Elevated glucose promotes MMP13 and ADAMTS5 production by osteoarthritic chondrocytes under oxygenated but not hypoxic conditions.

Type 2 diabetes is linked with increased incidence and severity of osteoarthritis. The purpose of this study was to determine the effect of extracellular glucose within the normal blood glucose and hyperglycemic range on catabolic enzyme production by chondrocytes isolated from osteoarthritic (OA) and macroscopically normal (MN) human cartilage under oxygenated (18.9% oxygen) and hypoxic (1% oxygen) conditions. OA and MN chondrocytes were maintained in 4, 6, 8, or 10 mM glucose for 24 h. Glucose consumption, GLUT1 glucose transporter levels, MMP13 and ADAMTS5 production, and levels of RUNX2, a transcriptional regulator of MMP13, ADAMTS5, and GLUT1, were assessed by enzyme-linked assays, RT-qPCR and/or western blot. Under oxygenated conditions, glucose consumption and GLUT1 protein levels were higher in OA but not MN chondrocytes in 10 mM glucose compared to 4 mM. Both RNA and protein levels of MMP13 and ADAMTS5 were also higher in OA but not MN chondrocytes in 10 mM compared to 4 mM glucose under oxygenated conditions. Expression of RUNX2 was overall lower in MN than OA chondrocytes and there was no consistent effect of extracellular glucose concentration on RUNX2 levels in MN chondrocytes. However, protein (but not RNA) levels of RUNX2 were elevated in OA chondrocytes maintained in 10 mM versus 4 mM glucose under oxygenated conditions. In contrast, neither RUNX2 levels or MMP13 or ADAMTS5 expression were increased in OA chondrocytes maintained in 10 mM compared to 4 mM glucose in hypoxia. Elevated extracellular glucose leads to increased glucose consumption and increased RUNX2 protein levels, promoting production of MMP13 and ADAMTS5 by OA chondrocytes in oxygenated but not hypoxic conditions. These findings suggest that hyperglycaemia may exacerbate chondrocyte-mediated cartilage catabolism in the oxygenated superficial zone of cartilage in vivo in patients with undertreated type 2 diabetes, contributing to increased OA severity.

Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare.

Gout is caused by monosodium urate (MSU) crystal deposition within joints. This leads to acute episodes of inflammation ("gout flares") driven by NLRP3 inflammasome activation in macrophages. Gout flares are frequently present during late night/early morning. The reason for this timing is unclear. Recent evidence suggests the NLRP3 inflammasome is under circadian control. The purpose of this study was to determine whether MSU crystals cause changes in the circadian clock in macrophages leading to time-of-day differences in NLRP3 inflammasome activation. Levels of circadian clock components were measured in undifferentiated "monocytic" and PMA-differentiated "macrophagic" THP-1 cells cultured with/without MSU crystals. Caspase-1 activity was measured to assess NLRP3 inflammasome activity. MSU crystal exposure resulted in minimal effects on clock genes in THP-1 monocytes but BMAL1, CRY1, PER2, and REV-ERBα showed altered expression with reduced protein levels of BMAL1 and REV-ERBα in THP-1 macrophages. REV-ERBα activation or BMAL1 over-expression resulted in reduced MSU crystal-induced caspase-1 activity. BMAL1 knockdown resulted in a further increase in MSU crystal-induced caspase-1 activity, but only at times of day when BMAL1 levels were naturally high. MSU crystal-induced NLRP3 inflammasome activation was greatest at the time of day when BMAL1 levels were naturally low. MSU crystals alter the expression of circadian clock components in THP-1 macrophages leading to loss of BMAL1 and REV-ERBα-mediated repression of NLRP3 inflammasome activity and time-of-day differences in susceptibility to inflammasome activation. Our findings suggest that the nocturnal risk of gout flare is at least partially a consequence of altered circadian control of immune cell function.

Phenotype and energy metabolism differ between osteoarthritic chondrocytes from male compared to female patients: Implications for sexual dimorphism in osteoarthritis development?

OBJECTIVES: The prevalence and severity of knee osteoarthritis (OA) are greater in females than males. The purpose of this study was to determine whether there is an underlying difference in the biology of OA chondrocytes between males and females. METHODS: Chondrocytes were obtained following knee arthroplasty from male and female patients with primary OA. Phenotype marker expression, glucose and fat consumption, and rates of glycolysis and oxidative phosphorylation were compared between females and males. RNAi was used to determine the consequences of differential expression of Sry-box transcription factor 9 (SOX9) and PGC1α between males and females. RESULTS: OA chondrocytes from male donors showed elevated ribonucleic acid (RNA) and protein levels of SOX9, elevated COL2A1 protein synthesis, higher glucose consumption, and higher usage of glycolysis compared to females. OA chondrocytes from females had higher PGC1α protein levels, higher fat consumption, and higher oxidative energy metabolism than males. Knockdown of SOX9 reduced expression of COL2A1 to a greater extent in male OA chondrocytes than females whereas knockdown of PGC1α reduced COL2A1 expression in females but not males. Expression of ACAN and the glycolytic enzyme PGK1 was also reduced in males but not females following SOX9 knockdown. CONCLUSIONS: OA chondrocyte phenotype and energy metabolism differ between males and females. Our results indicate transcriptional control of COL2A1 differs between the two. Differences in chondrocyte biology between males and females imply the underlying mechanisms involved in OA may also differ, highlighting the need to consider sex and gender when investigating pathogenesis and potential treatments for OA.

Concentration-dependent effects of leptin on osteoarthritis-associated changes in phenotype of human chondrocytes.

Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.

Differential Effects of Hypoxia versus Hyperoxia or Physoxia on Phenotype and Energy Metabolism in Human Chondrocytes from Osteoarthritic Compared to Macroscopically Normal Cartilage.

Chondrocyte phenotype and energy metabolism are altered in osteoarthritis (OA). However, most studies characterising the change in human chondrocyte behaviour in OA have been conducted in supraphysiological oxygen concentrations. The purpose of this study was to compare phenotype and energy metabolism in chondrocytes from macroscopically normal (MN) and OA cartilage maintained in 18.9% (standard tissue culture), 6% (equivalent to superficial zone of cartilage in vivo) or 1% oxygen (equivalent to deep zone of cartilage in vivo). MMP13 production was higher in chondrocytes from OA compared to MN cartilage in hyperoxia and physoxia but not hypoxia. Hypoxia promoted SOX9, COL2A1 and ACAN protein expression in chondrocytes from MN but not OA cartilage. OA chondrocytes used higher levels of glycolysis regardless of oxygen availability. These results show that differences in phenotype and energy metabolism between chondrocytes from OA and MN cartilage differ depending on oxygen availability. OA chondrocytes show elevated synthesis of cartilage-catabolising enzymes and chondrocytes from MN cartilage show reduced cartilage anabolism in oxygenated conditions. This is relevant as a recent study has shown that oxygen levels are elevated in OA cartilage in vivo. Our findings may indicate that this elevated cartilage oxygenation may promote cartilage loss in OA.

Re-thinking osteoarthritis pathogenesis: what can we learn (and what do we need to unlearn) from mouse models about the mechanisms involved in disease development.

Efforts to develop effective disease-modifying drugs to treat osteoarthritis have so far proved unsuccessful with a number of promising drug candidates from pre-clinical studies failing to show efficacy in clinical trials. It is therefore timely to re-evaluate our current understanding of osteoarthritis pathogenesis and the similarities and differences in disease development between commonly used pre-clinical mouse models and human patients. There is substantial heterogeneity between patients presenting with osteoarthritis and mounting evidence that the pathways involved in osteoarthritis (e.g. Wnt signalling) differ between patient sub-groups. There is also emerging evidence that the pathways involved in osteoarthritis differ between the STR/ort mouse model (the most extensively studied mouse model of spontaneously occurring osteoarthritis) and injury-induced osteoarthritis mouse models. For instance, while canonical Wnt signalling is upregulated in the synovium and cartilage at an early stage of disease in injury-induced osteoarthritis mouse models, this does not appear to be the case in the STR/ort mouse. Such findings may prove insightful for understanding the heterogeneity in mechanisms involved in osteoarthritis pathogenesis in human disease. However, it is important to recognise that there are differences between mice and humans in osteoarthritis pathogenesis. A much more extensive array of pathological changes are evident in osteoarthritic joints in individual mice with osteoarthritis compared to individual patients. There are also specified differences in the pathways involved in disease development. For instance, although increased TGF-ß signalling is implicated in osteoarthritis development in both mouse models of osteoarthritis and human disease, in mice, this is mainly mediated through TGF-ß3 whereas in humans, it is through TGF-ß1. Studies in other tissues have shown TGF-ß1 is more potent than TGF-ß3 in inducing the switch to SMAD1/5 signalling that occurs in osteoarthritic cartilage and that TGF-ß1 and TGF-ß3 have opposing effects on fibrosis. It is therefore possible that the relative contribution of TGF-ß signalling to joint pathology in osteoarthritis differs between murine models and humans. Understanding the similarities and differences in osteoarthritis pathogenesis between mouse models and humans is critical for understanding the translational potential of findings from pre-clinical studies.

REST, RCOR1 and RCOR2 expression is reduced in osteoarthritic chondrocytes and contributes to increasing MMP13 and ADAMTS5 expression through upregulating HES1.

Expression of key transcriptional regulators is altered in chondrocytes in osteoarthritis (OA). This contributes to an increase in production of cartilage-catabolizing enzymes such as MMP13 and ADAMTS5. RCOR1 and RCOR2, binding partners for the transcriptional repressor REST, have previously been found to be downregulated in OA chondrocytes although their function in chondrocytes is unclear. HES1 is a known REST/RCOR1 target gene and HES1 has been shown to promote MMP13 and ADAMTS5 expression in murine OA chondrocytes. The purpose of this study was to determine whether reduced REST/RCOR levels leads to increased HES1 expression in human OA chondrocytes and whether HES1 also promotes ADAMTS5 and MMP13 expression in these cells. Chondrocytes were isolated from osteoarthritic and adjacent macroscopically normal cartilage obtained from patients undergoing total knee arthroplasty. RNA and protein levels of REST, RCOR1 and RCOR2 were lower, but levels of HES1 higher, in chondrocytes isolated from osteoarthritic compared to macroscopically normal cartilage. Over-expression of either REST, RCOR1 or RCOR2 resulted in reduced HES1 levels in OA chondrocytes whereas knockdown of REST, RCOR1 or RCOR2 led to increased HES1 expression in chondrocytes from macroscopically normal cartilage. In OA chondrocytes, ADAMTS5 and MMP13 expression were reduced following HES1 knockdown, but further enhanced following HES1 over-expression. Levels of phosphorylated CaMKII were higher in chondrocytes from OA cartilage consistent with previous findings that HES1 only promotes gene transcription in the presence of active CaMKII. These findings identify the REST/RCOR/HES1 pathway as a contributing factor leading to increased ADAMTS5 and MMP13 expression in OA chondrocytes.

The growth hormone receptor interacts with transcriptional regulator HMGN1 upon GH-induced nuclear translocation.

Growth hormone (GH) actions are mediated through binding to its cell-surface receptor, the GH receptor (GHR), with consequent activation of downstream signalling. However, nuclear GHR localisation has also been observed and is associated with increased cancer cell proliferation. Here we investigated the functional implications of nuclear translocation of the GHR in the human endometrial cancer cell-line, RL95-2, and human mammary epithelial cell-line, MCF-10A. We found that following GH treatment, the GHR rapidly translocates to the nucleus, with maximal localisation at 5-10 min. Combined immunoprecipitation-mass spectrometry analysis of RL95-2 whole cell lysates identified 40 novel GHR binding partners, including the transcriptional regulator, HMGN1. Moreover, microarray analysis demonstrated that the gene targets of HMGN1 were differentially expressed following GH treatment, and co-immunoprecipitation showed that HMGN1 associates with the GHR in the nucleus. Therefore, our results suggest that GHR nuclear translocation might mediate GH actions via interaction with chromatin factors that then drive changes in specific downstream transcriptional programs.

Differential Expression of Steroid Hormone Receptors and Ten Eleven Translocation Proteins in Endometrial Cancer Cells.

Steroid hormones govern the complex, cyclic changes of the endometrium, predominantly through their receptors. An interplay between steroid hormones and epigenetic mechanisms controls the dynamic endometrial gene regulation. Abnormalities in expression of genes and enzymes associated with steroid hormone signaling, contribute to a disturbed hormonal equilibrium. Limited evidence suggests the involvement of TET (Ten Eleven Translocation)-mediated DNA hydroxymethylation in endometrial cancer, with some data on the use of TET1 as a potential prognostic and diagnostic biomarker, however the mechanisms guiding it and its regulation remains unexplored. This study aims to explore the changes in the expressions of TETs and steroid hormone receptors in response to estrogen and progesterone in endometrial cancer cells. Gene expression was examined using real-time PCR and protein expression was quantified using fluorescent western blotting in endometrial cancer cell lines (AN3 and RL95-2). Results indicate that TET1 and TET3 gene and protein expression was cell-specific in cancer cell-lines. Protein expression of TET1 was downregulated in AN3 cells, while TET1 and TET3 expressions were both upregulated in RL95-2 cells in response to estrogen-progesterone. Further, a decreased AR expression in AN3 cells and an increased ERα and ERß protein expressions in RL95-2 cells was seen in response to estrogen-progesterone. PR gene and protein expression was absent from both cancer cell-lines. Overall, results imply that expressions of steroid hormones, steroid-hormone receptors and TETs are co-regulated in endometrial cancer-cells. Further studies are needed to interpret how these mechanisms fit in with DNMTs and DNA methylation in regulating endometrial biology. Understanding the role of TETs and hydroxymethylation in steroid hormone receptor regulation is crucial to comprehend how these mechanisms work together in a broader context of epigenetics in the endometrium and its pathologies.

3D interactions with the growth hormone locus in cellular signalling and cancer-related pathways.

Growth hormone (GH) is a peptide hormone predominantly produced by the anterior pituitary and is essential for normal growth and metabolism. The GH locus contains five evolutionarily related genes under the control of an upstream locus control region that coordinates tissue-specific expression of these genes. Compromised GH signalling and genetic variation in these genes has been implicated in various disorders including cancer. We hypothesised that regulatory regions within the GH locus coordinate expression of a gene network that extends the impact of the GH locus control region. We used the CoDeS3D algorithm to analyse 529 common single nucleotide polymorphisms (SNPs) across the GH locus. This algorithm identifies colocalised Hi-C and eQTL associations to determine which SNPs are associated with a change in gene expression at loci that physically interact within the nucleus. One hundred and eighty-one common SNPs were identified that interacted with 292 eGenes across 48 different tissues. One hundred and forty-five eGenes were regulated in trans. eGenes were found to be enriched in GH/GHR-related cellular signalling pathways including MAPK, PI3K-AKT-mTOR, ERBB and insulin signalling, suggesting that these pathways may be co-regulated with GH signalling. Enrichment was also observed in the Wnt and Hippo signalling pathways and in pathways associated with hepatocellular, colorectal, breast and non-small cell lung carcinoma. Thirty-three eQTL SNPs identified in our study were found to be of regulatory importance in a genome-wide Survey of Regulatory Elements reporter screen. Our data suggest that the GH locus functions as a complex regulatory region that coordinates expression of numerous genes in cis and trans, many of which may be involved in modulating GH function in normal and disease states.

TNF-α Differentially Regulates Cell Cycle Genes in Promyelocytic and Granulocytic HL-60/S4 Cells.

Tumor necrosis factor alpha (TNF-α) is a potent cytokine involved in systemic inflammation and immune modulation. Signaling responses that involve TNF-α are context dependent and capable of stimulating pathways promoting both cell death and survival. TNF-α treatment has been investigated as part of a combined therapy for acute myeloid leukemia due to its modifying effects on all-trans retinoic acid (ATRA) mediated differentiation into granulocytes. To investigate the interaction between cellular differentiation and TNF-α, we performed RNA-sequencing on two forms of the human HL-60/S4 promyelocytic leukemia cell line treated with TNF-α. The ATRA-differentiated granulocytic form of HL-60/S4 cells had an enhanced transcriptional response to TNF-α treatment compared to the undifferentiated promyelocytes. The observed TNF-α responses included differential expression of cell cycle gene sets, which were generally upregulated in TNF-α treated promyelocytes, and downregulated in TNF-α treated granulocytes. This is consistent with TNF-α induced cell cycle repression in granulocytes and cell cycle progression in promyelocytes. Moreover, we found evidence that TNF-α treatment of granulocytes shifts the transcriptome toward that of a macrophage. We conclude that TNF-α treatment promotes a divergent transcriptional program in promyelocytes and granulocytes. TNF-α promotes cell cycle associated gene expression in promyelocytes. In contrast, TNF-α stimulated granulocytes have reduced cell cycle gene expression, and a macrophage-like transcriptional program.