Linda J. Sandell

Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes.

In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions.

Stimulation of BMP-2 Expression by Pro-Inflammatory Cytokines IL-1 and TNF-α in Normal and Osteoarthritic Chondrocytes

Background: Destruction of cartilage in osteoarthritis is a direct effect of an imbalance between catabolic and anabolic activities in the tissue. While a great deal is known about catabolism, we sought to determine the biochemical basis of the anabolic activity.Methods: Cartilage was isolated from normal and osteoarthritic patients and subjected to both cell and explant culture. mRNA expression levels of the growth and differentiation factors bone morphogenetic protein-2 (BMP-2), BMP-4, BMP-6, cartilage-derived morphogenetic protein-1 (CDMP-1), connective tissue growth factor (CTGF), and activin were determined. BMP-2 was localized in osteoarthritic cartilage by immunohistochemistry. To determine the mechanism of BMP-2 stimulation, chondrocytes were cultured with TGF-&bgr; (transforming growth factor-&bgr;), insulin-like growth factor-1 (IGF-1), interleukin-1&bgr; (IL-1&bgr;), and tumor necrosis factor-&agr; (TNF-&agr;). The BMP-2 response was monitored by quantitative real-time polymerase chain reaction to ascertain mRNA levels and by Western blot analysis, BMP-2 protein quantitation, and immunohistochemistry to determine protein levels.Results: BMP-2 was found to be up-regulated in osteoarthritic chondrocytes and cartilage. In cell culture, IL-1&bgr; and TNF-&agr; increased BMP-2 mRNA and protein levels by eightfold and fifteenfold, respectively, whereas IGF-1 and TGF-&bgr;1 had no effect. In cartilage explant cultures, IL-1&bgr; and TNF-&agr; increased BMP-2 levels both intracellularly and extracellularly. Functional relevance was suggested by co-localization of BMP-2 and newly synthesized type-II procollagen within the same cells.Conclusions: BMP-2 acts as a stimulus of anabolic activities in normal and osteoarthritic chondrocytes. Furthermore, the pro-inflammatory cytokines IL-1&bgr; and TNF-&agr;, known to be present in synovium and cartilage of patients with osteoarthritis, stimulate the production of active BMP-2.Clinical Relevance: These results suggest that, in addition to their role in catabolism of cartilage in osteoarthritis, the pro-inflammatory cytokines IL-1&bgr; and TNF-&agr; may stimulate anabolic events through production of BMP-2. BMP-2, in turn, increases the synthesis of chondrocyte extracellular molecules, aggrecan and type-II collagen. Consequently, local inflammatory events may contribute to the remodeling or repair of cartilage.

Reexpression of type IIA procollagen by adult articular chondrocytes in osteoarthritic cartilage

OBJECTIVE To test for the reexpression of the chondroprogenitor splice variant of the gene COL2A1, type IIA procollagen (containing a cysteine-rich NH2 propeptide), in adult articular chondrocytes in osteoarthritic (OA) joint disease. METHODS In situ hybridization and immunohistochemical localization were performed on normal and OA articular cartilage specimens. The presence of type IIA procollagen messenger RNA (mRNA) expression was confirmed by Northern blot analysis. RESULTS In normal articular cartilage, no expression of mRNA or presence of type IIA procollagen was found. In OA articular cartilage, focally intense staining for type IIA protein was detected. Consistent with this, chondrocytes, particularly in the middle zones of articular cartilage, expressed type IIA procollagen mRNA. OA repair cartilage typically showed a broad zone of cells expressing type IIA mRNA and protein. CONCLUSION Type IIA procollagen is reexpressed by adult articular chondrocytes in OA cartilage degeneration, indicating the potential reversion of the cells to a chondroprogenitor cellular phenotype. The absence of type IIA mRNA and protein in normal adult articular cartilage and its onset in the diseased state suggests type IIA procollagen as a marker of OA.

Value of biomarkers in osteoarthritis: current status and perspectives.

Osteoarthritis affects the whole joint structure with progressive changes in cartilage, menisci, ligaments and subchondral bone, and synovial inflammation. Biomarkers are being developed to quantify joint remodelling and disease progression. This article was prepared following a working meeting of the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis convened to discuss the value of biochemical markers of matrix metabolism in drug development in osteoarthritis. The best candidates are generally molecules or molecular fragments present in cartilage, bone or synovium and may be specific to one type of joint tissue or common to them all. Many currently investigated biomarkers are associated with collagen metabolism in cartilage or bone, or aggrecan metabolism in cartilage. Other biomarkers are related to non-collagenous proteins, inflammation and/or fibrosis. Biomarkers in osteoarthritis can be categorised using the burden of disease, investigative, prognostic, efficacy of intervention, diagnostic and safety classification. There are a number of promising candidates, notably urinary C-terminal telopeptide of collagen type II and serum cartilage oligomeric protein, although none is sufficiently discriminating to differentiate between individual patients and controls (diagnostic) or between patients with different disease severities (burden of disease), predict prognosis in individuals with or without osteoarthritis (prognostic) or perform so consistently that it could function as a surrogate outcome in clinical trials (efficacy of intervention). Future avenues for research include exploration of underlying mechanisms of disease and development of new biomarkers; technological development; the ‘omics’ (genomics, metabolomics, proteomics and lipidomics); design of aggregate scores combining a panel of biomarkers and/or imaging markers into single diagnostic algorithms; and investigation into the relationship between biomarkers and prognosis.

Etiology of osteoarthritis: genetics and synovial joint development

Osteoarthritis (OA) has a considerable hereditary component and is considered to be a polygenic disease. Data derived from genetic analyses and genome-wide screening of individuals with this disease have revealed a surprising trend: genes associated with OA tend to be related to the process of synovial joint development. Mutations in these genes might directly cause OA. In addition, they could also determine the age at which OA becomes apparent, the joint sites involved, the severity of the disease and how rapidly it progresses. In this Review, I propose that genetic mutations associated with OA can be placed on a continuum. Early-onset OA is caused by mutations in matrix molecules often associated with chondrodysplasias, whereas less destructive structural abnormalities or mutations confer increased susceptibility to injury or malalignment that can result in middle-age onset. Finally, mutations in molecules that regulate subtle aspects of joint development and structure lead to late-onset OA. In this Review, I discuss the genetics of OA in general, but focus on the potential effect of genetic mutations associated with OA on joint structure, the role of joint structure in the development of OA—using hip abnormalities as a model—and how understanding the etiology of the disease could influence treatment.

Cloning of a Retinoic Acid-sensitive mRNA Expressed in Cartilage and during Chondrogenesis

Retinoic acid (RA) is known to play a role in various aspects of skeletal development in vivo, including morphogenesis, growth plate maturation, and apoptosis. In cell culture, RA treatment of chondrocytes suppresses the differentiated phenotype characterized by production of type II collagen and aggrecan. In an effort to discover molecules involved in regulation of the chondrocyte phenotype or related to developmental processes such as chondrogenesis, mRNAs from bovine chondrocytes cultured with and without RA were amplified by reverse transcription-polymerase chain reaction (PCR) and compared by differential display. PCR products whose expression was inhibited by RA treatment were cloned. One cDNA encodes a molecule we call cartilage-derived retinoic acid-sensitive protein (CD-RAP), and its properties are described here. The full-length bovine CD-RAP mRNA was cloned after amplification by the rapid amplification of cDNA ends procedure, and a part of the rat CD-RAP mRNA was amplified by reverse transcription-PCR using sequence-specific primers. The bovine CD-RAP mRNA contains an open reading frame of 130 amino acids. CD-RAP mRNA expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage primordia and cartilage. The inhibition of CD-RAP mRNA expression by RA in vitro was time- and dose-dependent and was tested over concentrations from 10 to 10M. Southern blot analysis of genomic DNA indicated that CD-RAP was encoded by a single copy gene and that no other genes were closely related. What appears to be the human homologue of CD-RAP was recently isolated and cloned from a melanoma cell line and shown to function as a growth inhibitory protein (Blesch, A., Boberhoff, A.-K., Apfel, R., Behl, C., Hessdoerfer, B., Schmitt, A., Jachimcza, P., Lottspeich, F., Buettner, R., and Bogdahn, U.(1994) Cancer Res. 54, 5695-5701). Neither CD-RAP nor this protein showed any homology to known proteins. We speculate that, in vivo, CD-RAP functions during cartilage development and maintenance.

Trans-activation of the Mouse Cartilage-Derived Retinoic Acid-Sensitive Protein Gene by Sox9

The transcription factor Sox9 is capable of enhancing type II collagen gene expression and may play a crucial role in chondrogenesis. To determine whether Sox9 is an inducer of the chondrocyte phenotype, we investigated the role of Sox9 in transcription of another cartilage gene encoding the cartilage‐derived retinoic acid–sensitive protein (CD‐RAP). CD‐RAP is specifically expressed during chondrogenesis. We show here that Sox9 protein is able to bind to a SOX consensus sequence in the CD‐RAP promoter. Mutation of the SOX motif led to decreased transcription of a CD‐RAP promoter construct in chondrocytes. Overexpression of SOX9 resulted in a dose‐dependent increased activity of CD‐RAP promoter‐driven reporter gene in both chondrocytes and nonchondrogenic cells. A truncated SOX9, which contains a binding domain but no trans‐activation function, inhibited CD‐RAP promoter activity. Overexpression of SOX9 increased the level of endogenous CD‐RAP mRNA in chondrocytes, but was unable to induce endogenous gene expression in 10T1/2 mesenchymal cells or BALB/c‐3T3 fibroblasts. These results suggest that Sox9 is a general transcriptional regulator of cartilage‐specific genes. However, Sox9 does not appear to be able to induce the chondrocyte phenotype in nonchondrogenic cells, implying that other factors are involved in chondrogenesis.

Egr-1 mediates transcriptional repression of COL2A1 promoter activity by interleukin-1beta.

Following induction and activation of the early growth response (Egr)-1 transcription factor in human chondrocytes, interleukin-1β (IL-1β) suppresses the expression of the type II collagen gene (COL2A1), associated with induction of Egr-1 binding activity in nuclear extracts. The COL2A1 proximal promoter contains overlapping binding sites for Egr-1 and Sp1 family members at −119/−112 bp and −81/−74 bp. Mutations that block binding of Sp1 and Sp3 to either site markedly reduce constitutive expression of the core promoter. IL-1β-induced Egr-1 binds strongly to the −119/−112 bp site, and mutations that block Egr-1 binding prevent inhibition by IL-1β. Cotransfection with pCMV-Egr1 potentiates the inhibition of COL2A1 promoter activity by IL-1β, whereas overexpression of dominant-negative Egr-1 mutant, Wilms tumor-1 (WT1)/Egr1, Sp1, or Sp3 reverses the inhibition by IL-1β. Cotransfection of pGL2-COL2/Gal4, in which we substituted the critical residue for Egr-1 binding with a Gal4 binding domain and a pCMV-Gal4-Egr1 chimera permits an inhibitory response to IL-1β that is reversed by overexpression of Gal4-CBP. Our results indicate that IL-1β-induced activation of Egr-1 binding is required for inhibition of COL2A1 proximal promoter activity and suggest that Egr-1 acts as a repressor of a constitutively expressed collagen gene by preventing interactions between Sp1 and the general transcriptional machinery.

Identification of the proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization and immunohistochemistry

Versican is a large chondroitin sulfate proteoglycan (CSPG) initially identified in cultured human fibroblasts. Previous studies have shown that there is a versican-like molecule in cultured monkey smooth muscle cells. In this study, we have cloned and sequenced the large CSPG from cultured monkey smooth muscle cells, fetal and juvenile monkey aorta, and human fetal aorta. The cDNA sequence from human fetal aorta is completely homologous to the human fibroblast versican. We obtained 2.5 kb of cDNA sequence from monkey aortic RNA and cultured monkey smooth muscle cell RNA. This sequence covers three distinct domains of versican (hyaluronic acid binding domain, glycosaminoglycan attachment domain and protein binding domain) and demonstrates over 90% homology to the human versican sequence. In situ hybridization histochemistry indicates that the versican RNA transcript is located in the epithelium throughout the tunica media of the aorta. Western blot analysis and immunohistochemistry also confirm the presence of versican in human and monkey aorta.

Type IIA Procollagen Amino Propeptide Is Localized in Human Embryonic Tissues

Type II procollagen is synthesized in two forms generated by the alternative splicing of its precursor mRNA. The alternatively spliced domain, exon 2, encodes the 69-amino-acid cysteinerich region of the NH2 propeptide. Studies of mRNA expression have shown that the longer form, designated Type IIA procollagen, is synthesized by chondroprogenitor cells and various noncartilaginous tissues. The shorter form, Type IIB procollagen, is synthesized by differentiated chondrocytes. As the initial step in our investigations of the function of the Type IIA procollagen, the protein domain corresponding to exon 2 was created as a recombinant fusion protein and used to raise antibodies in rabbits. The resulting antiserum was specific for Type IIA procollagen NH2 propeptide as shown by ELISA, Western blotting, and immunofluorescent co-localization with the triple-helical domain of Type II collagen. Type IIA procollagen was identified in tissue culture medium of 54-day human fetal ribs. Confocal microscopy was used to localize the Type IIA NH2 propeptide in Day 50 and 53 human embryos. In the digital rays of the developing hand, where only Type IIA procollagen mRNA was detected, Type IIA procollagen NH2 propeptide was observed in the extracellular matrix. The presence of Type IIA procollagen NH2 propeptide was observed in the cartilage of the developing long bones of the lower arm and vertebral bodies even though these tissues synthesize Type IIB mRNA at this developmental stage. Type IIA procollagen NH2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH2 propeptide was also localized in noncartilaginous tissues known to synthesize Type IIA mRNA, such as the intervertebral area, perichondrium, notochordal sheath, and neuroepithelium of the otic vesicle. In most tissues, co-localization with antiserum against the triple-helical domain of Type II collagen was observed. Positive immunoreactivity with the Type IIA NH2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH2 propeptide antibodies with the triple-helical domain of the collagen molecule suggests that Type IIA procollagen is intact in the extracellular matrix of these tissues. Taken together, these results strongly suggest that around cells that synthesize Type IIA procollagen mRNA, Type IIA procollagen NH2 propeptide is secreted and deposited into the extracellular matrix. In light of these results, we predict that Type IIA procollagen plays a role in differentiation of tissues that augments its purely architectural function. (J Histochem Cytochem 45:1469–1480, 1997)