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...

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We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates chondrocyte hypertrophy at least partly through activation of Runx2 which in turn may induce col10a1 expression.

Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p < 0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection. ß