Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes.
ABSTRACT: Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment.
Project description:Berberine, a plant alkaloid used in Chinese medicine, has broad cell-protective functions in a variety of cell lines. Chondrocyte apoptosis contributes to the pathogenesis of cartilage degeneration in osteoarthritis (OA). However, little is known about the effect and underlying mechanism of berberine on OA chondrocytes. Here, we assessed the effects of berberine on cartilage degeneration in interleukin-1? (IL-1?)-stimulated rat chondrocytes and in a rat model of OA. The results of an MTT assay and western blotting analysis showed that berberine attenuated the inhibitory effect of IL-1? on the cell viability and proliferating cell nuclear antigen expression in rat chondrocytes. Furthermore, berberine activated Akt, which triggered p70S6K/S6 pathway and up-regulated the levels of aggrecan and Col II expression in IL-1?-stimulated rat chondrocytes. In addition, berberine increased the level of proteoglycans in cartilage matrix and the thickness of articular cartilage, with the elevated levels of Col II, p-Akt and p-S6 expression in a rat OA model, as demonstrated by histopathological and immunohistochemistry techniques. The data thus strongly suggest that berberine may ameliorate cartilage degeneration from OA by promoting cell survival and matrix production of chondrocytes, which was partly attributed to the activation of Akt in IL-1?-stimulated articular chondrocytes and in a rat OA model. The resultant chondroprotective effects indicate that berberine merits consideration as a therapeutic agent in OA.
Project description:Osteoarthritis (OA) is a degenerative disease characterized by deterioration of articular cartilage. Recent studies have demonstrated the importance of some microRNAs in cartilage damage. The aim of this study was to identify and characterize the expression of microRNA-634 (miR-634) in normal and OA chondrocytes, and to determine its role in OA pathogenesis. Human normal and OA chondrocytes obtained from patients were cultured in vitro. Transfection with miR-634 mimic or inhibitor was employed to investigate the effect of miR-634 on chondrocyte survival and matrix synthesis, and to identify miR-634 target. The results indicated that miR-634 was expressed at lower level in high grade OA chondrocyte compared with normal chondrocytes. Overexpression of miR-634 could inhibit cell survival and matrix synthesis in high grade OA chondrocytes. Furthermore, miR-634 targeted PIK3R1 gene that encodes the regulatory subunit 1 of class I PI3K (p85?) and exerted its inhibitory effect on the phosphorylation of Akt, mTOR, and S6 signal molecules in high grade OA chondrocytes. Therefore, the data suggested that miR-634 could suppress survival and matrix synthesis of high grade OA chondrocytes through targeting PIK3R1 gene to modulate the PI3K/Akt/S6 and PI3K/Akt/mTOR/S6 axes, with important implication for validating miR-634 as a potential target for OA therapy.
Project description:Autophagy maintains cellular homoeostasis. The enhancement of autophagy in chondrocytes could prevent osteoarthritis (OA) progression in articular cartilage. Peroxisome proliferator-activated receptor α (PPARα) activation may also protect articular chondrocytes against cartilage degradation in OA. However, whether the protective effect of activated PPARα is associated with autophagy induction in chondrocytes is not determined. In this study, we investigated the effect of PPARα activation by its agonist, WY14643, on the protein expression level of Aggrecan and ADAMTS5, and the protein expression level of autophagy biomarkers, including LC3B and P62, using Western blotting analysis in isolated mouse chondrocytes pre-treated with lipopolysaccharides (LPS, mimicking OA chondrocytes) with or without the autophagy inhibitor chloroquine diphosphate salt. Furthermore, Akt and ERK phosphorylation was detected in LPS-treated chondrocytes in response to WY14643. In addition, the effect of intra-articularly injected WY14643 on articular cartilage in a mouse OA model established by the destabilization of the medial meniscus was assessed using the Osteoarthritis Research Society International (OARSI) histopathology assessment system, along with the detection of Aggrecan, ADAMTS5, LC3B and P62 protein levels using immunohistochemistry assay. The results indicated that PPARα activation by WY14643 promoted proteoglycan synthesis by autophagy enhancement in OA chondrocytes in vivo and in vitro concomitant with the elevation of Akt and ERK phosphorylation. Therefore, autophagy could contribute to the chondroprotection of PPARα activation by WY14643, with the implication that PPARα activation by WY14643 may be a potential approach for OA therapy.
Project description:Osteoarthritis (OA) is a common multifactorial degenerative articular disease among the aging population. The current investigation aimed to elucidate the function of microRNA-495 (miR-495) in the development of OA. We found that miR-495 was upregulated in the cartilage of OA patients. Transfection of a miR-495 mimic into rat primary chondrocytes, human chondrocytes (HC) and SW1353 chondrosarcoma cells inhibited AKT1 expression, proliferation and scratch wound closure and induced apoptosis. Transfection of a miR-495 inhibitor produced an opposite effect. Furthermore, the production of cartilage degeneration-related substances was modified by miR-495. Luciferase reporter gene assay revealed that AKT1 is directly repressed by miR-495. Moreover, the levels of AKT1, p-S6 and p-mTOR diminished in chondrocytes overexpressing miR-495. AKT1 overexpression amplified p-S6 and p-mTOR levels as well as abolished miR-495 mimic-induced apoptosis and inhibition of proliferation. In the surgically induced rat OA model, apoptosis of chondrocytes and cartilage degeneration were remedied by the administration of a miR-495 antagomir. Moreover, there was an increased expression of AKT1. These findings indicate that miR-495 induces OA by targeting AKT1 and regulating the AKT/mTOR pathway. Therefore, miR-495 may be a prospective target for OA treatment.
Project description:Aberrant Wnt signaling may contribute to osteoarthritis (OA) but the Wnt family members involved have not been fully identified. The purpose of this study was to investigate the role of Wnt5a as a potential mediator of cartilage destruction in OA.Immunohistochemistry to detect Wnt5a was performed using normal and OA human articular cartilage. Cultured normal human chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus or recombinant Wnt5a protein with or without pretreatment using a panel of signaling inhibitors. Expression of Wnt5a, anabolic genes and catabolic genes were determined by quantitative real-time PCR. Production of Wnt5a protein and matrix metalloproteinases (MMPs) as well as activation of signaling proteins were analyzed by immunoblotting.Wnt5a was present in human articular cartilage with OA changes and its expression and secretion were increased in FN-f stimulated chondrocytes. FN-f stimulated Wnt5a production through the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) pathways. Wnt5a reduced aggrecan gene expression after 48 h of treatment. Wnt5a seemed to promote MMP1, -3, and -13 expression as well as MMP1 and MMP13 protein production in normal human chondrocytes. Wnt5a inhibitor peptides did not affect FN-f induced MMP production. Wnt5a activated ?-catenin independent signaling including calmodulin-dependent protein kinase II (CaMKII), JNK, p38, ERK1/2, p65 and Akt. Inhibition of JNK, p38, ERK, PI-3 kinase and CaMKII by specific signaling inhibitors suppressed Wnt5a mediated MMP1 and MMP13 production.Wnt5a is present in human OA cartilage and can promote chondrocyte catabolic activity through non-canonical Wnt signaling, which suggests a potential role in OA.
Project description:Osteoarthritis (OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus. Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre ERT2 developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice. Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore, inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.
Project description:The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1(-/-) and Sulf-2(-/-) mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf(-/-) mice. Articular cartilage and cultured chondrocytes from Sulf(-/-) mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.
Project description:Wnt7a is a protein that plays a critical role in skeletal development. However, its effect on cartilage homeostasis under pathological conditions is not known. In this study, we found a unique inverse correlation between Wnt7a gene expression and that of MMP and IL-1? in individual human OA cartilage specimens. Upon ectopic expression in primary human articular chondrocytes, Wnt7a inhibited IL-1?-induced MMP and iNOS gene expression. Western blot analysis indicated that Wnt7a induced both canonical Wnt signaling and NFAT and Akt non-canonical signaling. Interestingly, inhibiting the canonical and Akt pathway did not affect Wnt7a activity. However, inhibiting the NFAT pathway impaired Wnt7a's ability to inhibit MMP expression, suggesting that Wnt7a requires NFAT signaling to exert this function. In vivo, intraarticular injection of lentiviral Wnt7a strongly attenuated articular cartilage damage induced by destabilization of the medial meniscus (DMM) OA-inducing surgery in mice. Consistently, Wnt7a also inhibited the progressive increase of joint MMP activity in DMM animals. These results indicate that Wnt7a signaling inhibits inflammatory stimuli-induced catabolic gene expression in human articular chondrocytes and is sufficient to attenuate MMP activities and promote joint cartilage integrity in mouse experimental OA, demonstrating a novel effect of Wnt7a on regulating OA pathogenesis.
Project description:The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLC?1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLC?1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLC?1, p-PLC?1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLC?1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLC?1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLC?1 on MMP-13 may be partly attributed to the inhibition of the PLC?1/mTOR axis on the PLC?1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLC?1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.
Project description:Degeneration of articular cartilage is central to the pathology of osteoarthritis (OA). However, the molecular mechanisms leading to these irreversible changes are still poorly understood. This study was undertaken to investigate how changes in the chondrocyte translational apparatus may contribute to the development and progression of knee OA.Articular cartilage from the knees of normal healthy subjects and patients with OA was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and nonlesional areas of the human OA cartilage were used to estimate the relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament of rats to investigate changes in the translational apparatus associated with OA. The role of interleukin-1? (IL-1?) signaling was assessed in vitro using rat articular chondrocytes. In human or rodent knee cartilage, messenger RNA expression was analyzed by quantitative polymerase chain reaction, and protein levels were determined by immunohistochemistry and Western blotting.Several novel traits of OA chondrocytes were identified, including up-regulation of the serine/threonine kinases Akt-2 and Akt-3 at the posttranscriptional level and an increased rate of total protein synthesis, likely attributable to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a known repressor of cap-dependent translation. Inactivation of 4E-BP1 was dependent on the activity of mechanistic target of rapamycin and was crucial for the up-regulation of protein synthesis in general and expression of matrix metalloproteinase 13 and ADAMTS-5 in particular. In addition, treatment of articular chondrocytes with IL-1? led to inactivation of 4E-BP1 and up-regulation of protein synthesis.Precise control of protein synthesis is vital for cartilage homeostasis, and its dysregulation contributes to the molecular pathology of OA. The results of this study therefore identify a novel set of potential therapeutic targets to ameliorate the effects of knee OA.