Wnt induction of chondrocyte hypertrophy through the RUNX2 transcription factor
Center for Musculoskeletal Research, University of Rochester, Rochester, New York, USA. Journal of Cellular Physiology
(Impact Factor: 3.84).
07/2006; 208(1):77-86. DOI: 10.1002/jcp.20656
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.
Available from: Yufeng Dong
- "WI)asdescribedpreviously(Dongetal.,2006)andpRL-SV40 wildtyperenillawasusedasinternalcontrol. "
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ABSTRACT: While Notch signaling plays a critical role in the regulation of cartilage formation, its downstream targets are unknown. To address this we performed gain and losses of function experiments and demonstrate that Notch inhibition of chondrogenesis acts via up-regulation of the transcription factor Twist1. Upon Notch activation, murine limb bud mesenchymal progenitor cells in micromass culture displayed an inhibition of chondrogenesis. Twist1 was found to be exclusively expressed in mesenchymal progenitor cells at the onset stage of chondrogenesis during Notch activation. Inhibition of Notch signaling in these cells significantly reduced protein expression of Twist1. Furthermore, the inhibition effect of NICD1 on MPC chondrogenesis was markedly reduced by knocking down of Twist1. Constitutively active Notch signaling significantly enhanced Twist1 promoter activity; whereas mutation studies indicated that a putative NICD/RBPjK binding element in the promoter region is required for the Notch-responsiveness of the Twist1 promoter. Finally, chromatin immunoprecipitation assays further confirmed that the Notch intracellular domain influences Twist1 by directly binding to the Twist1 promoter. These data provide a novel insight into understanding the molecular mechanisms behind Notch inhibition of the onset of chondrogenesis.
Copyright © 2015. Published by Elsevier Ireland Ltd.
Available from: Tyler Pizzute
- "The higher expression of hypertrophic chondrocyte markers, type X collagen and MMP-13 (Kirsch and von der Mark 1992; Nurminskaya and Linsenmayer 1996) in OA suggests a correlation between hypertrophy and OA. Some studies have demonstrated that Wnt signaling promotes chick chondrocyte hypertrophy through the induction of the bone and cartilage-related transcription factor Runx2. Dong et al. (2006) reported that β-catenin is able to induce RUNX2 and COL10A1 transcription as the molecular mechanism through which Wnt signaling regulates chondrocyte hypertrophy. Protein levels of β-catenin, which accumulates in OA chondrocytes, are extremely low in differentiated articular chondrocytes; however, low levels of β-catenin are up-regulated during phenotypic loss after a serial monolayer culture. "
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ABSTRACT: Chondrogenesis is a developmental process that is controlled and coordinated by many growth and differentiation factors, in addition to environmental factors that initiate or suppress cellular signaling pathways and the transcription of specific genes in a temporal-spatial manner. As key signaling molecules in regulating cell proliferation, homeostasis and development, both mitogen-activated protein kinases (MAPK) and the Wnt family participate in morphogenesis and tissue patterning, playing important roles in skeletal development, especially chondrogenesis. Recent findings suggest that both signals are also actively involved in arthritis and related diseases. Despite the implication that crosstalk between MAPK and Wnt signaling has a significant function in cancer, few studies have summarized this interaction and its regulation of chondrogenesis. In this review, we focus on MAPK and Wnt signaling, referencing their relationships in various types of cells and particularly to their influence on chondrogenesis and cartilage development. We also discuss the interactions between MAPK and Wnt signaling with respect to cartilage-related diseases such as osteoarthritis and explore potential therapeutic targets for disease treatments.
Available from: sciencedirect.com
- "It has been reported that Xenopus ortholog XRNF185, with 91% identical to human RNF185, interacts with Xdsh , the ortholog of human disheveled/(Dvl ), a central mediator of Wnt signaling pathway . Wnt signaling has been demonstrated as a family of potent regulators of MSCs commitment to chondrogenic and osteogenic phenotypes    . Previous study has shown that osteoblast is one of the targets of Wnt/b-catenin signaling during bone formation and Wnt/b-catenin signaling is critical for osteoblasts to complete their differentiation cycle and synthesize bone . "
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ABSTRACT: Osteoblast plays a pivotal role in bone metabolism and bone remodeling by mediating bone formation and regulating the activity of osteoclast. Clarifying the regulators and regulation mechanisms of osteogenic differentiation of mesenchymal stem cells (MSCs) and pre-osteoblasts will provide tremendous promise for bone repair and bone regeneration. RNF185 was identified as a candidate of endogenous suppressors of osteogenic specification in human mesenchymal stem cells (hMSCs). Here we show that RNF185 down regulates osteogenic differentiation of mouse calvaria-derived MC3T3-E1 cells, confirmed by quantitative real-time-PCR (qRT-PCR) and alkaline phosphatase (ALP) activity. Further we confirm that RNF185 interacts with dishevelled2 (Dvl2), a key mediator of Wnt signaling pathway. Overexpression of RNF185 decreased the exogenous and endogenous level of Dvl2, promotes the ubiquitination and degradation of Dvl2 and inhibits Wnt signaling, which is evident from the down-regulation of β-catenin mediated transcriptional activity. And Dvl2 reverses the effect of RNF185 on osteogenic differention of MC3T3-E1 cells. Taken together, our results indicate that RNF185 negatively regulates osteogenesis through the degradation of Dvl2 and down-regulation of canonical Wnt signaling pathway and suggest a possible therapeutic target in osteoporosis.
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