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Articular cartilage deterioration commonly occurs following traumatic joint injury. Patients with posttraumatic osteoarthritis (PTA) experience pain and stiffness in the involved joint causing limited mobility and function. The mechanism by which PTA occurs has not been fully delineated. The goal of this study was to determine if a single high-energy impact load could cause the development of PTA in 3-month-old NZ White rabbits. Each rabbit underwent the application of a single, rapid, high-energy impact load to the posterior aspect of their right medial femoral condyle using a previously validated mechanism. At regular intervals (0, 1, 6 months) the injured cartilage was harvested and analyzed for the presence of PTA. Each specimen was assessed histologically for cell and tissue morphology and chondrocyte metabolism, including BMP-2 production and synthesis of extracellular matrix (type II procollagen mRNA). Cartilage from the contralateral sham limb, as well as uninjured cartilage from the experimental limb served as internal controls for each animal. Significant changes were found in the morphology of the cartilage including proteoglycan loss along with decreased BMP-2 and type II procollagen mRNA staining. These findings confirm that a single high-energy impact load can cause the development of PTA by disrupting the extracellular matrix and by causing a decrease in chondrocyte metabolism.

The aim of this study was to create a gene expression profile to better define the phenotype of human adipose-derived stromal cells (HADSCs) during in vitro chondrogenesis, osteogenesis and adipogenesis. A novel aspect of this work was the analysis of the same subset of genes during HADSC differentiation into all three lineages. Chondrogenic induction resulted in increased mRNA expression of Sox transcription factors, COL2A1, COL10A1 , Runx2, and Osterix . This is the first report demonstrating significant upregulation in expression of osteogenesis-related transcription factors Runx2 and Osterix by TGF-β3 induction of HADSCs during in vitro chondrogenesis. These findings suggest that the commonly-used chondrogenic induction reagents promote differentiation suggestive of hypertrophic chondrocytes and osteoblasts. We conclude that alternative strategies are required to induce efficient articular chondrocyte differentiation in order for HADSCs to be of clinical use in cartilage tissue engineering.

Objective Inflammatory processes in rheumatoid arthritis are primarily regulated by the cytokines tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β). Previous studies in our laboratory have shown that IL-1β represses expression of the cartilage characteristic genes, cartilage-derived retinoic acid–sensitive protein (cd-rap) and type II collagen (COL2A1); this mechanism of repression involves activation of a CCAAT/enhancer binding protein (c/EBP) site within promoter regions. The aim of this study was to investigate novel TNFα-mediated mechanisms that regulate the expression of cd-rap. Methods Rat chondrosarcoma cells were transiently transfected with complementary DNA constructs encoding cd-rap, in the presence of TNFα. The expression of c/EBPβ, SOX9, and p300 in rat chondrosarcoma cells and primary human articular chondrocytes after treatment with TNFα was examined by reverse transcription–polymerase chain reaction and Western blotting. The effect of TNFα on endogenous binding of c/EBPβ or SOX9 to the cd-rap promoter was examined by chromatin immunoprecipitation assays. Results We identified a new c/EBP binding site in the cd-rap promoter (from position −1059 bp to position −1046 bp). Binding of c/EBP to this site was regulated by TNFα but not IL-1β, resulting in down-regulation of cd-rap expression. This effect was reversed by mutational inactivation of the c/EBP motif. In addition, the activation potential of SOX9 and CREB binding protein/p300 on the cd-rap promoter was enhanced after mutation of the new c/EBP binding site, indicating that blockage of this site would increase transcription. Conclusion TNFα regulates the expression and/or DNA-binding potential of key positive-acting and negative-acting transcription factors that control the expression of the cartilage matrix gene, cd-rap.

Type II collagen, like other fibrillar collagens, is synthesized as a procollagen containing amino (NH2)-and carboxyl (COOH)-terminal extension peptides. Based on cDNA cloning of human (Baldwin, C. T., Reginato, A. M., Smith, C., Jimenez, S. A., and Prockop, D. J. (1989) Biochem. J. 262, 521-528) and rat (Kohno, K., Martin, G. R., and Yamada, Y. (1984) J. Biol. Chem. 259, 13668-13673) type II procollagen, it was concluded that much of the NH2-terminal propeptide seen in pro-alpha 1(I) was missing. Analysis of human genomic clones for type II collagen revealed an additional exon encoding a 69-amino acid cysteine-rich domain in the NH2-terminal propeptide. This exon (exon 2) is expressed in the mRNA population of chondrocytes isolated from human fetal skeleton and notochord, juvenile costal cartilage, and bovine articular cartilage. Oligonucleotide probes spanning specific exon boundaries were used to detect two populations of procollagen mRNA by Northern blot analysis. Amplification of cDNA templates using polymerase chain reaction provided direct evidence for two distinct pro-alpha 1(II) collagen mRNAs. DNA sequence analysis showed that the two mRNAs resulted from the alternative splicing of exon 2. The protein domain encoded by exon 2 is conserved between the fibrillar collagens and two other extracellular matrix proteins, thrombospondin and von Willebrand factor. In fibrillar collagens, this protein domain may play a regulatory role in fibrillogenesis and feedback inhibition of collagen biosynthesis. Consequently, the differential expression of this protein domain could alter the biosynthesis or fibril formation of type II collagen. In addition, the expression of exon 2 may be a marker for a distinct population of chondrocytes.

We have examined posttranslational modifications which are responsible for converting an apparently single precursor (Hering, T. M., and Sandell, L. J. (1988) J. Biol. Chem. 263, 1030-1036) to the two major forms of link protein in bovine articular cartilage. Resistance to endoglycosidases H and F suggests that Asn-linked oligosaccharides of link protein secreted by bovine chondrocytes in culture are of the complex or hybrid type. There is no evidence for O-linked oligosaccharides. There is no apparent precursor-product relationship between link protein (LP)1 and LP2, since after a short pulse with [3H]leucine two forms are present, consistent with the existence of two glycosylation sites. An immunoprecipitate of LP1 from pulse-labeled chondrocytes was observed to show a decrease in electrophoretic mobility and increased microheterogeneity during transit through the Golgi, whereas LP2 did not change. During processing both LP1 and LP2 become endoglycosidase H resistant. LP1, but not LP2, can be biosynthetically labeled with [35S]sulfate. Incorporation of [35S]sulfate is inhibited by tunicamycin, indicating that the sulfate is associated with Asn-linked carbohydrate. Sulfation may be important for normal processing, secretion, or degradation of link protein and with sialylation may confer considerable charge heterogeneity upon LP1. We conclude that there are considerable biochemical differences between glycoproteins LP1 and LP2 which may provide a basis for functional differences.

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1Pcko). S1Pcko mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1Pcko mice. Extraction of type II collagen is substantially lower from S1Pcko cartilage. In S1Pcko mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1Pcko chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

Chondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Several regulatory factors have been identified that play a role in chondrogenesis, including the positive transacting factors of the SOX family such as SOX9, SOX5, and SOX6, as well as negative transacting factors such as C/EBP and delta EF1. However, a complete understanding of the intricate regulatory network that governs the tissue-specific expression of cartilage genes is not yet available. We have taken a computational approach to identify cis-regulatory, transcription factor (TF) binding motifs in a set of cartilage characteristic genes to better define the transcriptional regulatory networks that regulate chondrogenesis. Our computational methods have identified several TFs, whose binding profiles are available in the TRANSFAC database, as important to chondrogenesis. In addition, a cartilage-specific SOX-binding profile was constructed and used to identify both known, and novel, functional paired SOX-binding motifs in chondrocyte genes. Using DNA pattern-recognition algorithms, we have also identified cis-regulatory elements for unknown TFs. We have validated our computational predictions through mutational analyses in cell transfection experiments. One novel regulatory motif, N1, found at high frequency in the COL2A1 promoter, was found to bind to chondrocyte nuclear proteins. Mutational analyses suggest that this motif binds a repressive factor that regulates basal levels of the COL2A1 promoter.

Although osteoarthritis (OA) is essentially a degenerative disease, anabolic activity of chondrocytes is significantly upregulated. Enhanced anabolism in OA chondrocytes was first reported more than 30 years ago [17,41, 47] and has since been confirmed by many studies. Often referred to as OA hyperanabolism, upregulation of synthetic activity is not limited to cartilage, but also affects other tissues inside and around the joint. For example, formation of osteophytes and sclerotic changes of the subchondral bone are pathologic events that symbolize vigorous anabolic activity. Modern techniques have given us a more comprehensive idea about the metabolic changes in OA. Proteomic analysis has confirmed that synthesis of type II collagen is enhanced in OA cartilage [35], and microarray analysis has provided an overall picture of the metabolic changes that take place in the disease process [7]. To date, the significance of anabolism in the pathology of OA is not well understood. Due to the dramatic changes in the metabolic activity of the chondrocytes in OA cartilage, the significance of anabolism in the pathology may be underestimated. Just like many other phenomena, anabolism in OA could have two faces. The enhanced synthesis of cartilage matrix components may prevent disease progression by counteracting catabolic events and initiating a repair process. On the other hand, the induction of pathologic proteins and possible disturbance in proper matrix synthesis observed in the disease may promote disease progression. This chapter provides an overview of the anabolism in OA, with emphasis on recent progress.

While a great deal of information is available on the cellular and molecular biology of cartilage degradation, less is known about anabolism in normal cartilage and degenerating cartilage. A consistent amount of evidence is now available on the neo-synthesis of matrix molecules and enzymes in OA: the entire cell metabolism appears to be increased leading to the hypothesis that chondrocytes in OA are actually "activated". This chapter will focus on anabolic events that are now known to occur in articular cartilage. We will begin to view articular cartilage as a complex three-dimensional tissue in which local events may be different. We will also be interested in viewing the development of degenerative arthritis as a continuum from functionally normal tissue to degeneration.

Objectives: Despite clinical evidence demonstrating that meniscus tears are early events in the initiation and propagation of knee osteoarthritis (OA), the biology of how meniscus injury leads to changes in the articular cartilage is not well studied. Therefore, we compared the molecular biology of articular and meniscal cartilage within the same knees undergoing partial meniscectomy to advance our understanding of early molecular events in the knee that contribute to the development of OA following a meniscus tear. The purpose of this study was to test the hypothesis that meniscal and articular cartilage from knees undergoing arthroscopic partial meniscectomy will demonstrate molecular signature representative of early OA. Methods: Patients (n=12) with a known isolated medial meniscus tear without any knee chondrosis or radiographic OA were consented prior to surgery. During arthroscopic partial meniscectomy, a sample of their injured meniscus and a small fragment of their articular cartilage off the medial femoral condyle were procured. The transcriptome signatures measured through Affymetrix microarray were compared between the two tissues and underlying biological processes were explored computationally. Results: 3566 gene transcripts, representing several underlying biological processes, including extracellular matrix organization, wound healing, cell adhesion, chemotaxis, blood vessel morphogenesis, and angiogenesis, were differentially expressed between meniscus and articular cartilage. Conclusion: Articular cartilage and meniscus exhibit molecular events associated with OA at the time of partial meniscectomy despite no gross evidence of degeneration in the knee. These findings identify early effects of meniscus injury on meniscus and articular cartilage, which likely lead to the development of knee OA.

Medial collateral ligament (MCL) injuries heal by a wound repair scar response controlled by a complex cellular and cytokine environment. Many enzymes participate in wound repair, particularly the matrix metalloproteinases. We hypothesize macrophage metalloelastase (MME/MMP-12) deficiency results in impaired healing of MCL injury. One hundred fifty MME-deficient and 150 WT (MME+/+) mice underwent knee MCL transection with the opposite knee as a sham operated control. Mice were sacrificed at 3, 7, 28, 42, and 56 days. At each of the five time points, 15 mice were utilized for biological and 15 were utilized for biomechanical testing. Outcome measures were the presence of macrophages to represent the inflammatory phase of wound healing, collagen synthesis to assay for matrix repair, and biomechanical testing for repair strength. Immunohistochemistry demonstrated significantly fewer macrophages in cut MCLs from MME-deficient mice versus wild-type (WT) mice at 3, 7, 28, and 42 days (all p

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-alpha induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3'-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3'-untranslated regions, and transient transfection experiments demonstrated that TNF-alpha increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-alpha modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-kappaB.

Rotator cuff repair is known to have a high failure rate. Little is known about the natural healing process of the rotator cuff repair site, hence little can be done to improve the tendon's ability to heal. The purpose of this study was to investigate the collagen formation at the early repair site and to localize TGFbeta-1 and 3 during early healing and compare their levels to cell proliferation and histological changes. Bilateral supraspinatus tendons were transected and repaired in 60 rats. Specimens were harvested and evaluated at 0, 1, 3, 7, 10, 28, and 56 days. Histological sections were evaluated for cell morphology. Immunohistochemistry and in situ hybridization was performed to localize protein and mRNA for collagen types I and III and TGFbeta-1 and 3. Proliferating cell nuclear antigen (PCNA) assay was performed to measure cell proliferation, and cells were counted to determine cell density. Biomechanical properties were evaluated. Repair tissue demonstrated an initial inflammatory response with multinucleated cells present at 1 and 3 days, and lymphocytes and plasma cells presents at 7 and 10 days. Capillary proliferation began at 3 days and peaked at 10 days. Ultimate force increased significantly over the time period studied. Collagen I protein and mRNA significantly increased at 10 days, and reached a plateau by 28 and 56 days. Collagen III showed a similar trend, with an early increase, and remained high until 56 days. TGFbeta-1 was localized to the forming scar tissue and showed a distinct peak at 10 days. TGFbeta-3 was not seen at the healing insertion site. Cell proliferation and density followed the same trend as TGFbeta-1. A wound healing response does occur at the healing rotator cuff insertion site, however, the characteristics of the tendon after healing differ significantly from the uninjured tendon insertion site at the longest time-point studied. A distinctive collagen remodeling process occurred with an initial increase in the formation of collagen types I and III followed by a decrease toward baseline levels seen at time 0. Growth factor TGFbeta-1 was localized to repair tissue and coincided with a peak in cell proliferation and cellularity. Repair sites remained unorganized histologically and biomechanically inferior in comparison to previously described uninjured insertion sites.

Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein whose function is unknown. Mutations in COMP cause pseudoachondroplasia and multiple epiphyseal dysplasia, two skeletal dysplasias which are associated with intracellular retention of COMP in chondrocytes. In contrast, COMP null mice are normal suggesting gene redundancy or that the detrimental effect is associated with mutant COMP rather than the absence of functional COMP. To define the elements that regulate COMP transcription and tissue-specificity, we have evaluated the human COMP promoter driving fusion gene expression in vitro and in vivo. COMP promoter activity is higher in rat chondrosarcoma cells (RCS) than in a fibroblast cell line. In RCS cells, expression of a reporter gene containing 1.7 kb of the human COMP promoter was three-fold higher than all shorter COMP promoter constructs. In transgenic mice, 1.7 kb of the human COMP promoter is active early in development in the limbs, spine, and eye. As development progresses, promoter activity diminishes in the eye and migrates from the center to the ends of the long bones. On the other hand, while 375 bp of the human COMP promoter is sufficient for proper tissue-specific expression, levels are less than those found with the 1.7-COMP promoter. The expression pattern of both promoters recapitulates endogenous cartilage COMP expression in mice. Our findings indicate that the elements required for chondrocyte-specific expression lie within 375 bp of the translational start site, while DNA enhancer elements are located between 1.0 to 1.7 kb.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a small secreted matrix protein expressed in developing and adult cartilage and by chondrocytes in culture. We have previously shown that the expression of Cd-rap, like many other cartilage matrix proteins, is repressed by interleukin 1beta and that the transcription factor CCAAT/enhancer-binding protein (C/EBP) beta plays an important role in the interleukin 1beta-induced repression (Okazaki, K., Li, J., Yu, H., Fukui, N., and Sandell, L. J. (2002) J. Biol. Chem. 277, 31526-31533). The co-activators CREB-binding protein (CBP) and p300 are transcriptional co-regulators that participate in the activities of many different transcription factors including C/EBP. Here we show that CBP/p300 can reverse the inhibitory effect of C/EBP and moreover can stimulate expression of Cd-rap. The mechanism of this effect is shown to involve a unique synergy whereby CBP/p300 stimulate Cd-rap gene expression by at least two mechanisms. First, binding of CBP/p300 to C/EBPbeta leads to sequestration of C/EBP eliminating DNA binding and subsequent repression; second, binding of CBP/p300 to the transcriptional activator Sox9 increases Sox9 DNA binding to the Cd-rap promoter leading to further stimulation of gene transcription. This is an example of a complementary transcriptional network whereby two very different mechanisms act together to confer a functional increase in transcription. This new paradigm is likely generally applicable to cartilage genes as Col2a1 cartilage collagen gene responds similarly.

Chordin-like cysteine-rich (CR) repeats (also referred to as von Willebrand factor type C (VWC) modules) have been identified in approximately 200 extracellular matrix proteins. These repeats, named on the basis of amino acid conservation of 10 cysteine residues, have been shown to bind members of the transforming growth factor-beta (TGF-beta) superfamily and are proposed to regulate growth factor signaling. Here we describe the intramolecular disulfide bonding, solution structure, and dynamics of a prototypical chordin-like CR repeat from procollagen IIA (CR(ColIIA)), which has been previously shown to bind TGF-beta1 and bone morphogenetic protein-2. The CR(ColIIA) structure manifests a two sub-domain architecture tethered by a flexible linkage. Initial structures were calculated using RosettaNMR, a de novo prediction method, and final structure calculations were performed using CANDID within CYANA. The N-terminal region contains mainly beta-sheet and the C-terminal region is more irregular with the fold constrained by disulfide bonds. Mobility between the N- and C-terminal sub-domains on a fast timescale was confirmed using NMR relaxation measurements. We speculate that the mobility between the two sub-domains may decrease upon ligand binding. Structure and sequence comparisons have revealed an evolutionary relationship between the N-terminal sub-domain of the CR module and the fibronectin type 1 domain, suggesting that these domains share a common ancestry. Based on the previously reported mapping of fibronectin binding sites for vascular endothelial growth factor to regions containing fibronectin type 1 domains, we discuss the possibility that this structural homology might also have functional relevance.

Type II collagen, the most abundant protein of cartilage matrix, is synthesized as a procollagen molecule including the N-(PIINP) and C-(PIICP) propeptides at each end. Type II procollagen is produced in two forms as the result of alternative RNA splicing. One form (IIA) includes and the other form (IIB) excludes a 69-amino acid cysteine-rich globular domain encoded by exon 2 in PIINP. During the process of synthesis, these N-propeptides are removed by specific proteases and released in the circulation, and their levels are believed to reflect type II collagen synthesis. In this chapter we describe the development of a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of the IIA form of PIINP (PIIANP) in serum based on a polyclonal antibody raised against recombinant human exon 2 fusion protein of type II procollagen. We show that this ELISA is highly specific for circulating PIIANP and has adequate technical precision. In patients with knee osteoarthritis and rheumatoid arthritis, serum PIIANP was decreased by 53% (p < 0.0001) and 35% (p < 0.001), respectively, suggesting that type IIA collagen synthesis is altered in these arthritic diseases. The measurement of serum PIIANP may be useful for the clinical investigation of patients with joint diseases.

The processes of apoptosis and epithelial-mesenchymal transformation have been identified as two major mechanisms by which secondary palatal shelves achieve fusion. The aim of this study was to investigate alterations in these mechanisms by changing the physical distance between paired palatal shelves in an in vitro model of palatogenesis. Wild-type palatal pairs were dissected from E13.5 CD1 mouse embryos and allowed to grow in tissue culture for 48 hours at various intershelf distances. During the fusion process, medial edge epithelial cell fate was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, to evaluate apoptosis, and carboxyfluorescence (carboxy-2,7'-dichlorofluorescein diacetate succinimidyl ester) labeling, to measure transformation to mesenchymal cells. Palatal pairs separated in culture greater than or equal to 0.4 mm failed to fuse. TUNEL staining showed that the number of apoptotic cells in the palatal shelves increased as the intershelf distance increased, becoming marked in shelves that did not achieve fusion. The amount of epithelial-mesenchymal transformation, however, decreased with increasing intershelf distance. These results suggest that the contribution of epithelial-mesenchymal transformation and apoptosis to palatal shelf development and fusion can be altered by physical proximity. Therefore, one mechanism behind clefting in utero may result from an imbalance in epithelial-mesenchymal transformation and apoptosis as observed in vitro where palatal shelves are challenged to fuse by physical separation. This effect could be significant in the understanding and treatment of developmental palatal abnormalities. Perhaps in utero manipulation of intershelf spacing or epithelial-mesenchymal transformation and/or apoptosis could reverse the clefting paradigm.

This study investigated the role of transcription factor AP-2α in chondrocyte differentiation in vitro. AP-2α mRNA declined during differentiation, and overexpression of AP-2α inhibited differentiation. The results demonstrated that AP-2α plays a negative role in chondrocyte differentiation. Introduction: Transcription factor AP-2α has been detected in growth plate and articular chondrocytes and has been shown to regulate cartilage matrix gene expression in vitro. However, the precise functional role of AP-2α in chondrocyte differentiation is not known. In this study, we assessed the expression and the function of AP-2α in chondrocyte differentiation of ATDC5 cells. Materials and Methods: Chondrocyte differentiation of ATDC5 cells was induced with insulin or transforming growth factor β (TGF-β). Proteoglycan production was assessed by alcian blue staining, and expression levels of chondrocyte marker genes and AP-2 gene family were determined by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). Overexpression of AP-2α in ATDC5 cells was accomplished by retroviral infection. Infected cells were selected for G418 resistance and pooled for further analysis. Results and Conclusions: Quantitative real time RT-PCR analysis showed that among the four members of the AP-2 gene family, AP-2α mRNA was the most abundant. AP-2α mRNA levels progressively declined during the differentiation induced by either insulin or TGF-β treatment. Retroviral expression of AP-2α in ATDC5 cells prevented the formation of cartilage nodules, suppressed the proteoglycan production, and inhibited the expression of type II collagen, aggrecan, and type X collagen. Expression profile analysis of key transcription factors involved in chondrogenesis showed that overexpression of AP-2α maintained the expression of Sox9 but suppressed the expression of Sox5 and Sox6. Taken together, we provide, for the first time, molecular and cellular evidence suggesting that AP-2α is a negative regulator of chondrocyte differentiation.

Mice lacking type II collagen fail to develop intervertebral discs. The present study describes the distribution of the developmentally expressed type IIA procollagen molecule, as well as types I and III collagens, in human IV disc specimens ranging from 42 to 101 days gestation. Type IIA procollagen contains the alternatively spliced exon 2 which encodes a 69-amino-acid cysteinerich domain. By radioactive in situ hybridization and fluorescence immunohistochemistry, we identified changes in the localization patterns of type IIA procollagen, particularly between days 54 and 101. At day 54, the developing disc was divided into the outer annulus containing types I and III collagens, the inner annulus containing type IIA procollagen and the notochord consisting of all three fibrillar collagens. Specifically, the IIA N-terminal propeptide was localized in the extracellular matrix at day 54 but, by day 101, was only observed in the cytoplasm of the inner annulus cells. A functional role for the IIA N-terminal propeptide during this specific stage of disc development seems apparent. This function may involve regulation of growth factors since the exon 2-encoded domain of type IIA procollagen has previously been shown to bind to bone morphogenetic protein-2 and transforming growth factor-β. We aim to explore this mechanism further.

The control of extracellular matrix (ECM) production is important for the development, maintenance, and repair of cartilage tissues. Matrix molecule synthesis is generally regulated by the rate of gene transcription determined by DNA transcription factors. We have shown that transcription factors Sox9, AP-2, and [delta]EF1 are able to alter the rate of CD-RAP transcription in vitro: Sox9 upregulates, AP-2 exhibits biphasic effects, and [delta]EF1 represses expression of the CD-RAP gene. To correlate these in vitro activities in vivo, transcription factors were co-immunolocalized with ECM proteins in three different cartilage tissues in which the rates of biosynthesis are quite different: articular, meniscal, and growth plate. Immunoreactivities of type II collagen and CD-RAP were higher in growth plate than in either the articular or meniscal cartilages and correlated positively with Sox9 protein. Sox9 staining decreased with hypertrophy and was low in articular and meniscal cartilages. In contrast, AP-2 and [delta]EF1 were low in proliferating chondrocytes but high in lower growth plate, articular, and meniscal cartilages. This increase was also accompanied by intense nuclear staining. These immunohistochemical results are the first to localize both [delta]EF1 and AP-2 to adult articular, meniscal, and growth plate cartilages and provide in vivo correlation of previous molecular biological studies.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a new protein that was isolated from bovine articular chondrocytes and human melanoma cell lines (melanoma inhibitory protein or MIA). In normal tissue its expression is limited to cartilage, and in morbid tissue to melanoma, chondrosarcoma, and breast cancer. Serum levels of CD-RAP/MIA correlate with the progression of malignant melanoma, but there have been no reports on chondrosarcoma. Here, it was first demonstrated by RT-PCR and immunohistological methods that CD-RAP was expressed in tissue from a Swarm rat chondrosarcoma that was used as an experimental model. The course following tumor transplantation and changes in serum CD-RAP after tumor excision were then observed to investigate whether serum CD-RAP could be used as a marker of tumor activity. Consequently, serum CD-RAP in control rats tended to decrease as the animal grew, whereas it rose in proportion to tumor proliferation in rats that had received a tumor graft. Serum CD-RAP levels dropped rapidly following excision of the tumor in a group of tumor-excised rats. In those rats which had a recurrence following excision of the tumor, serum CD-RAP rose prior to the appearance of the tumor. Serum CD-RAP thus sensitively reflected tumor onset and proliferation, so that it appeared to be an effective marker of tumor activity for Swarm rat chondrosarcoma.

Knowledge of how growth factors are regulated in the extracellular matrix is crucial for understanding processes of tissue development and repair. A complex series of processes occurs in the extracellular matrix to control the activity and distribution of bone morphogenetic proteins (BMPs), which are important factors during cartilage and bone development. It is known that tissue regeneration is a recapitulation of processes in embryonic development and morphogenesis. Thus, in the case of cartilage repair during osteoarthritis, we can apply our knowledge of BMP function/regulation during embryogenesis to developing therapeutic strategies that will induce and even prolong natural tissue repair.

Cartilage is comprised of a large amount of functional extracellular matrix that is made and maintained by a small number of chondrocytes, the sole resident cell type. Normal cartilage exists in a relatively steady state: that is, the anabolic processes (those that result in the synthesis of cartilage matrix components) are in equilibrium with the catabolic processes (those that result in the normal turnover of matrix molecules). If the functional extracellular matrix is disturbed by physical or molecular means, the cells respond in an attempt to repair the matrix. This stimulated activity does not result in repair due to the extent and complexity of the extracellular matrix. Eventually, the newly synthesized and activated catabolic enzymes degrade the matrix components. This review presents the cellular and molecular mechanisms that account for this activity and provides some possible solutions.

In many embryonic tissues, type IIA procollagen is synthesized and deposited into the extracellular matrix containing the NH(2)-propeptide, the cysteine-rich domain of which binds to bone morphogenic proteins. To investigate whether matrix metalloproteinases (MMPs) synthesized during development and disease can cleave the NH(2) terminus of type II procollagens, we tested eight types of enzymes. Recombinant trimeric type IIA collagen NH(2)-propeptide encoded by exons 1-8 fused to the lectin domain of rat surfactant protein D was used as a substrate. The latter allowed trimerization of the propeptide domain and permitted isolation by saccharide affinity chromatography. Although MMPs 1, 2, and 8 did not show cleavage, MMPs 3, 7, 9, 13, and 14 cleaved the recombinant protein both at the telopeptide region and at the procollagen N-proteinase cleavage site. MMPs 7 and 13 demonstrated other cleavage sites in the type II collagen-specific region of the N-propeptide; MMP-7 had another cleavage site close to the COOH terminus of the cysteine-rich domain. To prove that an MMP can cleave the native type IIA procollagen in situ, we demonstrated that MMP-7 removes the NH(2)-propeptide from collagen fibrils in the extracellular matrix of fetal cartilage and identified the cleavage products. Because the N-proteinase and telopeptidase cleavage sites are present in both type IIA and type IIB procollagens and the telopeptide cleavage site is retained in the mature collagen fibril, this processing could be important to type IIB procollagen and to mature collagen fibrils as well.

Study design The expression of cartilage-derived retinoic acid-sensitive protein (CD-RAP) was measured in cerebrospinal fluid from patients with spinal diseases. Objectives To quantify the levels of CD-RAP in human cerebrospinal fluid and to clarify its character. Summary of background data Cartilage-derived retinoic acid-sensitive protein is a newly discovered, secreted molecule that is expressed during the chondrogenesis phase of endochondral bone formation and in articular cartilage. In recent studies CD-RAP has been detected in the serum of patients with melanoma and breast cancer, and it has been used to monitor tumor activity. However, the function of CD-RAP is unknown, and the expression of CD-RAP in human cerebrospinal fluid has never been reported. Methods The concentration of CD-RAP in human cerebrospinal fluid was measured by enzyme-linked immunosorbent assay with antihuman CD-RAP antibodies. Cerebrospinal fluid samples were collected from two groups of patients. Group 1, the control group, consisted of 40 patients: 22 with trauma and 18 with gynecologic diseases. Group 2 consisted of 172 patients with spinal diseases: 5 with meningioma, 5 with neurinoma, 5 with arachnoid cyst, 30 with cervical spondylotic myelopathy, 35 with lumbar disc herniation, 56 with lumbar canal stenosis, and 36 with scoliosis. Results The concentration of CD-RAP in the control group was 16.5 +/- 8.3 ng/mL. The concentrations of CD-RAP in Group 2 were: 35.3 +/- 14.7 ng/mL in meningioma, 23.5 +/- 7.41 ng/mL in neurinoma, 26.0 +/- 22.2 ng/mL in arachnoid cyst, 41.7 +/- 22.3 ng/mL in cervical myelopathy, 27.8 +/- 14.7 ng/mL in lumbar disc herniation, 36.5 +/- 18.4 ng/mL in lumbar canal stenosis, and 13.4 +/- 7.48 ng/mL in scoliosis. The concentrations of CD-RAP in cervical myelopathy, lumbar canal stenosis, and lumbar disc herniation were significantly higher than in the control group (P Conclusions The CD-RAP concentration was low in the control group, whereas it was significantly higher in spinal diseases that cause spinal stenosis. CD-RAP is expressed in cerebrospinal fluid as a result of damage to or stressing of neural structures and could be a marker for spinal diseases.

The reaction patterns of chondrocytes in osteoarthritis can be summarized in five categories: (1) proliferation and cell death (apoptosis); changes in (2) synthetic activity and (3) degradation; (4) phenotypic modulation of the articular chondrocytes; and (5) formation of osteophytes. In osteoarthritis, the primary responses are reinitiation of synthesis of cartilage macromolecules, the initiation of synthesis of types IIA and III procollagens as markers of a more primitive phenotype, and synthesis of active proteolytic enzymes. Reversion to a fibroblast-like phenotype, known as "dedifferentiation", does not appear to be an important component. Proliferation plays a role in forming characteristic chondrocyte clusters near the surface, while apoptosis probably occurs primarily in the calcified cartilage.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a secreted protein primarily expressed in chondrocytes. Pathologically, CD-RAP is detected in melanoma, chondrosarcoma and breast cancer. As an approach to define the transcriptional regulatory domains responsible for induction of chondrocyte activity in vivo, we generated transgenic mice harboring various fragments of the mouse CD-RAP promoter linked to the Escherichia coli β-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining indicates that 2251 bp of the CD-RAP 5′-flanking sequence generates β-galactosidase activity in all cartilage in embryos and adult animals. In addition, we also detected transient X-gal staining in mammary gland primordium from day 11.5 to 15.5 of gestation. Histological examination revealed that the transgene is located in the chondrocytes of cartilage and the epithelial cells of mammary buds. The cartilage transgene expression pattern is consistent with that of endogenous CD-RAP gene expression. The presence of β-galactosidase in the mammary buds led us to the demonstration of a unique pattern of transient endogenous expression of CD-RAP in the mammary bud. The finding of transient CD-RAP expression in mammary buds suggests that it may play a role in the organogenesis of mammary glands.

Type II procollagen is synthesized in long (type IIA) and short (type IIB) forms because of alternative splicing of mRNA; the long form containing an additional cysteine-rich domain in the amino-propeptide. An antiserum (IIA) that recognizes this domain was used for immunolocalization studies on adult bovine vitreous at light and electron microscopic levels and for Western blot analyses. The immunolocalization studies revealed labelling by the IIA antiserum of the vitreous collagen fibrils. This labelling was removed by prior extraction of the fibrils with 6 M guanidine hydrochloride (GuHCl) and the extract was shown to contain pN-type IIA procollagen. Adult vitreous collagen fibrils are coated with pN-type IIA procollagen, a finding with potential implications for vitreous collagen fibril structure and function.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) and mRNA were examined in the mouse fracture model by immunohistochemistry and Northern blot analysis and compared with the expression of type II collagen. We also studied the expression of the transcription factor Sox9, reported to enhance type II collagen and CD-RAP gene expression in vitro. CD-RAP was first detected in immature chondrocytes on day 5. Intense signals for CD-RAP were found in fracture cartilage on days 7 and 9. CD-RAP decreased at the phase of endochondral ossification. Throughout fracture healing, CD-RAP was detected in cartilage and not in bone or fibrous tissue, thus CD-RAP may be a molecular marker of cartilage formation during fracture healing. Northern blot analysis revealed similar changes in CD-RAP and type II collagen mRNA levels. However, with respect to protein levels, CD-RAP decreased faster than type II collagen implying the stability is lower than type II collagen. Increased levels of Sox9 mRNA and protein were detected on day 5 and coincided with the initial increase of CD-RAP and type II collagen mRNAs. Sox9 mRNA levels declined with the progress of chondrocyte hypertrophy, followed by a concomitant decrease in CD-RAP and type II collagen mRNA levels. These changes in Sox9 expression compared with the cartilage-specific genes (CD-RAP and type II collagen) suggest that cell differentiation during fracture healing may be controlled by specific transcriptional factors which regulate phenotypic changes of the cells.

Objective. To define the relative contributions of procollagen IIA and IIB messenger RNA (mRNA) in the 8-fold elevation of total (IIA + IIB) type II procollagen mRNA in cartilage from joints with early experimental osteoarthritis (OA). Methods. Unilateral cruciate ligament transection was performed on skeletally mature animals. Total RNA was extracted from articular cartilage from control joints and from joints with experimental OA. Northern blot hybridization with oligoprobes was used to distinguish between types IIA and IIB mRNA. Results. Levels of IIA mRNA were similar in control and experimental cartilage. Type IIB mRNA accounted for the increase in total type II mRNA expression in OA joints. Conclusion. Although OA chondrocytes adapt to making immature forms of aggrecan proteoglycan, they do not revert to the prechondrocyte stage characterized by IIA mRNA expression.

Knowledge about the genes that encode extracellular matrix (ECM) proteins is expanding every day. Genes encoding long-known matrix proteins are being isolated while new genes for ECM proteins are being discovered using techniques that directly target the mRNA population. The regulation of expression of most genes occurs through the promoter domain upstream of the coding sequence, although this information may be supplemented by far upstream DNA sequences or DNA in the introns of the gene. Since this important information is embedded in the DNA, the study of gene structure is crucial to the analysis of gene regulation. With all of this new information, it will be possible to characterize common elements in genes that are coordinately regulated, and eventually target individual genes for control by exogenous agents such as drugs.

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum. mRNAs for aggrecan, types I and II collagen, and beta-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by 35SO4 uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.

The developmental sequence of the embryonic joint has been well studied morphologically. There are, however, no definitive studies of cell function during joint development. In order to begin to understand the differentiation events that contribute to joint formation, we examined the expression of collagen mRNAs encoding types I, IIA, IIB, and XI. In situ hybridization was performed on chicken embryo hind limb buds and digits from day 7 to day 18 (Hamburger and Hamilton stages 31-44). In the day 7 (stage 31) limb bud, there was a condensation of mesenchyme forming the primitive tarsal and metatarsal bones that showed abundant expression of type IIA procollagen message, but no type IIB or type alpha 1(XI) message. By day 8 (stage 33), co-expression of types IIA, and type XI procollagen mRNAs was observed in the condensations, with expression of IIB restricted to early chondrocytes with metachromatically staining matrix. At this stage, DNA fragmentation characteristic of apoptosis was observed in cells near the midline of the interzone region between the developing anlagen, and in areas between and around the individual digits of the paddle. The presumptive apoptotic cells were more numerous at day 9 (stage 35), and were not found in the developing joint at subsequent time points, including the initiation of spatial cavitation of the joint. From days 11-18, type IIA procollagen mRNA was expressed in flattened cells at the surface of the anlagen, and in the perichondrium and in the developing joint capsule; type IIB mRNA message was found only in chondrocytes. Type XI mRNA was expressed by all type II-expressing cells. Alpha 1(I) mRNA was expressed early by cells of the interzone and capsule, but as cavitation progressed, the type I expressing cells of the interzone merged with the superficial layer of the articular surface. Thus, at the time of joint cavitation, there was a distinct pattern of expression of procollagen messages at the articular surface, with type I being outermost, followed by morphologically similar cells expressing type IIA, then chondrocytes expressing type IIB. The progenitor cells expressing type IIA message define a new population of cells. These cell populations contribute to the molecular heterogeneity of the articular cartilage, and these same populations likely exist in the developing joints of other species. The transient transcription of type II and type XI collagen genes, characteristic of chondrocytes, by cells in the joint capsule demonstrates that these cells may have chondrogenic potential.