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. Inhibitors of ERK and NFkappaB could significantly block the induction of MMP-1 and -13 (p<0.05) and the repression of collagen type II (p<0.01). The inhibitor for p38 MAPK was able to block partially MMP-1 and -13 up-regulation (p<0.01), but did not significantly inhibit collagen type II repression. Our data suggest that ERK and NFkB pathways are particularly important for IL-1beta regulating collagen type II and MMP-1 and -13 expression and that p38, but not JNK is additionally involved in MMP-1 and -13 induction.

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. METHODS: Gene expression levels were detected by real-time polymerase chain reaction from isolated (nonpassaged) chondrocytes (normal [n = 6]; osteoarthritic [n = 7]) after stimulation with 0.01 ng, 0.1 ng, 1 ng, and 10 ng/ml IL-1beta. RESULTS: In normal adult articular chondrocytes the expression of both aggrecan and type II collagen genes was significantly down-regulated, whereas matrix-degrading proteases (except MMP-2), as well as the investigated cytokines, were induced by IL-1beta in a dose-dependent manner. The strongest regulation was found for IL-6 and LIF. Osteoarthritic chondrocytes showed strongly increased levels of catabolic enzymes and mediators, but were less responsive to further stimulation with IL-1beta. CONCLUSION: Our study confirms that IL-1beta activity is critically dependent on both the applied concentration and the reactivity of the cells stimulated. The responsiveness appears to be significantly reduced in late-stage osteoarthritic chondrocytes. However, these cells show high basic expression levels of catabolic enzymes and mediators. Thus, it remains open whether our data indicate that osteoarthritic chondrocytes are per se not responsive to IL-1beta or are already so strongly stimulated (e.g., by IL-1) during the disease process that they are refractory to further stimulation.

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. Immunolocalization of the I-Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen. RESULTS: Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I-Smads was found. In cultured articular chondrocytes, stimulation with IL-1beta showed up-regulation of Smad7, whereas Smad6 was down-regulated. CONCLUSION: Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I-Smads in articular cartilage in vivo. No evidence was found that up-regulation or down-regulation of I-Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL-1beta suggests a potentially important role of IL-1beta signaling in chondrocytes, via indirect influencing of the BMP/TGFbeta signaling cascade.

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. The aim of our study was to evaluate in vitro and in vivo whether IL-6 and LIF are differentially expressed in normal human and osteoarthritic adult articular chondrocytes and to investigate the potential intracellular signaling pathways of IL-1 involved in these gene regulation events. IL-6 and LIF mRNA expressions were found only at low levels in normal adult articular cartilage. Neither IL-6 nor LIF was strongly over-expressed in osteoarthritic cartilage degeneration. Clearly, both IL-6 and LIF can be very efficiently induced by IL-1beta in articular chondrocytes in vitro. However, this induction was somewhat less in osteoarthritic cells, which were overall activated in terms of expression of both cytokines without stimulation. Experiments using pathway selective inhibitors showed that intracellular signaling of IL-1beta for IL-6 and LIF is mediated by a mixture of the IL-1 signaling cascades. However, the ERK-pathway appeared to be particularly important and might be, therefore, of particular potential if one intends to block induction of these molecules by IL-1 in arthritic joint disease.

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. This technology offers outstanding sensitivity and accuracy in terms of determination of the amount of cDNA molecules. However, this method cannot account for factors such as efficiency of RNA isolation and reverse transcription conditions. Thus, normalization of the acquired data is required, with all its limitations as described.

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. Methods. Isolated adult human articular chondrocytes were stimulated for 48h with HGF (1, 10, and 100ng/ml). Synthesis of proteoglycans was determined by [(35)S]sulfate incorporation. mRNA levels for anabolic and catabolic genes as well as c-met and (functional) hgf were quantified using real-time PCR. Additionally, in situ mRNA expression levels of hgf and c-met were quantitatively measured from RNA directly isolated from normal and osteoarthritic adult human articular cartilage. Results. Proteoglycan synthesis in adult human articular chondrocytes was not stimulated by HGF nor was a selection of catabolic genes (collagenases and aggrecanases). Normal adult articular chondrocytes expressed only very low levels of hgf mRNA. Slightly higher levels of hgf were detected in chondrocytes isolated from osteoarthritic cartilage. Significant c-met expression was detected in both sample types. Conclusion. Despite the expression of its receptor c-met and its presence in articular cartilage, HGF does not appear to be a potent player in cartilage matrix turnover, at least not in terms of anabolic and catabolic gene expression in normal adult articular cartilage.

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). METHODS: Conventional and online polymerase chain reaction (PCR) technology was used to determine ADAMTS-1 mRNA expression levels of ADAMTS-1. Protein was localized using immunostaining with different polyclonal antibodies. RESULTS: Conventional and online semiquantitative PCR showed significant levels of ADAMTS-1 mRNA expression in normal and OA chondrocytes in vivo and in vitro. Only a slight increase was observed in OA cartilage. After stimulation with IL-1beta a downregulation of ADAMTS-1 was observed, whereas IGF did not appear to change mRNA expression levels in vitro. The in vivo mRNA expression results were confirmed by the presence of significant protein staining with antibodies for ADAMTS-1 in normal and OA chondrocytes as well as Western blotting analysis. Whereas a significantly stronger stain was seen in normal articular cartilage in the upper zones, in OA cartilage as well the middle zone showed enhanced staining. CONCLUSION: Our results confirm the expression and presence of ADAMTS-1 in articular cartilage. However, they also point out that ADAMTS-1 appears to be constitutively expressed by adult articular chondrocytes and overall is not strongly upregulated in OA. Thus our data suggest that ADAMTS-1 is the first matrix-degrading enzyme downregulated by the catabolic factor IL-1beta in vitro.

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. Conventional and quantificative PCR analysis showed significant levels of RhoB expression in normal articular cartilage. Immunolocalization and confocal laser scanning microscopy showed strong cytoplasmic signals for RhoB in normal chondrocytes. In osteoarthritic cartilage, a significantly lower expression of RhoB was detectable. In vitro experiments showed a quick (and transient) up-regulation of RhoB after stimulation with interleukin-1beta and serum. Our study suggests that RhoB is constitutively expressed and essential for adult articular chondrocytes, but significantly down-regulated in osteoarthritic chondrocytes. One intriguing speculation might be that the down-regulation of RhoB in osteoarthritic chondrocytes is at least partly a prerequisite for the sustained pre- or para-apoptotic phenotype of osteoarthritic chondrocytes, because RhoB is known to be one important molecule in the induction of apoptotic cell death in response to DNA damage and osteoarthritic chondrocytes are known to have significant DNA damage. Alternatively, RhoB could be involved in the activation or deactivation and the destabilization of the functional phenotype of chondrocytes in osteoarthritic joint degeneration Thirdly, RhoB is associated with the cell cycle, which is re-initiated in osteoarthritis.

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. Here we show that normal adult human articular chondrocytes in vivo contain high SOX9 mRNA levels, which are decreased in osteoarthritic cartilage. Surprisingly, no positive correlation between SOX9 and COL2A1 expression was observed--to the contrary, the expression of COL2A1 was significantly increased in the diseased cells. Immunolocalization confirmed the presence of SOX9 protein in normal and osteoarthritic chondrocytes without showing significant differences in both SOX9 quantity and subcellular localization in osteoarthritic compared to normal cartilage tissue. Interestingly, laser scanning confocal microscopy showed that the subcellular distribution of SOX9 in adult chondrocytes was not restricted to the nucleus as observed in fetal chondrocytes, but was also detected within the cytoplasm, with no differences in subcellular SOX9 distribution between normal and OA cartilage. This is consistent with the lack of positive correlation between SOX9 and COL2A1 expression in adult articular chondrocytes. Also, no positive correlation between SOX9 and COL2A1 expression was observed in vitro after challenge of chondrocytes with Il-1beta, which is a strong (negative) regulator of COL2A1 expression, or with IGF-I, which stimulates COL2A1 expression. These results suggest that SOX9 is not the key regulator of COL2A1 promoter activity in human adult articular chondrocytes. However, SOX9 might still be involved in maintaining the chondrocytic phenotype in normal and osteoarthritic cartilage.

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. At first sight, our results confirmed previously published data showing hardly any expression of collagen genes in normal and significantly enhanced expression in osteoarthritic cartilage. This included collagen types II, III, and IIA, but also collagen types I(alpha1) and X. However, if one considers the ratios of the various markers of chondrocytic differentiation in comparison to collagen type II, the main synthetic product of differentiated chondrocytes, no shift in the cellular phenotype was detectable. In fact, expression ratios remained constant or were even decreased in osteoarthritic cartilage. Our results confirm that normal adult human articular chondrocytes display hardly any expression activity of the collagen types investigated, whereas osteoarthritic chondrocytes show very increased synthetic activity. The largely unchanged ratios of collagen subtypes investigated indicate that no general shift in the cellular phenotype does occur in osteoarthritic cartilage as suggested by previous investigations.

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. Articular chondrocytes were cultured in alginate beads and short-term high-density monolayer cultures with and without stimulation by IL-1. In addition, immunolocalization of the receptor-associated Smads (R-Smads) was performed on sections of normal and diseased articular cartilage. Reverse-transcription (RT)-PCR analysis showed a moderate expression of all Smads investigated in normal, early degenerative, and late stage osteoarthritic cartilage. Immunolocalization detected the R-Smads in most chondrocytes on the protein level in all specimen groups investigated. In vitro, the Smads were also expressed and partly up-regulated by Il-1beta in alginate bead culture. Of note, for Smad 1, two truncated splice variants were expressed by articular chondrocytes missing exon 4 as well as exons 3 and 4. Our study showed that BMP-receptor Smads 1, 5, and 8 as well as common Smad (C-Smad) 4 are expressed and present in human normal and osteoarthritic articular chondrocytes corroborating the importance of BMPs and BMP signaling for articular cartilage. This study is the first to describe splicing variants for Smad 1. Smads 1, 4, and 5 are up-regulated in vitro by Il-1beta, suggesting a linkage of the Il-1 and BMP-signaling pathways within the chondrocytes. None of the Smads were grossly up- or down-regulated in osteoarthritic chondrocytes, suggesting that differences in overall expression levels of the investigated Smad proteins are not relevant for metabolic activity of articular chondrocytes in vivo.

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. RESULTS: In vivo, MMP-1 was detectable only at very low levels in any condition. MMP-13 expression was low in normal and early degenerative cartilage but was strongly up-regulated in late-stage OA specimens. MMP-1 and MMP-13 were expressed much higher in vitro than in vivo and were up-regulated by IL-1beta. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly down-regulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1beta. ADAM-TS5 and MMP-14 were expressed in all sample groups. Expression of ADAM-TS4 was very low in vivo and was induced in vitro after stimulation by IL-1beta. CONCLUSION: Our expression data clearly support MMP-13 as the major collagenase in OA cartilage. The most strongly expressed aggrecanase was ADAM-TS5. ADAM-TS4 was expressed only at a very low level in normal cartilage and was only slightly up-regulated in OA cartilage, casting doubt on this enzyme being the relevant aggrecanase of articular cartilage. Results of our study show that expression of many enzymes is significantly different in vitro and in vivo and suggest that IL-1beta stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.