaluated the effect of NFκB expression on the Osx promoter. results that NFκB not the inhibitory effect of as blockade of NFκB did not TNF expression of NFκB to the using a promoter an independent nuclear signal a potent function in the proximal Osx promoter. This was not activated by as TNF not a sufficient of nuclear NFκB to the site. the of transcriptional observed using the NFκB expression that the an of Osx expression and OB differentiation. an functional under that levels of NFκB is activated by a of including of the TNF that have not been for effects on OB U. 2003; PubMed Scopus Google Scholar). In addition, the of NFκB to Osx transcription could by of on key and shown to action at X. Farmer P. Rubin J. Nanes M.S. J. Cell. 2004; PubMed Scopus Google Scholar). a of NFκB at could to the inflammatory stimulus of TNF via is to the of NFκB in Osx expression and OB differentiation. Bone remodeling is a that the rate of with of new OB from the precursor to the stimulus with estrogen at or in inflammatory TNF the of OB and the of bone formation to shift toward a The inhibition of Osx by TNF may contribute to the of suppressed OB differentiation. We Dr. D. for the expression Dr. D. for the Dr. (University of for and Dr. M. (University of for