Chondrocyte Gene Research Articles

Endoplasmic reticulum stress-mediated apoptosis contributes to a skeletal dysplasia resembling platyspondylic lethal skeletal dysplasia, Torrance type, in a novel Col2a1 mutant mouse line

In humans, mutations in the COL2A1 gene encoding the α1(II) chain of type II collagen, create many clinical phenotypes collectively termed type II collagenopathies. However, the mechanisms generating this diversity remain to be determined. Here we identified a novel Col2a1 mutant mouse line by screening a large-scale N-ethyl-N-nitrosourea mutant mouse library. This mutant possessed a p.Tyr1391Ser missense mutation in the C-propeptide coding region, and this mutation was located in positions corresponding to the human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). As expected, p.Tyr1391Ser homozygotes exhibited lethal skeletal dysplasias resembling PLSD-T, including extremely short limbs and severe dysplasia of the spine and pelvis. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by an abnormally expanded endoplasmic reticulum (ER) and the up-regulation of ER stress-related genes in chondrocytes. Chondrocyte apoptosis was severely induced in the growth plate of the homozygotes. These findings strongly suggest that ER stress-mediated apoptosis caused by the accumulated mutant proteins in ER contributes to skeletal dysplasia in Co12a1 mutant mice and PLSD-T patients.

The function and interrelationship between GDF5 and ERG-010 during chondrogenesis in vitro.

Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton. Both GDF5 and ERG are proposed as regulators of chondrocyte differentiation during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Erg expression was identified in mouse knee joints at E13.5. Expression analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence and absence of GDF5 and ERG. At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant GDF5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by GDF5. Erg regulation by GDF5 in induced mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2 and/or Ube2b. Combined, these data suggest a possible pathway linking GDF5, ERG and downstream factors in the processes of chondrocyte differentiation during articular joint formation.

Sp1 upregulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes.

Type XI collagen is a cartilage-specific extracellular matrix, and is important for collagen fibril formation and skeletal morphogenesis. We have previously reported that NF-Y regulated the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes (Hida et. al. In Vitro Cell. Dev. Biol. Anim. 2014). However, the mechanism of the Col11a1 gene regulation in chondrocytes has not been fully elucidated. In this study, we further characterized the proximal promoter activity of the mouse Col11a1 gene in chondrocytes. Cell transfection experiments with deletion and mutation constructs indicated that the downstream region of the NF-Y binding site (-116 to +1) is also necessary to regulate the proximal promoter activity of the mouse Col11a1 gene. This minimal promoter region has no TATA box and GC-rich sequence; we therefore examined whether the GC-rich sequence (-96 to -67) is necessary for the transcription regulation of the Col11a1 gene. Luciferase assays using a series of mutation constructs exhibited that the GC-rich sequence is a critical element of Col11a1 promoter activity in chondrocytes. Moreover, in silico analysis of this region suggested that one of the most effective candidates was transcription factor Sp1. Consistent with the prediction, overexpression of Sp1 significantly increased the promoter activity. Furthermore, knockdown of Sp1 expression by siRNA transfection suppressed the proximal promoter activity and the expression of endogenous transcript of the mouse Col11a1 gene. Taken together, these results indicate that the transcription factor Sp1 upregulates the proximal promoter activity of the mouse Col11a1 gene in chondrocytes.

Identification of differentially methylated regions in new genes associated with knee osteoarthritis

Epigenetic changes in articular chondrocytes are associated with osteoarthritis (OA) disease progression. Numerous studies have identified differentially methylated cytosines in OA tissues; however, the consequences of altered CpG methylation at single nucleotides on gene expression and phenotypes are difficult to predict. With the objective of detecting novel genes relevant to OA, we conducted a genome-wide assessment of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). DNA was extracted from visually damaged and normal appearing, non-damaged human knee articular cartilage from the same joint and then subjected to reduced representation bisulfite sequencing. DMRs were identified using a genome-wide systematic bioinformatics approach. A sliding-window of 500bp was used for screening the genome for regions with clusters of DMSs. Gene expression levels were assessed and cell culture demethylation experiments were performed to further examine top candidate genes associated with damaged articular cartilage. More than 1000 DMRs were detected in damaged osteoarthritic cartilage. Nineteen of these contained five or more DMSs and were located in gene promoters or first introns and exons. Gene expression assessment revealed that hypermethylated DMRs in damaged samples were more consistently associated with gene repression than hypomethylated DMRs were with gene activation. Accordingly, a demethylation agent induced expression of most hypermethylated genes in chondrocytes. Our study revealed the utility of a systematic DMR search as an alternative to focusing on single nucleotide data. In particular, this approach uncovered promising candidates for functional studies such as the hypermethylated protein-coding genes FOXP4 and SHROOM1, which appear to be linked to OA pathology in humans and warrant further investigation.

Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes.

Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Expression of genes associated with cellular motility and migration was affected by anti-LAMA4 treatment. LAMA4-integrin signalling affects chondrocyte morphology and gene expression in vitro, thereby contributing to cluster formation in human osteoarthritic chondrocytes.

Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis

Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5′-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes

AimThe aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk.Materials and MethodsOsteoarthritic (OA) human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM). The production of aggrecan, matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-6 and nitric oxide (NO) and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC) or non-sclerotic (NSC) subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture.ResultsIn chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008). MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01). TIMP-1 production was slightly increased at 3 μM (p = 0.02) and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05). IL-6 and PGE2 production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes.ConclusionsCarnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially mediated via inhibition of IL-6 in osteoblasts as similar results were obtained with anti-IL-6 antibody.

Abstract 2999: DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression

Abstract Circadian rhythms are tightly regulated by clock genes. It has been reported that clock genes plays important roles in biological function of normal cells. However, it is not well understood the function of clock genes in tumor cells. We showed that basic helix-loop-helix (bHLH) transcription factors, differentiated embryonic chondrocyte gene 1 (DEC1/BHLHE40/Sharp2/Stra13) and DEC2 (BHLHE41/Sharp1), are clock genes and play important roles in circadian rhythm, cell proliferation, apoptosis, hypoxia response, various stresses and epithelial-to-mesenchymal transition (EMT) in tumor cells. Various stresses, such as exposure to transforming growth factor-beta (TGF-β), hypoxia, cytokines, serum-free, and anti-tumor drugs affect DEC expression. The increased or decreased DEC expression by such stresses is involved in tumor progression. The evidence that DEC2 has circadian expression in implanted mouse sarcoma cells and negatively regulates VEGF expression under hypoxia. DEC1 has pro-apoptotic effects in human breast cancer cells, whereas DEC2 has anti-apoptotic effects. DEC1 induced EMT by TGF-β in human pancreatic cancer cells, whereas DEC2 suppressed EMT. Immunohistochemical analysis showed that DEC1 is highly expressed in tumor cells and the expression is involved in malignancy. On the other hand, DEC2 is little expressed in tumor cells compared with adjacent non-tumor cells. These findings suggest that DEC1 and DEC2 have different roles in tumor progression under circadian rhythm. DEC2 regulates the circadian rhythm and VEGF expression by negative feed-back system, and DEC1 and DEC2 regulates EMT by positive and negative feed-back system in tumor cells. So, DEC acts as “accelerator” and “brake” in tumor progression. In this study, we show current progress of the DEC genes in tumor progression. Citation Format: Fuyuki Sato, Yasuteru Muragaki. DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2999. doi:10.1158/1538-7445.AM2015-2999

Age-independent effects of hyaluronan amide derivative and growth hormone on human osteoarthritic chondrocytes

Aim: Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment.Material and methods: Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01–10 μg/ml) alone or in combination with HAD (1 mg/ml).Results: We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients.Conclusions: hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.

Second Place

Background: Statins (HMG-CoA Reductase inhibitors) reduce the incidence and progression of osteoarthritis of the knee. Nitrogen-containing bisphosphonates (N-BPs) and statins have similar mechanisms of action, but the effects of N-BPs on articular cartilage in osteoarthritis are not understood. We sought to determine if the bisphosphonate alendronate (ALN) has anti-inflammatory effects on articular chondrocytes in vitro. Methods: An in vitro model was developed for sustaining harvested rat chondrocytes in an alginate gel matrix. Using this model with human chondrocytes, osteoarthritis was simulated using interleukin-1β (IL-1β) stimulation. Cells were treated with ALN (10-100 μM) and IL-1β (0.01 and 0.1 ng/mL) for 24 and 48 h. RNA was isolated to quantify the effects of IL-1β and ALN on chondrocyte gene expression for cartilage matrix synthesis (aggrecan, collagen type I and II) and degradation (MMP13, ADAMTS4, ADAMTS5). Results: Collagen type I, collagen type II, and aggrecan expression decreased with IL-1β stimulation in human chondrocytes, which was not reversed by ALN treatments. MMP13, ADAMTS4, and ADAMTS5 expression increased with IL-1β stimulation, and the increases in MMP13 and ADAMTS5 were reduced by ALN treatments after 24 h. Conclusions: ALN reversed increases in chondrocyte MMP13 and ADAMTS5 expression due to IL-1β stimulation in vitro, and ALN may play a role in preventing cartilage matrix degradation in osteoarthritis.

Epigenetic regulation of chondrocyte differentiation

Summary Chondrocytes play an essential role in endochondral bone development, which is requisite for mammalian skeletal development. During endochondral bone development, chondrocytes undergo well-organized stages of sequential differentiation, including proliferation and hypertrophy, a process harmoniously modulated by various transcription factors. Epigenetics, including DNA methylation and histone modification, has recently emerged as an essential regulatory system for gene expression, not only in physiological conditions, but also in human disease. During chondrocyte differentiation, transcriptional co-regulators, which form transcriptional protein complexes with specific transcription factors, are predominantly involved in this epigenetic process and cooperatively regulate chondrocyte gene expression. Importantly, several studies indicate that epigenetic regulators correlate with cartilage-related diseases, such as osteoarthritis, and are noted as a potential therapeutic target. Here, current studies of epigenetic regulation of chondrocyte differentiation are reviewed and novel aspects for the molecular mechanisms involved in chondrogenesis are introduced.

Genome-wide mapping of DNA hydroxymethylation in osteoarthritic chondrocytes.

To examine the genome-wide distribution of hydroxymethylated cytosine (5hmC) in osteoarthritic (OA) and normal chondrocytes in order to investigate the effect on OA-specific gene expression. Cartilage was obtained from OA patients undergoing total knee arthroplasty or from control patients undergoing anterior cruciate ligament reconstruction. Genome-wide sequencing of 5hmC-enriched DNA was performed in a small cohort of normal and OA chondrocytes to identify differentially hydroxymethylated regions (DhMRs) in OA chondrocytes. Data from the genome-wide sequencing of 5hmC-enriched DNA were intersected with global OA gene expression data to define subsets of genes and pathways potentially affected by increased 5hmC levels in OA chondrocytes. A total of 70,591 DhMRs were identified in OA chondrocytes as compared to normal chondrocytes, 44,288 (63%) of which were increased in OA chondrocytes. The majority of DhMRs (66%) were gained in gene bodies. Increased DhMRs were observed in ∼50% of genes previously implicated in OA pathology including MMP3, LRP5, GDF5, and COL11A1. Furthermore, analyses of gene expression data revealed gene body gain of 5hmC appears to be preferentially associated with activated, but not repressed, genes in OA chondrocytes. This study provides the first genome-wide profiling of 5hmC distribution in OA chondrocytes. We had previously reported a global increase in 5hmC levels in OA chondrocytes. Gain of 5hmC in the gene body is found to be characteristic of activated genes in OA chondrocytes, highlighting the influence of 5hmC as an epigenetic mark in OA. In addition, this study identifies multiple OA-associated genes that are potentially regulated either singularly by gain of DNA hydroxymethylation or in combination with loss of DNA methylation.

The in vivo and in vitro effect of inhibitors of brd4 and cdk9 on early phase of post traumatic osteoarthritis

Purpose: Joint trauma is a risk factor for osteoarthritis (OA), and about 50% of patients with ACL or meniscal injury develop posttraumatic OA (PTOA) within 10-20 years. The acute response to joint trauma increases transcription of pro-inflammatory cytokines and proteinases such as matrix metalloproteinases (MMPs), which trigger the onset of OA changes. Bromodomain protein 4 (Brd4) and cyclin-dependent kinase 9 (CDK9) control the rate-limiting step of the transcription of primary response genes, including most pro-inflammatory genes, by positively regulating mRNA elongation with phosphorylating and releasing the RNA polymerase II. The purpose of this study is to investigate the effects of small molecule inhibitors of Brd4 (JQ1) and CDK9 (Flavopiridol) on the activation of inflammatory genes using chondrocytes and cartilage tissue under inflammatory stimuli, and a mouse PTOA model. Methods: • Treatment of chondrocytes Human chondrocytes were cultured 5 hours with inflammatory stimuli (either 10ng/ml IL1b, 10ng/ml TNFa, or 100ng/ml IL6 and 60ng/ml IL6 receptor), with or without drugs. Treatment conditions were: 1) vehicle only (Ctrl), 2) no drug with cytokine, 3) Hi JQ1 (1200nM) with cytokine, 4) Hi Flavopiridol (250nM) with cytokine, 5) Combination of Lo JQ1 (250nM) and Lo Flavopiridol (60nM) with cytokine. Total RNA was extracted and analyzed by real time RT-PCR and microarray. • Treatment of cartilage explants Bovine cartilage explants were isolated and randomly assigned to 5 groups cultured as described above, with the cytokine being 10ng/ml IL1b. Glycosaminoglycan (GAG) released into the culture media was measured. • PTOA animal model The right knees of mice were injured with a mechanical compression, which causes rupture of the anterior cruciate ligament and leads to PTOA. Immediately after injury, mice were treated daily with JQ1 and/or Flavopiridol. Treatment conditions were: 1) vehicle only (Ctrl), 2) Hi JQ1 (50mg/kg), 3) Hi Flavopiridol (7.5mg/kg), 4) Combination of Lo JQ1 (17mg/kg) and Lo Flavopiridol (2.5mg/kg) Flavopiridol. • MMPSense Assay To assess MMP activity, MMPSense 750 was injected to mice and the intensity of signal from the MMPSense in the knees was measured by in vivo imaging. Results: • The mRNA expression levels of pro-inflammatory genes (iNOS, Cox2) and catabolic genes (MMP-1, -3, -9, and -13, and ADAMTS4) were significantly induced by all 3 inflammatory cytokines, and this induction was suppressed by all 3 drug treatments. The combination of both drugs at lower doses suppressed gene expression similarly or more strongly than single high doses of each individual drug. • Microarray showed that expression, 873 genes were induced >1.5-fold by IL1b compared to baseline. IL-1b treatment in the presence of either JQ1 or Flavopiridol alone prevented the induction of many genes. However, a combination of both drugs prevented the induction of most IL-1b response genes. (Fig.1) • IL1b treatment of cartilage explants induced significant release of GAG within 3-6 days. GAG release was effectively prevented when IL-1b treatment in the presence of either or both drugs. (Fig2) • In PTOA mouse model, knee injury caused significant increases of IL1b and IL6 expression in the injured joint. All 3 treatments showed effect to prevent increases of these cytokines and drug combination was more effective than single drugs. (Fig3A, B) MMP activity in injured knee was suppressed by all 3 treatment similarly at 24h and 48h after injury. (Fig3C, D) Conclusions: JQ1 and Flavopiridol are each able to effectively repress a panel of pro-inflammatory and catabolic genes in chondrocytes induced by inflammatory stimulus. We found that the combination of the 2 drugs showed a synergistic interaction, with similar or better repression achieved at reduced drug doses. Although previous reports indicated that Brd4 and CDK9 control mRNA transcription by regulating a common checkpoint, microarray analysis showed that there were also inflammatory genes only affected by each drug individually. Ex vivo and in vivo study also demonstrated the combination of lower dose of the drugs has similar or better intensity of effect. This indicates that using both drugs together may be able to suppress inflammation after trauma with preventing side effects induced by overdose, leading to novel treatment for PTOA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

MicroRNA-33a regulates cholesterol synthesis and cholesterol efflux related genes in osteoarthritic chondrocytes

Several studies have shown that osteoarthritis (OA) is strongly associated with metabolism-related disorders, highlighting OA as the fifth component of the metabolic syndrome (MetS). On the basis of our previous findings on dysregulation of cholesterol homeostasis in OA, we were prompted to investigate whether microRNA-33a (miR-33a), one of the master regulators of cholesterol and fatty acid metabolism, plays a key role in OA pathogenesis. Articular cartilage samples were obtained from 14 patients with primary OA undergoing total knee replacement surgery. Normal cartilage was obtained from nine individuals undergoing fracture repair surgery. Bioinformatics analysis was used to identify miR-33a target genes. miR-33a and sterol regulatory element-binding protein 2 (SREBP-2) expression levels were investigated using real-time PCR, and their expression was also assessed after treatment with transforming growth factor-β1 (TGF-β1) in cultured chondrocytes. Akt phosphorylation after treatment with both TGF-β1 and miR-33a inhibitor or TGF-β1 and miR-33a mimic was assessed by Western blot analysis. Furthermore, we evaluated the effect of miR-33a mimic and miR-33a inhibitor on Smad7, a negative regulator of TGF-β signaling, on cholesterol efflux-related genes, ATP-binding cassette transporter A1 (ABCA1), apolipoprotein A1 (ApoA1) and liver X receptors (LXRα and LXRβ), as well as on matrix metalloproteinase-13 (MMP-13), using real-time PCR. We found that the expression of miR-33a and its host gene SREBP-2 was significantly elevated in OA chondrocytes compared with normal chondrocytes. Treatment of cultured chondrocytes with TGF-β1 resulted in increased expression of both miR-33a and SREBP-2, as well as in rapid induction of Akt phosphorylation, whereas TGF-β-induced Akt phosphorylation was enhanced by miR-33a and suppressed by inhibition of miR-33a, as a possible consequence of Smad7 regulation by miR-33a. Moreover, treatment of normal chondrocytes with miR-33a resulted in significantly reduced ABCA1 and ApoA1 mRNA expression levels and significantly elevated MMP-13 expression levels, promoting the OA phenotype, whereas miR-33a’s suppressive effect was reversed using its inhibitor. Our findings suggest, for the first time to our knowledge, that miR-33a regulates cholesterol synthesis through the TGF-β1/Akt/SREBP-2 pathway, as well as cholesterol efflux-related genes ABCA1 and ApoA1, in OA chondrocytes, pointing to its identification as a novel target for ameliorating the OA phenotype.

The DOT1L protein and gene network in chondrocytes identifies H3K79 histone methylation as a key regulator of WNT and other growth factor cascades

Purpose: DOT1L is the only known H3K79 histone methyltransferase and has been linked to epigenetic regulation of the Wingless-like (Wnt) signaling pathway. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signaling. Here, we further investigated the biology of DOT1L in cartilage health and disease. Specifically, our objective was to define the transcriptional complexes and transcriptome associated with DOT1L in articular cartilage, and to link these DOT1L targets and interactors with the molecular characteristics of the articular chondrocyte during the loss of phenotype in a dedifferentiation setup. Finally, we aimed for a translational validation of our data in cartilage samples from OA patients. Methods: Human articular chondrocytes (hACs) stimulated or not with LiCl were used to carefully map the presence or absence of DOT1L protein complexes suggested earlier in leukemia cells, using a series of specific immunoprecipitation experiments. To elucidate the transcriptional network of Dot1l, we performed a microarray of hACs from 5 non-OA fracture patients treated with a specific DOT1L inhibitor (EPZ5676) or vehicle control. The differentially expressed genes were explored with the limma package. String-DB and Panther were used for gene network analyses. Based on the microarray analysis, selected genes were confirmed by quantitative PCR. To establish the role of DOT1L in the maintenance of hACs phenotype, hACs were cultured in monolayer in the presence or absence of EPZ5676, and RNA and protein were isolated at serial passages (P0 to P5). We analyzed by quantitative PCR the gene expression of known genes important for chondrocyte biology and/or genes that appeared in the microarray of DOT1L inhibition. Activation of the Wnt canonical and non-canonical signaling cascades was analyzed by Western blot. For translational validation we analyzed the differential gene expression profile of damaged as compared to intact cartilage areas within the same joint of patients with hip OA. Results: The presence of different DOT1L elongation complexes was confirmed in hACs. The CoIP experiments revealed that DOT1L interacts directly with ENL, AF10 and active beta-catenin (more strongly when cells were treated with LiCl to stabilize beta-catenin) and with AF4 and AF5 (only with cells were treated with LiCl). The formation of these complexes was disrupted when DOT1L was inhibited. In the microarray, 1937 genes significantly changed (p<0.05) in DOT1L inhibited samples compared to vehicle treated samples (1048 genes were down-regulated, 889 up-regulated). In EPZ5676 treated samples, chondrocyte differentiation-associated genes, such as ACAN, RGS5, GDF10 and LOXL2 were down-regulated; OA-related genes, such as MMP1, CCL7, CCL8 and GPNMB were up-regulated; and WNT target genes, ligands and antagonists such as LEF1, WNT5A and DKK1 were significantly up-regulated. In the de-differentiation experiment, in vitro expanded hACs showed progressive changes in gene expression. Genes involved in relevant pathways for cartilage biology such as the WNT, NOTCH and BMP pathways exhibited significant changes that were highly accentuated by DOT1L inhibition. We successfully validated these targets of interest in samples from patients with OA condition. Conclusions: Our transcriptomic, protein and gene interaction approach provides novel insights into the DOT1L molecular network and its putative role in osteoarthritis and cartilage. These data further support an important role for DOT1L in joint homeostasis as a key regulator of WNT signaling and other growth factor cascades in the joint.

Quantitative tracking of passage and 3D culture effects on chondrocyte and fibrochondrocyte gene expression.

Success in cartilage and fibrocartilage tissue engineering relies heavily on using an appropriate cell source. Many different cell sources have been identified, including primary and stem cells, along with experimental strategies to obtain the required number of cells or to induce chondrogenesis. However, no definitive method exists to quantitatively evaluate the similarity of the resulting cell phenotypes to those of the native cells between candidate strategies. In this study, we develop an integrative approach to enable such evaluations by deriving, from gene expression profiles, two quantitative metrics representing the nearest location within the range of native cell phenotypes and the deviation from it. As an example application to evaluating potential cell sources for cartilage or meniscus tissue engineering, we examine phenotypic changes of juvenile and adult articular chondrocytes and fibrochondrocytes across multiple passages and subsequent 3D culture. A substantial change was observed in cell phenotype due to the isolation process itself, followed by a clear progression toward the outer meniscal cell phenotype with passage. The new metrics also indicated that 3D culture moderately reduced the passage-induced deviation from the native meniscal phenotypes for juvenile chondrocytes and adult fibrochondrocytes, which was not obvious through examination of individual gene expressions. However, brief 3D culture alone did not move any of the cells towards an inner meniscal phenotype, the most relevant target for meniscal tissue engineering. This integrative approach of examining and combining multiple gene expressions can be used to evaluate various other tissue-engineering strategies to direct cells toward the desired phenotype. Copyright © 2015 John Wiley & Sons, Ltd.

MicroRNA-33a regulates cholesterol synthesis and cholesterol efflux-related genes in osteoarthritic chondrocytes.

IntroductionSeveral studies have shown that osteoarthritis (OA) is strongly associated with metabolism-related disorders, highlighting OA as the fifth component of the metabolic syndrome (MetS). On the basis of our previous findings on dysregulation of cholesterol homeostasis in OA, we were prompted to investigate whether microRNA-33a (miR-33a), one of the master regulators of cholesterol and fatty acid metabolism, plays a key role in OA pathogenesis.MethodsArticular cartilage samples were obtained from 14 patients with primary OA undergoing total knee replacement surgery. Normal cartilage was obtained from nine individuals undergoing fracture repair surgery. Bioinformatics analysis was used to identify miR-33a target genes. miR-33a and sterol regulatory element-binding protein 2 (SREBP-2) expression levels were investigated using real-time PCR, and their expression was also assessed after treatment with transforming growth factor-β1 (TGF-β1) in cultured chondrocytes. Akt phosphorylation after treatment with both TGF-β1 and miR-33a inhibitor or TGF-β1 and miR-33a mimic was assessed by Western blot analysis. Furthermore, we evaluated the effect of miR-33a mimic and miR-33a inhibitor on Smad7, a negative regulator of TGF-β signaling, on cholesterol efflux-related genes, ATP-binding cassette transporter A1 (ABCA1), apolipoprotein A1 (ApoA1) and liver X receptors (LXRα and LXRβ), as well as on matrix metalloproteinase-13 (MMP-13), using real-time PCR.ResultsWe found that the expression of miR-33a and its host gene SREBP-2 was significantly elevated in OA chondrocytes compared with normal chondrocytes. Treatment of cultured chondrocytes with TGF-β1 resulted in increased expression of both miR-33a and SREBP-2, as well as in rapid induction of Akt phosphorylation, whereas TGF-β-induced Akt phosphorylation was enhanced by miR-33a and suppressed by inhibition of miR-33a, as a possible consequence of Smad7 regulation by miR-33a. Moreover, treatment of normal chondrocytes with miR-33a resulted in significantly reduced ABCA1 and ApoA1 mRNA expression levels and significantly elevated MMP-13 expression levels, promoting the OA phenotype, whereas miR-33a’s suppressive effect was reversed using its inhibitor.ConclusionsOur findings suggest, for the first time to our knowledge, that miR-33a regulates cholesterol synthesis through the TGF-β1/Akt/SREBP-2 pathway, as well as cholesterol efflux-related genes ABCA1 and ApoA1, in OA chondrocytes, pointing to its identification as a novel target for ameliorating the OA phenotype.

DEC2 expression is positively correlated with HIF-1 activation and the invasiveness of human osteosarcomas.

BackgroundOsteosarcoma is the most common malignancy of bone. HIF-1 (hypoxia-inducible factor 1) activation is critical for the metabolic reprogramming and progression of solid tumors, and DEC2 (differentiated embryonic chondrocyte gene 2) has been recently reported to suppress HIF-1 in human breast and endometrial cancers. However, the roles of HIF-1 and DEC2 in human osteosarcomas remain unclear.MethodsWe evaluated the correlation of DEC2 and HIF-1 expression to the prognosis, and studied the roles of DEC2 and HIF-1 activation in the invasiveness of osteosarcoma. Multiple approaches including immunohistochemical staining of clinical osteosarcoma tissues, siRNA-based knockdown and other molecular biology techniques were used. Particularly, by using a repetitive trans-well culture-based in vitro evolution system, we selected a more invasive subpopulation (U2OS-M) of osteosarcoma cells from U2OS and used it as a model to study the roles of DEC2 and HIF-1 in the invasiveness of osteosarcoma.ResultsWe found that the expression of DEC2 was positively correlated with HIF-1α levels, and HIF-1α expression positively correlated with poor prognosis in osteosarcomas. DEC2 knockdown in osteosarcoma cell lines (U2OS, MNNG and 143B) attenuated HIF-1α accumulation and impaired the up-regulation of HIF-1 target genes in response to hypoxia. Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels. Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia. Finally, we found that HIF-1 activation is sufficient to upregulate DEC2 expression in osteosarcoma cells.ConclusionTaken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma. This implies that DEC2 may contribute to the progression and metastasis of human osteosarcoma by sensitizing tumor cells to hypoxia. On the other hand, HIF-1 activation may contribute to the expression of DEC2 in osteosarcoma. This is the first demonstration of a novel DEC2-HIF-1 vicious cycle in osteosarcoma and a tumor-type specific role for DEC2.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0135-8) contains supplementary material, which is available to authorized users.

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Age-related differential gene and protein expression in postnatal cartilage canal and osteochondral junction chondrocytes

Wnt/β-catenin, Indian hedgehog (Ihh)/Parathyroid-related peptide (PTHrP) and retinoid signaling pathways regulate cartilage differentiation, growth, and function during development and play a key role in endochondral ossification. The objective of this study was to elucidate the gene and protein expression of signaling molecules of these regulatory pathways in chondrocytes surrounding cartilage canals and the osteochondral junction during neonatal and pre-adolescent development. This study revealed cell-specific and age-related differences in gene and protein expression of signaling molecules of these regulatory pathways. A trend for higher gene expression of PTHrP along the cartilage canals and Ihh along the osteochondral junction suggests the presence of paracrine feedback in articular–epiphyseal cartilage. Differential expression of canonical (β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signaling (Wnt-5b, Wnt-11) and their inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding the cartilage canals and osteochondral junction provides evidence of the complex interactions occurring during endochondral ossification.