Exuberant expression of chemokine genes by adult human articular chondrocytes in response to IL-1beta.

OBJECTIVE: To provide a more complete picture of the effect of interleukin-1 beta (IL-1beta) on adult human articular chondrocyte gene expression, in contrast to the candidate gene approach. DESIGN: Chondrocytes from human knee cartilage were cultured in medium containing IL-1beta. Changes in gene expression were analyzed by microarray and reverse transcriptase-polymerase chain reaction analysis. The ability of transforming growth factor beta-1 (TGF-beta1), fibroblast growth factor (FGF)-18, and bone morphogenetic protein 2 (BMP-2) to alter the effects of IL-1beta was analyzed. Computational analysis of the promoter regions of differentially expressed genes for transcription factor binding motifs was performed. RESULTS: IL-1beta-treated human chondrocytes showed significant increases in the expression of granulocyte colony stimulating factor-3, endothelial leukocyte adhesion molecule 1 and leukemia inhibitory factor as well as for a large group of chemokines that include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL8, CCL20, CCL3L1, CX3CL1 and the cytokine IL-6. As expected, the mRNA for matrix metalloproteinase (MMP)-13 and BMP-2 also increased while mRNA for the matrix genes COL2A1 and aggrecan was down-regulated. A subset of chemokines increased rapidly at very low levels of IL-1beta. The phenotype induced by IL-1beta was partially reversed by TGF-beta1, but not by BMP-2. In the presence of IL-1beta, FGF-18 increased expression of ADAMTS-4, aggrecan, BMP-2, COL2A1, CCL3, CCL4, CCL20, CXCL1, CXCL3, CXCL6, IL-1beta, IL-6, and IL-8 and decreased ADAMTS-5, MMP-13, CCL2, and CCL8. Computational analysis revealed a high likelihood that the most up-regulated chemokines are regulated by the transcription factors myocyte enhancer binding factor-3 (MEF-3), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-kappaB). CONCLUSION: IL-1beta has a diverse effect on gene expression profile in human chondrocytes affecting matrix genes as well as chemokines and cytokines. TGF-beta1 has the ability to antagonize some of the phenotype induced by IL-1beta.

Oxidative stress induces expression of osteoarthritis markers procollagen IIA and 3B3(-) in adult bovine articular cartilage.

OBJECTIVE: Oxidative stress occurs when the metabolic balance of a cell is disrupted through exposure to excess pro-oxidant. Whilst it is known that unregulated production or exposure to exogenous sources of pro-oxidants induces chondrocyte cell death and degrades matrix components in vitro, relatively little is known of the effects of pro-oxidants on articular cartilage in situ. The objective of this study was to determine if a single exposure to the pro-oxidant hydrogen peroxide (H(2)O(2)) induces a degenerative phenotype. METHODS: Articular cartilage explants were obtained from skeletally mature bovine steers and exposed to a single dose of hydrogen peroxide (0.1-1.0 mM) and cultured for up to 21 days. Cell death, and sulfated glycosaminoglycan loss into the medium and gene expression were quantitatively determined. Adoption of an abnormal chondrocyte phenotype was analyzed through the expression of 3B3(-), nitrotyrosine and procollagen type IIA epitopes in cartilage explants. RESULTS: Cell death occurred primarily at the surface zone of cartilage in a dose-dependent manner in H(2)O(2) treated explants, and supplementation of standard serum-free medium with insulin-selenium-transferrin significantly reduced cell death (>fourfold). Nitric oxide synthase-2 gene expression and proteoglycan loss increased in oxidant treated explants in a concentration-dependent manner. Antibody labeling to 3B3(-), procollagen type IIA and nitrotyrosine was present in all treated explants but absent in untreated explants. CONCLUSIONS: This study demonstrates that a single exposure to high levels of pro-oxidant causes the expression of genes and antibody epitopes that are associated with early degenerative changes observed in experimental osteoarthritis.

Mouse CD-RAP/MIA gene: structure, chromosomal localization, and expression in cartilage and chondrosarcoma.

A cDNA encoding a novel protein has been previously isolated from two independent sources: melanoma cell cultures and chondrocytes. The protein from human melanoma cell lines and tumors is called melanoma inhibitory activity (MIA) (Blesch et al. [1994] Cancer Res. 54:5695-5701) and the protein from primary bovine chondrocytes and cartilaginous tissues is called cartilage-derived retinoic acid-sensitive protein (CD-RAP) (Dietz and Sandell [1996] J. Biol. Chem. 271:3311-3316). In order to investigate the gene regulation and function of CD-RAP/MIA, the mouse gene locus was isolated and analyzed. Developmental expression was determined by in situ hybridization to mouse embryos. Expression was limited to cartilaginous tissues and was initiated with the advent of chondrogenesis, remaining abundant throughout development. The mouse gene was isolated and sequenced from a 129Sv library and sequenced directly from an additional strain, B6C3Fe. The mouse CD-RAP/MIA gene is 1.5 kbp and consists of four exons. The promoter sequence of the gene contains many potential regulatory domains including 8 basic helix-loop-helix protein-binding domains and an AT-rich domain, both motifs shown to be present in the cartilage-specific enhancer of the type II procollagen gene. Other potential cis-acting motifs include binding sites for GATA-1, NF-IL6, PEA3, w-elements, NF kappa B, Zeste and Sp1. The gene, called cdrap, was localized to the end of an arm of chromosome 7 at the same site as the transforming growth factor beta 1 (Tgf-beta 1) and the glucose phosphate isomerase 1 (Gpi 1) genes. Potential mouse mutants that mapped to the same region of chromosome 7 were identified. Two of the potential mutants with skeletal phenotypes were sequenced, pudgy (pu) and extra toes with spotting (XsJ); however, no mutations were found in the coding sequence. To determine whether CD-RAP/MIA is associated with tumors of cartilage, mRNAs from a variety of rodent tissues and cell lines were screened. Expression was detected in a rodent tumor, the Swarm rat chondrosarcoma and a chondrosarcoma cell line derived from it, but not in other tissues or tumors of non-cartilage origin. Immunolocalization revealed CD-RAP/MIA protein localized in cartilage only. These results show that the normal expression of CD-RAP/MIA is limited to cartilage; however, pathologically, it is expressed both in melanoma and chondrosarcoma. The restricted expression of CD-RAP/MIA may provide an opportunity to monitor cartilage metabolic activity as well as the tumor activity of melanoma and chondrosarcoma.

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(-8) to 10(-6) M. 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.

Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension.

To define the influence of time in culture on gene expression for extracellular matrix proteins we have examined the progression of changes in gene expression for chondrocyte extracellular matrix proteins from the time the chondrocytes are initially isolated from their native cartilaginous matrix through 13 days of high-density culture. We have also determined the effect of matrix depletion and shape change by enzymatically resuspending cells after 6 days in culture and sampling the replated cells at intervals up to 13 days. Northern blots of chondrocyte RNA were hybridized with probes for collagen alpha 1(II), alpha 1(I), aggrecan, link protein, and decorin mRNA. The steady-state level of alpha 1(II) collagen mRNA dropped to 45% of the initial value within 24 h, with a further decrease to 21% by Day 3. A similar decline occurred, but less rapidly in ascorbate supplemented cultures with values of 78%, at 24 h and 62% at Day 3. Very low levels of alpha 1(I) collagen mRNA were detectible in cells maintained for 2 weeks without ascorbate supplementation. Type I collagen alpha 1(I) mRNA was not detected in freshly isolated chondrocytes or at the earliest times in culture but was increasingly abundant from Days 5-13 in the presence of ascorbate. Ascorbic acid supplementation altered the pattern of aggrecan expression over time. Without ascorbate there was an increase in steady-state aggrecan mRNA with time in culture, but in the presence of ascorbate, aggrecan mRNA levels peaked at early culture times and progressively diminished. Decorin steady-state mRNA levels in cultures not supplemented with ascorbic acid steadily increased over time in culture following a lag of several days. In cultures treated with ascorbate, however, there was a progressive increase in decorin steady-state mRNA levels from the first day in culture. Resuspending chondrocytes by digestion of the cell layer with pronase and collagenase at Day 6, which resulted in a transient shape change as well as matrix depletion, resulted in a greater than 2-fold increase in alpha 1(II) mRNA at Day 7 in ascorbate supplemented cultures. Only with ascorbate was there a small increase in decorin mRNA at Day 7, after resuspension. Aggrecan mRNA, however, showed a 3-fold increase without ascorbate and a 10-fold increase with ascorbate within 24 h of resuspension. Similarly, link protein steady-state mRNA showed an 8-fold increase without ascorbate and a 9-fold increase with ascorbate within 24 h after resuspension.