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...

No abstract

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

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic-related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis ident...

Objective. To determine whether Smurf2, an E3 ubiquitin ligase known to inhibit transforming growth factor ␤ (TGF␤) signaling, is expressed in human osteoarthritic (OA) cartilage and can initiate OA in mice.Methods. Human OA cartilage was obtained from patients undergoing knee arthroplasty. Samples were graded histologically using the Mankin scale and were examined immunohistochemically for Smurf2 expression. A transgene driven by the collagen 2␣1 promoter was used to overexpress Smurf2 in mice. Smurf2 overexpression in mouse sternal chondrocytes was confirmed by reverse transcription-polymerase chain reaction and Western blotting. Changes in articular cartilage area, chondrocyte number, and chondrocyte diameter were assessed histomorphometrically using OsteoMeasure software. Alterations in type X collagen and matrix metalloproteinase 13 (MMP-13) in articular chondrocytes were examined by in situ hybridization and immunohistochemistry, respectively. Joint bone phenotypes were evaluated by microfocal computed tomography. The effects of Smurf2 overexpression on TGF␤ signaling were examined using a luciferase-based reporter and immunoprecipitation/Western blotting.Results. Human OA cartilage strongly expressed Smurf2 as compared with nonarthritic human cartilage. By 8 months of age, Smurf2-transgenic mice exhibited decreased articular cartilage area, fibrillation, clefting, eburnation, subchondral sclerosis, and osteophytes. Increased expression of type X collagen and MMP-13 were also detected in articular cartilage from transgenic mice. Transgenic sternal chondrocytes showed reduced TGF␤ signaling as well as decreased expression and increased ubiquitination of pSmad3.Conclusion. Smurf2 is up-regulated during OA in humans, and Smurf2-transgenic mice spontaneously develop an OA-like phenotype that correlates with decreased TGF␤ signaling and increased pSmad3 degradation. Overall, these results suggest a role of Smurf2 in the pathogenesis of OA.Arthritis is the number one cause of disability in the US (1). It has been projected that by 2020, 59.4 million Americans will be affected (18.2%) (2). Osteoarthritis (OA), the most common form of arthritis, is a noninflammatory degenerative joint disease characterized by articular chondrocyte dysfunction, articular cartilage degradation, osteophyte formation, and subchondral sclerosis (3). There is limited understanding of the seminal molecular and/or cellular events in articular cartilage degeneration, and there are few therapeutic options for OA patients. Thus, understanding these events would have a tremendous impact on the development of more-effective therapeutic paradigms.Biochemical, genetic, and mechanical factors contribute to OA progression (4).

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis.Introduction: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. Materials and Methods: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGF␤ and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. Results: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGF␤ potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virusmediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. Conclusion: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-␤ signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3 −/− chondrocytes. BMP signaling is increased in TGF-␤ deficiency and is required for accelerated chondrocyte maturation.Introduction: Disruption of TGF-␤ signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. Materials and Methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3 −/− mice. Expressions of maturational markers, as well as genes involved in TGF-␤ and BMP signaling were examined. Chondrocytes were treated with TGF-␤ and BMP-2, and effects on maturation-related genes and BMP/TGF-␤ responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. Results: Expression of colX and other maturational markers was markedly increased in Smad3−/− chondrocytes. Smad3 −/− chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-␤1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-␤ toward BMP signaling. In Smad3 −/− chondrocytes, alternative TGF-␤ signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-␤ pathways reduced colX expression and alkaline phosphatase activity in TGF-␤-treated Smad3 −/− cultures, but only partially. In contrast, Smad3 −/− chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-␤ signaling with important implications for osteoarthritis and cartilage diseases.