Expression of the Osteoblast Differentiation Factor RUNX2 (Cbfa1/AML3/Pebp2αA) Is Inhibited by Tumor Necrosis Factor-α

Abstract

The transcription factor RUNX2 (Cbfa1/AML3/Pebp-2␣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 IC 50 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) 1 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 D 3 , 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 (1...

Lymphoid Enhancer Factor-1 and ॆ-Catenin Inhibit Runx2-dependent Transcriptional Activation of the Osteocalcin Promoter

“…Previous studies have identified numerous proteins that either enhance Runx2 function at the post-transcriptional level (13)(14)(15)(16) or modulate Runx2 activity by regulating the expression/ transcription of the Runx2 promoter (61)(62)(63)(64)(65). Our data indicate that additional mechanisms that negatively regulate Runx2 at the post-transcriptional level also exist.…”

TNF-α's effects on proliferation and apoptosis in human mesenchymal stem cells depend on RUNX2 expression

“…(42,43) In a context of high concentration of growth factors and low expression of RUNX2, TNF-a may inhibit osteoblastic differentiation through TNFR1 independently of apoptosis. (42,44) In these conditions, inhibition of proliferation without any effect on apoptosis induced by TNF-a could be explained by the effects of high percent of FCS on RUNX2 level.…”

Parathyroid Hormone Induction of the Osteocalcin Gene

“…Although the functions of OCN are poorly understood, gene deletion studies suggest possible functions in bone remodeling (10). The expression of OCN is regulated by a number of calcitropic hormones and growth factors including 1,25-dihydroxy vitamin D 3 (11)(12)(13), glucocorticoids (14,15), PTH (16), bone morphogenetic proteins (17), basic fibroblast growth factor 2 (18), tumor necrosis factor-␣ (19), and transforming growth factor ␤ (20). A number of transcription factors that bind to specific regions of the osteocalcin gene promoter have also been identified.…”