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Chondrocyte Phenotype (chondrocyte + phenotype)
Selected AbstractsFrom collagen chemistry towards cell therapy , a personal journeyINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2007Michael E. Grant Summary The Fell,Muir Award requires the recipient to deliver a lecture and a review manuscript which provides a personal overview of significant scientific developments in the field of matrix biology over the period of the recipient's career. In this context, this review considers the collagen family of structural proteins and the advances in biochemical, molecular biological and genetic techniques which led to the elucidation of the structure, synthesis and function of this important group of extracellular matrix constituents. Particular attention is focussed on early research on the identification and assembly of the soluble precursors of collagen types I and II, and the identification of the precursor of basement membrane collagen type IV. In subsequent studies investigating the maintenance of the chick chondrocyte phenotype in culture, the influence of the extracellular milieu was found to influence markedly both cell morphology and collagen gene expression. These studies led to the discovery of collagen type X whose expression is restricted to hypertrophic chondrocytes at sites of endochondral ossification. Such research provided a prelude to investigations of mammalian endochondral ossification which is known to be aberrant in a variety of human chondrodysplasias and is reactivated in bone fracture repair and in osteoarthritis. The cloning of bovine and then human collagen type X genes facilitated studies in relevant human diseases and contributed to the discovery of mutations in the COL10A1 gene in families with metaphyseal chondrodysplasia type Schmid. Clustering of mutations in the C-terminal domain of the type X collagen molecule has now been widely documented and investigations of the pathogenic mechanisms in animal models are beginning to suggest the prospect of novel treatment strategies. [source] Immortalized cell lines from mouse xiphisternum preserve chondrocyte phenotypeJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Manas K. Majumdar Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP-2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL-1 results in the increased expression of catabolic effectors including MMP-13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP-2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL-1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase-cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3-dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA. J. Cell. Physiol. 209: 551,559, 2006. © 2006 Wiley-Liss, Inc. [source] Microenvironment regulation of PRG4 phenotype of chondrocytesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2007Megan E. Blewis Abstract Articular cartilage is a heterogeneous tissue with superficial (S), middle (M), and deep (D) zones. Chondrocytes in the S zone secrete the lubricating PRG4 protein, while chondrocytes from the M and D zones are more specialized in producing large amounts of the glycosaminoglycan (GAG) component of the extracellular matrix. Soluble and insoluble chemicals and mechanical stimuli regulate cartilage development, growth, and homeostasis; however, the mechanisms of regulation responsible for the distinct PRG4-positive and negative phenotypes of chondrocytes are unknown. The objective of this study was to determine if interaction between S and M chondrocytes regulates chondrocyte phenotype, as determined by coculture in monolayer at different ratios of S:M (100:0, 75:25, 50:50, 25:75, 0:100) and at different densities (240,000, 120,000, 60,000, and 30,000 cells/cm2), and by measurement of PRG4 secretion and expression, and GAG accumulation. Coculture of S and M cells resulted in significant up-regulation in PRG4 secretion and the percentage of cells expressing PRG4, with simultaneous down-regulation of GAG accumulation. Tracking M cells with PKH67 dye in coculture revealed that they maintained a PRG4-negative phenotype, and proliferated less than S cells. Taken together, these results indicate that the up-regulated PRG4 expression in coculture is a result of preferential proliferation of PRG4-expressing S cells. This finding may have practical implications for generating a large number of phenotypically normal S cells, which can be limited in source, for tissue engineering applications. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:685,695, 2007 [source] Aberrant hypertrophy in Smad3-deficient murine chondrocytes is rescued by restoring transforming growth factor ,,activated kinase 1/activating transcription factor 2 signaling: A potential clinical implication for osteoarthritisARTHRITIS & RHEUMATISM, Issue 8 2010Tian-Fang Li Objective To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor , (TGF,) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. Methods Joint disease in Smad3-knockout (Smad3,/,) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3,/, mice. Results In Smad3,/, mice, an end-stage OA phenotype gradually developed. TGF,-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3,/, mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3,/, cells restored the normal p38 response to TGF,. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3,/, chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms ,, ,, and ,, but not ,. Conclusion Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3,phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA. [source] Induction of CD44 cleavage in articular chondrocytesARTHRITIS & RHEUMATISM, Issue 5 2010Nobunori Takahashi Objective The hyaluronan receptor CD44 provides chondrocytes with a mechanism for sensing and responding to changes in the extracellular matrix. The purpose of this study was to document the fragmentation and loss of CD44 and to determine the likely mechanisms involved. Methods A polyclonal anti-CD44 cytotail antibody was generated to detect CD44 fragmentation by Western blot analysis. Chondrocytes were isolated from human or bovine articular cartilage. Primary articular chondrocytes were treated with interleukin-1, (IL-1,), hyaluronan oligosaccharides, or phorbol myristate acetate or were passaged and subcultured in monolayer to induce dedifferentiation. Conditions that altered the capacity of CD44 to transit into lipid rafts, or pharmacologic inhibitors of metalloproteinase or ,-secretase activity were used to define the mechanism of fragmentation of CD44. Results Chondrocytes from osteoarthritic cartilage exhibited CD44 fragmentation as low molecular mass bands, corresponding to the CD44-EXT and CD44-ICD bands. Following dedifferentiation of chondrocytes or treatment of primary chondrocytes with hyaluronan oligosaccharides, IL-1,, or phorbol myristate acetate, CD44 fragmentation was enhanced. Subsequent culture of the dedifferentiated chondrocytes in 3-dimensional alginate beads rescued the chondrocyte phenotype and diminished the fragmentation of CD44. Fragmentation of CD44 in chondrocytes was blocked in the presence of the metalloproteinase inhibitor GM6001 and the ,-secretase inhibitor DAPT. Conclusion CD44 fragmentation, consistent with a signature pattern reported for sequential metalloproteinase/,-secretase cleavage of CD44, is a common metabolic feature of chondrocytes that have undergone dedifferentiation in vitro and osteoarthritic chondrocytes. Transit of CD44 into lipid rafts may be required for its fragmentation. [source] Hypoxia-inducible factor 1, inhibits the fibroblast-like markers type I and type III collagen during hypoxia-induced chondrocyte redifferentiation: Hypoxia not only induces type II collagen and aggrecan, but it also inhibits type I and type III collagen in the hypoxia-inducible factor 1,,dependent redifferentiation of chondrocytesARTHRITIS & RHEUMATISM, Issue 10 2009Elise Duval Objective Autologous chondrocyte implantation requires expansion of cells ex vivo, leading to dedifferentiation of chondrocytes (loss of aggrecan and type II collagen to the profit of type I and type III collagens). Several approaches have been described for redifferentiation of these cells. Among them, low oxygen tension has been exploited to restore the differentiated chondrocyte phenotype, but molecular mechanisms of this process remain unclear. However, under conditions of hypoxia, one of the major factors involved is hypoxia-inducible factor 1, (HIF-1,). The purpose of this study was to investigate the role of HIF-1, during human chondrocyte redifferentiation. Methods We used complementary approaches to achieving HIF-1, loss (inhibition by cadmium ions and dominant-negative expression) or gain (ectopic expression and cobalt ion treatment) of function. Expression of chondrocyte, as well as fibroblast-like, phenotype markers was determined using real-time reverse transcription,polymerase chain reaction and Western blot analyses. Binding activities of HIF-1, and SOX9, a pivotal transcription factor of chondrogenesis, were evaluated by electrophoretic mobility shift assays and by chromatin immunoprecipitation assay. Results We found that hypoxia and HIF-1, not only induced the expression of SOX9, COL2A1, and aggrecan, but they simultaneously inhibited the expression of COL1A1, COL1A2, and COL3A1. In addition, we identified the binding of HIF-1, to the aggrecan promoter, the first such reported demonstration of this binding. Conclusion This study is the first to show a bimodal role of HIF-1, in cartilage homeostasis, since HIF-1, was shown to favor specific markers and to impair dedifferentiation. This suggests that manipulation of HIF-1, could represent a promising approach to the treatment of osteoarthritis. [source] Ligands for retinoic acid receptors are elevated in osteoarthritis and may contribute to pathologic processes in the osteoarthritic jointARTHRITIS & RHEUMATISM, Issue 6 2009Mark R. Davies Objective Vitamin A derivatives, including all- trans -retinoic acid (ATRA), have a well-established role during skeletal development and limb formation and have been shown to have profound effects on chondrocyte phenotype. The aim of this study was to elucidate the effects of retinoids and components of the retinoid metabolic pathway on chondrocyte phenotype in the tibiofemoral joints of patients with osteoarthritis (OA), to show that the retinoids can have multiple effects relevant to the OA disease process. Methods Human explant tissue and a chondrocyte-like cell line were treated with ATRA, and the responses of 4 key markers of chondrocyte phenotype were analyzed. In addition, the effects of ATRA on a number of novel genes associated with OA were assessed using a low-density microarray containing 80 disease marker genes. Results Vitamin A metabolite levels were elevated in synovial fluid, serum, and cartilage from patients with OA. Expression profiling of a retinoic acid receptor , coactivator protein, P/CAF, demonstrated elevated expression in patients with OA, suggesting the potential for increased signaling via the retinoid receptors in the disease. ATRA increased the levels of matrix metalloproteinase 13 and aggrecanase activity in human cartilage explants and in a human chondrocyte cell line. Furthermore, ATRA altered the expression of a wide range of relevant genes, including the types I, II, IX, and XI collagen genes, toward a nonchondrogenic and OA-like phenotype. Conclusion These results suggest that retinoid signaling could have a central role in OA, and that components of the pathway may provide potential disease biomarkers or targets for therapeutic intervention. [source] Lentivirus-mediated knockdown of aggrecanase-1 and -2 promotes chondrocyte-engineered cartilage formation in vitroBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Zheng-Hui Wang Abstract Chondrocyte-based tissue engineering has emerged as a promising approach for repair of injured cartilage tissues that have a poor self-healing capacity. However, this technique faces a major limitation: dedifferentiation of chondrocytes occurs following several passages in culture. Aggrecan, a major component of cartilage extracellular matrix, plays an essential role in chondrocyte differentiation. The aim of this study is to determine whether inhibition of chondrocyte aggrecanases, key degradative enzymes for aggrecan in cartilage, could benefit chondrocyte differentiation and the preservation of chondrocyte phenotype within a long-term period. Lentivirus-mediated RNA interference (RNAi) was employed to target both aggrecanase-1 and -2 in primary rat chondrocytes, and the transduced cells were seeded into chitosan,gelatin three-dimensional scaffolds. Histological, morphological, and biochemical analyses were performed at 1,8 weeks post-implantation to study chondrocyte survival, differentiation, and function. We found that lentivirus-mediated RNAi notably decreased the abundance of aggrecanase transcripts in chondrocytes but did not affect cell viability. Most importantly, compared to the control constructs seeded with untransduced chondrocytes, the aggrecanase inhibition increased chondrocyte proliferation and reinforced the production of glycosaminoglycans and total collagen, indicative of chondrocyte differentiation. The mRNA expression of chondrocyte marker genes (collagen II and aggrecan) was enhanced by aggrecanase silencing relative to the control. Together our data demonstrate that inhibition of endogenous aggrecanases facilitates chondrocyte differentiation and chondrocyte-engineered cartilage formation in vitro. The combination of lentiviral delivery system and genetic manipulation techniques provides a useful tool for modulation of chondrocyte phenotype in cartilage engineering. Biotechnol. Bioeng. 2010;107:730,736. © 2010 Wiley Periodicals, Inc. [source] Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium,derived mesenchymal stem cellsARTHRITIS & RHEUMATISM, Issue 5 2009Yoichi Ezura Objective Human synovium,derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium,derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis. Methods Human synovium,derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription,polymerase chain reaction. Methylation status was also examined in human articular cartilage. Results Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture. Conclusion The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium,derived MSCs. [source] Lack of oxygen in articular cartilage: consequences for chondrocyte biologyINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 2 2010Jérôme E. Lafont Summary Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia-mediated chondrocyte function in the native articular cartilage. [source] | |||||||||||||