Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1α in articular chondrocytes: involvement of HIF-1α in the pathogenesis of osteoarthritis

Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis – including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production – under hypoxic conditions. We speculated that HIF-1α may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1α mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1β and H(2)O(2 )induce the expression of HIF-1α in OA chondrocytes under normoxic and hypoxic conditions (O(2 )<6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1α-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1α mRNA was higher in the degenerated regions than in the intact regions. Both IL-1β and H(2)O(2 )accelerated mRNA and protein levels of HIF-1α in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1α expression. HIF-1α-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1α-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1α expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1β, and oxidative stress induce the expression of HIF-1α in chondrocytes. Our results suggest an important role of stress-induced HIF-1α in the maintenance of chondrocyte viability in OA articular cartilage

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H(2)O(2)) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage

Sphingosine-1-phosphate attenuates proteoglycan aggrecan expression via production of prostaglandin E(2 )from human articular chondrocytes

BACKGROUND: Sphingosine-1-phosphate (S1P), a downstream metabolite of ceramide, induces various bioactivities via two distinct pathways: as an intracellular second messenger or through receptor activation. The receptor for S1P (S1PR) is the family of Endothelial differentiation, sphingolipid G-protein-coupled receptor (EDG). We have here attempted to reveal the expression of EDG/S1PR in human articular chondrocytes (HAC), exploring the implications of S1P in cartilage degradation. METHODS: Articular cartilage specimens were obtained from patients with rheumatoid arthritis (RA), osteoarthritis (OA) or traumatic fracture (representing normal chondrocytes) who underwent joint surgery. Isolated HAC were cultured in vitro by monolayer and stimulated with S1P in the presence or absence of inhibitors of signaling molecules. Stimulated cells and culture supernatants were collected and subjected to analyses using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). RESULTS: All of the tested HAC samples showed positive results in terms of EDG/S1PR expression in basal condition. When HAC was stimulated with S1P, a significant increase in prostaglandin (PG) E(2 )production was observed together with enhanced expression of cyclooxygenase (COX)-2. S1P stimulated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in HAC, and the PGE(2 )induction was abrogated by PD98059 and SB203580. Pertussis toxin inhibited the PGE(2 )induction from HAC by S1P, suggesting an essential role for Gi protein. S1P also attenuated the expression of proteoglycan aggrecan, a component of cartilage matrix, in HAC at transcriptional level. CONCLUSION: It was suggested that the S1P-induced PGE(2 )was at least in part involved in the aggrecan-suppressing effect of S1P, seeing as COX inhibitors attenuated the effect. Accordingly, S1P might play an important role in cartilage degradation in arthritides