The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Henry M Kronenberg - One of the best experts on this subject based on the ideXlab platform.
-
dicer dependent pathways regulate chondrocyte proliferation and differentiation
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Tatsuya Kobayashi, Ernestina Schipani, Andrew P Mcmahon, Bradley S Cobb, Stephen J Rodda, Matthias Merkenschlager, Henry M KronenbergAbstract:Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of Chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating Chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic Chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null Chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.
-
a raf and b raf are dispensable for normal endochondral bone development and parathyroid hormone related peptide suppresses extracellular signal regulated kinase activation in hypertrophic Chondrocytes
Molecular and Cellular Biology, 2008Co-Authors: Sylvain Provot, Gregory Nachtrab, Jennifer L Paruch, Adele Pin Chen, Alcino J Silva, Henry M KronenbergAbstract:Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in Chondrocytes. Though B-Raf is expressed in proliferative Chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with Chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic Chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.
-
pth pthrp receptor delays chondrocyte hypertrophy via both runx2 dependent and independent pathways
Developmental Biology, 2006Co-Authors: Ungil Chung, Dehong Yang, Richard F Bringhurst, Gerard Karsenty, Henry M KronenbergAbstract:Abstract The transcription factor, Runx2, promotes chondrocyte hypertrophy, whereas parathyroid hormone-related protein (PTHrP) delays this process. To examine whether PTHrP suppresses chondrocyte hypertrophy via Runx2-dependent or -independent pathways, Runx2 expression and chondrocyte differentiation were analyzed using bones from embryonic limbs of wild type and Runx2(−/−) mice. Treatment of cultured rudiments with PTH dramatically suppresses Runx2 mRNA levels in hypertrophic Chondrocytes. PTH-induced delay of chondrocyte hypertrophy was observed in cultured tibiae from both Runx2(−/−) and wild-type embryos. This delay was also seen after PTH administration to limbs from wild type and Runx2(−/−) mice expressing Runx2 in Chondrocytes via a collagen 2 promoter-driven transgene. To further explore Runx2-dependent and -independent effects of PTHrP, we examined embryonic tibiae and femurs from littermates null for PTHrP, Runx2, or both genes. Runx2(−/−) femurs exhibited no vascular invasion or Chondrocytes expressing collagen type X or osteopontin mRNA. In contrast, Runx2(−/−)/PTHrP(−/−) mice exhibited limited vascular invasion and some Chondrocytes expressing collagen X or osteopontin mRNA. In both tibia and femur, Runx2(−/−)/PTHrP(−/−) mice exhibited expanded regions of proliferating Chondrocytes when compared to the same regions in PTHrP(−/−) mice. These data indicate that the delayed hypertrophy induced by PTHrP is mediated by both Runx2-dependent and -independent mechanisms.
-
absence of transcription factor c maf causes abnormal terminal differentiation of hypertrophic Chondrocytes during endochondral bone development
Developmental Biology, 2003Co-Authors: Helen E Maclean, Henry M Kronenberg, James I Kim, Melvin J Glimcher, Jinxi Wang, Laurie H GlimcherAbstract:Abstract In this study, we report that the transcription factor c-Maf is required for normal chondrocyte differentiation during endochondral bone development. c-maf is expressed in hypertrophic Chondrocytes during fetal development (E14.5–E18.5), with maximal expression in the tibia occurring at E15.5 and E16.5, in terminally differentiated Chondrocytes. In c-maf -null mice, fetal bone length is decreased ∼10%, and hypertrophic chondrocyte differentiation is perturbed. There is an initial decrease in the number of mature hypertrophic Chondrocytes at E15.5 in c-maf -null tibiae, with decreased expression domains of collagen X and osteopontin, markers of hypertrophic and terminal hypertrophic Chondrocytes, respectively. By E16.5, there is an expanded domain of late hypertrophic, osteopontin -positive Chondrocytes in the c-maf −/−. This accumulation of hypertrophic Chondrocytes persists and is still observed at 4 weeks of age. These data suggest that c-Maf facilitates the initial chondrocyte terminal differentiation and influences the disappearance of hypertrophic Chondrocytes. BrdU and TUNEL analyses show normal proliferation rate and apoptosis in the c-maf -null. There is a specific decrease in MMP-13 expression at E15.5 in the c-maf -null. MMP-13 is known to be regulated by AP-1 and may also be a target of c-Maf. Thus, cartilage is a novel system in which c-Maf acts during development, where c-Maf is required for normal chondrocyte differentiation.
-
pthrp and indian hedgehog control differentiation of growth plate Chondrocytes at multiple steps
Development, 2002Co-Authors: Tatsuya Kobayashi, Ungil Chung, Gerard Karsenty, Ernestina Schipani, Michael Starbuck, Takenobu Katagiri, Dale L Goad, Beate Lanske, Henry M KronenbergAbstract:In developing murine growth plates, Chondrocytes near the articular surface (periarticular Chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar Chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar Chondrocytes, Ihh action in the periarticular Chondrocytes was upregulated because of ectopically differentiated hypertrophic Chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular Chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular Chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.
Qian Chen - One of the best experts on this subject based on the ideXlab platform.
-
subcellular relocation of histone deacetylase 4 regulates growth plate chondrocyte differentiation through ca2 calmodulin dependent kinase iv
American Journal of Physiology-cell Physiology, 2012Co-Authors: Yingjie Guan, Paul Haines, Richard M. Terek, Xu Yang, Qian Chen, Tingcun ZhaoAbstract:Regulatory mechanisms of chondrocyte differentiation in the growth plate are incompletely understood. Here, we find that histone deacetylase 4 (HDAC4) is located in the nucleus of Chondrocytes in the proliferation zone and relocates to the cytoplasm of Chondrocytes in the prehypertrophic zone in vivo. This suggests that the relocation of HDAC4 from the nucleus to the cytoplasm may play a role during chondrocyte differentiation. Expression of active CaMKIV in Chondrocytes promotes HDAC4 relocation into cytoplasm in primary Chondrocytes. Conversely, HDAC4 relocation is blocked by a Ca2+/calmodulin-dependent kinase IV (CaMKIV) inhibitor. This indicates that CaMKIV signaling plays an important role in regulating HDAC4 relocation. In addition, CaMKIV is required for HDAC4 phosphorylation, which is required for HDAC4 association with the cytoplasmic protein 14-3-3. Active CaMKIV also stimulates runt-related transcription factor-2 (RunX2) and type X collagen (Col X) promoter activities and overcomes repression of these promoter activities by HDAC4. Furthermore, CaMKIV increases gene expression of the chondrocyte differentiation markers Ihh and Col X. Our results demonstrate that CaMKIV induces chondrocyte differentiation through regulation of HDAC4 subcellular relocation, from the nucleus to the cytoplasm, which results in increased activity of RunX2 and transition of Chondrocytes from the proliferative to the prehypertrophic stage. Thus, CaMKIV plays an important regulatory role during chondrocyte differentiation.
-
Pericellular matrilins regulate activation of Chondrocytes by cyclic load-induced matrix deformation
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2006Co-Authors: Katsuaki Kanbe, Xu Yang, Lei Wei, Changqi Sun, Qian ChenAbstract:Pericellular matrix is at the ideal location to be involved in transmitting mechanical signals from the microenvironment to a cell. We found that changes of the content of matrilins that link various pericellular molecules surrounding Chondrocytes affect mechanical stimulation of chondrocyte proliferation and gene expression. Thus, pericellular matrilins may play a role in chondrocyte mechanotransduction. Introduction: Chondrocytes reside in a capsule of pericellular matrix (chondron), which has been hypothesized to play a critical role in transducing mechanical signals to the cell. In this study, we test the hypothesis that the levels of matrilin (MATN)-1 and -3, major components of the chondrocyte pericellular matrix network, regulate activation of chondrocyte proliferation and differentiation by cyclic load–induced matrix deformation. Materials and Methods: Functional matrilins were decreased by expressing a dominant negative mini-MATN in primary Chondrocytes or by using MATN1-null Chondrocytes. The abundance of matrilins was also increased by expressing a wildtype MATN1 or MATN3 in Chondrocytes. Chondrocytes were cultured in a 3D sponge subjected to cyclic deformation at 1 Hz. Chondrocyte gene expression was quantified by real-time RT-PCR and by Western blot analysis. Matrilin pericellular matrix assembly was examined by immunocytochemistry. Results: Elimination of functional matrilins from pericellular matrix abrogated mechanical activation of Indian hedgehog signaling and abolished mechanical stimulation of chondrocyte proliferation and differentiation. Excessive or reduced matrilin content decreased mechanical response of Chondrocytes. Conclusions: Normal content of matrilins is essential to optimal activation of Chondrocytes by mechanical signals. Our data suggest that the sensitivity of Chondrocytes to the changes in the microenvironment can be adjusted by altering the content of matrilins in pericellular matrix. This finding supports a critical role of pericellular matrix in chondrocyte mechano-transduction and has important implications in cartilage tissue engineering and mechanical adaptation.
-
mechanoregulation of chondrocyte proliferation maturation and hypertrophy ion channel dependent transduction of matrix deformation signals
Experimental Cell Research, 2000Co-Authors: Qiuqian Wu, Qian ChenAbstract:Abstract Mechanical stress-induced matrix deformation plays a fundamental role in regulating cellular activities; however, little is known about its underlying mechanisms. To understand the effects of matrix deformation on Chondrocytes, we characterized primary Chondrocytes cultured on three-dimensional collagen scaffoldings, which can be loaded mechanically with a computer-controlled “Bio-Stretch” device. Cyclic matrix deformation greatly stimulated proliferation of immature Chondrocytes, but not that of hypertrophic Chondrocytes. This indicates that mechanical stimulation of chondrocyte proliferation is developmental stage specific. Synthesis of cartilage matrix protein (CMP/matrilin-1), a mature chondrocyte marker, and type X collagen, a hypertrophic chondrocyte marker, was up-regulated by stretch-induced matrix deformation. Therefore, genes of CMP and type X collagen are responsive to mechanical stress. Mechanical stimulation of the mRNA levels of CMP and type X collagen occurred exactly at the same time points when these markers were synthesized by nonloading cells. This indicates that cyclic matrix deformation does not alter the speed of differentiation, but affects the extent of differentiation. The addition of the stretch-activated channel blocker gadolinium during loading abolished mechanical stimulation of chondrocyte proliferation, but did not affect the up-regulation of CMP mRNA by mechanical stretch. In contrast, the calcium channel blocker nifedipine inhibited both the stretch-induced proliferation and the increase of CMP mRNA. This suggests that stretch-induced matrix deformation regulates chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals and calcium channels involved in transducing the signals for both proliferation and differentiation.
-
progression and recapitulation of the chondrocyte differentiation program cartilage matrix protein is a marker for cartilage maturation
Developmental Biology, 1995Co-Authors: Qian Chen, David M Johnson, Dominik R Haudenschild, Paul F GoetinckAbstract:Abstract During endochondral bone formation, Chondrocytes in the cartilaginous anlage of long bones progress through a spatially and temporally regulated differentiation program before being replaced by bone. To understand this process, we have characterized the differentiation program and analyzed the relationship between Chondrocytes and their extracellular environment in the regulation of the program. Our results indicate that, within an epiphyseal growth plate, the zone of proliferating Chondrocytes is not contiguous with the zone of hypertrophic Chondrocytes identified by the transcription of the type X collagen gene. We find that the postproliferative Chondrocytes which make up the zone between the zones of proliferation and hypertrophy specifically transcribe the gene for cartilage matrix protein (CMP). This zone has been termed the zone of maturation. The identification of this unique population of Chondrocytes demonstrates that the chondrocyte differentiation program consists of at least three stages. CMP translation products are present in the matrix surrounding the nonproliferative Chondrocytes of both the zones of maturation and hypertrophy. Thus, CMP is a marker for postmitotic Chondrocytes. As a result of the changes in gene expression during the differentiation program, Chondrocytes in each zone reside in an extracellular matrix with a unique macromolecular composition. Chondrocytes in primary cell culture can proceed through the same differentiation program as they do in the cartilaginous rudiments. In culture, a wave of differentiation begins in the center of a colony and spreads to its periphery. The cessation of proliferation coincides with the appearance of CMP and eventually the cells undergo hypertrophy and synthesize type X collagen. These results reveal distinct switches at the proliferative–maturation transition and at the maturation–hypertrophy transition during chondrocyte differentiation and indicate that Chondrocytes synthesize new matrix molecules and thus modify their preexisting microenvironment as differentiation progresses. However, when “terminally” differentiated hypertrophic Chondrocytes are released from their surrounding environment and incubated in pellet culture, they stop type X collagen synthesis, resume proliferation, and reinitiate aggrecan synthesis. Eventually they cease proliferation and reinitiate CMP synthesis and finally type X collagen. Thus they are capable of recapitulating all three stages of the differentiation programin vitro.The data suggest a high degree of plasticity in the chondrocyte differentiation program and demonstrate that the progression and maintenance of this program is regulated, at least in part, by the extracellular environment which surrounds a differentiating chondrocyte during endochondral bone formation.
Farshid Guilak - One of the best experts on this subject based on the ideXlab platform.
-
the role of the cytoskeleton in the viscoelastic properties of human articular Chondrocytes
Journal of Orthopaedic Research, 2004Co-Authors: Wendy R Trickey, Parker T Vail, Farshid GuilakAbstract:Biomechanical factors are believed to play an important role in regulating the metabolic activity of Chondrocytes in articular cartilage. Previous studies suggest that cytoskeletal proteins such as actin, vimentin, and tubulin influence cellular mechanical properties, and may therefore influence the mechanical interactions between the chondrocyte and the surrounding tissue matrix. In this study, we investigated the role of specific cytoskeletal components on the mechanical properties of individual Chondrocytes isolated from normal or osteoarthritic hip articular cartilage. Chondrocytes were exposed to a range of concentrations of chemical agents that disrupt the primary cytoskeletal elements (cytochalasin D for F-actin microfilaments, acrylamide for vimentin intermediate filaments, and colchicine for microtubules). Chondrocyte mechanical properties were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. Chondrocyte stiffness (elastic modulus) was significantly increased with osteoarthritis. With increasing cytochalasin D treatment, chondrocyte stiffness decreased by up to 90% and apparent viscosity decreased by up to 80%. The effect of cytochalasin D was greater on normal Chondrocytes than those isolated from osteoarthritic cartilage. Treatment with acrylamide also decreased the moduli and viscosity, but only at the highest concentration tested. No consistent changes in cell mechanical properties were observed with colchicine treatment. These findings suggest that microfilaments and possibly intermediate filaments provide the viscoelastic properties of the chondrocyte, and changes in the structure and properties of these cytoskeletal elements may reflect changes in the chondrocyte with osteoarthritis.
-
alterations in the young s modulus and volumetric properties of Chondrocytes isolated from normal and osteoarthritic human cartilage
Journal of Biomechanics, 1999Co-Authors: Wendy R Jones, Ping H Tingbeall, Greta M Lee, Scott S Kelley, R M Hochmuth, Farshid GuilakAbstract:The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of Chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human Chondrocytes and to determine the Young's modulus of Chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated Chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic Chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that Chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.
-
the deformation behavior and mechanical properties of Chondrocytes in articular cartilage
Osteoarthritis and Cartilage, 1999Co-Authors: Farshid Guilak, Wendy R Jones, Ping H TingbeallAbstract:Abstract Introduction: Chondrocytes in articular cartilage utilize mechanical signals to regulate their metabolic activity. A fundamental step in determining the role of various biophysical factors in this process is to characterize the local mechanical environment of the chondrocyte under physiological loading. Methods: A combined experimental and theoretical approach was used to quantify the in-situ mechanical environment of the chondrocyte. The mechanical properties of enzymatically-isolated Chondrocytes and their pericellular matrix (PCM) were determined using micropipette aspiration. The values were used in a finite element model of the chondron (the chondrocyte and its PCM) within articular cartilage to predict the stress-strain and fluid flow microenvironment of the cell. The theoretical predictions were validated using three-dimensional confocal microscopy of chondrocyte deformation in situ . Results: Chondrocytes were found to behave as a viscoelastic solid material with a Young's modulus of approximately 0.6kPa. The elastic modulus of the PCM was significantly higher than that of the chondrocyte, but several orders of magnitude lower than that of the extracellular matrix. Theoretical modeling of cell-matrix interactions suggests the mechanical environment of the chondrocyte is highly non-uniform and is dependent on the viscoelastic properties of the PCM. Excellent agreement was observed between the theoretical predictions and the direct measurements of chondrocyte deformation, but only if the model incorporated the PCM. Conclusions: These findings imply that the PCM plays a functional biomechanical role in articular cartilage, and alterations in PCM properties with aging or disease will significantly affect the biophysical environment of the chondrocyte.
-
compression induced changes in the shape and volume of the chondrocyte nucleus
Journal of Biomechanics, 1995Co-Authors: Farshid GuilakAbstract:Abstract Changes in cell shape and volume are believed to play a role in the process of mechanical signal transduction by Chondrocytes in articular cartilage. One proposed pathway through which chondrocyte deformation may be transduced to an intracellular signal is through cytoskeletally mediated deformation of intracellular organelles, and more specifically, of the cell nucleus. In this study, confocal scanning laser microscopy was used to perform in situ three-dimensional morphometric analyses of the nuclei of viable condrocytes during controlled compression of articular cartilage explants from the canine patellofemoral groove. Unconfined compression of the tissue to a 15% surface-to-surface strain resulted in a significant decrease of chondrocyte height and volume by 14.7 ± 6.4 and 11.4 ± 8.4%, respectively, and of nuclear height and volume by 8.8 ± 6.2% and 9.8 ± 8.8%, respectively. Disruption of the actin cytoskeleton using cytochalasin D altered the relationship between matrix deformation and changes in nuclear height and shape, but not volume. The morphology and deformation behavior of the Chondrocytes were not affected by cytochalasin treatment. These results suggest that the actin cytoskeleton plays an important role in the link between compression of the extracellular matrix and deformation of the chondrocyte nuclei and imply that Chondrocytes and their nuclei undergo significant changes in shape and volume in vivo .
Ungil Chung - One of the best experts on this subject based on the ideXlab platform.
-
pth pthrp receptor delays chondrocyte hypertrophy via both runx2 dependent and independent pathways
Developmental Biology, 2006Co-Authors: Ungil Chung, Dehong Yang, Richard F Bringhurst, Gerard Karsenty, Henry M KronenbergAbstract:Abstract The transcription factor, Runx2, promotes chondrocyte hypertrophy, whereas parathyroid hormone-related protein (PTHrP) delays this process. To examine whether PTHrP suppresses chondrocyte hypertrophy via Runx2-dependent or -independent pathways, Runx2 expression and chondrocyte differentiation were analyzed using bones from embryonic limbs of wild type and Runx2(−/−) mice. Treatment of cultured rudiments with PTH dramatically suppresses Runx2 mRNA levels in hypertrophic Chondrocytes. PTH-induced delay of chondrocyte hypertrophy was observed in cultured tibiae from both Runx2(−/−) and wild-type embryos. This delay was also seen after PTH administration to limbs from wild type and Runx2(−/−) mice expressing Runx2 in Chondrocytes via a collagen 2 promoter-driven transgene. To further explore Runx2-dependent and -independent effects of PTHrP, we examined embryonic tibiae and femurs from littermates null for PTHrP, Runx2, or both genes. Runx2(−/−) femurs exhibited no vascular invasion or Chondrocytes expressing collagen type X or osteopontin mRNA. In contrast, Runx2(−/−)/PTHrP(−/−) mice exhibited limited vascular invasion and some Chondrocytes expressing collagen X or osteopontin mRNA. In both tibia and femur, Runx2(−/−)/PTHrP(−/−) mice exhibited expanded regions of proliferating Chondrocytes when compared to the same regions in PTHrP(−/−) mice. These data indicate that the delayed hypertrophy induced by PTHrP is mediated by both Runx2-dependent and -independent mechanisms.
-
pthrp and indian hedgehog control differentiation of growth plate Chondrocytes at multiple steps
Development, 2002Co-Authors: Tatsuya Kobayashi, Ungil Chung, Gerard Karsenty, Ernestina Schipani, Michael Starbuck, Takenobu Katagiri, Dale L Goad, Beate Lanske, Henry M KronenbergAbstract:In developing murine growth plates, Chondrocytes near the articular surface (periarticular Chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar Chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar Chondrocytes, Ihh action in the periarticular Chondrocytes was upregulated because of ectopically differentiated hypertrophic Chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular Chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular Chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.
-
the chondrogenic transcription factor sox9 is a target of signaling by the parathyroid hormone related peptide in the growth plate of endochondral bones
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Wendong Huang, Ungil Chung, Henry M KronenbergAbstract:Abstract In the growth plate of endochondral bones, parathyroid hormone (PTH)-related peptide (PTHrP) regulates the rate of chondrocyte maturation from prehypertrophic Chondrocytes to hypertrophic Chondrocytes. Using an antibody specific for Sox9 phosphorylated at serine 181 (S181), one of the two consensus protein kinase A phosphorylation sites of Sox9, we showed that the addition of PTHrP strongly increased the phosphorylation of SOX9 in COS7 cells transfected with both SOX9- and PTH/PTHrP receptor-expressing vectors. PTHrP also increased the SOX9-dependent activity of chondrocyte-specific enhancers in the gene for type II collagen (Col2a1) in transient transfection experiments. This increased enhancer activity did not occur with a Sox9 mutant harboring serine-to-alanine substitutions in its two consensus protein kinase A phosphorylation sites. Consistent with these results, PTHrP also increased Col2a1 mRNA levels in rat chondrosarcoma cells as well as 10T1/2 mesenchymal cells transfected with a PTH/PTHrP receptor expressing plasmid. No phosphorylation of Sox9 at S181 was detected in prehypertrophic Chondrocytes of the growth plate or any Chondrocytes of PTH/PTHrP receptor null mutants. In contrast in wild-type mouse embryos, previous immunohistochemistry experiments indicated that Sox9 phosphorylated at S181 was detected almost exclusively in Chondrocytes of the prehypertrophic zone. Sox9, regardless of the phosphorylation state, was present in all Chondrocytes of both genotypes except hypertrophic Chondrocytes. Our results indicated that Sox9 is a target of PTHrP signaling in prehypertrophic Chondrocytes in the growth plate. We hypothesize that Sox9 mediates at least some effects of PTHrP in the growth plate and that the PTHrP-dependent increased transcriptional activity of Sox9 helps maintain the chondrocyte phenotype of cells in the prehypertrophic zone and inhibits their maturation to hypertrophic Chondrocytes.
-
the parathyroid hormone parathyroid hormone related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Ungil Chung, Beate Lanske, Kaechoong Lee, Henry M KronenbergAbstract:During vertebrate limb development, growth plate Chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (−/−) cells, and analyzed cell–cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating Chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant Chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type Chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic Chondrocytes. Further, ectopic hypertrophic Chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.
A C Hall - One of the best experts on this subject based on the ideXlab platform.
-
rapid in situ chondrocyte death induced by staphylococcus aureus toxins in a bovine cartilage explant model of septic arthritis
Osteoarthritis and Cartilage, 2013Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, J P Winstanley, E Czarniak, C Doherty, A C HallAbstract:Summary Objective To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus . Methods Bovine cartilage explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus -induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring cartilage wet and dry weights. Results S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ Chondrocytes to S. aureus 8325-4 toxins found SZ Chondrocytes to be more susceptible to the toxins than DZ Chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. Conclusions Toxins released by S. aureus have a rapid and fatal action on in situ Chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.
-
bicarbonate dependent phi regulation by Chondrocytes within the superficial zone of bovine articular cartilage
Journal of Cellular Physiology, 2007Co-Authors: Victoria L Simpkin, Dianne H Murray, Andrew P Hall, A C HallAbstract:: Control of chondrocyte pH (pH(i)) determines articular cartilage matrix metabolism. However, the transporters of Chondrocytes in situ throughout cartilage zones are unclear, and we tested the hypothesis that chondocytes within the superficial zone (SZ) utilise a HCO(3) (-)-dependent system absent from other zones. Imaging of single BCECF-labelled cells was used to monitor the pH(i) of in situ Chondrocytes within the cartilage zones, and also that of cells isolated from the SZ or full depth (FD) explants. Resting pH(i) and intrinsic buffering power (beta(i)) in HEPES-buffered saline was not different between SZ and DZ cells, however the pH(i) of SZ Chondrocytes was lower in HCO(3) (-) saline. Ammonium pre-pulse was used to acid-load cells and pH(i) recovery by in situ or isolated SZ Chondrocytes shown to be totally dependent on HCO(3) (-). pH(i) recovery rate was significantly (P 0.05). Na(+)-dependent HCO(3) (-)-(NBC) transporters were identified in SZ Chondrocytes by fluorescence immunohistochemistry suggesting that this system might account for the HCO(3) (-)-dependent recovery of pH(i). Bovine articular cartilage Chondrocytes possess a HCO(3) (-)-dependent transporter which plays a key role in pH(i) regulation in cells in the SZ, but not in Chondrocytes within deeper cartilage zones.
-
viability and volume of in situ bovine articular Chondrocytes changes following a single impact and effects of medium osmolarity
Osteoarthritis and Cartilage, 2005Co-Authors: Peter G Bush, Peter D Hodkinson, Georgina L Hamilton, A C HallAbstract:Summary Objective Mechanical stress above the physiological range can profoundly influence articular cartilage causing matrix damage, changes to chondrocyte metabolism and cell injury/death. It has also been implicated as a risk factor in the development of osteoarthritis (OA). The mechanism of cell damage is not understood, but chondrocyte volume could be a determinant of the sensitivity and subsequent response to load. For example, in OA, it is possible that the chondrocyte swelling that occurs renders the cells more sensitive to the damaging effects of mechanical stress. This study had two aims: (1) to investigate the changes to the volume and viability of in situ Chondrocytes near an injury to cartilage resulting from a single blunt impact, and (2) to determine if alterations to chondrocyte volume at the time of impact influenced cell viability. Methods Explants of bovine articular cartilage were incubated with the fluorescent indicators calcein-AM and propidium iodide permitting the measurement of cell volume and viability, respectively, using confocal laser scanning microscopy (CLSM). Cartilage was then subjected to a single impact (optimally 100g from 10cm) delivered from a drop tower which caused areas of chondrocyte injury/death within the superficial zone (SZ). The presence of lactate dehydrogenase (LDH; an enzyme released following cell injury) was used to determine the effects of medium osmolarity on the response of Chondrocytes to a single impact. Results A single impact caused discrete areas of chondrocyte injury/death which were almost exclusively within the SZ of cartilage. There appeared to be two phases of cell death, a rapid phase lasting ∼3min, followed by a slower progressive ‘wave of cell death' away from the initial area lasting for ∼20min. The volume of the majority (88.1±5.99% (n=7) of the viable Chondrocytes in this region decreased significantly ( P Conclusions A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen Chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ Chondrocytes to mechanical stress.