tretinoin and Osteoarthritis

Osteoarthritis (OA) is a highly prevalent debilitating joint disease for which there are currently no licensed disease-modifying treatments. The pathogenesis of OA is complex, involving genetic, mechanical, biochemical, and environmental factors. Cartilage injury, arguably the most important driving factor in OA development, is able to activate both protective and inflammatory pathways within the tissue. Recently, >100 genetic risk variants for OA have been identified through Genome Wide Association Studies, which provide a powerful tool to validate existing putative disease pathways and discover new ones. Using such an approach, hypomorphic variants within the aldehyde dehydrogenase 1 family member A2 (

Topics: Cartilage; Cartilage, Articular; Genome-Wide Association Study; Humans; Osteoarthritis; Retinoids; Tretinoin

More than 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease-modifying treatments for this disabling condition. Common polymorphic variants in

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Anti-Inflammatory Agents; Cartilage, Articular; Chondrocytes; Knee Joint; Mice; Osteoarthritis; Retinal Dehydrogenase; Tretinoin

To investigate the role of activated protein C (APC) in cartilage degradation.. Chondrocyte expression of protein C, endothelial protein C receptor (EPCR), and thrombomodulin (TM) were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). APC was immunolocalized in developing joints and in osteoarthritic (OA) cartilage from humans. The effect of APC on aggrecan and collagen degradation was examined in explant cultures of ovine cartilage in control cultures and in cultures stimulated with interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNFalpha), or retinoic acid (RetA), using colorimetric assays and Western blotting. Chondrocyte expression of matrix metalloproteinases (MMPs), ADAMTS, and tissue inhibitor of metalloproteinases (TIMPs) was measured by RT-PCR. MMP-2 and MMP-9 activity was evaluated by gelatin zymography and MMP-13 by fluorogenic assay.. Positive cellular immunostaining for APC was found at sites of MMP activity in developing joints and in OA, but not normal, cartilage. Chondrocytes expressed messenger RNA for protein C, EPCR, and TM, with the latter 2 levels increased by IL-1alpha and TNFalpha stimulation. APC augmented aggrecan release and initiated collagen breakdown in IL-1alpha-treated and TNFalpha-treated cartilage, but not in normal or in RetA-treated cartilage. APC-stimulated aggrecan and collagen breakdown were due to MMP activity but were not associated with modulation of MMP, ADAMTS, or TIMP expression. APC resulted in MMP-13 activation in cartilage cultures. APC could not directly activate proMMP-13, but it was associated with increased MMP-2 and MMP-9 activity.. APC may be a relevant activator of MMPs in cartilage and may play a role in progressive cartilage degradation in arthritis.

Topics: Aggrecans; Animals; Antigens, CD; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagen; Disease Models, Animal; Endothelial Protein C Receptor; Humans; Interleukin-1alpha; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Osteoarthritis; Protein C; Proteoglycans; Receptors, Cell Surface; RNA, Messenger; Sheep; Thrombomodulin; Tretinoin; Tumor Necrosis Factor-alpha

In order to elucidate the mechanism of cartilage degradation in osteoarthritis (OA), we established a cell assay system. Under the stimulation of all-trans retinoic acid (ATRA), the human chondrosarcoma cell line HCS-2/8 increased proteoglycan release from inactivated bovine nasal cartilage (BNC) and the results suggested the involvement of membrane-bound metalloproteinase(s). Therefore, we focused on the induction of a disintegrin and metalloproteinase (ADAM) superfamily upon ATRA stimulation. Of all ADAMs tested, only ADAM28 was induced by ATRA in HCS-2/8 cells and also in human primary chondrocytes. We found that transfection of ADAM28 or its alternatively spliced soluble form augmented proteoglycan release in the cell assay; however, a mutant soluble form in which a portion of the disintegrin domain was deleted did not have proteoglycan-releasing activity, implying the importance of the domain for enzyme localization and substrate recognition for cartilage degradation in OA.

Topics: ADAM Proteins; Alternative Splicing; Animals; Biological Assay; Cattle; Cell Line; Chlorocebus aethiops; Chondrocytes; COS Cells; Humans; Metalloproteases; Osteoarthritis; Phylogeny; Proteoglycans; Transfection; Tretinoin

To evaluate the expression and function of the orphan nuclear receptor Rev-ErbAalpha in articular cartilage and to investigate its role in osteoarthritis (OA).. Expression of Rev-ErbAalpha was analyzed at both the messenger RNA and protein levels in human and bovine articular cartilage and chondrocytes by real-time polymerase chain reaction (TaqMan) and immunocytochemical techniques. The effects of cartilage catabolic and anabolic agents on the expression of Rev-ErbAalpha were evaluated by TaqMan analysis. Overexpression was achieved by either adenoviral transduction or treatment with a peroxisome proliferator-activated receptor alpha agonist, whereas expression was suppressed by antisense oligonucleotides.. Among the 48 known nuclear receptors, Rev-ErbAalpha was found to be the most highly expressed in OA cartilage. It is known to function as a transcription repressor. Treatment of articular chondrocytes with known catabolic agents resulted in the induction of Rev-ErbAalpha, whereas stimulation with anabolic agents led to a decrease in expression. Overexpression of the nuclear receptor was associated with an increase in the expression of matrix-degrading enzymes such as matrix metalloproteinase 13 and aggrecanase. In contrast, a decrease in Rev-ErbAalpha expression led to a concomitant reduction in the activity of matrix-degrading enzymes.. This study is the first to demonstrate that Rev-ErbAalpha is highly expressed in OA articular chondrocytes and that its expression is modulated by known cartilage catabolic and anabolic stimuli. We also demonstrated that modulation of Rev-ErbAalpha expression in chondrocytes may be a novel means of regulating the expression and production of multiple matrix-degrading enzymes. These observations suggest that Rev-ErbAalpha may be a novel therapeutic target for OA.

Topics: ADAM Proteins; ADAMTS5 Protein; Animals; Antineoplastic Agents; Cartilage; Cattle; Cells, Cultured; Chondrocytes; DNA-Binding Proteins; Down-Regulation; Extracellular Matrix Proteins; Gene Expression; Humans; Insulin-Like Growth Factor I; Interleukin-1; Matrix Metalloproteinase 13; Nuclear Receptor Subfamily 1, Group D, Member 1; Oligonucleotides, Antisense; Osteoarthritis; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transforming Growth Factor beta; Tretinoin; Tumor Necrosis Factor-alpha; Up-Regulation

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.

Topics: Alkaline Phosphatase; Amino Acid Sequence; Animals; Anthraquinones; Biological Transport; Blotting, Northern; Cartilage; Cell Differentiation; Cells, Cultured; Chickens; Chondrocytes; Cloning, Molecular; Diphosphates; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Immunoblotting; Immunohistochemistry; Membrane Proteins; Models, Biological; Molecular Sequence Data; Osteoarthritis; Phosphates; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sequence Homology, Amino Acid; Signal Transduction; Time Factors; Transcription Factor Pit-1; Transcription Factors; Tretinoin; Up-Regulation

To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA).. We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated.. ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE(373-374)ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines.. Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE(373-374)ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.

Topics: ADAM Proteins; ADAMTS4 Protein; Aggrecans; Animals; Cartilage, Articular; Disease Progression; Extracellular Matrix Proteins; Interleukin-1; Lectins, C-Type; Metalloendopeptidases; Mice; Mice, Knockout; Mice, Transgenic; Osteoarthritis; Procollagen N-Endopeptidase; Proteoglycans; Tretinoin

Altered chondrocyte differentiation, including development of chondrocyte hypertrophy, mediates osteoarthritis and pathologic articular cartilage matrix calcification. Similar changes in endochondral chondrocyte differentiation are essential for physiologic growth plate mineralization. In both articular and growth plate cartilages, chondrocyte hypertrophy is associated with up-regulated expression of certain protein-crosslinking enzymes (transglutaminases (TGs)) including the unique dual-functioning TG and GTPase TG2. Here, we tested if TG2 directly mediates the development of chondrocyte hypertrophic differentiation. To do so, we employed normal bovine chondrocytes and mouse knee chondrocytes from recently described TG2 knockout mice, which are phenotypically normal. We treated chondrocytes with the osteoarthritis mediator IL-1 beta, with the all-trans form of retinoic acid (ATRA), which promotes endochondral chondrocyte hypertrophy and pathologic calcification, and with C-type natriuretic peptide, an essential factor in endochondral development. IL-1 beta and ATRA induced TG transamidation activity and calcification in wild-type but not in TG2 (-/-) mouse knee chondrocytes. In addition, ATRA induced multiple features of hypertrophic differentiation (including type X collagen, alkaline phosphatase, and MMP-13), and these effects required TG2. Significantly, TG2 (-/-) chondrocytes lost the capacity for ATRA-induced expression of Cbfa1, a transcription factor necessary for ATRA-induced chondrocyte hypertrophy. Finally, C-type natriuretic peptide, which did not modulate TG activity, comparably promoted Cbfa1 expression and hypertrophy (without associated calcification) in TG2 (+/+) and TG2 (-/-) chondrocytes. Thus, distinct TG2-independent and TG2-dependent mechanisms promote Cbfa1 expression, articular chondrocyte hypertrophy, and calcification. TG2 is a potential site for intervention in pathologic calcification promoted by IL-1 beta and ATRA.

Topics: Alkaline Phosphatase; Animals; Calcinosis; Cartilage, Articular; Cattle; Cells, Cultured; Chondrocytes; Collagen Type X; Collagenases; Core Binding Factor Alpha 1 Subunit; Extremities; Gene Expression; GTP-Binding Proteins; Hypertrophy; Interleukin-1; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Mice, Knockout; Natriuretic Peptide, C-Type; Neoplasm Proteins; Osteoarthritis; Protein Glutamine gamma Glutamyltransferase 2; Transcription Factors; Transglutaminases; Tretinoin

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1 alpha or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.

Topics: ADAM Proteins; ADAMTS5 Protein; Aggrecans; Amino Acid Sequence; Animals; Arthritis, Rheumatoid; Blotting, Western; Cartilage; Cattle; Culture Media, Conditioned; Extracellular Matrix Proteins; Gene Expression Regulation; Heparin; Humans; Interleukin-1; Lectins, C-Type; Metalloendopeptidases; Molecular Weight; Osteoarthritis; Peptide Fragments; Proteoglycans; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, Protein; Synovial Membrane; Tretinoin

To assess the presence of fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion.. Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis.. MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor alpha stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13.. These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.

Topics: Aged; Animals; Base Sequence; Blotting, Northern; Cartilage, Articular; Collagenases; DNA Primers; Gene Expression; Humans; Interleukin-1; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Matrix Metalloproteinase 8; Middle Aged; Osteoarthritis; Polymerase Chain Reaction; RNA, Messenger; Swine; Tretinoin; Tumor Necrosis Factor-alpha

This article demonstrates that both the bulk water self-diffusion coefficient (D) and the spatially resolved variation in D for lesion canine cartilage due to osteoarthritis is increased by about 25% over that of surrounding cartilage. This increase in D can be mimicked by enzymatic degradation of cartilage with trypsin, hyaluronidase, and collagenase, or by mechanical means. However, it is established here using excised disks of living cartilage whose proteoglycan and collagen contents were manipulated by biochemical intervention in tissue culture that the diffusion measurement is not sensitive to the proteoglycan content of cartilage. Instead, self-diffusion appears to monitor mesoscopic (nonspecific) tissue damage. These results show that D, measured in a spatially resolved manner by pulsed field gradient nuclear magnetic resonance imaging, can localize regions of cartilage degradation.

Topics: Animals; Cartilage, Articular; Collagen; Diffusion; Dogs; Hyaluronoglucosaminidase; In Vitro Techniques; Male; Osteoarthritis; Proteoglycans; Tretinoin; Trypsin; Water

We studied whether cyclic loading is harmful to degraded cartilage. Sets of four cartilage-bearing sesamoid bones were dissected from 5-year old cows. One bone from each set was cultured for 17 h in control medium to serve as an ex vivo control. The three others were cultured for 1 week in control medium to which 0, 10 or 300 ng/mL retinoic acid (RAc), which depletes the cartilage matrix of proteoglycans, had been added. Two were then cultured for another week in control medium. During the last week, one of the two was subjected to a cyclic load (1 MPa, 0.2 Hz). Following treatment with RAc, glycosaminoglycan content and synthesis were significantly decreased, as confirmed by safranin O staining and autoradiography. They were further diminished by loading during the second week of culture. Increased amounts of 3-B-3(-)epitope were found in cartilage that had been treated with 300 ng/mL RAc and then loaded. While loading cartilage matrix that was only slightly degraded proved to be damaging, loading severely degraded cartilage matrix apparently induced osteoarthritic-like changes.

Topics: Animals; Autoradiography; Cartilage, Articular; Cattle; Cells, Cultured; Extracellular Matrix; Female; Glycosaminoglycans; Keratolytic Agents; Osteoarthritis; Phenazines; Stress, Mechanical; Tretinoin; Weight-Bearing

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNF alpha, and LPS strongly induced expression of LIF/D-factor mRNA in both clonal MC3T3-E1 cells and primary osteoblast-like cells. Neither parathyroid hormone nor 1 alpha,25-dihydroxyvitamin D3 stimulated expression of LIF/D-factor mRNA. LIF/D-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of LIF/D-factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other bone-resorbing cytokines such as IL-1 and IL-6 involved in the bone resorption seen in the joints of RA patients. LIF/D-factor specifically bound to MC3T3-E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D-factor dose-dependently suppressed incorporation of [3H]thymidine into MC3T3-E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors.

Topics: Acid Phosphatase; Animals; Arthritis, Rheumatoid; Bone and Bones; Bone Resorption; Cells, Cultured; Dose-Response Relationship, Drug; Female; Growth Inhibitors; Interleukin-1; Interleukin-6; Leukemia Inhibitory Factor; Lipopolysaccharides; Lymphokines; Mice; Mice, Inbred C57BL; Osteoarthritis; Osteoblasts; Pregnancy; RNA, Messenger; Synovial Fluid; Tretinoin; Tumor Necrosis Factor-alpha

The aim of this study was to test the hypothesis that fibronectin in cartilage of osteoarthritic joints accumulates in increased quantities in response to a reduction in proteoglycan content in the matrix. We determined fibronectin content in an ELISA system, uptake of plasma 125I-fibronectin from the medium, and glycosaminoglycan content by a dye method. The proteoglycan content of canine articular cartilage was reduced by co-incubation of the cartilage with canine synovium, partially purified catabolin, or retinoic acid. Data suggested that proteoglycan loss alone was not sufficient to result in the increased fibronectin accumulation that has been reported in the deteriorating articular cartilage of osteoarthritic joints. Cartilage that lost as much as 75% of its proteoglycan content did not have increased fibronectin content and did not take up more fibronectin from the external medium. A related goal of this study was to evaluate the percentage of water in proteoglycan-depleted but disease-free cartilage, as the percentage of water is known to be elevated in osteoarthritic cartilage. Data indicated that the percentage of water in the depleted cartilage increased in proportion to the decrease in proteoglycan content, but the total amount of water in the cartilage did not increase; water content appeared to be unchanged. Under the conditions used, the proteoglycan content was reduced and the percentage of water was elevated, but fibronectin content was not increased. Thus, the cartilage incubated in vitro did not mimic osteoarthritic cartilage.

Topics: Animals; Body Water; Cartilage, Articular; Culture Media; Culture Techniques; Dogs; Ethanol; Extracellular Matrix; Fibronectins; Glycosaminoglycans; Iodine Radioisotopes; Osteoarthritis; Proteoglycans; Synovial Membrane; Tretinoin

Independent studies have previously shown that mononuclear cell supernatants stimulate the release of plasminogen activator and latent collagenase from synovial cell monolayer cultures. Simultaneous secretion of these enzymes could be an important pathway for tissue destruction under inflammatory conditions, since plasminogen activator can cause activation of latent collagenase in the presence of plasminogen. We investigated the kinetics of release of the two enzymes from synovial cells in response to the addition of mononuclear cell supernatants and retinoic acid. Synovial cells derived from osteoarthritic and rheumatoid arthritic patients responded similarly. Plasminogen activator is released within a few hours of stimulation, and secretion usually stops when the stimulus is removed. In contrast, significant amounts of collagenase are secreted only after an initial lag period of 1--2 days, and secretion is sustained long after removal of mononuclear cell supernatant. Another difference in regulation of the secretion of these two neutral proteinases is that the addition of all-trans retinoic acid to the same synovial cell culture elevates plasminogen activator secretion while suppressing that of latent collagenase. Differential regulation of these enzymes under conditions of chronic inflammation may allow for continual accumulation of latent enzyme(s) which are activated during short periods of plasminogen activator release.

Topics: Arthritis, Rheumatoid; Blood Proteins; Cells, Cultured; Culture Media; Humans; In Vitro Techniques; Microbial Collagenase; Monocytes; Osteoarthritis; Plasminogen Activators; Synovial Membrane; Tretinoin