Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor

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‐β‐induced expression of Sox9 and col2a1. Over‐expression of β‐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 β‐catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical β‐catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of β‐catenin‐TCF/Lef on Runx2 promoter. Co‐transfection of lymphoid enhancer factor (Lef1) and β‐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 β‐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/β‐catenin signaling is regulated by TGF‐β 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. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.

[1]  Kozo Nakamura,et al.  The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner. , 2005, Biochemical and biophysical research communications.

[2]  E. Schwarz,et al.  Wnt‐mediated regulation of chondrocyte maturation: Modulation by TGF‐β , 2005, Journal of cellular biochemistry.

[3]  T. Komori.  Regulation of skeletal development by the Runx family of transcription factors , 2005, Journal of cellular biochemistry.

[4]  Robert A. Winn,et al.  Restoration of Wnt-7a Expression Reverses Non-small Cell Lung Cancer Cellular Transformation through Frizzled-9-mediated Growth Inhibition and Promotion of Cell Differentiation* , 2005, Journal of Biological Chemistry.

[5]  Tomohiro Otani,et al.  Developmental regulation of Wnt/beta-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification. , 2005, The Journal of biological chemistry.

[6]  Walter Birchmeier,et al.  Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. , 2005, Developmental cell.

[7]  Xizhi Guo,et al.  Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. , 2005, Developmental cell.

[8]  C. Lengner,et al.  Nkx3.2-mediated Repression of Runx2 Promotes Chondrogenic Differentiation* , 2005, Journal of Biological Chemistry.

[9]  G. Stein,et al.  Overlapping expression of Runx1(Cbfa2) and Runx2(Cbfa1) transcription factors supports cooperative induction of skeletal development , 2005, Journal of cellular physiology.

[10]  R. Tuan,et al.  Long‐term in vitro analysis of limb cartilage development: Involvement of Wnt signaling , 2004, Journal of cellular biochemistry.

[11]  L. Topol,et al.  Wnt/β-catenin signaling is sufficient and necessary for synovial joint formation , 2004 .

[12]  E. Schwarz,et al.  Parathyroid hormone-related peptide (PTHrP) inhibits Runx2 expression through the PKA signaling pathway. , 2004, Experimental cell research.

[13]  Ajamete Kaykas,et al.  WNT and β-catenin signalling: diseases and therapies , 2004, Nature Reviews Genetics.

[14]  P. S. Klein,et al.  The Frizzled family: receptors for multiple signal transduction pathways , 2004, Genome Biology.

[15]  P. McCrea,et al.  Interactions between Sox9 and β-catenin control chondrocyte differentiation , 2004 .

[16]  E. Schwarz,et al.  Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes , 2003, Journal of cellular biochemistry.

[17]  Jeffrey D. Axelrod,et al.  A Second Canon , 2003 .

[18]  Yoshiaki Kawano,et al.  Secreted antagonists of the Wnt signalling pathway , 2003, Journal of Cell Science.

[19]  E. Schwarz,et al.  Retinoic acid stimulates chondrocyte differentiation and enhances bone morphogenetic protein effects through induction of Smad1 and Smad5. , 2003, Endocrinology.

[20]  L. Topol,et al.  Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation , 2003, Development.

[21]  G. Xiao,et al.  Regulation of the osteoblast‐specific transcription factor, Runx2: Responsiveness to multiple signal transduction pathways , 2003, Journal of cellular biochemistry.

[22]  K. Miyazono,et al.  RUNX transcription factors as key targets of TGF-β superfamily signaling , 2003 .

[23]  Xi He,et al.  Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. , 2003, Genes & development.

[24]  Changshan Wu,et al.  The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis. , 2002, Developmental biology.

[25]  S. Mundlos,et al.  Role of Runx genes in chondrocyte differentiation. , 2002, Developmental biology.

[26]  G. Stein,et al.  1,25-(OH)2-vitamin D3 suppresses the bone-related Runx2/Cbfa1 gene promoter. , 2002, Experimental cell research.

[27]  S. K. Zaidi,et al.  Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  N. Kanatani,et al.  Skeletal Malformations Caused by Overexpression of Cbfa1 or Its Dominant Negative Form in Chondrocytes , 2001, The Journal of cell biology.

[29]  Tohru Itoh,et al.  WNT Signals Control FGF-Dependent Limb Initiation and AER Induction in the Chick Embryo , 2001, Cell.

[30]  G. Stein,et al.  Smad-Runx Interactions During Chondrocyte Maturation , 2001, The Journal of bone and joint surgery. American volume.

[31]  Christine Hartmann,et al.  Wnt-14 Plays a Pivotal Role in Inducing Synovial Joint Formation in the Developing Appendicular Skeleton , 2001, Cell.

[32]  E. Schwarz,et al.  Smad2 and 3 Mediate Transforming Growth Factor-β1-Induced Inhibition of Chondrocyte Maturation* *The work was supported by National Health Services Grant AR-38945 (to R.J.O.) and an Orthopaedic Research Education Foundation Award (to C.M.F.). , 2000, Endocrinology.

[33]  Stephen N. Jones,et al.  Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene , 2000, Journal of cellular physiology.

[34]  C. Hartmann,et al.  Dual roles of Wnt signaling during chondrogenesis in the chicken limb. , 2000, Development.

[35]  Randall T Moon,et al.  Mechanism and function of signal transduction by the Wnt/β-catenin and Wnt/Ca2+ pathways , 1999, Oncogene.

[36]  C. Grimsrud,et al.  BMP‐6 Is an Autocrine Stimulator of Chondrocyte Differentiation , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[37]  S. Nomura,et al.  Maturational disturbance of chondrocytes in Cbfa1‐deficient mice , 1999, Developmental dynamics : an official publication of the American Association of Anatomists.

[38]  S. Mundlos,et al.  Regulation of chondrocyte differentiation by Cbfa1 , 1999, Mechanisms of Development.

[39]  J. Loureiro,et al.  The Wnts , 1999, Current Biology.

[40]  P. LuValle,et al.  A BMP responsive transcriptional region in the chicken type X collagen gene. , 1998, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[41]  C. Tabin,et al.  Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud. , 1998, Science.

[42]  Jörg Stappert,et al.  β‐catenin is a target for the ubiquitin–proteasome pathway , 1997 .

[43]  S. Mundlos,et al.  Cbfa1, a Candidate Gene for Cleidocranial Dysplasia Syndrome, Is Essential for Osteoblast Differentiation and Bone Development , 1997, Cell.

[44]  G. Karsenty,et al.  Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation , 1997, Cell.

[45]  Makoto Sato,et al.  Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts , 1997, Cell.

[46]  A. Brown,et al.  Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro. , 1997, Developmental biology.

[47]  R. Kosher,et al.  Wnt-5a and Wnt-7a are expressed in the developing chick limb bud in a manner suggesting roles in pattern formation along the proximodistal and dorsoventral axes , 1993, Mechanisms of Development.

[48]  M. Kühl.  The WNT/calcium pathway: biochemical mediators, tools and future requirements. , 2004, Frontiers in bioscience : a journal and virtual library.

[49]  S. K. Zaidi,et al.  Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors. , 2004, Critical reviews in eukaryotic gene expression.

[50]  Xizhi Guo,et al.  Wnt/beta-catenin signaling is sufficient and necessary for synovial joint formation. , 2004, Genes & development.

[51]  P. McCrea,et al.  Interactions between Sox9 and beta-catenin control chondrocyte differentiation. , 2004, Genes & development.

[52]  Randall T Moon,et al.  WNT and beta-catenin signalling: diseases and therapies. , 2004, Nature reviews. Genetics.

[53]  T. Komori,et al.  Runx2 expression and action in chondrocytes are regulated by retinoid signaling and parathyroid hormone-related peptide (PTHrP). , 2003, Osteoarthritis and cartilage.

[54]  Randall T Moon,et al.  A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. , 2003, Developmental cell.

[55]  K. Miyazono,et al.  RUNX transcription factors as key targets of TGF-beta superfamily signaling. , 2003, Current opinion in genetics & development.

[56]  L. Topol,et al.  Wnt 5 a and Wnt 5 b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation , 2003 .

[57]  G. Stein,et al.  Identification of novel protein/DNA interactions within the promoter of the bone‐related transcription factor Runx2/Cbfa1 , 2002, Journal of cellular biochemistry.

[58]  Vicki L Church,et al.  Wnt signalling during limb development. , 2002, The International journal of developmental biology.

[59]  T. Komori.  Runx2, A multifunctional transcription factor in skeletal development , 2002, Journal of cellular biochemistry.

[60]  R Kemler,et al.  beta-catenin is a target for the ubiquitin-proteasome pathway. , 1997, The EMBO journal.