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Primary Human Chondrocytes (primary + human_chondrocyte)

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Medical Sciences	100%

Selected Abstracts

Discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes,

THE JOURNAL OF PATHOLOGY, Issue 2 2009
Andreas R Klatt
Abstract We deciphered constituent parts of a signal transduction cascade that is initiated by collagen II and results in the release of various pro-inflammatory cytokines, including interleukin-6 (IL-6), in primary human chondrocytes. This cascade represents a feed-forward mechanism whereby cartilage matrix degradation is exacerbated by the mutually inducing effect of released collagen II fragments and pro-inflammatory cytokines. We previously proposed discoidin domain receptor 2 as a central mediator in this event. Since this cascade plays a prominent role in the pathogenesis of osteoarthritis, our study further investigates the hypothesis that discoidin domain receptor 2 is a candidate receptor for collagen II, and that transcription factor NF,B, lipid kinase PI3K, and the MAP kinases are constituent parts of this very signal transduction cascade. To accomplish this, we selectively knocked down the molecules of interest in primary human chondrocytes, induced the specified cascade by incubating primary human chondrocytes with collagen II, and observed the outcome, specifically the changes in interleukin-6 release. Knockdown was performed by siRNA-mediated gene silencing in the case of discoidin domain receptor 2 (DDR2) or by using specific inhibitors for the remainder of the molecules. Results indicated that discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes and that MAP kinases p38, JNK and ERK, as well as transcription factor NF,B, are integral components of intracellular collagen II signalling. Given the detrimental role of these molecules in osteoarthritis, our findings provide new targets for more specific therapeutics, which may have fewer side effects than those currently applied. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source]

Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors

ARTHRITIS & RHEUMATISM, Issue 8 2010
Rosa Maria Borzí
Objective To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and ,-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. Results Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, ,-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13,remodeled or ,degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusion MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors. [source]

SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathway

ARTHRITIS & RHEUMATISM, Issue 5 2010
Viktoria Gagarina
Objective The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans. Methods SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin-like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis. Results Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor ,/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3-kinase, phosphoinosite-dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1-mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down-regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased. Conclusion For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down-regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage. [source]