Expression In Human Chondrocytes Research Articles

Inhibitory Effect of a Tankyrase Inhibitor on Mechanical Stress-Induced Protease Expression in Human Articular Chondrocytes.

We investigated the effects of a Tankyrase (TNKS-1/2) inhibitor on mechanical stress-induced gene expression in human chondrocytes and examined TNKS-1/2 expression in human osteoarthritis (OA) cartilage. Cells were seeded onto stretch chambers and incubated with or without a TNKS-1/2 inhibitor (XAV939) for 12 h. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 8% elongation, 30 min) was applied and the gene expression of type II collagen a1 chain (COL2A1), aggrecan (ACAN), SRY-box9 (SOX9), TNKS-1/2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and matrix metalloproteinase-13 (MMP-13) were examined by real-time PCR. The expression of ADAMTS-5, MMP-13, nuclear translocation of nuclear factor-κB (NF-κB), and β-catenin were examined by immunocytochemistry and Western blotting. The concentration of IL-1β in the supernatant was examined by enzyme-linked immunosorbent assay (ELISA). TNKS-1/2 expression was assessed by immunohistochemistry in human OA cartilage obtained at the total knee arthroplasty. TNKS-1/2 expression was increased after CTS. The expression of anabolic factors were decreased by CTS, however, these declines were abrogated by XAV939. XAV939 suppressed the CTS-induced expression of catabolic factors, the release of IL-1β, as well as the nuclear translocation of NF-κB and β-catenin. TNKS-1/2 expression increased in mild and moderate OA cartilage. Our results demonstrated that XAV939 suppressed mechanical stress-induced expression of catabolic proteases by the inhibition of NF-κB and activation of β-catenin, indicating that TNKS-1/2 expression might be associated with OA pathogenesis.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

Galectin network in osteoarthritis: galectin-4 programs a pathogenic signature of gene and effector expression in human chondrocytes in vitro

Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1β (IL-1β) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.

RANKL expression in chondrocytes and its promotion by lymphotoxin-α in the course of cartilage destruction during rheumatoid arthritis.

We investigated the expression and localization of the receptor activator nuclear factor κB ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-α on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-α concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-α, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-α, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-α concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-α increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-α, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-α stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-α could promote the expression of RANKL in human chondrocytes.

Activation of NOTCH1 by Shear Force Elicits Immediate Cytokine Expression in Human Chondrocytes.

Osteoarthritis is caused by overloading of joints and is characterized by inflammation-induced disruption of cartilage structure. Current treatment strategy aims to relieve inflammation and prevent further deterioration of joint function. However, how mechanical force leads to inflammation and deterioration of chondrocyte function still remains incompletely understood. To explore the force-regulated molecular mechanism, an in vitro hydraulic shear force experiment to simulate the condition of force loading was required. The result demonstrated that multiple cytokines and immune regulators, including interleukin 8, interferon β, TRAF1 and TNFAIP3, were significantly increased by shear force within two hours of treatment. Moreover, JAG1 and HES1 were drastically upregulated as well, suggesting that NOTCH1 signaling is activated by shear force. Short-term expression of NOTCH1 intracellular domain activated a similar set of cytokines, indicating that NOTCH1 responds to shear force and activates downstream genes. When incubated under the medium conditioned by NOTCH1-activated chondrocyte, osteoblasts expressed higher levels of interferon β and interferon λ. Together, our results indicated that NOTCH1 functions as a force sensor and promotes expression of cytokines and immune regulators from shear-force bearing chondrocytes.

Magnolol Alleviates IL-1β-Induced Dysfunction of Chondrocytes Through Repression of SIRT1/AMPK/PGC-1α Signaling Pathway.

Osteoarthritis is a common chronic joint disease related with mitochondrial dysfunction, damage, and synthetic defects in chondrocytes. Magnolol is a lignin extracted from Magnolia officinalis with antioxidant and anti-inflammation functions. This study aims to investigate the function of magnolol on mitochondrial dysfunction, oxidative stress, and inflammation in human primary chondrocytes. Chondrocytes were stimulated with IL-1β to mimic the pathogenesis of osteoarthritis. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA was employed to examine the concentration of inflammatory cytokine IL-8. Protein expression of SIRT1/pAMPK/PGC-1α, metabolism-related proteins and Cox2 were examined by Western blot. Mitochondrial function, reactive oxygen species concentration, superoxide dismutase activity, and NF-κB activity were analyzed using commercial kit, respectively. We demonstrated that magnolol increased SIRT1/AMPK/PGC-1α expression in human chondrocytes. Magnolol could alleviate IL-1β-induced mitochondrial dysfunction and oxidative stress through SIRT1/AMPK/PGC-1α signaling pathway in human chondrocytes. In addition, magnolol maintained the anabolism and catabolism of extracellular matrix balance by SIRT1/AMPK/PGC-1α signaling pathway. Furthermore, magnolol alleviated IL-1β-induced inflammation in human chondrocytes. Magnolol alleviates IL-1β-induced dysfunction of chondrocytes through repressing SIRT1/AMPK/PGC-1α signaling pathway, which provides a potential new therapeutic strategy for human osteoarthritis.

MicroRNA-103 contributes to osteoarthritis development by targeting Sox6

Osteoarthritis is a degenerative joint disease, worldwide, and its underlying molecular mechanisms are still poorly understood. MicroRNAs are important regulators of diverse biological processes, including osteoarthritis. In this study, we showed that miR-103 was deregulated in osteoarthritis patients. We performed CCK8 and colony formation assay and found that miR-103 inhibited chondrocyte proliferation. We also found that miR-103 inhibited chondrocyte formation and maturation by RT-PCR, western blotting, and immunocytochemistry. Inhibition of miR-103 suppressed production of the catabolic factors and pro-inflammatory cytokines induced by IL-1β in chondrocytes. Finally, we found that Sox6 was a direct target of miR-103 and participated in osteoarthritis development. In summary, we demonstrated that miR-103 contributed to osteoarthritis development by directly targeting and inhibiting the expression of Sox6. Regulation of miR-103 expression in human chondrocytes may be an effective treatment for osteoarthritis.

NOD2 signaling pathway is involved in fibronectin fragment-induced pro-catabolic factor expressions in human articular chondrocytes

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptor-interacting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilage-degrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways.

CITED2 mediates the cross-talk between mechanical loading and IL-4 to promote chondroprotection.

Osteoarthritis (OA) pathogenesis is mediated largely through the actions of proteolytic enzymes such as matrix metalloproteinase (MMP) 13. The transcriptional regulator CITED2, which suppresses the expression of MMP13 in chondrocytes, is induced by interleukin (IL)-4 in T cells and macrophages, and by moderate mechanical loading in chondrocytes. We tested the hypothesis that CITED2 mediates cross-talk between IL-4 signaling and mechanical loading-induced pathways that result in chondroprotection, at least in part, by downregulating MMP13. IL-4 induced CITED2 gene expression in human chondrocytes in a dose- and time-dependent manner through JAK/STAT signaling. Mechanical loading combined with IL-4 resulted in additive effects on inducing CITED2 expression and downregulating of MMP13 in human chondrocytes in vitro. In vivo, IL-4 gene knockout (KO) mice exhibited reduced basal levels of CITED2 expression in chondrocytes. While moderate treadmill running induced CITED2 expression and reduced MMP13 expression in wild-type mice, these effects were blunted (for CITED2) or abolished (for MMP13) in chondrocytes of IL-4 gene KO mice. Moreover, intra-articular injections of mouse recombinant IL-4 combined with regular cage activity mitigated post-traumatic OA to a greater degree compared to immobilized mice treated with IL-4 alone. These data suggest that using moderate loading to enhance IL-4 may be a potential therapeutic strategy for chondroprotection in OA.

Cytokine-induced cysteine- serine-rich nuclear protein-1 (CSRNP1) selectively contributes to MMP1 expression in human chondrocytes.

Irreversible cartilage collagen breakdown by the collagenolytic matrix metalloproteinases (MMPs)-1 and MMP-13 represents a key event in pathologies associated with tissue destruction such as arthritis. Inflammation is closely associated with such pathology and occurs in both rheumatoid and osteoarthritis making it highly relevant to the prevailing tissue damage that characterises these diseases. The inflammation-induced activating protein-1 (AP-1) transcription factor is an important regulator of both MMP1 and MMP13 genes with interplay between signalling pathways contributing to their expression. Here, we have examined the regulation of MMP1 expression, and using in vivo chromatin immunoprecipitation analyses we have demonstrated that cFos bound to the AP-1 cis element within the proximal MMP1 promoter only when the gene was transcriptionally silent as previously observed for MMP13. Subsequent small interfering RNA-mediated silencing confirmed however, that cFos significantly contributes to MMP1 expression. In contrast, silencing of ATF3 (a prime MMP13 modulator) did not affect MMP1 expression whilst silencing of the Wnt-associated regulator cysteine- serine-rich nuclear protein-1 (CSRNP1) resulted in substantial repression of MMP1 but not MMP13. Furthermore, following an early transient peak in expression of CSRNP1 at the mRNA and protein levels similar to that seen for cFOS, CSRNP1 expression subsequently persisted unlike cFOS. Finally, DNA binding assays indicated that the binding of CSRNP1 to the AP-1 consensus-like sequences within the proximal promoter regions of MMP1 and MMP13 was preferentially selective for MMP1 whilst activating transcription factor 3 (ATF3) binding was exclusive to MMP13. These data further extend our understanding of the previously reported differential regulation of these MMP genes, and strongly indicate that although cFos modulates the expression of MMP1/13, downstream factors such as CSRNP1 and ATF3 ultimately serve as transcriptional regulators in the context of an inflammatory stimulus for these potent collagenolytic MMPs.

SFMBT2 positively regulates SOX9 and chondrocyte proliferation.

SRY‑box9(SOX9) is the master regulator of the chondrocyte phenotype, which is essential for differentiating chondrogenic mesenchymal condensations into chondrocytes, and is involved in regulating every stage of chondrocyte differentiation. SOX9 deletion in chondrocytes at the late stages of cartilage development results in decreased chondrocyte proliferation; inhibited expression of cartilage matrix genes, including Indian hedgehog and the downstream parathyroid hormone‑related protein; and premature conversion of proliferating chondrocytes into hypertrophic chondrocytes, which mineralize their matrix prematurely. Therefore, SOX9 is considered vital for the majority of phases of chondrocyte lineage, from early condensations to the differentiation of proliferating chondrocytes, leading to chondrocyte hypertrophy. It has been reported that SOX9 expression is decreased in osteoarthritis(OA) cartilage. Regeneration or repair of cartilage degradation in OA remains a challenge. Previous studies have indicated that overexpression of SOX9 can promote cartilage repair and can be used as a potential therapeutic agentat the early stages of human OA. The present study identified Scm‑like with four malignant brain tumor domains2(SFMBT2) as a novel regulator of SOX9 expression in human chondrocytes. Our previous study revealed that SFMBT2 is negatively regulated in OA cartilage, and decreased levels of SFMBT2 contribute to the catabolic phenotype of chondrocytes. The present study detected increased expression levels of SFMBT2 in early cartilage development and during the early phases of chondrogenesis. Overexpression of SFMBT2 in C28/I2 cells upregulated SOX9 expression in a dose‑dependent manner. Furthermore, SFMBT2 positively regulated C28/I2 cell proliferation and restored the decreased levels of SOX9 in chondrocytes following tumor necrosis factor‑α treatment. Additional studies may reveal novel insights into the molecular mechanism involved and the potential role of SFMBT2 in cartilage repair and OA management.

MiR-497 Is Implicated in the Process of Chondrogenesis and Inhibits IHH Gene Expression in Human Chondrocytes.

The aim of this study was to examine differences in microRNA-497 (miR-497) expression during cartilage tissue formation and to test whether miR-497 directly interferes with Indian hedgehog (IHH) gene and inhibits IHH expression in human chondrocytes. At different cartilage development stages and different time points in bone matrix gelatin-induced endochondral ossification (BMG-ECO) rat models, the expression of miR-497 and the Ihh gene was monitored at the mRNA level. Bioinformatic analysis, gene mutation, dual luciferase reporter gene assays and gene expression assays at both the mRNA and protein levels in human chondrocytes were subsequently performed to validate the interaction between miR-497 and the IHH gene. The mRNA expression of miR-497 or the Ihh gene in BMG-ECO rats showed significant differences between the cartilage development stages and between different time points, and the trends in the expression of miR-497 and Ihh were reversed. Bioinformatic and dual luciferase reporter gene assays demonstrated a direct interaction between miR-497 and the IHH gene. Differential mRNA and protein expression profiles of the IHH gene in human chondrocytes after 48 hours of transfection with miR-497 mimics and a negative control indicated that miR-497 inhibited IHH expression. Our study provided new clues for further functional and molecular mechanism studies of miR-497 in chondrogenesis and demonstrated a potential target for clinical therapy for cartilage degenerative disease.

2 dyn/cm2 shear force upregulates kruppel-like factor 4 expression in human chondrocytes to inhibit the interleukin-1β-activated nuclear factor-κB.

The shear force effect on human chondrocytes is time and magnitude dependent. Recently, kruppel-like factor (KLF) 4 has been identified as a pleiotropic protein and its activity in cells is dependent on different stimuli and/or cell types. The role of KLF4 in chondrocytes is still unclear and there has been no report determining whether shear force regulates KLF4 levels in chondrocytes. Hence, this study was carried out to investigate the role of KLF4 in human chondrocytes under shear force stimulation and the underlying mechanism. Human primary and SW1353 chondrocytes were used in this study. The shear forces at 2, 5, or 15 dyn/cm2 intensity were applied to both types of human chondrocytes. The specific small interfering RNAs, activators, and inhibitors were used to study the detailed mechanism of shear force. The presented results showed that 2, but not 5 and 15, dyn/cm2 shear force increases KLF4 expression in human primary and SW1353 chondrocytes. Extracellular signal-regulated kinase 5 induced peroxisome proliferator-activated receptor γ transcription activity to increase KLF4 transcription. Moreover, the KLF4 induction in human chondrocytes in response to 2 dyn/cm2 shear force could attenuate interleukin (IL)-1β-stimulated nuclear factor-κB activation. These results elucidate the role of KLF4 in antagonizing the effect of IL-1β in human chondrocytes under 2 dyn/cm2 shear force stimulation and provide a possible mechanism to demonstrate the protection of moderate forces or exercises in cartilage.

Corrigendum to: Effects of Arthrocen, an avocado/soy unsaponifiables agent, on inflammatory mediators and gene expression in human chondrocytes.

[This corrects the article DOI: 10.1002/2211-5463.12176.].

Protein kinase D3 modulates MMP1 and MMP13 expression in human chondrocytes.

Many catabolic stimuli, including interleukin-1 (IL-1) in combination with oncostatin M (OSM), promote cartilage breakdown via the induction of collagen-degrading collagenases such as matrix metalloproteinase 1 (MMP1) and MMP13 in human articular chondrocytes. Indeed, joint diseases with an inflammatory component are characterised by excessive extracellular matrix (ECM) catabolism. Importantly, protein kinase C (PKC) signalling has a primary role in cytokine-induced MMP1/13 expression, and is known to regulate cellular functions associated with pathologies involving ECM remodelling. At present, substrates downstream of PKC remain undefined. Herein, we show that both IL-1- and OSM-induced phosphorylation of protein kinase D (PKD) in human chondrocytes is strongly associated with signalling via the atypical PKCι isoform. Consequently, inhibiting PKD activation with a pan-PKD inhibitor significantly reduced the expression of MMP1/13. Specific gene silencing of the PKD isoforms revealed that only PKD3 (PRKD3) depletion mirrored the observed MMP repression, indicative of the pharmacological inhibitor specifically affecting only this isoform. PRKD3 silencing was also shown to reduce serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation of all three mitogen-activated protein kinase groups. This altered signalling following PRKD3 silencing led to a significant reduction in the expression of the activator protein-1 (AP-1) genes FOS and JUN, critical for the induction of many MMPs including MMP1/13. Furthermore, the AP-1 factor activating transcription factor 3 (ATF3) was also reduced concomitant with the observed reduction in MMP13 expression. Taken together, we highlight an important role for PKD3 in the pro-inflammatory signalling that promotes cartilage destruction.

EZH2 inhibition reduces IL-1β-mediated inflammation and increases anabolic gene expression in human chondrocytes

EZH2 inhibition reduces IL-1β-mediated inflammation and increases anabolic gene expression in human chondrocytes

Depletion of aquaporin 1 decreased ADAMTS‑4 expression in human chondrocytes.

Inflammation serves an important role in the progression of osteoarthritis (OA), and IL-1β may act as a catabolic factor on cartilage, reducing the synthesis of primary cartilage components type II collagen and aggrecan. Aquaporin 1 (AQP1) is a 28-kDa water channel formed of six transmembrane domains on the cell membrane. AQP1 is highly expressed in the anus, gallbladder and liver, and is moderately expressed in the hippocampus, ependymal cells of the central nervous system and articular cartilage. It was hypothesized that AQP1 may be highly expressed in OA cartilage and that it may increase the expression of catabolic factors during inflammatory OA progression. Therefore, the present study evaluated AQP1 functions in human OA articular chondrocytes. Primary chondrocytes were isolated from human hip and knee cartilage tissues, cultured and transfected with AQP1-specific small interfering RNA with or without subsequent IL-1β treatment. In vitro explant culture from hip cartilages were also prepared. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to assess the expression of AQP genes in human articular cartilage, AQP1 immunohistochemistry of the cartilages and explant culture, as well as RT-quantitative PCR, western blotting and immunocytochemistry/immunofluorescence of OA chondrocytes to evaluate the expression of AQP1, and catabolic and anabolic factors. RT-PCR results demonstrated that AQP0, 1, 3, 7, 9, and 11 were expressed in OA chondrocytes. Immunohistochemistry revealed that AQP1 was highly expressed in the superficial to middle zones of OA articular cartilages. Additionally, AQP1 mRNA was significantly higher in OA cartilage and IL-1β treatment significantly increased AQP1 expression in hip explant cartilage. Furthermore, AQP1 downregulation decreased a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 expression in OA chondrocytes, though it did not affect other associated genes. Immunofluorescence showed that AQP1 and ADAMTS-4 were co-localized. These findings indicated that AQP1 depletion may decrease ADAMTS-4 expression in human OA chondrocytes. Therefore, regulating AQP1 expression may be a strategy to suppress catabolic factors during OA progression.

MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3.

Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism.Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1β (IL-1β)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured.Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3′-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1β-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects.Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.

2 dyn/cm2 Shear Force Upregulates Kruppel-like Factor 4 Expression in Human Chondrocytes to Inhibit the Interleukin-1beta-activated NF-kappaB

2 dyn/cm2 Shear Force Upregulates Kruppel-like Factor 4 Expression in Human Chondrocytes to Inhibit the Interleukin-1beta-activated NF-kappaB

Carvacrol ameliorates inflammatory response in interleukin 1β-stimulated human chondrocytes.

Carvacrol, a monoterpenic phenol present in Origanumvulgare (oregano) and Thymusvulgaris (thyme), possesses anti‑inflammatory effects; however, little is known about the effects and underlying mechanism of carvacrol on chondrocytes in osteoarthritis (OA). The present study aimed to investigate the protective effects of carvacrol against inflammation in interleukin 1β (IL‑1β)‑stimulated human chondrocytes. The results indicated that carvacrol inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production, and decreased the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX‑2). Carvacrol also suppressed the protein expression levels of matrix metalloproteinase (MMP)‑3 and MMP‑13 in IL‑1β‑stimulated human OA chondrocytes. Furthermore, carvacrol suppressed the activation of nuclear factor (NF)‑κB signaling pathway in IL‑1β‑induced human chondrocytes. In conclusion, the present results demonstrated that carvacrol was able to inhibit IL‑1β‑induced NO and PGE2 production, as well as iNOS, COX‑2 and MMPs expression in human chondrocytes by suppressing the activation of NF‑κB signaling pathway. Thus, carvacrol may have potential therapeutic functions for the treatment of OA.