Macroscopic Synovial Inflammation Correlates with Symptoms and Cartilage Lesions in Patients Undergoing Arthroscopic Partial Meniscectomy: A Clinical Study.

BACKGROUND: The aim of the study was to examine the relationship among patients' characteristics, intraoperative pathology and pre/post-operative symptoms in a cohort of patients undergoing arthroscopic partial meniscectomy for symptomatic meniscal tears. METHODS: Clinical data were collected (age, sex, body mass index, time to surgery, trauma). Intraoperative cartilage pathology was assessed with Outerbridge score. Meniscal tears were graded with the ISAKOS classification. Synovial inflammation was scored using the Macro-score. Patient symptoms were assessed pre/post-operatively using the KOOS instrument. RESULTS: In the series of 109 patients (median age 47 years), 50% of the meniscal tears were traumatic; 85% of patients showed mild to moderate synovitis; 52 (47.7%) patients had multiple cartilage defects and 31 (28.4%) exhibited a single focal chondral lesion. Outerbridge scores significantly correlated with patient age, BMI and synovial inflammation. There was a correlation between severity of chondral pathology and high-grade synovial hyperplasia. Pre-operative KOOS correlated with BMI, meniscal degenerative changes and symptom duration. Obesity, time to surgery, presence of high-grade synovial hyperplasia and high-grade cartilage lesions were independent predictors of worse post-operative pain and function. CONCLUSION: We demonstrated that pre-operative symptoms and post-operative outcomes correlate with synovitis severity and cartilage pathology, particularly in old and obese patients that underwent arthroscopic partial meniscectomy. Importantly, patients with a degenerative meniscal pattern and with longer time to surgery experienced more severe cartilage damage and, consequentially, pain and dysfunction. These findings are fundamental to identify patients suitable for earlier interventions.

Do Synovial Inflammation and Meniscal Degeneration Impact Clinical Outcomes of Patients Undergoing Arthroscopic Partial Meniscectomy? A Histological Study.

The menisci exert a prominent role in joint stabilization and in the distribution of mechanical loading. Meniscal damage is associated with increased risk of knee OA. The aim of this study was to characterize the synovial membrane and meniscal tissues in patients undergoing arthroscopic partial meniscectomy for meniscal tear and to evaluate association with clinical outcomes. A total of 109 patients were recruited. Demographic and clinical data were collected. Visual Analogic Scale (VAS) measuring pain and Knee injury and Osteoarthritis Outcome Score (KOOS) were recorded at baseline and at 2-years follow-up. Histological and immunohistochemical characterizations were performed on synovial membranes and meniscal tissues. More than half of the patients demonstrated synovial mononuclear cell infiltration and hyperplasia. Synovial fibrosis was present in most of the patients; marked vascularity and CD68 positivity were observed. Inflammation had an impact on both pain and knee symptoms. Patients with synovial inflammation had higher values of pre-operative VAS and inflammation. Higher pre-operative pain was observed in patients with meniscal MMP-13 production. In conclusion, multivariate analysis showed that synovial inflammation was associated with pre-operative total KOOS scores, knee symptoms, and pain. Moreover, meniscal MMP-13 expression was found to be associated with pre-operative pain in multivariate analysis. Thus, targeting inflammation of the synovial membrane and meniscus might reduce clinical symptoms and dysfunction at the time of surgery.

LOXL2 promotes aggrecan and gender-specific anabolic differences to TMJ cartilage.

In the United States, 5-12% of adults have at least one symptom of temporomandibular joint (TMJ) disorders, including TMJ osteoarthritis (TMJ-OA). However, there is no chondroprotective agent that is approved for clinical application. We showed that LOXL2 is elevated in the regenerative response during fracture healing in mice and has a critical role in chondrogenic differentiation. Indeed, LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses, and attenuates NF-kB signaling. The specific goal of the study was to evaluate if adenoviral delivery of LOXL2 is anabolic to human and mouse TMJ condylar cartilage in vivo and evaluate the protective and anabolic effect on cartilage-specific factors. We employed two different models to assess TMJ-OA. In one model, clinical TMJ-OA cartilage from 5 different samples in TMJ-OA cartilage plugs were implanted subcutaneously in nude mice. Adenovirus LOXL2 -treated implants showed higher mRNA levels of LOXL2, ACAN, and other anabolic genes compared to the adenovirus-Empty-treated implants. Further characterization by RNA-seq analysis showed LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. In order to evaluate if LOXL2-induced functional and sex-linked differences, both male and female four-month-old chondrodysplasia (Cho/+) mice, which develop progressive TMJ-OA due to a point mutation in the Col11a1 gene, were subjected to intraperitoneal injection with Adv-RFP-LOXL2 every 2 weeks for 12 weeks. The data showed that adenovirus delivery of LOXL2 upregulated LOXL2 and aggrecan (Acan), whereas MMP13 expression was slightly downregulated. The fold change expression of Acan and Runx2 induced by Adv-RFP-LOXL2 was higher in females compared to males. Interestingly, Adv-RFP-LOXL2 injection significantly increased Rankl expression in male but there was no change in females, whereas VegfB gene expression was increased in females, but not in males, as compared to those injected with Adv-RFP-Empty in respective groups. Our findings indicate that LOXL2 can induce specifically the expression of Acan and other anabolic genes in two preclinical models in vivo. Further, LOXL2 has beneficial functions in human TMJ-OA cartilage implants and promotes gender-specific anabolic responses in Cho/+ mice with progressive TMJ-OA, suggesting its merit for further study as an anabolic therapy for TMJ-OA.

Labral calcification plays a key role in hip pain and symptoms in femoroacetabular impingement.

BACKGROUND: Hip osteoarthritis (HOA) is the most common hip disorder and a major cause of disability in the adult population, with an estimated prevalence of end-stage disease and total hip replacement. Thus, the diagnosis, prevention, and treatment of the early stages of the disease in young adults are crucial to reduce the incidence of end-stage HOA. The purpose of this study was to determine whether (1) a relationship among the inflammatory status of labrum and synovium collected from patients with femoroacetabular impingement (FAI) would exist; and (2) to investigate the associations among the histopathological features of joint tissues, the pre-operative symptoms and the post-operative outcomes after arthroscopic surgery. METHODS: Joint tissues from 21 patients undergoing hip arthroscopy for FAI were collected and their histological and immunohistochemical features were correlated with clinical parameters. RESULTS: Synovial mononuclear cell infiltration was observed in 25% of FAI patients, inversely correlated with the hip disability and osteoarthritis outcome score (HOOS) pain and function subscales and with the absolute and relative change in total HOOS. All the labral samples showed some pattern of degeneration and 67% of the samples showed calcium deposits. The total labral score was associated with increased CD68 positive cells in the synovium. The presence of labral calcifications, along with the chondral damage worsened the HOOS post-op symptoms (adjusted R-square = 0.76 p = 0.0001). CONCLUSIONS: Our study reveals a relationship between the histologic labral features, the synovial inflammation, and the cartilage condition at the time of FAI. The presence of labral calcifications, along with the cartilage damage and the synovitis negatively affects the post-operative outcomes in patients with FAI.

Lysyl Oxidase-Like 2 Protects against Progressive and Aging Related Knee Joint Osteoarthritis in Mice.

BACKGROUND: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. METHODS: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1ß in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. RESULTS: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1ß-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. CONCLUSION: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.

Bystander effectors of chondrosarcoma cells irradiated at different LET impair proliferation of chondrocytes.

While the dose-response relationship of radiation-induced bystander effect (RIBE) is controversial at low and high linear energy transfer (LET), mechanisms and effectors of cell-to-cell communication stay unclear and highly dependent of cell type. In the present study, we investigated the capacity of chondrocytes in responding to bystander factors released by chondrosarcoma cells irradiated at different doses (0.05 to 8 Gy) with X-rays and C-ions. Following a medium transfer protocol, cell survival, proliferation and DNA damages were quantified in bystander chondrocytes. The bystander factors secreted by chondrosarcoma cells were characterized. A significant and major RIBE response was observed in chondrocyte cells (T/C-28a2) receiving conditioned medium from chondrosarcoma cells (SW1353) irradiated with 0.1 Gy of X-rays and 0.05 Gy of C-ions, resulting in cell survivals of 36% and 62%, respectively. Micronuclei induction in bystander cells was observed from the same low doses. The cell survival results obtained by clonogenic assays were confirmed using impedancemetry. The bystander activity was vanished after a heat treatment or a dilution of the conditioned media. The cytokines which are well known as bystander factors, TNF-α and IL-6, were increased as a function of doses and LET according to an ELISA multiplex analysis. Together, the results demonstrate that irradiated chondrosarcoma cells can communicate stress factors to non-irradiated chondrocytes, inducing a wide and specific bystander response related to both doses and LET.

Morphological and ultrastructural analysis of normal, injured and osteoarthritic human knee menisci.

The human meniscus plays a crucial role for transmission and distribution of load across the knee, as well as shock absorption, joint stability, lubrication, and congruity. The aim of this study was to compare the complex geometry, and unique ultrastructure and tissue composition of the meniscus in healthy (control) and pathological conditions to provide understanding of structural changes that could be helpful in the future design of targetted therapies and improvement of treatment indications. We analyzed meniscus samples collected from 3 healthy multi-organ donors (median age, 66 years), 5 patients with traumatic meniscal tear (median age, 41 years) and 3 patients undergoing total knee replacement (TKR) for end-stage osteoarthritis (OA) (median age, 72 years). We evaluated the extracellular matrix (ECM) organization, the appearance and distribution of areas of calcification, and modifications of cellular organization and structure by electron microscopy and histology. The ECM structure was similar in specimens from traumatic meniscus tears compared to those from patients with late-stage OA, showing disorganization of collagen fibers and increased proteoglycan content. Cells of healthy menisci showed mainly diffuse chromatin and well preserved organelles. Both in traumatic and in OA menisci, we observed increased chromatin condensation, organelle degeneration, and cytoplasmic vacuolization, a portion of which contained markers of autophagic vacuoles. Areas of calcification were also observed in both traumatic and OA menisci, as well as apoptotic-like features that were particularly prominent in traumatic meniscal tear samples. We conclude that meniscal tissue from patients with traumatic meniscal injury demonstrate pathological alterations characteristic of tissue from older patients undergoing TKR, suggesting that they have high susceptibility to develop OA.

Correction to: Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model.

Following publication of the original article [1], the authors reported an error in Figs. 2C and 5C.

Inflammatory molecules produced by meniscus and synovium in early and end-stage osteoarthritis: a coculture study.

The aim of this study was to identify the molecules and pathways involved in the cross-talk between meniscus and synovium that may play a critical role in osteoarthritis (OA) pathophysiology. Samples of synovium and meniscus were collected from patients with early and end-stage OA and cultured alone or cocultured. Cytokines, chemokines, metalloproteases, and their inhibitors were evaluated at the gene and protein levels. The extracellular matrix (ECM) changes were also investigated. In early OA cultures, higher levels of interleukin-6 (IL-6) and IL-8 messenger RNA were expressed by synovium and meniscus in coculture compared with meniscus cultured alone. RANTES release was significantly increased when the two tissues were cocultured compared with meniscus cultured alone. Increased levels of matrix metalloproteinase-3 (MMP-3) and MMP-10 proteins, as well as increased release of glycosaminoglycans and aggrecan CS846 epitope, were observed when synovium was cocultured with meniscus. In end-stage OA cultures, increased levels of IL-8 and monocyte chemoattractant protein-1 (MCP-1) proteins were released in cocultures compared with cultures of meniscus alone. Chemokine (C-C motif) ligand 21 (CCL21) protein release was higher in meniscus cultured alone and in coculture compared with synovium cultured alone. Increased levels of MMP-3 and 10 proteins were observed when tissues were cocultured compared with meniscus cultured alone. Aggrecan CS846 epitope release was increased in cocultures compared with cultures of either tissue cultured alone. Our study showed the production of inflammatory molecules by synovium and meniscus which could trigger inflammatory signals in early OA patients, and induce ECM loss in the progressive and final stages of OA pathology.

Evaluation of surfactant proteins A, B, C, and D in articular cartilage, synovial membrane and synovial fluid of healthy as well as patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Surfactant Proteins (SPs) are well known from lung and form, along with phospholipids, a surface-active-layer at the liquid-air-interface of the alveolar lining. They play a major protective role by lowering surface tension, activating innate and adaptive immune defense at the lung mucosal interface, especially during infection. We analyzed the regulation of SPs in human and mouse articular chondrocytes, synoviocytes, and synovial fluid under healthy and inflammatory conditions, as well as in tissues of patients suffering from osteoarthritis and rheumatoid arthritis. METHODS: Immunohistochemistry, RT-PCR, qRT-PCR, ELISA, Western blotting were performed in cell cultures and tissue samples to determine localization, regulation, and concentration of SPs. RESULTS: All four SPs, were expressed by healthy human and mouse articular chondrocytes and synoviocytes and were also present in synovial fluid. Treatment with inflammatory mediators like IL-1ß and TNF-α led to short-term upregulation of individual SPs in vitro. In tissues from patients with osteoarthritis and rheumatoid arthritis, protein levels of all four SPs increased significantly compared to the controls used. CONCLUSION: These results show the distribution and amount of SPs in tissues of articular joints. They are produced by chondrocytes and synoviocytes and occur in measurable amounts in synovial fluid. All four SPs seem to be differently regulated under pathologic conditions. Their physiological functions in lowering surface tension and immune defense need further elucidation and make them potential candidates for therapeutic intervention.

Elf3 Contributes to Cartilage Degradation in vivo in a Surgical Model of Post-Traumatic Osteoarthritis.

The E-74 like factor 3 (ELF3) is a transcription factor induced by inflammatory factors in various cell types, including chondrocytes. ELF3 levels are elevated in human cartilage from patients with osteoarthritis (OA), and ELF3 contributes to the IL-1ß-induced expression of genes encoding Mmp13, Nos2, and Ptgs2/Cox2 in chondrocytes in vitro. Here, we investigated the contribution of ELF3 to cartilage degradation in vivo, using a mouse model of OA. To this end, we generated mouse strains with cartilage-specific Elf3 knockout (Col2Cre:Elf3f/f) and Comp-driven Tet-off-inducible Elf3 overexpression (TRE-Elf3:Comp-tTA). To evaluate the contribution of ELF3 to OA, we induced OA in 12-week-old Col2Cre:Elf3f/f and 6-month-old TRE-Elf3:Comp-tTA male mice using the destabilization of the medial meniscus (DMM) model. The chondrocyte-specific deletion of Elf3 led to decreased levels of IL-1ß- and DMM-induced Mmp13 and Nos2 mRNA in vitro and in vivo, respectively. Histological grading showed attenuation of cartilage loss in Elf3 knockout mice compared to wild type (WT) littermates at 8 and 12 weeks following DMM surgery that correlated with reduced collagenase activity. Accordingly, Elf3 overexpression led to increased cartilage degradation post-surgery compared to WT counterparts. Our results provide evidence that ELF3 is a central contributing factor for cartilage degradation in post-traumatic OA in vivo.

E74-Like Factor (ELF3) and Leptin, a Novel Loop Between Obesity and Inflammation Perpetuating a Pro-Catabolic State in Cartilage.

BACKGROUND/AIMS: The E74-like factor 3 (ELF3) is an inflammatory mediator that participates in cartilage destruction in osteoarthritis. Leptin and other adipokines negatively impact articular cartilage, triggering catabolic and inflammatory responses in chondrocytes. Here, we investigated whether leptin induces ELF3 expression in chondrocytes and the signaling pathway involved in this process. METHODS: We determined mRNA and protein levels of ELF3 by RT-qPCR and Western blotting using cultured human primary chondrocytes and the human T/C-28a2 chondrocyte cell line. Further, we measured luciferase activities of different reporter constructs, and we assessed the contribution of leptin to the induction of ELF3 mRNA by knocking down hLEPR gene expression using siRNA technology. RESULTS: Leptin synergizes with IL-1ß in inducing ELF3 expression in chondrocytes. We also found that PI3K, p38, and JAK2 signaling pathways are at play in the leptin-driven induction of ELF3. Moreover, we confirm the participation of NFΚB in the leptin/IL-1ß synergistic induction of ELF3. CONCLUSION: Here we show, for the first time, the regulation of ELF3 expression by leptin, suggesting that this transcription factor likely mediates the inflammatory responses triggered by leptin in articular chondrocytes.

Phlpp inhibitors block pain and cartilage degradation associated with osteoarthritis.

Phlpp protein phosphatases are abnormally abundant within human osteoarthritic articular chondrocytes and may contribute to the development of osteoarthritis. Mice lacking Phlpp1 were previously shown to be resistant to post-traumatic osteoarthritis. Here a small molecule with therapeutic properties that inhibits Phlpp1 and Phlpp2 was tested for its ability to slow post-traumatic OA in mice and to stimulate anabolic pathways in human articular cartilage from OA joints. PTOA was induced in male C57Bl/6 mice by surgically destabilizing the meniscus. Seven weeks after surgery, mice received a single intra-articular injection of the Phlpp inhibitor NSC117079 or saline. Mechanical allodynia was measured with von Frey assays, mobility was tracked in an open field system, and cartilage damage was assessed histologically. A single intra-articular injection of the Phlpp inhibitor NSC117079 attenuated mechanical allodynia and slowed articular cartilage degradation in joints with a destabilized meniscus. Animals treated with the Phlpp inhibitor 7 weeks after injury maintained normal activity levels, while those in the control group traveled shorter distances and were less active 3 months after the joint injury. NSC117079 also increased production of cartilage extracellular matrix components (glycosaminoglycans and aggrecan) in over 90% of human articular cartilage explants from OA patients and increased phosphorylation of Phlpp1 substrates (AKT2, ERK1/2, and PKC) in human articular chondrocytes. Our results indicate that Phlpp inhibitor NSC117079 is a novel osteoarthritis disease modifying drug candidate that may have palliative affects. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1487-1497, 2018.

Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis.

BACKGROUND: Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA). METHODS: LOXL2 was visualized in tissues from human knee and hip joints and temporomandibular joints (TMJ) by immunofluorescence. The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays, microarray analysis, and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. Finally, LOXL2-induced effects on collagen type 2 α1 (COL2A1) and phospho-SMAD2/3 were evaluated by immunofluorescence analysis. RESULTS: LOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-ß1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1ß and TNF-α downregulated LOXL2, in human chondrocytes. Viral transduction of LOXL2 in OA chondrocytes increased the mRNA levels of chondroitin sulfate proteoglycan (CSPG4), aggrecan (ACAN), sex determining region Y-box containing gene 9 (SOX9), and COL2A1 but reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinase (MMP)1, MMP3, and MMP13. Further, forced expression of LOXL2 promoted chondrogenic lineage-specific gene expression, increased the expression of COL2A1 in the presence of TNF-α, and inhibited chondrocyte apoptosis. LOXL2 expression also inhibited IL-1ß-induced phospho-NF-κB/p65 and TGF-ß1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. Finally, immunofluorescence staining revealed co-localization of LOXL2 with SOX9 in the nuclei of cells in the implants, decreased phospho-SMAD2/3, and increased COL2A1 staining. CONCLUSION: Our results suggest that although LOXL2 is upregulated in cartilage affected by OA, this may be a protective response that promotes anabolism while inhibiting specific catabolic responses in the pathophysiology of OA.

DNA methylation of the RUNX2 P1 promoter mediates MMP13 transcription in chondrocytes.

The Runt-related transcription factor 2 (RUNX2) is critical for bone formation as well as chondrocyte maturation. Matrix metalloproteinase (MMP)-13 is a major contributor to cartilage degradation in osteoarthritis (OA). We and others have shown that the abnormal MMP13 gene expression in OA chondrocytes is controlled by changes in the DNA methylation status of specific CpG sites of the proximal promoter, as well as by the actions of different transactivators, including RUNX2. The present study aimed to determine the influence of the methylation status of specific CpG sites in the RUNX2 promoter on RUNX2-driven MMP13 gene expression in OA chondrocytes. We observed a significant correlation between MMP13 mRNA levels and RUNX2 gene expression in human OA chondrocytes. RUNX2 overexpression enhanced MMP13 promoter activity, independent of the MMP13 promoter methylation status. A significant negative correlation was observed between RUNX2 mRNA levels in OA chondrocytes and the percentage methylation of the CpG sites in the RUNX2 P1 promoter. Accordingly, the activity of the wild type RUNX2 promoter was decreased upon methylation treatment in vitro. We conclude that RUNX2 gene transcription is regulated by the methylation status of specific CpG sites in the promoter and may determine RUNX2 availability in OA cartilage for transactivation of genes such as MMP13.

ELF3 modulates type II collagen gene (COL2A1) transcription in chondrocytes by inhibiting SOX9-CBP/p300-driven histone acetyltransferase activity.

AIM: We showed previously that E74-like factor 3 (ELF3) protein levels are increased in osteoarthritic (OA) cartilage, that ELF3 accounts for inflammatory cytokine-driven MMP13 gene expression, and that, upon induction by interleukin-1ß, ELF3 binds to the COL2A1 promoter and suppresses its activity in chondrocytes. Here, we aimed to further investigate the mechanism/s by which ELF3 represses COL2A1 transcription in chondrocytes. METHODS AND RESULTS: We report that ELF3 inhibits Sox9-driven COL2A1 promoter activity by interfering with the activator functions of CBP/300 and Sox9. Co-transfection of the pGL2B-COL2A1 (-577/+3428 bp) reporter construct with Sox9 and with Sox5 and/or Sox6 increased COL2A1 promoter activity, and ELF3 overexpression significantly reduced the promoter transactivation. Co-transfection of ELF3 with the pLuc 4x48 enhancer construct, containing the 89-bp COL2A1 promoter and lacking the previously defined ELF3 binding sites, decreased both basal and Sox9-driven promoter activity. Co-transfection of ELF3 with a Gal4 reporter construct also inhibited Gal4-Sox9-driven transactivation, suggesting that ELF3 directly interacts with Sox9. Using truncated Sox9 fragments, we found that ELF3 interacts directly with the HMG domain of Sox9. Importantly, overexpression of ELF3 significantly decreased Sox9/CBP-dependent HAT activity. Finally, we show evidence that increased ELF3 mRNA expression in OA chondrocytes correlates with hypermethylation of the proximal promoter, suggesting that ELF3 transcription is subjected to epigenetic control in OA disease. CONCLUSION: Our results highlight the contribution of ELF3 to transcriptional regulation of COL2A1 and its potential role in OA disease, and uncover epigenetic mechanisms at play in the regulation of ELF3 and its downstream targets in articular chondrocytes.

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

We previously reported that perlecan, a heparan-sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage-synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2-/- -Tg (Hspg2-/- ;Col2a1-Hspg2Tg/- ) mice, in which the perlecan-knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic-, osteogenic-, and adipogenic-potentials were examined in the Hspg2-/- -Tg SMCs compared to the control SMCs prepared from wild-type Hspg2+/+ -Tg (Hspg2+/+ ;Col2a1-Hspg2Tg/- ) littermates. In a culture condition permitting proliferation, both control and Hspg2-/- -Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2-/- -Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2-/- -Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837-846, 2017.

E74-like factor 3 and nuclear factor-κB regulate lipocalin-2 expression in chondrocytes.

KEY POINTS: E74-like factor 3 (ELF3) is a transcription factor regulated by inflammation in different physio-pathological situations. Lipocalin-2 (LCN2) emerged as a relevant adipokine involved in the regulation of inflammation. In this study we showed for the first time the involvement of ELF3 in the control of LCN2 expression and its cooperation with nuclear factor-κB (NFκB). Our results will help to better understand of the role of ELF3, NFκB and LCN2 in the pathophysiology of articular cartilage. ABSTRACT: E74-like factor 3 (ELF3) is a transcription factor induced by inflammatory cytokines in chondrocytes that increases gene expression of catabolic and inflammatory mediators. Lipocalin 2 (LCN2) is a novel adipokine that negatively impacts articular cartilage, triggering catabolic and inflammatory responses in chondrocytes. Here, we investigated the control of LCN2 gene expression by ELF3 in the context of interleukin 1 (IL-1)-driven inflammatory responses in chondrocytes. The interaction of ELF3 and nuclear factor-κB (NFκB) in modulating LCN2 levels was also explored. LCN2 mRNA and protein levels, as well those of several other ELF3 target genes, were determined by RT-qPCR and Western blotting. Human primary chondrocytes, primary chondrocytes from wild-type and Elf3 knockout mice, and immortalized human T/C-28a2 and murine ATDC5 cell lines were used in in vitro assays. The activities of various gene reporter constructs were evaluated by luciferase assays. Gene overexpression and knockdown were performed using specific expression vectors and siRNA technology, respectively. ELF3 overexpression transactivated the LCN2 promoter and increased the IL-1-induced mRNA and protein levels of LCN2, as well as the mRNA expression of other pro-inflammatory mediators, in human and mouse chondrocytes. We also identified a collaborative loop between ELF3 and NFκB that amplifies the induction of LCN2. Our findings show a novel role for ELF3 and NFκB in the induction of the pro-inflammatory adipokine LCN2, providing additional evidence of the interaction between ELF3 and NFκB in modulating inflammatory responses, and a better understanding of the mechanisms of action of ELF3 in chondrocytes.

Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse post-traumatic osteoarthritis model.

INTRODUCTION: Epigallocatechin 3-gallate (EGCG), a polyphenol present in green tea, was shown to exert chondroprotective effects in vitro. In this study, we used a post-traumatic osteoarthritis (OA) mouse model to test whether EGCG could slow the progression of OA and relieve OA-associated pain. METHODS: C57BL/6 mice were subjected to surgical destabilization of the medial meniscus (DMM) or sham surgery. EGCG (25 mg/kg) or vehicle control was administered daily for four or eight weeks by intraperitoneal injection starting on the day of surgery. OA severity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical analysis to detect cleaved aggrecan and type II collagen, and expression of proteolytic enzymes matrix metalloproteinase (MMP)-13 and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS5). Real-time polymerase chain reaction (PCR) was performed to characterize the expression of genes critical for articular cartilage homeostasis. During the course of the experiments, tactile sensitivity testing (von Frey test) and open field assays were used to evaluate pain behaviors associated with OA, and expression of pain expression markers and inflammatory cytokines in the dorsal root ganglion (DRG) were determined by real-time PCR. RESULTS: Four and eight weeks after DMM surgery, the cartilage in EGCG-treated mice exhibited less Safranin O loss and cartilage erosion, and lower OARSI scores compared to vehicle-treated controls, which was associated with reduced staining for aggrecan and type II collagen cleavage epitopes, and reduced staining for MMP-13 and ADAMTS5 in the articular cartilage. Articular cartilage in the EGCG-treated mice also exhibited reduced levels of MMP-1, -3, -8, -13, ADAMTS5, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α mRNA and elevated gene expression of the MMP regulator Cbp/p300 Interacting Transactivator 2 (CITED2). Compared to vehicle controls, mice treated with EGCG exhibited reduced OA-associated pain, as indicated by higher locomotor behavior (i.e. distance traveled). Moreover, expression of chemokine receptor (CCR2), and pro-inflammatory cytokines IL-1ß and TNF-α in the DRG were significantly reduced to levels similar to sham-operated animals. CONCLUSIONS: This study provides the first evidence in an OA animal model that EGCG significantly slows OA disease progression and exerts a palliative effect.