Role of mitogen-activated protein kinases and NFkappaB on IL-1beta-induced effects on collagen type II, MMP-1 and 13 mRNA expression in normal articular human chondrocytes.

Interleukin-1ss is a pro-inflammatory cytokine that causes anti-anabolic and catabolic effects on articular chondrocytes via four major signaling pathways. In this study, we investigated the role of these pathways for the repression of collagen type II, and induction of MMP-1 and -13 by Il-1ss. Human adult chondrocytes were stimulated with IL-1beta together with selective inhibitors of the ERK, JNK, p38, and NFkappaB pathways.

Freshly isolated osteoarthritic chondrocytes are catabolically more active than normal chondrocytes, but less responsive to catabolic stimulation with interleukin-1beta.

Objective: Interleukin-1beta (IL-1beta) is one potentially important cytokine during cartilage destruction. The aim of this study was to investigate whether there are different effects of low and high concentrations of IL-1beta on the expression level of anabolic genes (type II collagen, aggrecan), catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-2, MMP-3, MMP-13, and ADAMTS-4), and cytokines (IL-1beta, IL-6, and leukemia inhibitory factor [LIF]) by articular chondrocytes (normal and osteoarthritic). Determination of whether there was a difference in reactivity between normal and osteoarthritic chondrocytes was also a goal of this study.

Bone morphogenetic protein and transforming growth factor beta inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin-1beta.

Objective: Bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFbeta signaling, inhibitory Smad6 (I-Smad6) and I-Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin-1beta (IL-1beta) stimulation in vitro.

Methods: RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology.

IL-1beta induction of IL-6 and LIF in normal articular human chondrocytes involves the ERK, p38 and NFkappaB signaling pathways.

Interleukin-1 (IL-1) is an important catabolic cytokine in rheumatoid and osteoarthritic joint disease. Besides inducing a catabolic response in articular chondrocytes it also strongly induces synergistic mediators such as leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). The molecular basis of this is so far hardly understood.

Quantification of mRNA expression levels in articular chondrocytes with PCR technologies.

Unlike any other technology in molecular biology, the polymerase chain reaction (PCR) has changed the technological armamentarium of molecular scientists working on cartilage, in terms of outstanding sensitivity and accuracy. Four approaches to determine mRNA expression levels by PCR amplification of specific cDNA sequences are currently in use and are discussed in this chapter: conventional PCR with end-point determination, conventional PCR in the logarithmic amplification phase, conventional PCR using internal competitive DNA fragments, and real-time PCR as offered by TaqMan technology and others. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels within cartilage and cultured chondrocytes, as in other tissues and cell types.

Hepatocyte growth factor/scatter factor is not a potent regulator of anabolic and catabolic gene expression in adult human articular chondrocytes.

Objective. Hepatocyte growth factor (HGF) has been reported to be present in articular cartilage and to be a potentially important inducing factor of anabolic and catabolic activity in chondrocytes. The aim of this study was to determine the expression levels of full length-functional-hgf and its receptor c-met in normal and osteoarthritic cartilage and the effect of HGF on anabolic and catabolic gene expression in adult human articular chondrocytes.

Down-regulation of the GTPase RhoB might be involved in the pre-apoptotic phenotype of osteoarthritic chondrocytes.

Anabolic activity, phenotypic alterations, and in particular survival of the chondrocytes are essential for the maintenance of proper articular cartilage and appears to fail during osteoarthritic cartilage degeneration. In this study, we investigated the presence and expression of RhoB in adult human articular cartilage and its regulation in osteoarthritic cartilage as well as in chondrocytes in vitro. RhoB belongs to the family of small GTPases, which are thought to be involved in a large range of activities important for eukaryotic cells.

ADAMTS-1, a gene product of articular chondrocytes in vivo and in vitro, is downregulated by interleukin 1beta.

Objective: Osteoarthritic (OA) cartilage degeneration and cartilage destruction in rheumatoid arthritis depends significantly on enzymatic degradation of cartilage proteoglycan aggrecan. A member of the ADAMTS family of proteases, ADAMTS-1 was described to have "aggrecanase" activity. We investigated the quantitative expression and distribution of ADAMTS-1 in healthy and OA cartilage and in cultured articular chondrocytes with and without stimulation by interleukin 1beta (IL-1beta) and insulin-like growth factor-I (IGF-I).

SOX9 expression does not correlate with type II collagen expression in adult articular chondrocytes.

Anabolic activity is a crucial activity of articular chondrocytes and its failure is one major reason of osteoarthritic cartilage degeneration. The intracellular factors responsible for the increase or decrease of anabolic activity of articular chondrocytes remain largely unknown. A recent candidate, the transcription factor SOX9, has elicited much interest as it is suggested to be a central factor in chondrocytic differentiation during development, including collagen type II (COL2A1) expression, the major anabolic gene product of chondrocytes.

Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytes.

Many studies have shown increased anabolic activity in osteoarthritic cartilage and have suggested changes in the cellular phenotypes of articular chondrocytes. Most of these studies relied on non-quantitative technologies, which did not allow the estimation of the relative importance of the different differentiation phenomena. In the present study, we developed and used quantitative PCR assays for collagen types I, II(total), IIA, III, and X as marker genes indicating cellular synthetic activity (collagen type II) as well as differentiation pattern of chondrocytes (collagen types I, IIA, III, and X) and quantified these genes in normal, early degenerative, and late stage osteoarthritic cartilage in parallel.

Bone morphogenetic protein-mediating receptor-associated Smads as well as common Smad are expressed in human articular chondrocytes but not up-regulated or down-regulated in osteoarthritic cartilage.

Bone morphogenetic proteins (BMPs) are supposed to be important for cartilage matrix anabolism. In this study, we investigated whether the intracellular mediators of BMP activity, Smads 1, 4, 5, and 8, are expressed in normal human articular chondrocytes in vivo and in vitro and whether alterations in expression and distribution pattern are found in osteoarthritic cartilage or in vitro after stimulation with interleukin (IL)-1, because down-regulation of these mediators could be responsible for the decrease of anabolic activity in osteoarthritic cartilage. RNA was isolated from normal and osteoarthritic human knee cartilage and analyzed by (quantitative) polymerase chain reaction (PCR) technology.

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro.

Objective: Osteoarthritic (OA) cartilage destruction depends on collagen- and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1beta (IL-1beta)-stimulated chondrocytes in vitro.

Methods: Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1beta.