MMP-9 Gene Expression Research Articles

Pooled Platelet-Rich Plasma Lysate Therapy Increases Synoviocyte Proliferation and Hyaluronic Acid Production While Protecting Chondrocytes From Synoviocyte-Derived Inflammatory Mediators

Platelet-rich plasma (PRP) preparations are being used with moderate success to treat osteoarthritis (OA) in humans and in veterinary species. Such preparations are hindered, however, by being autologous in nature and subject to tremendous patient and processing variability. For this reason, there has been increasing interest in the use of platelet lysate preparations instead of traditional PRP. Platelet lysate preparations are acellular, thereby reducing concerns over immunogenicity, and contain high concentrations of growth factors and cytokines. In addition, platelet lysate preparations can be stored frozen for readily available use. The purpose of this study was to evaluate the effects of a pooled allogeneic platelet-rich plasma lysate (PRP-L) preparation on equine synoviocytes and chondrocytes challenged with inflammatory mediators in-vitro to mimic the OA joint environment. Our hypothesis was that PRP-L treatment of inflamed synoviocytes would protect chondrocytes challenged with synoviocyte conditioned media by reducing synoviocyte pro-inflammatory cytokine production while increasing synoviocyte anti-inflammatory cytokine production. Synoviocytes were stimulated with either interleukin-1β (IL-1β) or lipopolysaccharide (LPS) for 24 h followed by no treatment or treatment with platelet-poor plasma lysate (PPP-L) or PRP-L for 48 h. Synoviocyte growth was evaluated at the end of the treatment period and synoviocyte conditioned media was assessed for concentrations of hyaluronic acid (HA), IL-1β, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Chondrocytes were then challenged for 48 h with synoviocyte conditioned media from each stimulation and treatment group and examined for gene expression of collagen types I (COL1A1), II (COL2A1), and III (COL3A1), aggrecan (ACAN), lubricin (PRG4), and matrix metallopeptidase 3 (MMP-3) and 13 (MMP-13). Treatment of inflamed synoviocytes with PRP-L resulted in increased synoviocyte growth and increased synoviocyte HA and IL-6 production. Challenge of chondrocytes with conditioned media from PRP-L treated synoviocytes resulted in increased collagen type II and aggrecan gene expression as well as decreased MMP-13 gene expression. The results of this study support continued investigation into the use of pooled PRP-L for the treatment of osteoarthritis and warrant further in-vitro studies to discern the mechanisms of action of PRP-L.

Abstract 886: Metformin effect on multidrug resistant leukemia cells: metalloproteinases 2 and 9

Abstract Besides the progress in studies of cancer, the rates of morbidity and mortality due to this disease still considerably elevated. Chronic myeloid leukemia (CML) is a hematological neoplasm, therefore chemotherapy is the main therapeutic approach used, however most patients develop resistance to chemotherapy. Several factors contribute to this scenario, among them the lack of selective treatment, the emergence of tumor cells resistant to the wide variety of cytotoxic agents during treatment and the formation of metastases, which accounts for the majority of cancer deaths. Therefore, application of new drugs in antitumor therapies has been constantly studied. Studies have shown that metformin, an oral euglycemic used in the treatment of type II diabetes, has potential use in the treatment of cancer, potentializing the effects of standard chemotherapy used in treatment. In this work the effects of metformin on the metastatic capacity of K562 (non-resistant leukemic cell), Lucena and FEPS (leukemic cells resistant to multiple drugs) were evaluated through the study of MMP2 and MMP9 metalloproteinases, enzymes essential for the metastatic process. Thus, the present project aimed to evaluate the gene expression and enzymatic activity of MMP2 and MMP9 metalloproteinases in CML cells, before and after treatment with metformin, seeking to verify the effects of metformin on the expression and activity of these enzymes. Gene expression was evaluated using quantitative PCR and the quantification of enzymatic activity was performed by the substrate degradation using the zymography method. The results showed that metformin has the potential to modulate the expression and activity of the studied metalloproteinases. Citation Format: Ligia P. Oliveira, Camila Perea, Caroline Baldi, Rodrigo F. Prata, Débora L. Renó, Luana P. Lima, Rodrigo Curvello, Ana Carolina S. Souza. Metformin effect on multidrug resistant leukemia cells: metalloproteinases 2 and 9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 886.

Gene Expression of MMP-1, MMP-2 and TNF-α in the Endometrium of Mares With Different Degrees of Fibrosis

Gene Expression of MMP-1, MMP-2 and TNF-α in the Endometrium of Mares With Different Degrees of Fibrosis

PBI-4547 Improves Glucose Metabolism and Insulin Resistance, and Reduces Liver Damage in a High-Fat Diet Mouse Model of Obesity and Metabolic Syndrome

Introduction and Aims: Obesity and its resulting metabolic disturbances are major health threats and are the main cause of a host of diseases, including nonalcoholic fatty liver disease (NAFLD). PBI-4547 is a novel first-in-class orally active antidiabetic compound which displays pleiotropic activities and has been shown to reduce NASH, diabetes, and renal fibrosis in different animal models. The aim of this study was to investigate the effects of PBI-4547 in a mouse model of high-fat diet-induced obesity and metabolic syndrome. Methods: C57BL/6 mice were fed with either a standard or a high-fat diet (HFD, Harlan, TD.06414) for 14 weeks. These mice were divided in three groups [normal chow, HFD + vehicle, and HFD + PBI-4547 (10 mg/kg, oral once a day)] and treated for an additional 6 weeks. Blood glucose, serum insulin level, serum triglyceride and adiponectin, liver histology, as well as liver and white adipose tissue (WAT) pro-inflammatory/fibrotic gene expression were examined. Results: In an oral glucose tolerance test, PBI-4547 increased glucose metabolism compared to the HFD control group. Insulin resistance (measured by HOMA-IR) and β cell function (HOMA1-β) were improved by PBI-4547. Serum triglyceride level was significantly reduced by PBI-4547 while adiponectin in serum was significantly increased. Histological analysis showed a significant reduction of hepatic steatosis and ballooning by PBI-4547. To further characterize the activity of PBI-4547, quantitative RT-PCR analysis of pro-fibrotic markers was performed, demonstrating that PBI-4547 reduced CTGF and MMP2 gene expression in the liver as well as CTGF, MCP-1, MMP2 and collagen type I gene expression in the WAT. Conclusions: Taken together, these results suggest that PBI-4547 offers the potential as a novel therapy for nonalcoholic fatty liver disease, obesity and associated metabolic syndrome. Disclosure M. Leduc: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. B. Grouix: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. M. Tremblay: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. L. Gervais: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. F. Sarra-Bournet: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. A. Felton: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. J. Simard: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. F.A. Leblond: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc. P. Laurin: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc.. Board Member; Self; Prometic Life Sciences Inc. L. Gagnon: Employee; Self; Prometic Biosciences Inc.. Stock/Shareholder; Self; Prometic Life Sciences Inc..

Molecular mechanism and cytotoxicity of allicin and all-trans retinoic acid against CD44+ versus CD117+ melanoma cells

BackgroundAll-trans retinoic acid (ATRA) is a differentiating agent that inhibits cancer cell growth during the cell cycle. However, despite its potent antitumor properties, some melanoma cells are resistant to ATRA therapy. PurposeHere, we hypothesized that allicin can sensitize malignant melanoma cells to ATRA treatment. To clarify this mechanism, we determined the sensitivity to ATRA, allicin and allicin/ATRA in CD44+ and CD117+ melanoma cell subpopulations. MethodsThe CD44+and CD117+cells were sorted from A375 melanoma cell line using the magnetic-activated cell sorting (MACS). The potential anticancer effects of ATRA, allicin and allicin/ATRA were examined using cell proliferation MTT assay. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of the treatments in controlling cancer cell proliferation was assessed by quantitative realtime polymerase chain reaction (RT-PCR). ResultsHere, we demonstrated that CD44+ melanoma cells were more resistant to allicin and ATRA than CD117+ cells. Importantly, we observed that allicin sensitized melanoma cell to ATRA-induced cell death. The combination treatment with allicin and ATRA significantly reduced the IC50 value obtained for ATRA alone in CD44+ melanoma cells. In CD44+ cells, the IC50 value of ATRA was 37.43 ± 0.54, while the IC50 value of allicin/ATRA treatment was 17.53 ± 0.2 µM. Allicin treatment resulted in significant increases in the percentage of cells at the G2/M and G0/G1 phases in the CD44+ and CD117+ cells, respectively. The combination treatment caused the inhibition of CD44+ and CD117+ melanoma cells at the S phases compared to ATRA alone. Allicin, ATRA, and allicin/ATRA increased the expression of cyclin D1 mRNA in both CD44+ and CD117+ cells. Allicin combination with ATRA increased the mRNA level of RARβ in CD117+ cells. Furthermore, allicin alone caused a remarkable reduction of MMP-9 mRNA expression in both CD44+ and CD117+ cells. In contrast, ATRA and the combination treatment significantly increased MMP-9 gene expression in CD44+ cells. ConclusionOverall, our results indicate that allicin reinforces the ATRA-mediated inhibitory effects on CD44+ and CD117+ melanoma cells and may provide a new approach for the treatment of malignant melanoma.

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s)

BackgroundExposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis.ResultsExposure-relevant concentrations of DEP (0.12 μg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 μg/cm2) induced low to moderate increases in IL-1α, IL-1β, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects.ConclusionLipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

Regulation of cardiac fibroblast MMP2 gene expression by scleraxis

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFβ occurs via a scleraxis-dependent mechanism: TGFβ induces recruitment of scleraxis to the MMP2 promoter, and TGFβ was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.

Matrix metalloproteinases and their inhibitors in human cumulus and granulosa cells as biomarkers for oocyte quality estimation

To study the molecular profile of metalloproteinases and their tissue inhibitors in granulosa and cumulus cells in a subset of fertile and infertile women. Molecular study with granulosa and cumulus cells. University hospital. Forty-four women undergoing assisted reproductive techniques for female infertility factor, with partners having a normal spermiogram and 15 normally fertile women with male partner affected by severe oligoasthenoteratozoospermia or nonobstructive azoospermia. Invitro fertilization. We investigated gene expression level of metalloproteinases (MMP2, MMP9, MMP11) and their tissue inhibitors (TIMP1, TIMP2) by means of quantitative reverse-transcription polymerase chain reaction, protein quantification by means of Western blot, and localization by means of immunofluorescence. We firstly validated HPRT1 as the most reliable housekeeping gene enabling correct gene expression analysis in both granulosa and cumulus cells. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP9, and MMP11 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these cells. MMP9 is specifically expressed only in granulosa, whereas MMP2 is more expressed in cumulus and granulosa cells in cases of reduced ovarian response and decreased fertilization rate. This study sheds light on MMP and TIMP expression in granulosa and cumulus cells, and it may help in understanding the fine regulation of oocyte maturation inside the follicle. Although further studies are needed to fully understand the molecular mechanisms involved in these processes, our findings may be useful in the identification of biomarkers of oocyte maturation, competence acquiring, and fertilization.

Inflammatory Response of Human Peripheral Blood Mononuclear Cells and Osteoblasts Incubated With Metallic and Ceramic Submicron Particles.

Inflammatory reactions associated with osteolysis and aseptic loosening are the result of wear particles generated at the articulating surfaces of implant components. The aim of the present study was to analyze the biological response of human osteoblasts and peripheral blood mononuclear cells (PBMCs) after exposure to metallic and alumina ceramic particles regarding cellular differentiation, cytokine release, and monocyte migration. Cells were exposed to particles (0.01 and 0.05 mg/ml) from an alumina matrix composite (AMC) ceramic and a CoCr28Mo6 alloy with an average size of 0.5 µm over 48 and 96 h. The expression rates of osteogenic (Col1A1, ALP) and pro-osteoclastic (RANK, Trap5b) differentiation markers as well as pro-osteolytic mediators (MMP-1, TIMP-1, IL-6, IL-8, MCP-1) were determined and soluble protein concentrations of active MMP-1, IL-6, IL-8, and pro-collagen type 1 in cell culture supernatants were evaluated. Additionally, the capacity of particle-treated osteoblasts to attract potentially pro-inflammatory cells to the site of particle exposure was investigated by migration assays using osteoblast-conditioned media. The cellular morphology and metabolism of human osteoblasts and adherent PBMCs were influenced by particle type and concentration. In human osteoblasts, Col1A1 expression rates and protein production were significantly reduced after exposing cells to the lower concentration of cobalt-chromium (CoCr) and AMC particles. Exposure to AMC particles (0.01 mg/ml) resulted in increased mRNA levels of RANK and Trap5b in adherent PBMCs. For MMP-1 gene expression, elevated levels were more prominent after incubation with CoCr compared to AMC particles in osteoblasts, which was not reflected by the protein data. Interleukin (IL)-6 and IL-8 mRNA and protein were induced in both cell types after treatment with AMC particles, whereas exposure to CoCr particles resulted in significantly upregulated IL-6 and IL-8 protein contents in PBMCs only. Exposure of osteoblasts to CoCr particles reduced the chemoattractant potential of osteoblast-conditioned medium. Our results demonstrate distinct effects of AMC and CoCr particles in human osteoblasts and PBMCs. Complex cell and animal models are required to further evaluate the impact of cellular interactions between different cell types during particle exposure.

Effects of simulated muscle exercise on chondrocyte gene expression in a 3D-alginate bead model system

Purpose: The joint organ is a complex system composed of bone, muscle, tendon, ligament, synovial and cartilage tissues. Myokines produced and released by muscle cells (myocytes) in response to muscular contractions have autocrine, paracrine and/or endocrine effects. Exercise is one of the recommended core treatments for all osteoarthritis patient; exercise-induced myokines appear to be involved in mediating both systemic as well as local anti-inflammatory effects. We hypothesized that myokines, produced by muscle stimulation mimicking exercise, will have anti-inflammatory and anabolic paracrine effects on chondrocytes. We tested this hypothesis by analyzing the effects of conditioned media from skeletal muscle myobundles (with and without electrical stimulation) on gene expression of chondrocytes in a 3D-alginate culture system. Methods: Electrically responsive, contractile human skeletal muscle myobundles were made from primary myogenic cells isolated from standard muscle biopsies obtained under IRB approval. After four-weeks in culture, when these human myobundles matured, electrical pulse simulation of myobundles was performed to simulate different exercise conditions: 1) Non-stimulated, 2) 1 Hz stimulation (simulated slow-twitch exercise), 3) 10 Hz stimulation (simulated fast-twitch exercise). Conditioned media (CM) from all 3 groups were collected each day for 7 days. Primary chondrocytes, collected as surgical waste, were isolated from normal appearing articular cartilage of the lateral femoral condyle from patients undergoing knee replacement for osteoarthritis (OA). Cartilage was diced, digested in 0.1% (weight/volume) collagenase type II for 16-24 h at 37 °C, and the cells were cultured in F12 media in a T-75 cm2 flask in 5% CO2 until confluent. Passage 1-4 chondrocytes were encapsulated in 1.2% alginate solution (2x10e6 cells/ml) by drop solidification into 102mM CaCl2 solution, then cultured for up to 7 days in DMEM/F12 culture media. Chondrocytes (3 biological replicates) in alginate were cultured for 48 hr in non-stimulated myobundle CM or the stimulated myobundle CM at a ratio of 1:1 to chondrocyte growth media. Quantitative PCR was performed on RNA isolated from alginate bead cultured chondrocytes for analysis of gene expression of MMP13, ADAMTS4, ADAMTS5, COL2A1, COL10A1 and ACAN and YWHAZ (housekeeping control). Results: Compared to non-stimulated CM, stimulated CM tended to reduce chondrocyte gene expression (normalized to the housekeeping control) of ACAN (p=0.086), ADAMTS5 (p=0.081) and MMP13 (p=0.111). Compared to 1 Hz stimulated CM, chondrocytes exposed for 48 hours to 10 Hz stimulated CM exhibited elevated gene expression (normalized to the control and the non-stimulated level of gene expression) of collagens II and X (p=0.045, p=0.01, Figure 1). Conclusions: Our study suggests that myokines produced by muscle stimulation mimicking exercise have anti-catabolic effects. Compared with low frequency stimulation, myokines produced in response to high frequency stimulation induced markers of chondrogenesis (collagen II) and late-stage chondrocyte hypertrophy (collagen X). This pilot study provides evidence for a paracrine effect of muscle on cartilage. Factors from muscle responsible for these effects warrant further investigation and identification to understand the physiologic interactions of muscle and cartilage.Table 1Chondrocyte gene expression in response to muscle conditioned media (CM).GeneChondrocyte gene expression (fold change in stimulated vs non-stimulated muscle CM)P valueChondrocyte gene expression (fold change in 10 Hz vs 1 Hz stimulated muscle CM)P valueCOL21.130.3961.420.045COL100.640.1731.120.010ACAN0.670.0861.040.387ADAMTS40.740.2831.040.442ADAMTS50.630.0811.090.210MMP130.630.1111.050.259 Open table in a new tab

Type IV Collagenase Inhibitor, BiPS, Alters Matrix Metalloproteinase 9 Expression in Sulfur Mustard Exposed Mouse Skin

Sulfur mustard (bis‐2‐chloroethylsulfide, SM), is a chemical warfare agent that penetrates the skin rapidly causing extensive blisters and constitutes a potentially serious threat to military and civilian populations. While its mechanisms of toxic action are yet poorly defined, animal models demonstrate disruption of the skin architecture at the dermal‐epidermal junction (DEJ). Collagen degradation in the basement membrane zone by matrix metalloproteinases (MMPs) further exacerbates vesication post SM exposure. The type IV collagenase (MMP9 and MMP2) may mediate the disruption of type IV collagen (Col IV), a main component of the basement membrane zone (BMZ) in skin wounds post SM exposure. N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide (BiPS) was tested as a highly selective MMP2/MMP9 inhibitor. The study aimed to evaluate the therapeutic effectiveness of topically delivered BiPS to protect against SM induced skin injury. We performed a time course study (24, 72, and 168 hrs post‐SM exposure) and a compound evaluation study (BiPS as a single pretreatment using the same time points post SM exposure) on mouse ear skin to determine improvement using histopathologic changes, MMP9 gene expression, and basement membrane protein expression as markers. SM induced characteristic skin structural damage including edema, inflammatory cell infiltration, and epidermal hyperplasia. MMP9 mRNA expression increased with time. Dual immunofluorescent studies using Col IV α1 and MMP9 antibodies showed BMZ breakdown of Col IV in the basement membrane and apparent MMP9 expression in the epidermis and the adjacent dermal matrix of the SM injured skin. In the compound evaluation study, the BiPS pretreatment group demonstrated significant reduction in edema and improvement in overall tissue structure by 72 hr post‐SM exposure. mRNA expression of MMP9 was significantly downregulated in the BiPS treated group by 168 hr post exposure. Immunofluorescent studies showed that significant reduction of MMP9 expression in the skin sections for all time points; the basement membrane appeared improved as indicated by a continuous (without disruption) expression of Col IV in the BMZ; which was similar to the unexposed control skin. These results suggest that BiPS may modulate MMP9 expression which may in turn improve basement membrane integrity after skin exposure to SM. BiPS, the MMP2/MMP9 inhibitor II, may present as a potential therapeutic countermeasure against SM induced skin injury.Support or Funding InformationES005022, T32ES007148, and NIAMS U54AR055073This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Implication of 14-3-3epsilon as a potential alarmin in bone/cartilage communication in osteoarthritis

Purpose: Osteoarthritis (OA) is the most frequent and disabling joint disease. OA is now considered as a whole-joint disease characterized by cartilage degradation, synovial inflammation and bone remodeling. Our team identified 14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. This protein acts as a potent MMP-3 and MMP-13-stimulatory factor in chondrocytes leading to catabolic phenotype. 14-3-3ε seems to share common characteristics with alarmins, endogenous molecules with intracellular functions that are released in the extracellular media after infection or tissue damage. Alarmins (such as S100 and HMGB1 involved in cartilage degradation) bind to receptors expressed on the surface of cells engaged in host defense and tissue repair. These receptors include the Toll-like receptors, TLR2 and TLR4, as well as RAGE. Moreover, innate alarmins can stimulate the activation of resident immune cells, particularly macrophages. Our aim is to investigate the interactions between 14-3-3ε and its potential receptors, TLR2, TLR4 and RAGE and his role as an alarmin particularly as a new molecular mediator determining macrophage polarization in OA joint. Methods: Primary cultures of chondrocytes were performed from murine cartilage of the C57BL/6J mice (5 days old), and from human cartilage samples, obtained from OA patients undergoing total joint replacement surgery. The receptors TLR2, TLR4 and RAGE were blocked by monoclonal antibodies and pharmacologic inhibitors in chondrocytes (15mn) and then stimulated by recombinant 14-3-3ε during 24h. Immunocytochemistry was realized on murine chondrocytes with receptors antibodies. Chondrocyte gene expression and release of MMP-3, MMP-13 and IL-6 were evaluated by RT-PCR and ELISA. The direct interaction of 14-3-3ε and TLR2 was measured using SPR on a Biacore 3000 instrument. The potential 14-3-3ε-binding sites on receptors were investigated by in silico computational analysis PyMOL. Polarisation M1/M2 of marcrophages was performed by activating THP1 cells (ATCC) with PMA during 24h and stimulating them 24h hours with IL-4, LPS or 14-3-3ε. Different markers of the M1/M2 types were analysed by ELISA and RT-qPCR to identify the polarisation of macrophages (M1: TNF, IL-6, IL-1b, MCP1, Nos2, M2: IL-10, TGFb, Arg-1, FIZZ1, YM2). Results: TLR2, TLR4 and RAGE receptors were found on murine articular chondrocytes by immunocytochemistry using monoclonal antibodies. Invalidation of TLR2, TLR4 or RAGE receptors by blocking antibody in articular chondrocytes reduced significantly mRNA expression and protein release of MMP-3, MMP-13 and IL-6 induced by 14-3-3ε. For MMP3 mRNA expression, the inhibition rate reached 78% with blocking antibodies and 98% with OxPAPC, a TLR2/TLR4 inhibitor. The inhibition rate reached 65% (blocking antibodies), 98% (OxPAPC) for MMP13 and 74% (blocking antibodies) and 93% (OxPAPC) for IL-6. ELISA experiments showed an inhibition rate of 84% (blocking antibodies) and 97% (OxPAPC) for MMP3. Similar rates are observed for IL-6 secretion (83% and 97%). These results should be confirmed on cultures of human chondrocytes. Human recombinant TLR2 and TLR4 were covalently immobilized on CM5 chip. Results showed a direct interaction between recombinant TLR2 and 14-3-3ε (83 RU) and between TLR4 and 14-3-3ε (41 RU). Modeling experiments seems to show a direct interaction between 14-3-3ε and TLR2 receptor through a phosphorylated residue (TYR). Polarization of macrophages stimulated by recombinant 14-3-3ε is ongoing and expression of M1 type is expected. Conclusions: We have discovered 14-3-3ε, a new catabolic factor, produced by the subchondral bone and able to induce a catabolic phenotype of chondrocytes. Taking together, our results could designate 14-3-3ε as a novel alarmin to be explored in OA either for therapeutic or for prognostic purposes and its interaction with receptors could be a novel target in OA.

ZCCHC6 (TUT7) deletion inhibit the expression of IL-6 and MMP-13 and reduces the severity of post-traumatic osteoarthritis in a murine model

Purpose: Approximately 20-50% of patients diagnosed with osteoarthritis (OA) develop the disease in joints that previously sustained trauma. RNA terminal uridyltransferases (TUTs), including Zcchc6 (zinc-finger CCHC-domain containing protein 6/TUT7), have been implicated in the post-transcriptional regulation of inflammatory mediators. However, role of Zcchc6 (TUT7) in the pathogenesis of OA or post-traumatic OA has not been explored. Methods: All murine studies were approved by the IACUC and the studies with deidentified, discarded human cartilage were approved by the IRB. Zcchc6 knockout mice were generated by gene targeting (KOMP, UC-Davis). Human or murine chondrocytes were prepared by the enzymatic digestion of the OA cartilage or knee cartilage respective and treated with recombinant human or mouse IL-1β (5ng/ml, R&D Systems, #: 201-LB-025 and #:401-ML-005 respectively) for in vitro studies. IL-6 and MMP13 gene and protein expression in chondrocytes was analyzed by qPCR and immunoblotting respectively. IL-6 mRNA stability was determined by Actinomycin-D chase experiments. For in vivo studies, post-traumatic OA in the knee joints of Zcchc6 deficient male mice and wild type littermates was induced by the surgical destabilization of medial meniscus (DMM) and mice were sacrificed after eight weeks of DMM and the severity of OA was determined per OARSI guidelines for murine OA. Contra lateral knee joints with sham surgery served as controls. Expression of MMP13 (Santa Cruz Biotechnology, #sc-30071), Type-II Collagen cleavage (IBEX Pharmaceuticals, #C1,2C) and Zcchc6 (Santa Cruz Biotechnology, #sc-137947) in vivo were analyzed by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed using the RNAScope technology. RNA immunoprecipitation (RIP) was performed using validated antibodies. siRNAs were used to deplete the expression of ZCCHC6. Results: Knee cartilage chondrocytes prepared from Zcchc6 KO mice showed increased levels of IL-6 and MMP13 mRNA expression compared to WT chondrocytes in response to IL-1β stimulation. Of interest was the observation that the protein levels of IL-6 and MMP13 were significantly downregulated in IL-1β stimulated Zcchc6 KO chondrocytes lysate and culture supernatants in comparison to WT chondrocytes. FISH and RIP analysis revealed that ZCCHC6 protein binds to IL-6 mRNAs in primary human OA chondrocytes under pathological conditions. Furthermore, IL-6 mRNA half-life was reduced by 50% in Zcchc6 KO murine chondrocytes and ZCCHC6 depleted human primary chondrocytes. The expression of Zcchc6 in the knee joints of wild type mice with DMM surgery was highly upregulated in comparison to control joints. Zcchc6 KO mice expressed low levels of MMP13 and showed lesser matrix degradation in the joints with DMM surgery compared to joints of the WT mice and the control contralateral joints and developed less severe OA as determined by Safranin-O-Fast green staining followed by OARSI scoring. Synovitis was also found to be decreased in the KO mice DMM joints in comparison to WT DMM joints. Conclusions: Our data demonstrate that Zcchc6 is upregulated in DMM joints and functions as a positive regulator of IL-6 and MMP13 expression in murine chondrocytes and cartilage in vivo. Severity of the posttraumatic OA and cartilage degradation was significantly low in Zcchc6 KO mice and correlated with reduced expression of MMP-13. These results identify ZCCHC6 as a potential therapeutic target for the treatment of OA.

A novel regulatory function of CDKN1A/p21 in TNFα-induced matrix metalloproteinase 9-dependent migration and invasion of triple-negative breast cancer cells

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.

Effects of stanozolol on normal and IL-1\u03b2-stimulated equine chondrocytes in vitro

BackgroundIntra-articular administration of stanozolol has shown promising results by improving the clinical management of lameness associated with naturally-occurring osteoarthritis (OA) in horses, and by decreasing osteophyte formation and subchondral bone reaction in sheep following surgically induced OA. However, there is limited evidence on the anti-inflammatory and modulatory properties of stanozolol on articular tissues. The objective of the current study was to evaluate the effects of stanozolol on chondrocyte viability and gene expression in normal equine chondrocytes and an inflammatory in vitro system of OA (interleukin-1β (IL-1β) treated chondrocytes).ResultsChondrocytes from normal metacarpophalangeal joints of skeletally mature horses were exposed to four treatment groups: (1) media only (2) media+IL-1β (3) media+IL-1β + stanozolol (4) media+stanozolol. Following exposure, chondrocyte viability and the expression of catabolic, anabolic and structural genes were determined. General linear models with Dunnet’s comparisons with Bonferroni’s adjustment were performed. Cell viability was similar in all groups. Stanozolol treatment reduced gene expression of MMP-13, MMP-1, IL-6 and COX-2 in both normal and IL-1β treated chondrocytes. Stanozolol treatment reduced ADAMTS4 gene expression in normal chondrocytes. Stanozolol reduced the expression of COL2A1.ConclusionsThe current study demonstrates stanozolol has chondroprotective effects through downregulation of genes for pro-inflammatory/catabolic cytokines and enzymes associated with OA. However, there is no evidence of increased cartilage stimulation through upregulation of the anabolic and structural genes tested.

Lithium chloride attenuates mitomycin C induced necrotic cell death in MDA-MB-231 breast cancer cells via HMGB1 and Bax signaling

The clinical use of potent anticancer drug mitomycin C (MMC) has limited due to side effects and resistance of cancer cells. The aim of this study was to investigate whether lithium chloride (LiCl), as a mood stabilizer, can affect the sensitivity of MDA-MB-231 breast cancer cells to mitomycin C.The cells were exposed to various concentrations of mitomycin C alone and combined with LiCl and the viability determined by trypan blue and MTT assays. Proteins were analyzed by western blot and mRNA expression of HMGB1 MMP9 and Bcl-2 were analyzed by RT-PCR. Flow cytometry was used to determine the cell cycle arrest and percent of apoptotic and necrotic cells. Concentration of Bax assessed by ELISA.Exposure of the cells to mitomycin C revealed IC50 value of 20 μM, whereas pretreatment of the cells with LiCl induced synergistic cytotoxicity and IC50 value declined to 5 μM. LiCl combined with mitomycin C significantly down-regulated HMGB1, MMP9 and Bcl-2 gene expression but significantly increased the level of Bax protein. In addition, the content of HMGB1 in the nuclei decreased and pretreatment with LiCl reduced the content of HMGB1 release induced by MMC. LiCl increased mitomycin C-induced cell shrinkage and PARP fragmentation suggesting induction of apoptosis in these cells. LiCl prevented mitomycin C-induced necrosis and changed the cell death arrest at G2/M-phase. Taking all together, it is suggested that LiCl efficiently enhances mitomycin C-induced apoptosis and HMGB1, Bax and Bcl-2 expression may play a major role in this process, the findings that provide a new therapeutic strategy for LiCl in combination with mitomycin C.

Glucocorticoid sensitivity and inflammatory status of peripheral blood mononuclear cells in patients with coronary artery disease

Objective: Mechanisms behind sustained inflammation in patients with coronary artery disease (CAD) are not clarified but hypothalamus-pituitary-adrenal (HPA) axis dysfunction may have a role. Here, we investigated whether inflammatory status of peripheral blood mononuclear cells (PBMCs) was associated with altered glucocorticoid sensitivity in CAD patients.Methods: In 55 CAD patients and 30 controls, mRNA levels of GR-α, GR-β, NF-κB, IκBα, MMP-9 and TIMP-1 were measured in PBMCs. Suppressive effects of dexamethasone on GR-α, GR-β, NF-κB, IκBα, MMP-9 and TIMP-1 mRNA levels were assessed in PBMCs ex vivo. Salivary cortisol was repeatedly measured over 3 days.Results: GR-α mRNA levels were higher in CAD patients than in controls, 0.50 (0.38–0.59) versus 0.26 (0.18–0.37), p < .001, while GR-β mRNA levels were equally low in both groups. GR-α mRNA expression was associated with inflammatory gene expression and, also, with flatter diurnal cortisol rhythm. In both patients and controls, dexamethasone suppressed gene expression of NF-κB, IκBα, MMP-9 and TIMP-1 (p < .001). Dexamethasone also reduced GR-α mRNA levels (p < .001), while LPS increased it (p < .001).Conclusions: PBMCs from CAD patients displayed an inflammatory gene expression profile. This was not explained by reduced glucocorticoid sensitivity. Instead, inflammation was associated with increased expression of GR-α mRNA, suggesting a hypocortisolemic state.Key messages • Peripheral blood mononuclear cells from patients with coronary artery disease (CAD) display an inflammatory gene expression profile. • This inflammatory state cannot be explained by reduced glucocorticoid sensitivity in CAD patients. • Instead, the inflammatory gene expression profile is associated with upregulated levels of glucocorticoid receptor-α mRNA, suggesting a hypocortisolemic state.

MiR-29b enhances prostate cancer cell invasion independently of MMP-2 expression

BackgroundThe ability to metastasize is one of the most important characteristics of neoplastic cells. An imbalance between the action of some matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs drives the invasion process. Some studies have suggested that MMP-2 is involved in metastasis, while other studies have reported that collagen production by cancer cells might also contribute to motility. However, decreased expression of microRNA-29b (miR-29b), which may control MMP-2 and collagen gene expression, has been shown in prostate cancer (PCa). The objectives of the present study were to clarify whether MMP-2 as well as collagens I and III (encoded by COL1A1 and COL3A1, respectively) are controlled by miR-29b and to determine whether metastasis is altered by this relationship.MethodsPCa DU145 and PC-3 cells were transfected with 100 μL of OPTI-MEM I containing 100 nmol of miR-29b (or its inhibitor) along with 1.5 μL of lipofectamine. Positive and negative controls were prepared using the same protocol. MMP-2, COL1A1 and COL3A1 messenger RNA (mRNA) levels were evaluated via real-time polymerase chain reaction (qRT-PCR). For qRT-PCR, 6 × 104 cells were used. Invasion studies were conducted with Matrigel assays, which simulate invasion of the extracellular matrix by neoplastic cells. After transfection of 3 × 104 cells, invasion was allowed to proceed for 48 h. Invasive cells were counted under an optical microscope. Each experiment was performed in triplicate.ResultsMMP-2 mRNA was not expressed in DU145 cells after transfection with miR-29b. After transfection of cells with the miR-29b inhibitor, COL1A1 (p = 0.02) and COL3A1 (p = 0.06) mRNA expression was increased in DU145 cells, and a large number of transfected DU145 and PC3 cells invaded the Matrigel membrane.ConclusionsIn vitro studies showed that reducing the amount of miR-29b may lead to higher PCa cell invasion via a process that is independent of MMP-2. Collagen expression, controlled by miR-29b, may facilitate this motility process. Thus, the present study suggests that collagen production plays an active role in metastasis control and restoration of miR-29b levels may decrease metastasis. Altogether, these findings support further exploration of drug therapy targeting this aspect of the metastasis circuit.

Association between the expression of MMP1 gene and prognosis in head and neck squamous cell carcinoma

Objective:The aim of this study is to investigate the expression of MMP1 and prognosis in patients with head and neck squamous cell carcinoma (HNSCC), and to identify the potential mechanism of MMP1 in HNSCC. Method:The RNA sequencing data and related clinical data of HNSCC were downloaded from the TCGA public database. The MMP1 gene expression data and corresponding clinical information in the samples were retrospectively analyzed; The data of gene microarray were used to verify the correlation between MMP1 gene and HNSCC. The disease free survival and overall survival of HNSCC were also analyzed; Gene set enrichment analysis was conducted to identify the potential mechanism of MMP1 in HNSCC. Result:Among the 332 HNSCC patients, the expression of MMP1 was significantly associated with lymphatic invasion and tumor grade (P<0.01). The higher the expression level of MMP1 was, the more susceptible the patient was to lymph node metastasis. The data confirmed that the expression of MMP1 in HNSCC was significantly higher than that in normal mucosa (P<0.05); HNSCC of patient in MMP1 high expression group proved to have worse disease free survival and overall survival than in MMP1 low expression group (P<0.05); Gene enrichment analysis indicates that the high expression of MMP1 gene might influence the biological process of tumor though epithelial mesenchymal transition, TGF-β signaling pathway, hypoxia, angiogenesis, Noth signaling pathway, and up-regulation of KRAS gene signaling pathway. Conclusion:The high expression of MMP1 was related with occurrence and development of HNSCC, which can be used as an independent risk factor and has a great clinical significance.

Significance of MMP-9 expression and MMP-9 polymorphism in prostate cancer.

The aim of the study was to assess the expression of the MMP-9 gene and -1562 C/T polymorphism in MMP-9 gene promoter in relation to clinicopathological parameters in predicting the clinical outcome of prostate cancer patients. A total of 82 patients with histopathologically diagnosed prostate cancer were enrolled in the study. MMP-9 gene expression was assessed by reverse transcription-PCR method. MMP-9 (-1562 C/T) polymorphism variants were determined by the polymerase chain reaction-based restriction fragment length polymorphism method. MMP-9 expression and MMP-9 -1562 polymorphism variants in relation to disease pathological stage (P = 0.006; P <0.0001, respectively), as well as to prognostic group (P = 0.019; P <0.0001, respectively), were statistically significant. Only MMP-9 -1562 polymorphism variants in relation to tumor differentiation grade (P = 0.044) were found to be statistically significant. Positive MMP-9 gene expression was associated with 5-year survival rate of prostate cancer patients with pathological stage III (P = 0.036) and for the patients in prognostic group III (P = 0.012). Patients with tumor differentiation grade G2 and with the identified CC variant had a significantly longer survival time than patients with the identified TT variant (P = 0.007). MMP-9 gene expression and MMP-9 -1562 polymorphism variants were associated with prostate cancer pathological stage and prognostic group. MMP-9 -1562 polymorphism CC variant was associated with prostate cancer tumor differentiation grade. Five-year survival analysis showed the relationship between MMP-9 gene expression and pathological stage III, as well as prognostic group III, whereas MMP-9 -1562 polymorphism variants, with tumor differentiation grade G2.