Cationic ribosomal proteins can inhibit pro-inflammatory action stimulated by LPS+HMGB1 and are hindered by advanced glycation end products.

We previously found that ribosomal protein L9 (RPL9) is a novel advanced glycation end product (AGE)-binding protein that can decrease pro-inflammatory TNF-α expression stimulated by lipopolysaccharide (LPS) plus high-mobility group box 1 (HMGB1), suggesting that RPL9 has a role in regulating LPS+HMGB1-stimulated inflammatory reactions. Among the various ribosomal proteins, it was found that RPS5 reproduced the regulatory activity of RPL9 on LPS+HMGB1-stimulated TNF-α expression in macrophage-like RAW264.7 cells. RPL9 and RPS5 share a common feature as cationic proteins. Polylysine, a cationic polypeptide, and a synthetic peptide of the cationic region from RPL9 also exhibited reducing activity on LPS+HMGB1-induced TNF-α expression. By pull-down assay, RPL9 and RPS5 were confirmed to interact with AGEs. When AGEs coexisted with LPS, HMGB1, plus RPL9 or RPS5, the reducing effect of TNF-α expression by these cationic ribosomal proteins was shown to be abrogated. The results suggest that cationic ribosomal proteins have a regulatory role in the pro-inflammatory response induced by LPS+HMGB1, and in the pathophysiological condition of accumulating AGEs, this regulatory effect is abolished, which exacerbates inflammation.

Different modulation of STING/TBK1/IRF3 signaling by advanced glycation end products.

Advanced glycation end products (AGEs) are a heterogeneous group of compounds that are non-enzymatically produced by reactions between carbonyl compounds and proteins. Many types of AGEs are produced according to the type or concentration of the reacting carbonyl compound. We have previously demonstrated that a glycolaldehyde-derived AGE suppresses stimulator of interferon gene (STING)/TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3), which is a component of the innate immune system. In this report, we investigated the effects of AGEs prepared by several carbonyl compounds on STING/TBK1/IRF3 signaling. AGEs used in the present study were numbered based on the carbonyl compound type: AGE1, derived from glucose; AGE2, derived from glyceraldehyde; AGE3, derived from glycolaldehyde; AGE4, derived from methylglyoxal; and AGE5, derived from glyoxal. AGEs derived from aldehyde (AGE2 and AGE3) and dicarbonyl compounds (AGE4 and AGE5) suppressed cyclic GMP-AMP (cGAMP)-induced activation of STING/TBK1/IRF3 signaling, with different suppression efficiencies observed. Lysine modification by carbonyl compounds was related to the efficiency of the suppressive effect on STING/TBK1/IRF3 signaling. Among the AGEs used, only AGE1 enhanced cGAMP-induced activation of STING/TBK1/IRF3 signaling. Enhancing the modulation of STING/TBK1/IRF3 signaling by AGE1 was mediated by toll-like receptor 4. These results indicated that modulation of STING/TBK1/IRF3 signaling by prepared AGEs is dependent on the type and concentration of the carbonyl compound present. Modulating STING/TBK1/IRF3 signaling by AGEs may involve modification of lysine residues in proteins.

Glycolaldehyde-derived advanced glycation end products promote macrophage proliferation via the JAK-STAT signaling pathway.

BACKGROUND: Advanced glycation end products (AGEs) are heterogeneous proinflammatory molecules produced by a non-enzymatic glycation reaction between reducing sugars (and their metabolites) and biomolecules with amino groups, such as proteins. Although increases in and the accumulation of AGEs have been implicated in the onset and exacerbation of lifestyle- or age-related diseases, including diabetes, their physiological functions have not yet been elucidated in detail. METHODS AND RESULTS: The present study investigated the cellular responses of the macrophage cell line RAW264.7 stimulated by glycolaldehyde-derived AGEs (Glycol-AGEs) known as representative toxic AGEs. The results obtained showed that Glycol-AGEs significantly promoted the proliferation of RAW264.7 cells at a low concentration range (1-10 µg/mL) in a concentration-dependent manner. On the other hand, neither TNF-α production nor cytotoxicity were induced by the same concentrations of Glycol-AGEs. The increases observed in cell proliferation by low concentrations of Glycol-AGEs were also detected in receptor triple knockout (RAGE-TLR4-TLR2 KO) cells as well as in wild-type cells. Increases in cell proliferation were not affected by various kinase inhibitors, including MAP kinase inhibitors, but were significantly suppressed by JAK2 and STAT5 inhibitors. In addition, the expression of some cell cycle-related genes was up-regulated by the stimulation with Glycol-AGEs. CONCLUSIONS: These results suggest a novel physiological role for AGEs in the promotion of cell proliferation via the JAK-STAT pathway.

Histamine promotes angiogenesis through a histamine H1 receptor-PKC-VEGF-mediated pathway in human endothelial cells.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

Glycolaldehyde-derived advanced glycation end products suppress STING/TBK1/IRF3 signaling via CD36.

AIMS: We have previously reported that advanced glycation end products derived from incubation of albumin with glycolaldehyde (glycol-AGE), lead to suppression of the toll-like receptor 4 (TLR4) signaling response to lipopolysaccharide. Glycol-AGE-induced suppression of TLR4 signaling is involved in the downregulation of CD14, which is an adaptor protein necessary for transferring lipopolysaccharide to TLR4. Therefore, glycol-AGEs impair the innate immune response through suppression of the upstream process in TLR4 signaling. However, the effect of glycol-AGEs on intracellular signaling related to the innate immune response remains unclear. This study aimed to examined the effect of glycol-AGEs on stimulator of interferon gene (STING) signaling in macrophages. MAIN METHODS: In differentiated THP-1 cells, which are a human monocytic leukemia cell line, cyclic GMP-AMP (cGAMP) transfection was used to activate STING signaling. The phosphorylation levels of TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3) were evaluated by western blot analysis. Downstream cytokine levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays. KEY FINDINGS: Glycol-AGEs suppressed cGAMP-induced phosphorylation of TBK1 and IRF3, as well as the production of cytokines regulated by IRF3. There was no effect of glycol-AGEs on the efficacy of cGAMP transfection. Treatment of a neutralizing antibody against CD36 prevented cGAMP-induced phosphorylation of TBK1 and IRF3, and also upregulation of interferon-ß and C-X-C motif chemokine ligand 10 in glycol-AGE-treated cells. SIGNIFICANCE: Glycol-AGEs negatively regulate cGAMP-induced activation of STING/TBK1/IRF3 signaling via CD36. Our findings suggest that glycol-AGEs lead to impairment of the innate immune response by suppressing intracellular signaling.

Nordihydroguaiaretic acid inhibits glyoxalase I, and causes the accumulation of methylglyoxal followed by cell-growth inhibition.

BACKGROUND: Methylglyoxal (MGO) is a known toxic byproduct of glycolysis, with MGO-induced cytotoxicity believed to contribute to the pathogenesis of several diseases. Glyoxalase I (GLO1) is a key enzyme for eliminating MGO in mammalian cells, therefore, compounds affecting GLO1 activity are potential therapeutic agents for MGO-induced disorders. Previously, we found nordihydroguaiaretic acid (NDGA) as a potent GLO1 inhibitor. METHODS: The inhibitory characteristics of NDGA were determined spectrophotometrically with recombinant GLO1. NDGA-induced growth-inhibition and accumulation of MGO-derived advanced glycation end products (AGEs) were examined in EA.hy926 cells. RESULTS: NDGA showed significant inhibition of GLO1 enzymatic activity in a dose-dependent manner. Its Ki value was estimated to be 146-fold lower than that of myricetin, a known GLO1 inhibitor. The co-addition of MGO with NDGA to the cells resulted in significant growth inhibition, suggesting that MGO accumulation, sufficient to affect cell growth, was caused by NDGA inhibiting GLO1. These findings were supported by the observations that the addition of aminoguanidine, a typical MGO scavenger, significantly reversed cell-growth inhibition by co-addition of MGO with NDGA, and that an increase in intracellular MGO-derived AGEs was observed during incubation with the co-addition of MGO with NDGA. CONCLUSION: NDGA was found to be a novel and potent inhibitor of GLO1. The co-addition of NDGA with MGO to the cells resulted in increased intracellular MGO accumulation followed by enhanced cell-growth inhibition.

Potential of a Novel Chemical Compound Targeting Matrix Metalloprotease-13 for Early Osteoarthritis: An In Vitro Study.

Osteoarthritis is a progressive disease characterized by cartilage destruction in the joints. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) play key roles in osteoarthritis progression. In this study, we screened a chemical compound library to identify new drug candidates that target MMP and ADAMTS using a cytokine-stimulated OUMS-27 chondrosarcoma cells. By screening PCR-based mRNA expression, we selected 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide as a potential candidate. We found that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated IL-1ß-induced MMP13 mRNA expression in a dose-dependent manner, without causing serious cytotoxicity. Signaling pathway analysis revealed that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated ERK- and p-38-phosphorylation as well as JNK phosphorylation. We then examined the additive effect of 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide in combination with low-dose betamethasone on IL-1ß-stimulated cells. Combined treatment with 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide and betamethasone significantly attenuated MMP13 and ADAMTS9 mRNA expression. In conclusion, we identified a potential compound of interest that may help attenuate matrix-degrading enzymes in the early osteoarthritis-affected joints.

Identification of ribosomal protein L9 as a novel regulator of proinflammatory damage-associated molecular pattern molecules.

BACKGROUND: We previously reported that advanced glycation endproducts (AGEs) increase the proinflammatory activity of high mobility group box-1 (HMGB1), a representative damage-associated molecular pattern molecule (DAMP), through their direct interaction. This suggested that AGEs activate other DAMPs and led us to search for novel DAMPs capable of interacting with AGEs. METHODS AND RESULTS: The chromatographic analysis using AGE-immobilized gel revealed the ribosomal protein family to be a factor with binding activity to AGEs. Ribosomal protein L9 (RPL9), a member of the ribosomal protein family, was found in the centrifugal supernatant of ruptured cells and in the serum of lipopolysaccharide (LPS)-stimulated sepsis model mice, exhibiting similar characteristic properties to HMGB1. Although HMGB1 potentiated LPS-stimulated TNF-α expression in macrophage-like RAW264.7 cells, RPL9 hardly exhibited this activity. Of note, RPL9 significantly suppressed the potentiated mRNA expression and protein production of TNF-α by HMGB1 plus LPS stimulation, suggesting its regulatory roles in DAMP-induced proinflammatory activity. Based on the differential scanning fluorimetric analysis, the direct interaction between RPL9 and HMGB1 may play a role in the suppressive effects of RPL9. CONCLUSIONS: This study suggested that RPL9 is a novel type of DAMP with a regulatory role in the proinflammatory response and provided insight into the pathophysiology of inflammatory diseases.

Involvement of multiple scavenger receptors in advanced glycation end product-induced vessel tube formation in endothelial cells.

Toxic advanced glycation end products (toxic AGEs) derived from glycolaldehyde (AGE3) have been implicated in the development of diabetic vascular complications such as retinopathy characterised by excessive angiogenesis. Different receptor types, such as receptor for AGEs (RAGE), Toll like receptor-4 and scavenger receptors, are expressed in endothelial cells and contribute to AGE-elicited alteration of cell function. In the present study, we examined the involvement of AGE-related receptors on AGE-induced angiogenesis in endothelial cells. The effects of pharmacological inhibitors or receptor neutralizing antibodies on AGE3-induced tube formation were investigated using the in vitro Matrigel tube formation assay in b.End5 cells (mouse endothelial cells). AGE3-induced signalling pathways and receptor expression changes were analysed by Western blot analysis and flow cytometry, respectively. Both FPS-ZM1, a RAGE inhibitor, and fucoidan, a ligand for scavenger receptors, suppressed AGE3-induced tube formation. Cocktails of neutralizing antibodies against the scavenger receptors CD36, CD163 and LOX-1 prevented AGE3-induced tube formation. AGE3 activated mTOR signalling, resulting in facilitation of tube formation. Activation of the AGE-RAGE pathway also led to the upregulation of scavenger receptors. Taken together, our findings suggest that the scavenger receptors CD36, CD163 and LOX-1 in conjunction with the RAGE receptor work together to mediate toxic AGE-induced facilitation of angiogenesis.

Osteopontin silencing attenuates bleomycin-induced murine pulmonary fibrosis by regulating epithelial-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-ß1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-ß1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-ß1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

Advanced glycation end-products reduce lipopolysaccharide uptake by macrophages.

Hyperglycaemia provides a suitable environment for infections and the mechanisms of glucose toxicity include the formation of advanced glycation end-products (AGEs), which comprise non-enzymatically glycosylated proteins, lipids, and nucleic acid amino groups. Among AGE-associated phenotypes, glycolaldehyde-derived toxic AGE (AGE-3) is involved in the pathogenesis of diabetic complications. Internalisation of endotoxin by various cell types contributes to innate immune responses against bacterial infection. An endotoxin derived from Gram-negative bacteria, lipopolysaccharide (LPS), was reported to enhance its own uptake by RAW264.7 mouse macrophage-like cells, and an LPS binding protein, CD14, was involved in the LPS uptake. The LPS uptake induced the activation of RAW264.7 leading to the production of chemokine CXC motif ligand (CXCL) 10, which promotes T helper cell type 1 responses. Previously, we reported that AGE-3 was internalised into RAW264.7 cells through scavenger receptor-1 Class A. We hypothesized that AGEs uptake interrupt LPS uptake and impair innate immune response to LPS in RAW264.7 cells. In the present study, we found that AGE-3 attenuated CD14 expression, LPS uptake, and CXCL10 production, which was concentration-dependent, whereas LPS did not affect AGE uptake. AGEs were reported to stimulate the receptor for AGEs and Toll-like receptor 4, which cause inflammatory reactions. We found that inhibitors for RAGE, but not Toll-like receptor 4, restored the AGE-induced suppression of CD14 expression, LPS uptake, and CXCL10 production. These results indicate that the receptor for the AGE-initiated pathway partially impairs the immune response in diabetes patients.

NF-Ä¸ß upregulates ADAMTS5 expression by direct binding after TNF-α treatment in OUMS-27 chondrosarcoma cell line.

Inflammation caused-aggrecan degradation is a critical event in the pathogenesis of osteoarthritis (OA). The aggrecanases like a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) are assumed to be key players in the aggrecan destruction. To develop the comprehensive therapy method for OA, it is essential to elucidate the activation mechanism of ADAMTS5 gene after stimulation of inflammatory cytokines like tumor necrosis factor-α (TNF-α). The cell lines of human chondrosarcoma (OUMS-27) and embryonic kidney (HEK293T) were incubated with tumor necrosis factor-α (TNF-α) for certain time periods, and the expression level of ADAMTS5 was measured in both mRNA and protein levels. Tissue-specific ADAMTS5 activation was founded to be induced after TNF-α treatment. Then, the constructs for the promoter region of ADAMTS5 were prepared and luciferase assay was conducted to understand the involvement mechanism of nuclear factor-kappa beta (NF-ĸß) in ADAMTS5 activation. It was demonstrated that NF-Ä¸ß induces the ADAMTS5 expression level by directly binding the promoter region of ADAMTS5. Although the TNF-α blocker is used for OA treatment, the development of a more comprehensive treatment strategy is an urgent need. Our experimental data contributes in terms of selecting NF-Ä¸ß as a target molecule. Up to date, NF-Ä¸ß has been proven to involve in the ADAMTS5 up-regulation after several pro-inflammatory cytokines stimulation. In conclusion, our findings make important contributions to the knowledge about the roles of NF-Ä¸ß in ADAMTS5 activation under inflammatory conditions. So, NF-Ä¸ß could be considered to be a potential target for OA treatment.

Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1.

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1ß and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

Functional analysis of ESM1 by siRNA knockdown in primary and metastatic head and neck cancer cells.

BACKGROUND: Genetic factors play a large role in cancer, and thus, there is a great desire to understand the effects of different genes in cancer and to also develop gene therapy for better treatments. Therefore, the development of alternative diagnosis and therapy modalities is of utmost importance. The aim of our study was to illuminate the role of ESM1 (endothelial cell-specific molecule-1, also known as Endocan) in proliferation and migration of head and neck cancer, thus helping to pave the way for new treatment modalities and predictive biomarkers. METHODS: ESM1 expression was shown with immunofluorescence assay using confocal laser scanning microscope in primary and metastatic head and neck cancer cells. ESM1 expression was knocked down by RNA interference in head and neck cancer cells. Knockdown efficiency was evaluated by quantitative real-time RT-PCR and Western blot. Cell proliferation and migration assays were performed by xCELLigence real-time cell analysis system. RESULTS: Immunofluorescence assay showed nuclear localization and high expression of ESM1 in primary and metastatic head and neck cancer cells. ESM1 mRNA and protein levels were significantly decreased in ESM1-knockdown cells compared to control. ESM1-knockdown cells showed reduced proliferation and migration activity when compared to control cells. CONCLUSION: These findings suggest that ESM1 has roles on proliferation and migration of head and neck cancer cells.

Matrix Metalloproteinases 2 and 9 Polymorphism in Patients With Myeloproliferative Diseases: A STROBE-Compliant Observational Study.

Chronic myeloproliferative disorders such as polycythemia vera (PV), essential thrombocytosis (ET), and idiopathic myelofibrosis arise from clonal proliferation of neoplastic stem cells in the bone marrow. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that have potential to degrade all types of extracellular matrix (ECM) and also play a role in remodeling of the ECM. It is known that MMPs play a role in bone marrow remodeling.The primary goal of our study is to explore the relationship between chronic myeloproliferative diseases and some of MMP gene polymorphisms. The demonstration of a relationship will help to understand whether these polymorphisms may be a potential early diagnosis marker of the diseases.Patients were selected from outpatient clinics of Turgut Ozal University Hospital, Ankara, Turkey, between December 2010 and May 2011. Twenty-eight patients that previously diagnosed and followed-up with PV, 17 with secondary polycythemia (SP), and 12 with ET were enrolled in the study, along with a control group of 22 healthy people.DNA was isolated from peripheral blood. Using polymerase chain reaction-restriction fragment length polymorphism method, MMP2 and MMP9 gene polymorphisms were analyzed with agarose gel electrophoresis. There was a statistically significant difference between the study groups and the control group in terms of Gln279Arg polymorphisms rates of MMP9. The highest MMP9 Gln279Arg polymorphism rate was observed in the ET group. But nobody from the control group had polymorphic MMP9. There was no statistically significant difference between the groups in terms of MMP2-735 C > T polymorphism rates.In conclusion, MMP9 gene Gln279Arg polymorphism was associated with ET, SP, and PV diseases. Hence, we believe that these gene polymorphisms may play a role in the mechanism of bone marrow fibrosis and may be a factor that increases the risk of thrombosis. Illumination of the molecular basis of the relationship between MMP-thrombosis and MMP-fibrosis provides a better understanding of the pathophysiology of PV and ET diseases and will allow new approaches to diagnosis and treatment.

Leptin induces ADAMTS-4, ADAMTS-5, and ADAMTS-9 genes expression by mitogen-activated protein kinases and NF-ĸB signaling pathways in human chondrocytes.

Elucidation of the causes of inflammation has vital importance in the development of new approaches for the treatment of arthritic diseases. The degradation of aggrecan by upregulated disintegrin and metalloproteinase with trombospondin motifs (ADAMTSs) is the key event in the development of both rheumatoid arthritis (RA) and osteoarthritis (OA). Increased levels of leptin in both RA and OA have been demonstrated, thus linking leptin to arthritic diseases, but the mechanism has not been clarified. This study investigated the putative role of signaling pathways (p38, JNK, MEK1, NF-ĸB, and PI3) involved in leptin-induced cartilage destruction. Normal human articular chondrocytes were cultured with recombinant human leptin at 100, 250, 500, and 1000 ng/mL doses for 6, 12, 24, and 48 h, after which ADAMTS-4, -5, and -9 genes expression were determined by real time-polymerase chain reaction (RT-PCR) and Western Blot methods. The signaling pathways involved in leptin-induced ADAMTSs upregulation were also investigated by using inhibitors of signaling pathways. It was demonstrated that ADAMTSs expression level was peaked at 1000 ng/mL doses for 48 hours, and MAPKs (p38, JNK, and MEK) and NF-ĸB signaling pathways involving in leptin triggered ADAMTSs upregulation. Obesity as a risk for RA and OA may contribute to the inflammation of both RA and OA diseases by secreting adipokines like leptin. We hypothesize that leptin is involved in the development of RA and OA accompanied with obesity by increasing ADAMTS-4, -5, and -9 genes expression via MAPKs and NF-ĸB signaling pathways.

Potential genetic biomarkers in the early diagnosis of Alzheimer disease: APOE and BIN1.

BACKGROUND/AIM: Alzheimer disease (AD) is triggered by interactions of multiple genetic and environmental factors. The APOE gene E4 allele is the best-known risk factor for AD, yet it represents a small ratio of genetic factors. According to genome-wide association studies, the BIN1 gene is the second important risk factor for AD, following the APOE gene. We aimed to identify a novel biomarker indicating susceptibility to AD by investigating APOE alleles and BIN1 gene polymorphisms in a Turkish population. MATERIALS AND METHODS: Fifty-three AD patients and 56 controls were included to examine polymorphism and allele frequency of the APOE and BIN1 genes. Genomic DNAs were isolated from whole blood by SDS/proteinase K treatment, phenol-chloroform extraction, and ethanol precipitation. RFLP was done for identification of polymorphisms in the APOE gene and allele-specific PCR was used for the BIN1 gene. RESULTS: Frequency of the APOE E4 allele was higher in the AD patient group, while the frequency of the E2 allele was higher in controls. The E4/E4 genotype was detected in the AD patient group, while this genotype was not observed in the controls. The frequencies of BIN1 alleles were similar in both groups. CONCLUSION: There was a strong association between AD and the APOE E4 allele, while no such relation was observed with BIN1 gene polymorphism.

Polymorphisms in MMP-2 and TIMP-2 in Turkish patients with prostate cancer.

AIM: Prostate cancer is the most commonly diagnosed malignancy and the second most common cause of cancer deaths in the Western male population. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) modulate the remodeling of the extracellular matrix (ECM). The imbalance between MMPs and TIMPs may lead to an emergence of pathological processes such as cancer. In this study, the association between TIMP-2 (-418 G/C) and MMP-2 (-1306 C/T) polymorphisms and prostate cancer in the Turkish population was investigated. MATERIALS AND METHODS: Sixty-one prostate cancer patients and 46 healthy subjects were included in the study. DNA was isolated from 2 mL of peripheral blood taken from subjects, and genotypes were analyzed by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The TIMP-2 -418 (GC) genotype was found in 15 cases (32.6%) in the control group and in 9 cases (14.8%) in the patients group, and statistical significance was determined (P = 0.037, OR = 0.346). The MMP-2 -1306 (CT) genotype was found 2.17 times more in the patient group than in the control group (P = 0.149, OR = 2.17). CONCLUSION: Our results show that the TIMP-2 -418 (GC) genotype had a putative protective effect against prostate cancer.

The effects of hypericin on ADAMTS and p53 gene expression in MCF-7 breast cancer cells.

PURPOSE: The purpose of this study was to determine the effects of hypericin on MCF-7 (Michigan Cancer Foundation- 7) breast cancer cells, as it is known to exert an antitumor effect on the expression and regulation of ADAMTS1, 3, 10 and the p53 gene in breast cancer cells. METHODS: MFC-7 cells were cultured and subjected separately to various doses (1, 5 and 7.5 µg /mL) hypericin. After 24 hrs, RNA was isolated and transcribed into cDNA. Expression analysis was performed by real time (RT)-PCR and cell survival was determined by the XTT assay. RESULTS: While the expression of ADAMTS1 in MFC-7 cells decreased to 0.04-fold after exposure to 1 µg /mL hypericin, the expression increased by 5.6- and 36-fold with 5 and 7.5 µg/mL, respectively. Furthermore, ADAMTS3 expression in MCF7 cells increased 3.9-fold with the use of 5 µg /mL of hypericin. These concentrations of hypericin did not lead to significant changes in the expression of ADAMTS10 and the p53 gene. Viability of cancer cells as evaluated by the XTT assay showed that hypericin concentration of 7.5 µg /mL led to increased apoptosis of cancer cells. CONCLUSION: The increase in ADAMTS1 expression may prevent metastasis or facilitate the development of an adjuvant factor with tumor-suppressive effects. Hypericin may therefore exert its antitumor and apoptotic effects in MFC-7 cells via ADAMTS1 and ADAMTS3.

Polymorphisms in the promoters of MMP-2 and TIMP-2 genes in patients with acne vulgaris.

Acne, a chronic inflammatory skin disease, can be seen at any age but it most often occurs in adolescents and young people. Several factors, including increased sebum production, abnormal cornification of the pilosebaceous units, proliferation of Propionibacterium acne, and extracellular matrix (ECM) remodeling, are thought to be associated with the pathogenesis of the acne. The remodeling of the ECM is regulated by a balance between matrix metalloproteinases (MMPs) and their inhibitors called tissue inhibitors of metalloproteinases (TIMPs). The current study investigated the potential association between MMP-2 (-1306 C/T) and TIMP-2 (-418 G/C) polymorphisms and the risk for acne in a Turkish population. The study was conducted with 85 subjects who presented to the Dermatology Department of Duzce University Hospital. DNA was isolated from 2 ml of peripheral blood taken from each subject, and their genotypes were analyzed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The CC, CT, and TT genotypes for MMP-2 (-1306 C/T) polymorphism were similar between the patient and control group (24 [55.8%], 17 [39.5%], and 2 [4.7%], respectively, vs. 21 [50%], 18 [42.9%], and 3 [7.1%], respectively). However, the distribution of the GG, GC, and CC genotypes for TIMP-2 (-418 G/C) polymorphism were different between the patient and control group (30 [69.8%], 9 [14.8%] and 4 [9.3%], respectively, vs. 26 [61.9%], 14 [33.3%], and 2 [4.8%], respectively). The results demonstrated that the TIMP-2 (-418 CC) genotype was nearly two times more common in the patient group compared to the control group (p=0.686, OR=1.45). It may be possible that the TIMP-2 (-418 CC) genotype increases the tendency to develop acne vulgaris by disrupting the balance between MMPs and TIMPs. Further investigations are needed to clarify more precisely the relationship between acne and MMP-TIMP genes.