[show abstract][hide abstract] ABSTRACT: The normal structure and function of articular cartilage are the result of a precisely balanced interaction between anabolic and catabolic processes. The transforming growth factor-beta (TGF-beta) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) exert a strong catabolic effect. Recent evidence has shown that IL-1beta, and TNF-alpha, and the TGF-beta signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-beta by IL-1beta or TNF-alpha signaling pathways occurs through regulation of activity and availability of mothers against DPP (Drosophila) human homologue (Smad) proteins.
Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in poly-(2-hydroxyethyl methacrylate)-coated dishes with either 10% fetal bovine serum media or serum-deprived media 6h before treatment with IL-1beta alone, TNF-alpha alone or IL-1beta followed by TGF-beta. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-kappaB) and Smad3/4 deoxyribonucleic acid (DNA) binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, type II collagen alpha 1 (COL2A1), aggrecan and SRY-related high mobility group-Box gene 9 (SOX-9) mRNA expression were measured by real-time polymerase chain reaction (PCR).
The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was downregulated by TNF-alpha (46%) or IL-1beta treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1beta or TNF-alpha. TGF-beta-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1beta in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1beta partially reversed the TGF-beta stimulation of Smad7 mRNA and protein levels in TGF-beta-treated human OA cells. In contrast, TGF-beta-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1beta.
IL-1beta or TNF-alpha exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-beta-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1beta partially reversed the increase in TGF-beta-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-beta signaling induced by IL-1beta or TNF-alpha in articular chondrocytes. The balance between the IL-1beta or TNF-alpha and the TGF-beta signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA.
Osteoarthritis and Cartilage 01/2008; 15(12):1367-77. · 4.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: The transcription factor SOX9 is essential for multiple steps during skeletal development, including mesenchymal cell chondrogenesis and endochondral bone formation. We recently reported that the human SOX9 proximal promoter region is regulated by the CCAAT-binding factor through two CCAAT boxes located within 100 bp of the transcriptional start site. Here we report that the human SOX9 proximal promoter is also regulated by the cyclic-AMP response element binding protein (CREB) and Sp1. We show by DNaseI protection and EMSA analysis that CREB and Sp1 interact with specific sites within the SOX9 proximal promoter region. By transient transfection analysis we also demonstrate that mutations of the CREB and Sp1 binding sites result in a profound reduction of SOX9 promoter activity. Chromatin immunoprecipitation (ChIP) assay demonstrated that both Sp1 and CREB interact with the SOX9 promoter in vivo. Finally, we demonstrate that IL-1beta treatment of chondrocytes isolated from human normal and osteoarthritic (OA) cartilage down-regulates SOX9 promoter activity, an effect accompanied by a reduction of Sp1 binding to the SOX9 proximal promoter.
Experimental Cell Research 05/2007; 313(6):1069-79. · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1alpha, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1alpha were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1alpha subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1alpha protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1alpha protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1alpha transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1alpha is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1alpha can be used as a phenotypic marker of nucleus pulposus cells.
Journal of Cellular Biochemistry 06/2006; 98(1):152-9. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.
Arthritis research & therapy 02/2006; 8(6):R164. · 4.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sox9 is an essential transcriptional regulator of chondrogenesis and chondrocyte-specific gene expression; however, the identity and function of transcription factors that regulate Sox9 gene expression are not well understood. Here, we have undertaken an analysis of the human Sox9 proximal promoter region in an effort to elucidate the function and identity of transcriptional regulators that are important for controlling Sox9 gene transcription. By transfection analysis, we show that elements residing between -256 bp and +67 bp are important for the overall level of Sox9 promoter activity. Previously, two CCAAT boxes were identified in the Sox9 mouse and human promoters (position -60 bp and -100 bp) by sequence analysis (Kanai, Y., Koopman, P., 1999. Structural and functional characterization of the mouse Sox9 promoter: implications for campomelic dysplasia. Hum. Mol. Genet., 8: 691-696). We demonstrate by electrophoretic mobility shift (EMSA) competition and supershift assays that the CCAAT-binding factor (CBF) can form a complex with both Sox9 CCAAT boxes in nuclear extracts from multiple cell lines. Transfection of human Sox9 promoter-luciferase constructs containing mutated or deleted CCAAT boxes demonstrated that both CCAAT boxes are important for Sox9 promoter activity in chondrogenic cell lines and primary chondrocytes. Chromatin immunoprecipitation (ChIP) experiments demonstrated that CBF interacts with the Sox9 promoter in vivo. Together, these studies show that the Sox9 promoter is regulated by CBF through its interaction with two functional CCAAT boxes.
[show abstract][hide abstract] ABSTRACT: Articular cartilage is a unique tissue in that it is avascular with its nutrition and oxygen supply being dependent on the diffusion of solutes through the synovial fluid and to and from the subchondral bone. The oxygen levels in articular cartilage, therefore, are assumed to be low. Oxygen is an important modulator of gene expression and this regulation occurs largely through the activation of the transcriptional complex hypoxia-inducible factor-1 (HIF-1). However, little is known about how articular cartilage regulates genes in response to O(2)tension and whether this regulation occurs through HIF-1.
The aim of this study was to investigate the expression profile of HIF-1alpha in normal and osteoarthritic (OA) chondrocytes under normoxic and hypoxic conditions, and in response to treatment with tumor necrosis factor alpha (TNFalpha).
Articular chondrocytes from human normal and OA knee cartilage were isolated and cultured in suspension under normoxic (21% O(2)) or hypoxic conditions (1% O(2)). Chondrocytes were also treated with TNF-alpha under normoxic conditions. Nuclear extracts and total RNA were prepared and HIF-1alpha protein and mRNA levels were assayed by immunoblotting and Northern hybridization. Localization of HIF-1alpha by immunofluorescence was performed on frozen sections of cartilage tissue by confocal microscopy.
HIF-1alpha expression was detectable in human normal and OA chondrocytes and cartilage by Northern analysis, immunoblotting and immunofluorescence under normoxic conditions. Culture of OA or normal chondrocytes under hypoxic conditions for up to 16h resulted in a modest stabilization and/or increase of HIF-1alpha expression. Treatment of articular chondrocytes with TNFalpha resulted in an increase in HIF-1alpha protein steady state levels under normoxic conditions. The increase in HIF-1alpha expression induced by TNFalpha was partially blocked by pretreatment of the chondrocytes with inhibitors of NFkappaB or p38 MAP kinase. We also observed the expression of HIF-2alpha mRNA in human chondrocytes.
HIF-1alpha is expressed in human normal and OA articular chondrocytes cultured under normoxic conditions. HIF-1alpha can be further induced or stabilized in articular chondrocytes by hypoxia or by treatment with TNFalpha. The relatively high constitutive expression of HIF-1alpha by chondrocytes may be an important adaptation to survival in the avascular-hypoxic environment of cartilage. Modulation of HIF-1alpha levels by TNF-alpha may have important implications for chondrocyte metabolism during degenerative joint disease. In addition, we detected for the first time the expression of HIF-2alpha mRNA in chondrocytes.
Osteoarthritis and Cartilage 05/2004; 12(4):336-45. · 4.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: To consider the potential of tumor necrosis factor (TNF) neutralization in rheumatologic disorders other than rheumatoid arthritis (RA).
Literature on the safety and efficacy of TNF inhibition in the treatment of non-RA arthropathies and systemic inflammatory diseases from American and European medical journals was reviewed.
Clinical trials, open-label studies, and case studies indicate great promise for TNF inhibitors alone or in combination with other protocols in the treatment of non-RA rheumatologic disorders. In randomized, double-blind, placebo-controlled trials of etanercept and open-label studies of infliximab in patients with psoriatic arthritis, these 2 TNF inhibitors resulted in an approximately 80% to 90% response rate. Double-blind, placebo-controlled, randomized trials also indicate an encouraging degree of efficacy in patients with ankylosing spondylitis. Preliminary evidence from open-label trials and case studies suggests that these TNF inhibitors also may be effective in the treatment of Behçet disease, Wegener granulomatosis, and sarcoidosis.
Neutralization of TNF may have an important role in the treatment of rheumatologic disorders other than RA.
Seminars in Arthritis and Rheumatism 09/2003; 33(1):1-18. · 3.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter activity. In transient-transfection analyses with promoter deletion constructs, it was found that full promoter activity in RCS cells depended on the region from -560 bp to +130 bp relative to the transcriptional start site (+1). Sequence analysis of the region from -890 bp to the transcriptional start predicted five putative SOX/Sry-binding sites. Mutation analysis revealed that two of three putative SOX/Sry binding sites within the -560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection experiments with a SOX9 expression plasmid revealed that a construct containing the five putative SOX/Sry-binding sites was transactivated 20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within the -560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from -560 to -357 bp, which does not contain any other conspicuous SOX9 sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a specific complex with one of the SOX/Sry-binding sites with in the -560 to +130 region.
Journal of Biological Chemistry 02/2003; 278(1):117-23. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA).
Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A+) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing approximately 5,000 known human genes and approximately 3,000 expressed sequence tags (ESTs).
A > or =2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1alpha, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes.
Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.
[show abstract][hide abstract] ABSTRACT: During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata.
[show abstract][hide abstract] ABSTRACT: To perform stable transfections of human chondrocytes under conditions that allow the maintenance of the chondrocyte-specific phenotype, and to examine the effects of the stable transfection of a mutated type II procollagen gene (COL2A1) on the structure of the cartilaginous extracellular matrix produced in vitro.
A type II procollagen minigene that lacks exons 16-27 was stably transfected into human fetal epiphyseal chondrocytes in vitro. Expression of the minigene was detected by reverse transcriptase-polymerase chain reaction, and the encoded protein was identified by Western blot with a human type II collagen-specific antibody. The cartilaginous extracellular matrix produced by the cultured transfected chondrocytes was characterized using histochemical staining, polarized light microscopy analysis, and transmission electron microscopy.
A shortened type II collagen encoded by the transfected minigene was biosynthesized and produced in the cultures of transfected cells. Histologic analyses demonstrated a more dense, negatively charged cartilaginous matrix in control cultures. In contrast, COL2A1 minigene-transfected cultures were more cellular, were populated with cells of irregular shape and less-chondrocytic appearance, contained abundant intracellular dense granules, and were surrounded by a less-dense matrix. Polarized light microscopy and transmission electron microscopy revealed a well-organized collagen fibrillar matrix in untransfected, control chondrocyte cultures, while the matrix in the transfected cultures was less birefringent and contained numerous truncated collagen fibrils.
The findings illustrate the feasibility of gene transfer into human fetal chondrocytes under conditions that allow the preservation of their specific phenotype, and also shed light on the function of type II collagen in the maintenance of the structural integrity of articular cartilage matrix.