Central role of SREBP-2 in the pathogenesis of osteoarthritis.

BACKGROUND: Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action. METHODOLOGY/PRINCIPAL FINDINGS: We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes' transfection with SREBP-2-G/C plasmid resulted in interleukin-1ß and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-ß (TGF-ß) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-ß alone can induce SREBP-2, we treated normal chondrocytes with TGF-ß and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-ß activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels. CONCLUSIONS/SIGNIFICANCE: We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-ß induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment.

The catabolic role of toll-like receptor 2 (TLR-2) mediated by the NF-κB pathway in septic arthritis.

Toll-like receptors (TLRs) are involved in mediating cell activation on stimulation with microbial components. Our objective was to investigate the role of TLR-2 mediated by the NF-κB pathway in septic arthritic chondrocytes. TLR-1, -2, and -6 mRNA expression levels were investigated in septic and normal chondrocytes using real-time reverse transcription-PCR. TLR-2 and MMP-13 mRNA and protein levels were measured using real-time PCR and Western blot analysis, respectively. Blocking TLR-2 mRNA expression was performed using small interfering RNA (siRNA) against TLR-2 and subsequently MMP-3, MMP-13, IL-1ß, and IL-6 mRNA levels, as well as p65 NF-κB, IkBα, and MMP-13 protein levels were evaluated using real-time PCR and Western blot analysis. IL-6 protein levels were measured using ELISA assay. We observed that TLR-1, -2, and -6 mRNA expression levels were significantly higher in septic compared to normal chondrocytes. MMP-13 mRNA and protein expressions were also significantly upregulated in septic arthritic cartilage. Blocking TLR-2 mRNA expression in septic chondrocytes resulted in significant increase of inactivated nonphosphorylated p65 NF-κB and IkBα protein levels and reduction in MMP-13, IL-1ß, and IL-6 expression. Our findings suggest the pro-inflammatory and catabolic role of TLR-2 mediated by the NF-κB pathway in septic arthritis. Modulation of TLR-mediated signaling may be a potential therapeutic strategy for the prevention of postinfectious cartilage degradation in articular joints.

Correlation of promoter hypermethylation in hTERT, DAPK and MGMT genes with cervical oncogenesis progression.

DNA hypermethylation occurs during the multistep process of cervical carcinogenesis. We investigated whether the methylation status in the promoter region of a potential oncogene, the human telomerase reverse transcriptase (hTERT), and the tumor suppressor genes death-associated protein kinase (DAPK) and O6-methylguanine DNA methyltransferase (MGMT), were able to distinguish the early from late stages of cervical oncogenesis. The methylation status in the promoter of these genes was analyzed using real-time MethyLight analysis in 115 cervical specimens, including normal, premalignant [atypical squamous epithelial cells (ASCUS), low-grade squamous intraepithelial lesions (LGSIL), high-grade squamous intraepithelial lesions (HGSIL)] and cancer specimens. Clinicopathological parameters (cytology, histology, grade, stage) were compared to the levels of promoter hypermethylation. We found that hTERT, MGMT and DAPK hypermethylation levels were increased during cervical oncogenesis progression. hTERT promoter hypermethylation was able to distinguish normal from cancer (p=0.008), normal from premalignant (p=0.036), as well as premalignant from cervical cancer cases (p=0.003). A significant association was also observed between all three genes and the grade of cervical cancer, with hTERT showing a better association (p<0.0001). Our data suggest that the combination of hTERT, MGMT, DAPK promoter hypermethylation could have a potential function as molecular biomarker of cervical oncogenesis progression.

Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks.

BACKGROUND: Osteoarthritis is a multifactorial disease characterized by destruction of the articular cartilage due to genetic, mechanical and environmental components affecting more than 100 million individuals all over the world. Despite the high prevalence of the disease, the absence of large-scale molecular studies limits our ability to understand the molecular pathobiology of osteoathritis and identify targets for drug development. METHODOLOGY/PRINCIPAL FINDINGS: In this study we integrated genetic, bioinformatic and proteomic approaches in order to identify new genes and their collaborative networks involved in osteoarthritis pathogenesis. MicroRNA profiling of patient-derived osteoarthritic cartilage in comparison to normal cartilage, revealed a 16 microRNA osteoarthritis gene signature. Using reverse-phase protein arrays in the same tissues we detected 76 differentially expressed proteins between osteoarthritic and normal chondrocytes. Proteins such as SOX11, FGF23, KLF6, WWOX and GDF15 not implicated previously in the genesis of osteoarthritis were identified. Integration of microRNA and proteomic data with microRNA gene-target prediction algorithms, generated a potential "interactome" network consisting of 11 microRNAs and 58 proteins linked by 414 potential functional associations. Comparison of the molecular and clinical data, revealed specific microRNAs (miR-22, miR-103) and proteins (PPARA, BMP7, IL1B) to be highly correlated with Body Mass Index (BMI). Experimental validation revealed that miR-22 regulated PPARA and BMP7 expression and its inhibition blocked inflammatory and catabolic changes in osteoarthritic chondrocytes. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that obesity and inflammation are related to osteoarthritis, a metabolic disease affected by microRNA deregulation. Gene network approaches provide new insights for elucidating the complexity of diseases such as osteoarthritis. The integration of microRNA, proteomic and clinical data provides a detailed picture of how a network state is correlated with disease and furthermore leads to the development of new treatments. This strategy will help to improve the understanding of the pathogenesis of multifactorial diseases such as osteoarthritis and provide possible novel therapeutic targets.

The growth differentiation factor 5 (GDF5) core promoter polymorphism is not associated with knee osteoarthritis in the Greek population.

Genetic factors have been shown to play an important role in the etiology of osteoarthritis (OA). A functional single nucleotide polymorphism (SNP) +104T/C; rs143383 in the 5' UTR of the GDF5 gene was recently associated with susceptibility to osteoarthritis in the Japanese and Chinese population. Our objective was to assess whether this SNP was also associated with knee OA in a Greek Caucasian population sample. The +104T/C SNP was genotyped in a total of 519 case-control cohort; 251 patients with idiopathic knee OA and 268 controls were used. No significant differences were found in genotype or allele frequencies of the +104T/C SNP of GDF5 gene between cases and controls (p < 0.05). Also, no significant differences in allelic and genotypic frequencies were found when the individuals were stratified by sex. Our data implied that the +104T/C; rs143383 GDF5 core promoter polymorphism is not a risk factor for OA etiology in Greek Caucasians. Our study highlights the heterogeneous nature of OA genetic susceptibility.

Association of interleukin-1A, interleukin-1B and interleukin-1 receptor antagonist gene polymorphisms with multiple myeloma.

Interleukin-1 (IL-1) is a cytokine involved in the maturation and proliferation of B cells and plays a significant role in the development of lytic bone lesions, a major clinical feature of multiple myeloma (MM) patients. Genes that regulate products involved in the immune system are highly polymorphic and contribute to inter-individual differences that can influence the genetic predisposition and progression of particular diseases and cancers. In this study, we investigated the correlation between the single nucleotide polymorphisms IL1A -889, IL1B -511, IL1B +3954, IL1RN Mspa1 +11100 and susceptibility to MM in 74 patients and 160 controls. We found that individuals possessing IL1A -889 CT polymorphism had a higher risk in developing MM. Moreover, genotypes IL1B -511 CC, IL1B +3954 CC, IL-1RN Mspa1 +11100 CC and the combination of IL1B +3954 CC with IL1B -511 CC or IL-1RN Mspa1 +11100 CC exerted a protective effect in individuals possessing them.

DNA methylation is not likely to be responsible for hTERT expression in premalignant cervical lesions.

Human telomerase reverse transcriptase (hTERT) mRNA expression seems to play an important role in cervical carcinogenesis. Analysis of the hTERT promoter region revealed the presence of a CpG island and a high overall GC content, suggesting a possible role for methylation in the regulation of hTERT gene expression. The present study was designed to evaluate the role of hTERT promoter methylation and E6/E7 human papilloma virus 16 (HPV-16) mRNA expression in hTERT regulation in premalignant cervical specimens. The methylation status of the hTERT promoter gene and hTERT mRNA quantification were investigated in 26 normal and 64 specimens of abnormal cytology using the MethyLight technique, Telo-TAGGG hTERT Quantification Kit and LightCycler technology. E6/E7 HPV-16 mRNA expression was also evaluated. No significant correlations were observed between hTERT mRNA expression and hTERT promoter methylation, as well as between telomerase activity and hTERT promoter methylation in normal and in premalignant cervical specimens. E6/E7 HPV-16 mRNA expression was observed in 72% of HPV-16-infected samples and was correlated with hTERT mRNA expression and telomerase activity (P < 0.05). This is the first study investigating the role of hTERT promoter methylation in hTERT mRNA expression and telomerase activity in premalignant lesions. The observed lack of correlation suggests that other mechanisms might be involved in the regulation of hTERT expression. The correlation between hTERT mRNA and E6/E7 mRNA expression confirms the role of HPV infection in hTERT regulation.

Quantitative determination of human telomerase reverse transcriptase messenger RNA expression in premalignant cervical lesions and correlation with human papillomavirus load.

Expression of human telomerase reverse transcriptase (hTERT) messenger RNA (mRNA) and human papillomavirus (HPV)-16 load were quantified using real-time polymerase chain reaction and correlated with cytological findings and the presence of HPV infection in cervical specimens. Human telomerase reverse transcriptase mRNA expression was evaluated in 15 (20.5%) of 73 specimens of atypical squamous epithelial cells of undetermined significance, in 62 (39.7%) of 156 low-grade squamous intraepithelial lesions (LGSILs), in 49 (96%) of 51 high-grade squamous intraepithelial lesions (HGSILs), and in 9 (20%) of 45 normal samples, whereas viral load was quantified in 52 (89.6%) of 58 samples infected with HPV-16. The mean levels of hTERT mRNA expression were 0.11 in normal tissue, 0.23 in atypical squamous epithelial cells of undetermined significance, 0.75 in LGSILs, and 2.5 in HGSILs. Thus, a significant increase in hTERT mRNA expression was observed with increasing degrees of cervical dysplasia. The HPV-16 load was significantly higher in samples of HGSIL than in those of LGSILs (P < .001). A significant correlation was observed between viral load and quantitative hTERT mRNA expression (r = 0.65; P < .05). Quantitative hTERT mRNA assessment showed 96% sensitivity and 100% negative predictive value for high-grade dysplasia, whereas the specificity and positive predictive value were 72% and 36.2%, respectively. It is suggested that quantitative hTERT has a very high sensitivity and negative predictive value, whereas the observed specificity was moderate, indicating that it cannot be used as a diagnostic marker but may be an adjunct in the management of women with high-grade cervical dysplasia. However, the final diagnosis must rely on the inclusion of clinical evaluation and additional assessment data.