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Chondrocyte Maturation (chondrocyte + maturation)
Selected AbstractsOverexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of ,-Catenin,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2008Qiuqian Wu MD Abstract Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of ,-catenin in Col2a1-Smurf2 embryos. The mechanism underlying Smurf2-mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction: Our recent finding that adult Col2a1-Smurf2 mice have an osteoarthritis-like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods: We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results: Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of ,-catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1-Smurf2 limbs. Conclusions: Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of ,-catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice. [source] Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2006Tian-Fang Li Abstract 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. [source] Targeted Expression of SHH Affects Chondrocyte Differentiation, Growth Plate Organization, and Sox9 Expression,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2004Sara Tavella Abstract The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation. Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates. Materials and Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization. Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter. Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones. [source] Identification of Novel Target Genes of the Bone-Specific Transcription Factor Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Michael Stock Abstract Fifteen putative transcriptional target genes regulated by the osteogenic transcription factor Runx2 were identified by cDNA microarray and differential hybridization techniques. Expression pattern and regulation of one gene, Pttg1ip, was analyzed in detail. Introduction: The transcription factor Runx2 is a key regulator of osteoblast development and plays a role in chondrocyte maturation. The identification of transcriptional target genes of Runx2 may yield insight into how osteoblastic differentiation is achieved on a molecular level. Materials and Methods: Using a differential hybridization technique (selective amplification through biotin and restriction-mediated enrichment [SABRE]) and cDNA microarray analysis, 15 differentially expressed genes were identified using mRNA from C3H 10T1/2 cells with constitutive and inducible overexpression of Runx2. Results and Conclusions: Among the 15 genes identified, 4 encode the extracellular matrix proteins Ecm1, Mgp, Fbn5, and Osf-2, three represent the transcription factors Esx1, Osr1, and Sox9, whereas others were Ptn, Npdc-1, Hig1, and Tem1. The gene for Pttg1ip was upregulated in Runx2-expressing cells. Pttg1ip is widely expressed during development, but at highest levels in limbs and gonads. The Pttg1ip promoter binds Runx2 in a sequence specific manner, and Runx2 is able to transactivate the Pttg1ip promoter in MC3T3-E1 cells. Therefore, Pttg1ip is likely to be a novel direct transcriptional target gene of Runx2. In conclusion, the genes identified in this study are important candidates for mediating Runx2 induced cellular differentiation. [source] Expression of FGFR3 with the G380R Achondroplasia Mutation Inhibits Proliferation and Maturation of CFK2 Chondrocytic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Janet E. Henderson Abstract A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression. [source] Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response elementJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Arthur J. Cohen Abstract Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half-sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half-sites function as two overlapping RAREs that share the middle half-site. Ablation of the middle half-site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half-sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Primary mouse embryonic fibroblasts: A model of mesenchymal cartilage formation,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher J. Lengner Cartilage formation is an intricate process that requires temporal and spatial organization of regulatory factors in order for a mesenchymal progenitor cell to differentiate through the distinct stages of chondrogenesis. Gene function during this process has best been studied by analysis of in vivo cartilage formation in genetically altered mouse models. Mouse embryonic fibroblasts (MEFs) isolated from such mouse models have been widely used for the study of growth control and DNA damage response. Here, we address the potential of MEFs to undergo chondrogenic differentiation. We demonstrate for the first time that MEFs can enter and complete the program of chondrogenic differentiation ex vivo, from undifferentiated progenitor cells to mature, hypertrophic chondrocytes. We show that chondrogenic differentiation can be induced by cell,cell contact or BMP-2 treatment, while in combination, these conditions synergistically enhance chondrocyte differentiation resulting in the formation of 3-dimensional (3-D) cartilaginous tissue ex vivo. Temporal expression profiles of pro-chondrogenic transcription factors Bapx1 and Sox9 and cartilaginous extracellular matrix (ECM) proteins Collagen Type II and X (Coll II and Coll X) demonstrate that the in vivo progression of chondrocyte maturation is recapitulated in the MEF model system. Our findings establish the MEF as a powerful tool for the generation of cartilaginous tissue ex vivo and for the study of gene function during chondrogenesis. © 2004 Wiley-Liss, Inc. [source] Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturationJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009Christine A. Clark Abstract While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2,treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-, to activate the TGF-,-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 785,792, 2009 [source] Regulation of embryonic endochondral ossification by Smurf2JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2008Qiuqian Wu Abstract Smurf2 is an E3 ubiquitin ligase that targets TGF-, receptor activated Smad2 and Smad3 for the proteasome in primary articular chondrocytes, thus stimulating their hypertrophic differentiation. Comparatively, how Smurf2 functions in growth plate chondrocytes in a developing long bone is an open question. In this study, we measured the mRNA levels of endogenous Smurf2 and type X collagen in chick growth plate at different embryonic stages to monitor the correlation between the level of Smurf2 expression and chondrocyte maturational stage. We found that high levels of Smurf2 were associated with the differentiative and proliferative stages, while Smurf2 levels were thereafter decreased as the chondrocytes matured toward hypertrophy. In addition, we injected Smurf2 -RCAS into chick wing buds at HH stage 20,23 and examined how the ectopic overexpression of Smurf2 in condensing chondrogenic mesenchyme affects the subsequent process of chondrocyte maturation and ossification during embryonic development. Histological analysis showed that overexpression of Smurf2 in a developing wing bud accelerated chondrocyte maturation and endochondral ossification, which may result from a decrease in TGF-, signaling in the infected chondrocytes with Smurf2 -RCAS. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:704,712, 2008 [source] Transforming growth factor-,1 induced alteration of skeletal morphogenesis in vivoJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2004Cristin M. Ferguson Abstract Transforming growth factor beta (TGF-,) is expressed in the growth plate and is an important regulator of chondrocyte maturation. Loss of function results in premature chondrocyte maturation both in vitro and in vivo. While TGF-, inhibits chondrocyte maturation in cell cultures, the effect of increased TGF-, has not been well characterized in an in vivo development model. Addition of Affi-gel agarose beads loaded with TGF-,1 (10 ng/,l) to developing stage 24,25 chick limb buds resulted in limb shortening and altered morphology. In situ hybridization studies showed down regulation of Indian hedgehog (ihh), bone morphogenetic protein 6 (bmp6), and collagen type X (colX) expression, markers of chondrocyte maturation, in TGF-,1 treated limbs. TGF-,1 also decreased chondrocyte proliferation in the developing anlage. The findings confirm a critical role for TGF-, during skeletal development. A more complete understanding of the role of TGF-, and its down-stream signals will lead to improved understanding and treatment of cartilage diseases. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Molecular aspects of healing in stabilized and non-stabilized fracturesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2001A. X. Le Bone formation is a continuous process that is initiated during fetal development and persists in adults in the form of bone regeneration and remodeling. These latter two aspects of bone formation are clearly influenced by the mechanical environment. In this study we tested the hypothesis that alterations in the mechanical environment regulate the program of mesenchymal cell differentiation, and thus the formation of a cartilage or bony callus, at the site of injury. As a first step in testing this hypothesis we produced stabilized and non-stabilized tibial fractures in a mouse model, then used molecular and cellular methods to examine the stage of healing. Using the "molecular map" of the fracture callus, we divided our analyzes into three phases of fracture healing: the inflammatory or initial phase of healing, the soft callus or intermediate stage, and the hard callus stage. Our results show that indian hedgehog(ihh), which regulates aspects of chondrocyte maturation during fetal and early postnatal skeletogenesis, was expressed earlier in an non-stabilized fracture callus as compared to a stabilized callus, ihh persisted in the non-stabilized fracture whereas its expression was down-regulated in the stabilized bone. IHH exerts its effects on chondrocyte maturation through a feedback loop that may involve bone morphogenetic protein 6 [bmp6; (S. Pathi, J.B. Rutenberg, R.L. Johnson, A. Vortkamp, Developmental Biology 209 (1999) 239,253)] and the transcription factor gli3, bmp6 and gli3 were re-induced in domain adjacent to the ihh -positive cells during the soft and hard callus stages of healing. Thus, stabilizing the fracture, which circumvents or decreases the cartilaginous phase of bone repair, correlates with a decrease in ihh signaling in the fracture callus. Collectively, our results illustrate that the ihh signaling pathway participates in fracture repair, and that the mechanical environment affects the temporal induction of ihh, bmp6 and gli3. These data support the hypothesis that mechanical influences affect mesenchymal cell differentiation to bone. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Genetic variation in the SMAD3 gene is associated with hip and knee osteoarthritisARTHRITIS & RHEUMATISM, Issue 8 2010Ana M. Valdes Objective Smad3 (or, MADH3) is a key intracellular messenger in the transforming growth factor , signaling pathway. In mice, Smad3 deficiency accelerates growth plate chondrocyte maturation and leads to an osteoarthritis (OA),like disease. We undertook this study to investigate the role of genetic variation in SMAD3 in the risk of large-joint OA in humans. Methods Ten tag single-nucleotide polymorphisms (SNPs) in the SMAD3 gene region were tested in a discovery set: 313 patients who had undergone total knee replacement, 214 patients who had undergone total hip replacement, and 520 controls from the UK. The SNP associated with both hip and knee OA was subsequently genotyped in 1,221 controls and 1,074 cases from 2 cohorts of patients with hip OA and 2,537 controls and 1,575 cases from 4 cohorts of patients with knee OA. Results A SNP (rs12901499) mapping to intron 1 of SMAD3 was associated with both knee and hip OA (P < 0.0022 and P < 0.021, respectively) in the discovery set. In all study cohorts, the major allele (G) was increased among OA patients relative to controls. A meta-analysis for knee OA yielded an odds ratio (OR) of 1.22 (95% confidence interval [95% CI] 1.12,1.34), P < 7.5 × 10,6. For hip OA, the OR was 1.22 (95% CI 1.09,1.36), P < 4.0 × 10,4. No evidence for heterogeneity was found (I2 = 0%). Conclusion Our data indicate that genetic variation in the SMAD3 gene is involved in the risk of both hip OA and knee OA in European populations, confirming the results from animal models on the potential importance of this molecule in the pathogenesis of OA. [source] | |||||||||||||