Expression of the Osteoblast Differentiation Factor RUNX2 (Cbfa1/ AML3/Pebp2 A) Is Inhibited by Tumor Necrosis

The transcription factor RUNX2 (Cbfa1/AML3/Pebp2 A) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor (TNF) on the expression of RUNX2 because TNF is known to inhibit differentiation of osteoblasts from pluripotent progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 clonal pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 steady state mRNA as measured by reverse transcription-PCR. The IC50 for TNF inhibition was 0.6 ng/ml. TNF suppression of RUNX2 mRNA was confirmed using Northern analysis. The effect of TNF was studied using isoform-specific primers that flanked unique regions of two major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting expression of the mRNA for the longer MASNS isoform by 50%. RUNX2 nuclear content was evaluated by electrophoretic mobility shift assay using a rat osteocalcin promoter binding sequence as probe and by Western analysis. TNF reduced nuclear RUNX2 protein. Inhibition of new protein synthesis with cycloheximide failed to prevent TNF inhibition of RUNX2 mRNA, suggesting that a newly translated protein did not mediate the TNF effect. RUNX2 mRNA half-life was 1.8 h and reduced to 0.9 h by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6-kb RUNX2 promoter-luciferase reporter in MC3T3-E1 cells. TNF caused a dose-dependent inhibition of transcription to 50% of control values. The inhibitory effect of TNF was preserved with deletions to nucleotide 108 upstream of the translational start site; however, localization downstream of nucleotide 108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. The disproportionate inhibition of RUNX2 nuclear protein suggests that additional post-transcriptional mechanisms may be occurring. Suppression of RUNX2 by TNF may decrease osteoblast differentiation and inhibit bone formation in TNF excess states. The inflammatory cytokine tumor necrosis factor(TNF) has been shown to contribute to bone loss through a variety of mechanisms that increase bone resorption and decrease bone formation. TNF has a major role as an inflammatory mediator in rheumatoid arthritis where increased bone resorption causes periarticular bone loss, and in postmenopausal osteoporosis in which there is generalized bone loss (1–5). In addition to the effects of TNF on bone resorption, TNF also inhibits the bone-forming function of osteoblasts. In mature osteoblasts TNF inhibits the expression of the skeletal matrix proteins type I collagen and osteocalcin, causes resistance to the genomic action of 1,25-dihydroxyvitamin D3, and increases the production of matrix metalloproteinases and pathologic paracrine factors (6–11). We have shown previously that TNF inhibits the differentiation of new osteoblasts from precursor cells (12). In the presence of low concentrations of TNF, fetal calvaria precursor cells fail to form a mineralized matrix or to express the bone-specific osteocalcin gene. Similarly, clonal MC3T3-E1 cells, which spontaneously differentiate to the osteoblast phenotype, remain undifferentiated after TNF treatment and fail to form a matrix that is competent for mineralization. Osteoblasts differentiate from pluripotent precursor cells that have the capacity to become adipocytes, skeletal muscle cells, tendon, or fibroblasts (13–17). During differentiation, a program of gene expression occurs that is characterized by sequential steps of proliferation, phenotype selection, skeletal gene expression, and finally apoptosis (18). A number of hormonal, paracrine, and autocrine signals regulate the steps that promote differentiation along an osteoblastic trajectory, rather than the selection of other cell phenotypes. The way in which TNF inhibits the program of osteoblast differentiation is unknown but could involve suppression of a critical regulator. RUNX2 (Cbfa1/AML3/Pebp2 A) is a runt related transcription factor that is essential for osteoblast differentiation (19– 22). RUNX2 regulates the expression of several osteoblastic genes including 1(I)collagen, osteopontin, bone sialoprotein, and the skeletal-specific osteocalcin gene (23, 24). The binding of nuclear RUNX2 to osteoblast-specific elements up-regulates skeletal genes and consequently the osteoblast phenotype. Mice engineered as nullizygous for both RUNX2 alleles are born with a completely cartilaginous skeleton (21). These experiments established the requirement for RUNX2 in osteoblast differentiation during embryogenesis. Studies in cultured * This work was supported by National Institutes of Health Grant R01 AR46452-01 and a Department of Veterans Affairs Merit Review grant (both to M. S. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed: Div. of Endocrinology and Metabolism, Veterans Affairs Medical Center (111), 1670 Clairmont Rd., Decatur, GA 30033. E-mail: [email protected]. 1 The abbreviations used are: TNF, tumor necrosis factor; CHX, cycloheximide; RT, reverse transcription; D-PBS, Dulbecco’s phosphatebuffered saline; MEM, minimal essential medium; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 4, Issue of January 25, pp. 2695–2701, 2002 Printed in U.S.A.