MMP-9 Gene Expression Research Articles

TGFβ1 single-nucleotide polymorphism C-509T alters mucosal cell function in pediatric eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic Th2 antigen driven disorder associated with tissue remodeling. Inflammation and remodeling lead to esophageal rigidity, strictures, and dysphagia. TGFβ1 drives esophageal remodeling including epithelial barrier dysfunction and subepithelial fibrosis. A functional SNP in the TGFβ1 gene that increases its transcription (C-509T) associates with elevated numbers of esophageal TGFβ1 expressing cells. We utilized esophageal biopsies and fibroblasts from TT genotype EoE children to understand if TGFβ1 influenced fibroblast and epithelial cell function in vivo. Genotype TT EoE esophageal fibroblasts had higher baseline TGFβ1, collagen1α1, periostin, and MMP-2 (p<.05) gene expression and distinct contractile properties compared with CC genotype (n=6 subjects per genotype). In vitro TGFβ1 exposure caused greater induction of target gene expression in genotype CC fibroblasts (p<.05). Esophageal biopsies from TT genotype subjects had significantly less epithelial membrane bound E-cadherin (p<.01) and wider cluster distribution at nanometer resolution. TGFβ1 treatment of stratified primary human esophageal epithelial cells and spheroids disrupted transepithelial resistance (p<.001) and E-cadherin localization (p<.0001). A TGFβ1-receptor-I inhibitor improved TGFβ1-mediated E-cadherin mislocalization. These data suggest that EoE severity can depend on genotypic differences that increase in vivo exposure to TGFβ1. TGFβ1 inhibition may be a useful therapy in subsets of EoE patients.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells

ABSTRACT Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.

Proinflammatory Effects of IL-1β Combined with IL-17A Promoted Cartilage Degradation and Suppressed Genes Associated with Cartilage Matrix Synthesis In Vitro.

Combinations of IL-1β and other proinflammatory cytokines reportedly promote the severity of arthritis. We aimed to investigate the effects of IL-1β combined with IL-17A on cartilage degradation and synthesis in in vitro models. Cartilage explant degradation was determined using sulfated glycosaminoglycans (S-GAGs) levels, matrix metalloproteinase (MMP13) gene expression, uronic acid, and collagen contents. Cell morphology and accumulation of proteoglycans were evaluated using hematoxylin-eosin and safranin O staining, respectively. In the pellet culture model, expressions of cartilage-specific anabolic and catabolic genes were evaluated using real-time qRT-PCR. Early induction of MMP13 gene expression was found concomitantly with significant S-GAGs release. During the prolonged period, S-GAGs release was significantly elevated, while MMP-13 enzyme levels were persistently increased together with the reduction of the cartilaginous matrix molecules. The pellet culture showed anabolic gene downregulation, while expression of the proinflammatory cytokines, mediators, and MMP13 genes were elevated. After cytokine removal, these effects were restored to nearly basal levels. This study provides evidence that IL-1β combined with IL-17A promoted chronic inflammatory arthritis by activating the catabolic processes accompanied with the suppression of cartilage anabolism. These suggest that further applications, which suppress inflammatory enhancers, especially IL-17A, should be considered as a target for arthritis research and therapy.

Effects of Papain-Based Gel Used For Caries Removal on Macrophages and Dental Pulp Cells.

Papain-based gel is used for chemical-mechanical caries removal and present antimicrobial and anti-inflammatory activities. However, its effects on dental pulp cells and on macrophages remains largely unknown. Therefore, the aim of this study was to investigate whether the papain-based gel Papacárie Duo® acts as an immunomodulator in lipopolysaccharide (LPS)-activated macrophages and its effects on dental pulp cells . J774.1 macrophage and OD-21 dental pulp cells were stimulated with 0.5% and 5% of Papacárie Duo®, following pre-treatment or not with LPS. After 24 h, a lactate dehydrogenase assay was used to measure cytotoxicity, a tetrazolium-based colorimetric assay (MTT) was used to measure cell viability, and qRT-PCR was used to analyze relative gene expression of Ptgs2, Il10, Tnf, Mmp9, Runx2, Ibsp and Spp1. Papacárie Duo® was cytotoxic and reduced cell viability at 5% but not at 0.5% in both cultures. In macrophages, Papacárie Duo® increased the expression Il10 and LPS-induced Ptgs2, but it did not affect Tnf or Mmp9. In OD-21 cells, Papacárie Duo® inhibited Runx2 and Ibsp expression, but stimulated Spp1 expression. Papain-based gel presented a concentration dependent cytotoxicity, without affecting cell viability, for dental pulp cells and macrophages. Interestingly, the gel presented an inhibitory effect on pulp cell differentiation but modulated the activation of macrophages stimulated with LPS. We speculate that in dental pulp tissue, Papacárie Duo® would impair reparative dentinogenesis but could activate macrophages to perform their role in defense and inflammation.

Antioxidant Effects of Red Fruit Oil on MMP-1 Gene Expression and Malondialdehyde Levels on Skin Exposed to UVB Rays

Background: Chronic exposure ultraviolet (UV)-B radiation causes reactive oxygen species (ROS) formation. Furthermore, ROS will induce the formation of malondialdehyde and increase matrix metalloproteinase (MMP)-1 expression. One strategy against the free radicals effects is by consuming antioxidants. This study aims to analyze the antioxidants effect of red fruit oil (RFO) on MMP-1 expression and malondialdehyde levels due to exposure to UVB rays.Materials and Methods: Thirty male Wistar rats were divided into 5 groups. The P0 group was not given treatment, the P1 group was only exposed to UVB light, the P2 group was exposed to UVB light and given 0.5 mL/200 g body weight (BW) of RFO, the P3 group was exposed to UVB light and given 1 mL/200 g BW of RFO, and group P4 exposed to UVB rays and given 2 mL/200 g BW of RFO. Experimental animals would be examined for MMP-1 expression and malondialdehyde level. RFO would be identified with β-carotene and tocopherol content.Results: Beta-carotene and tocopherol were detected in RFO. RFO reduced significantly MMP-1 expression (p&lt;0.05) in P2 group (0.73±1.27), P3 group (0.63±0.95), P4 group (9.56±20.97) compared group P1 (48.07±65.58). However, RFO did not reduce malondialdehyde levels (p&gt;0.05).Conclusion: Our research demonstrates RFO containing tocopherol and β-carotene can reduce the MMP-1 expression, but does not affect malondialdehyde levels due to exposure to UVB rays. An effective dose that can reduce malondialdehyde levels and MMP-1 expression is 1 mL/200 g BW.Keywords: red fruit oil, antioxidant, skin, MMP-1 expression, malondialdehyde, UVB rays, photoaging

Dieckol or phlorofucofuroeckol extracted from Ecklonia cava suppresses lipopolysaccharide-mediated human breast cancer cell migration and invasion

The phlorotannin compounds, dieckol and phlorofucofuroeckol (PFF), extracted from Ecklonia cava, have been suggested to exhibit anti-inflammatory, anti-adipogenic or anti-tumorigenic activity. However, role of dieckol or PFF in LPS/TLR-4 signaling that regulates cancer development still remains to be determined. Here we report that dieckol or PFF blocked lipopolysaccharide (LPS)-induced migration and invasion of human breast cancer MCF-7 or MDA-MB-231 cells by about 50% (p < 0.0001) or 60% (p = 0.0001), respectively. Also, PFF as well as dieckol markedly attenuated both of LPS-induced Toll-like receptor4 (TLR-4) expression and NF-κB promoter-driven transcriptional activity, which are essential for the migration and invasion. Moreover, LPS induction of NF-κB-dependent gene, matrix metalloproteinase9 (MMP-9) was markedly decreased about 2.6 or 2.5 fold by dieckol and PFF, respectively. In addition, LPS-induced MMP-2 gene expression was reduced about 1.7 or 2.1 fold by dieckol and PFF, respectively. Significantly, each inhibitor of MMP-9 or NF-κB notably reduced invasion of MDA-MB-231 cells in response to LPS by about 70% (p = 0.0115) or 40% (p < 0.0001), respectively. Collectively, these findings demonstrate that both of dieckol and PFF functions as a critical inhibitor of LPS-induced breast cancer cell invasion through downregulation of TLR-4-NF-κB-MMP-9 signaling axis, suggesting a potential role of the phlorotannins in inflammation-mediated breast cancer development.

Carbon Nanomaterials Stimulate HMGB1 Release From Macrophages and Induce Cell Migration and Invasion.

Carbon nanomaterials (CNMs) are widely used in industrial and medical sectors. The increasing exposure of CNMs necessitates the studies of their potential environmental and health effects. High-mobility group box-1 (HMGB1) is a nuclear DNA-binding protein, but when released from cells, may cause sustained inflammatory response and promote cell migration and invasion. In this work, we found that 7-day exposure of 2.5 mg/kg/day CNMs, including C60, single-walled carbon nanotubes, and graphene oxides significantly elevated the level of HMGB1 in blood and lung lavage fluids in C57BL/6 mice. Subsequently, cellular effects and underlying mechanism were explored by using Raw264.7. The results showed that noncytotoxic CNMs enhanced HMGB1 intracellular translocation and release via activating P2X7 receptor. Released HMGB1 further activated receptor for advanced glycation endproducts (RAGE) and downstream signaling pathway by upregulating RAGE and Rac1 expression. Simultaneously, CNMs prepared the cells for migration and invasion by modulating MMP2 and TIMP2 gene expression as well as cytoskeleton reorganization. Intriguingly, released HMGB1 from macrophages promoted the migration of nearby lung cancer cell, which can be efficiently inhibited by neutralizing antibodies against HMGB1 and RAGE. Taken together, our work demonstrated that CNMs stimulated HMGB1 release and cell migration/invasion through P2X7R-HMGB1-RAGE pathway. The revealed mechanisms might facilitate a better understanding on the inflammatory property and subsequent cell functional alteration of CNMs.

Silencing microRNA-330-5p increases MMP1 expression and promotes an invasive phenotype in oesophageal adenocarcinoma

BackgroundMany patients diagnosed with oesophageal adenocarcinoma (OAC) present with advanced disease and approximately half present with metastatic disease. Patients with localised disease, who are managed with curative intent, frequently undergo neoadjuvant chemoradiotherapy. Unfortunately, ~ 70% of patients have little or no response to chemoradiotherapy. We previously identified miR-330-5p as being the most significantly downregulated microRNA in the pre-treatment OAC tumours of non-responders to treatment, but that loss of miR-330-5p had a limited impact on sensitivity to chemotherapy and radiation in vitro. Here, we further examined the impact of miR-330-5p loss on OAC biology.MethodsmiR-330-5p was suppressed in OE33 OAC cells following stable transfection of a vector-driven anti-sense RNA. Whole transcriptome digital RNA-Seq was employed to identify miR-330-5p regulated genes, and qPCR was used for validation. Protein expression was assessed by protein array, Western blotting and zymography. Invasive potential was measured using a transwell assay system. Tumour xenograft growth profile studies were performed in immunocompromised CD1 mice.ResultsIn OE33 cells, suppression of miR-330-5p significantly altered expression of 42 genes, and several secreted proteases. MMP1 gene expression and protein secretion was significantly enhanced with miR-330-5p suppression. This corresponded to enhanced collagen invasion in vitro. In vivo, OE33-derived tumour xenografts with miR-330-5p suppression grew faster than controls.ConclusionsLoss of miR-330-5p expression in OAC tumours may influence tumour cell invasive capacity, tumour growth and therapeutic sensitivity via alterations to the tumour microenvironment.

Transfection of microRNA-143 mimic could inhibit migration of HN-5 cells through down-regulating of metastatic genes

Oral squamous cell cancer (OSCC) is one of the causes of death worldwide. The purpose of this project was to define the restoring of microRNA-143 in HN-5 cells and discover molecular apparatuses responsible for the anticancer processes. Firstly, expression levels of miR-143, K-Ras, MMP9 and C-Myc were evaluated in OSCC tissues. Then, microRNA-143 was transfected into HN-5 cells. The cytotoxic effects of microRNA-143 on HN-5 cells were evaluated. To estimate the effects of microRNA-143 on cell migration, wound healing assay was done. The expression levels of microRNA-143, K-Ras, MMP9, C-Myc, ADAMTS and CXCR4 were evaluated via the qRT-PCR method. microRNA-143 mimic inhibited cell migration in HN-5 cell line. microRNA-143 mimic decreased K-Ras, MMP9, C-My, ADAMTS and CXCR4 gene expression. microRNA-143 can inhibit HN-5 cells migration in vitro by down-regulating the expression of invasion-linked genes. Hence, microRNA-143 can be a new diagnostic biomarker and new therapeutic target for OSCC.

Expression Levels of JARID1B, Hes1 and MMP-9 Genes in CML Patients Treated with Imatinib Mesylate

To explore the relationship between the expression levels of JARID1B,Hes1 and MMP-9 genes and the stages of chronic myelogenous leukemia(CML) and the curative effect of imatinib mesylate (IM). Peripheral blood samples of 15 cases of CML in chronic phase and 10 cases of CML in progressive phase were collected from the Hematology Department of Taihe Hospital affiliated to Hubei University of Medicine and 15 cases of healthy people in the Physical Examination Center. CML patients were divided into effective group and ineffective group based on the efficacy after treatment with IM, then real-time PCR was used to detect the expression levels of JARID1B, Hes1 and MMP-9 mRNA, finally, the differences in the level of gene expression and their correlations with CML stages and IM curative efficacy were analysed. The expression levels of Hes1 and MMP-9 in initially diagnosed patients in chronic and progressive phase without IM treatment were significantly higher than those of health people（P＜0.05）. There was no significant difference in the expression level of JARID1B between chronic phase patients and health people（P＞0.05）, but the expression level of JARID1B in the progressive phase patients was higher than that of health people （P＜0.05）. The expression levels of JARID1B and Hes1 in the IM-effective group were not significantly different from those in the IM-ineffective group (P=0.85,P=0.82）, while the expression level of MMP-9 in the IM-effective group [JP2]was significantly lower than that in the IM-ineffective group（P＜0.05）. The expression levels of JARID1B Hes1 and MMP-9 relate with the different phase of CML; The expression levels of JARID1B and Hes1 have not significant relationship with IM curative efficacy, the MMP-9 gene expression level relates with IM curative efficacy.

Type II collagen peptide Coll2-1 is an actor of synovitis

We evaluated the ability of Coll2-1, a type II collagen peptide, to activate pro-inflammatory pathways in synovial cells and to induce arthritis in Lewis rats. Human synoviocytes and chondrocytes from knee OA patients were cultured for 24h with/without Coll2-1 and/or purified immunoglobulin G (AS0619) binding specifically this peptide, and/or CLI-095, a TLR-4 signaling inhibitor and/or apocynin and diphenyleneiodonium, Reactive oxygen species (ROS) production inhibitors. The Interleukin (IL)-8 and Vascular Endothelium Growth Factor (VEGF) expression, the IL-8 production, the IκB-α and p65 phosphorylation and ROS were evaluated. Coll2-1 peptide, bovine type II collagen (CIA), streptococcal cell wall (SCW) or saline solution were injected into Lewis rats. The Coll2-1 peptide was injected subcutaneously (SC; 20-200μg/100μl/animal) or intra-articularly (IA; 0.5-5μg/50μl/animal) and compared to CIA injected in SC (200μg/100μl/animal) and SCW in IA (5μg/50μl/animal). The animals were injected on day 0 and monitored for 28 days. Histological lesions assessment was performed using an arthritis score. Coll2-1 peptide significantly increased IL-8 gene expression and production by synoviocytes. AS0619 and CLI-095 significantly decreased IL-8 expression. Coll2-1 induced p65 and IκBα phosphorylation and oxidative stress inhibitors decreased it. In human chondrocytes culture, Coll2-1 significantly increased MMP-3 and VEGF gene expression. In Lewis rats, CIA, SCW or Coll2-1 injection triggered arthritis. Like CIA or SCW, Coll2-1 induced synovitis, loss of cartilage proteoglycans, cartilage structure lesion and subchondral bone remodeling. Coll2-1 activates synoviocytes to produce IL-8 and induces arthritis in rat. These findings suggest that neutralizing Coll2-1 could be a therapeutic approach of arthritis.

The Role of MicroRNA-21 in Venous Neointimal Hyperplasia: Implications for Targeting miR-21 for VNH Treatment.

The molecular mechanism of hemodialysis access arteriovenous fistula (AVF) failure due to venous neointimal hyperplasia (VNH) is not known. The role of microRNA-21 (miR-21) in VNH associated with AVF failure was investigated by performing in vivo and in vitro experiments. In situ hybridization results revealed that miR-21 expression increased and was associated with fibroblasts in failed AVFs from patients. In a murine AVF model, qRT-PCR gene expression results showed a significant increase in miR-21 and a decrease in miR-21 target genes in graft veins (GVs) compared to contralateral veins in mouse AVF. miR-21 knockdown in GVs was performed using a lentivirus-mediated small hairpin RNA (shRNA), and this improved AVF patency with a decrease in neointima compared to control GVs. Moreover, loss of miR-21 in GVs significantly decreased the Tgfβ1, Col-Ia, and Col-Iva genes. Immunohistochemistry demonstrated a significant decrease in myofibroblasts and proliferation with an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in miR-21-knockdown vessels, along with a decrease in hypoxia-inducible factor-1 alpha (HIF-1α) and phospho-SMAD2 (pSMAD-2) and phospho-SMAD3 (pSMAD-3) and an increase in phosphatase and tensin homolog (PTEN) staining. Hypoxic fibroblast knockdown for miR-21 showed a significant decrease in Tgfβ-1 expression and pSMAD-2 and -3 levels and a decrease in myofibroblasts. These results indicate that miR-21 upregulation causes VNH formation by fibroblast-to-myofibroblast differentiation.

Abstract 184: The role of matrix metalloproteinases (MMPs) and CTNNB1 mutations in endometrial carcinoma

Abstract Introduction: Advanced endometrial carcinoma (EC) carries a poor prognosis, especially for tumors with CTNNB1 somatic mutations. GOG-86P recently demonstrated that treatment of CTNNB1-mutated tumors with the anti-VEGFA antibody bevacizumab (bev) resulted in a better outcome, however, the mechanism for this response is unknown. We tested the hypothesis that overexpression of MMP proteins increases angiogenesis through VEGFA downstream signaling leading to improved clinical response in CTNNB1-mutated tumors. Methods: Data from the endometrial TCGA and CPTAC projects were used to examine the relationship between exon 3 CTNNB1 mutations and VEGFA expression. We used 3 EC cell lines with wildtype (WT) CTNNB1, Ishikawa, HEC59 and JHUEM7, and a single EC cell line with a mutated CTNNB1, HEC108. We compared our results to a colorectal cancer (CRC) model by using the WT cell line RKO and mutated cell line HCT116. We measured VEGFA, CTNNB1, MMP2, MMP7, MMP9 RNA and protein levels in these cell lines using RT-PCR, western blots and ELISA. We transiently and stably overexpressed CTNNB1 in WT CRC and EC cell lines and downregulated CTNNB1 using shRNA transduction in mutated cell lines. Results: An analysis of TCGA microsatellite stable, TP53-wildtype, endometrioid EC showed a 1.4-fold increase in VEGFA gene expression in tumors with CTNNB1 mutations compared to those without (P=0.01). CPTAC endometrial cancer proteomic data did not identify greater VEGFA protein expression in CTNNB1-mutated tumors. An analysis of known β-catenin targets in TCGA and CPTAC revealed significantly increased MMP7 and MMP9 gene and protein expression in CTNNB1-mutated tumors. Transient overexpression of CTNNB1 in WT EC and CRC cell lines increased VEGFA gene expression of 2-3 fold. Stable overexpression of physiologic levels of mutated CTNNB1 in WT EC and CRC lines resulted in significantly increased VEGFA and MMP7 gene expression and increased levels of secreted MMP7. Knockdown of CTNNB1 induced a significant decrease in VEGFA gene expression in HCT116, but not HEC108. MMP7 gene expression was paradoxically increased in CTNNB1-knockdown HCT116, but decreased in CTNNB1-knockdown HEC108. Conclusions: We have shown in silico and in vitro that CTNNB1 exon 3 mutations are associated with increased VEGFA and MMP7 expression. Knockdown experiments in mutated tumors in EC and CRC models suggest that control of angiogenesis and mechanism of action for bev may vary between tumor types supporting the role of CTNNB1 mutations as a biomarker in EC. Ongoing experiments will study the role of MMP7 in CTNNB1-mutated tumors. These findings are being translated into biomarker stratified clinical trials and individualized treatment for women with advanced EC. Citation Format: Amnon A. Berger, Douglas A. Levine, Emily Kawaler. The role of matrix metalloproteinases (MMPs) and CTNNB1 mutations in endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 184.

Effects of omega-3 fatty acids and aspirin on Porphyromonas gingivalis-induced periodontitis in rats.

Periodontitis is a common chronic inflammatory disease caused by bacteria which can result in periodontal tissue inflammation, as well as alveolar bone resorption. The purpose of this study was to evaluate the effects of omega-3 fatty acids plus aspirin (ASA) on ligature-induced periodontitis in rats. Ninety-six male Sprague-Dawley (SD) rats (age 6 weeks) were randomly divided into eight groups (n=12 each) and had ligatures placed for 7days, followed by daily treatment with specific drug regimens for 14days. The rats were sacrificed 20days after drug treatment, and their maxillary were subjected to histomorphometric analysis. RAW264.7 cells were cultured with lipopolysaccharide (LPS) or receptor activator (NF)-κB ligand (RANKL), and treated with various concentrations of omega-3 and ASA. Then, cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS) protein expression and receptor activator of nuclear factor κ B (RANK), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), MMP-2, and Cathepsin-K gene expression were detected. The administration of omega-3 fatty acids and aspirin significantly inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in serum of rats. Histomorphometric analysis showed omega-3 fatty acids plus aspirin promoted alveolar bone increase. Omega-3 fatty acids only, aspirin only, or omega-3 fatty acids plus aspirin also inhibited the protein expressions of COX-2 and iNOS in LPS-stimulated RAW264.7 cells. In addition, omega-3 combined with ASA also inhibited the RANKL-induced gene expressions of MMPs in dose-dependent manners. These results demonstrate that omega-3 fatty acids plus aspirin could decrease alveolar bone loss, while simultaneously increasing the protection against periodontal inflammation.

Derepression of matrix metalloproteinase gene transcription and an emphysema‐like phenotype in transcription factor Zbtb7c knockout mouse lungs

Dysregulated matrix metalloproteinase (MMP) gene expression is a major cause of the degradation of lung tissue that is integral to emphysema pathogenesis. Cigarette smoking (CS) increases MMP gene expression, a major contributor to emphysema development. We previously reported that Zbtb7c is a transcriptional repressor of several Mmp genes (Mmps-8, -10, -13, and -16). Here, we show that Zbtb7c knockout mice have mild emphysema-like phenotypes, including alveolar wall destruction, enlarged alveoli, and upregulated Mmp genes. Alveolar size and Mmp gene expression in Zbtb7c-/- mouse lungs are increased more severely upon exposure to CS, compared to those of Zbtb7c+/+ mouse lungs. These observations suggest that Zbtb7c degradation or absence may contribute to the pathogenesis of emphysema.

Down-regulation of genes related to photoaging and inflammation in UVBirradiated mice premature skin photoaging by linoleic acid-rich tempeh oil

Polyunsaturated fatty acids (PUFAs) are natural substances that are considered for skin protection caused by UVB exposure. The increased levels of photoaging and inflammation markers, i.e. matrix metalloproteinases (MMPs), cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) are correlated with the signs of photoaging and inflammation in the skin. Tempeh, one of the Indonesian fermented foods is a promising source of PUFAs for food applications including nutraceuticals and cosmeceuticals. In this study, PUFA-rich oil was extracted from white and black soybean tempeh and tested on its efficacy on down-regulating several genes related to photoaging and inflammation in UVB-irradiated premature skin aging in mice model by conducting hematoxylin and eosin (H&amp;E) staining and quantitative Real Time-PCR (qRT-PCR) assays. Tempeh oil was extracted in methanol and chloroform using Bligh and Dyer method, and PUFA constituents in tempeh oil were determined by pyrolysis gas chromatography-mass spectrometry (py/GC-MS) analysis. Mice skin dorsal was irradiated with UVB gradually for 4 weeks to induce premature photoaging. Mice were grouped into negative control; positive control; black soybean tempeh oil at 100 and 300 mg/kg; white soybean tempeh oil at 100 and 300 mg/kg; and omega 3 standard. Mice were orally supplemented with tempeh oil for 4 weeks along with UVB irradiation. The py/GC-MS profiling revealed that both tempeh oils contained major linoleic acid PUFA with 52.3% in white tempeh oil and 85.69% in black tempeh oil, respectively. H&amp;E staining demonstrated that UVB irradiation caused premature skin photoaging in mice, indicating by necrosis, epidermal atrophy, and tissue damage. Orally tempeh oil treatment improved tissue damage and increased the proliferation of hair follicle in the skin. Tempeh oil was found to be effectively inhibited the genes expression of MMP-1, MMP-3, MMP-9, COX-2 and iNOS in UVB-irradiated mice skin. Among these genes, tempeh oil from black and white soybean tempeh exerted a significant effect on down-regulating the expression of MMP-9 gene in UVB-irradiated mice skin. In conclusion, tempeh oil may offer the promising photoprotective ingredient in application of nutraceuticals and cosmeceuticals for protecting skin photoaging.

MICRORNA-222 AND MICRORNA-203 SIGNATURES IN ORAL SQUAMOUS CELL CARCINOMA: POTENTIAL ROLE IN PROGRESSION AND AS THERAPEUTIC TARGETS

Objectives To discuss the proposed role of microRNA-222 (miR-222) and microRNA-203 (miR-203) in oral squamous cell carcinoma in the progression and as possible therapeutic targets. Findings miR-222 is colocalized as a cluster in the short arm of chromosome X. Luciferase reporter gene assays in oral tongue squamous cell carcinoma (OTSCC) have shown that hsa-miR-222 regulates the MMP1 expression through both direct cis-regulatory mechanism (targeting MMP1 mRNA) and indirect trans-regulatory mechanism (indirect controlling of MMP1 gene expression by targeting SOD2). Hence, hsa-miR-222 might serve as a novel therapeutic target for OTSCC patients at risk of metastatic disease. miR-222 has been shown to regulate TRAIL resistance and enhancement of tumorigenicity through PTEN and TIMP3(Tissue inhibitor of metalloproteinase 3) downregulation. miR-222 has been implicated to target PUMA(p53 up-regulated modulator of apoptosis) to improve sensitization of UM1 cells to Cisplatin. miR-203 acts as a molecular switch between keratinocyte proliferation and differentiation in adult epidermis by targeting ΔNp63 mRNA. Following DNA damage, ΔNp63 downregulates and a possible activation of the apoptotic program in head and neck squamous cell carcinoma has been thought of. miR-203 has been shown to target EIF5A2 in colorectal cancer cells. Serving as a tumor suppressor gene, miR-203 has been thought to be a useful potential therapeutic target in colorectal cancer. miR-203 as a therapeutic target in oral squamous cell carcinoma needs further validation. Conclusion In tumour progression, several cellular pathways may be affected by a single microRNA since it can target multiple mRNAs. Much more light is to be shed by developing as well as by tracking the identified microRNA signatures in oral squamous cell carcinoma, to pave the way for their future clinical use in the diagnosis, management, and prognosis.

Montelukast, a Cysteinyl Leukotriene Receptor 1 Antagonist, Induces M2 Macrophage Polarization and Inhibits Murine Aortic Aneurysm Formation.

Background The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by atherosclerosis with chronic inflammation in the aortic wall. Montelukast is a selective cys-LT 1 receptor antagonist that can suppress atherosclerotic diseases. We evaluated the in vitro properties of montelukast and its in vivo activities in an angiotensin II–infused apolipoprotein E–deficient (apoE−/−) AAA mouse model. Methods The mouse monocyte/macrophage cell line J774A.1 was used in vitro. M1 macrophages were treated with montelukast, and gene expressions of inflammatory cytokines were measured. Macrophages were cultured with montelukast, then gene expressions of arginase-1 and IL (interleukin)-10 were assessed by quantitative polymerase chain reaction, arginase-1 was measured by fluorescence-activated cell sorting, and IL-10 concentration was analyzed by enzyme-linked immunosorbent assay. In vivo, one group (Mont, n=7) received oral montelukast (10 mg/kg/day) for 28 days, and the other group (Saline, n=7) was given normal Saline as a control for the same period. Aortic diameters, activities of matrix metalloproteinases (MMPs), cytokine concentrations, and the number of M2 macrophages were analyzed. Results Relative to control, montelukast significantly suppressed gene expressions of MMP-2, MMP-9, and IL-1β, induced gene expressions of arginase-1 and IL-10, enhanced the expression of the arginase-1 cell surface protein, and increased the protein concentration of IL-10. In vivo, montelukast significantly decreased aortic expansion (Saline vs Mont; 2.44 ± 0.15 mm vs 1.59 ± 0.20 mm, P<.01), reduced MMP-2 activity (Saline vs Mont; 1240 μM vs 755 μM, P<.05), and induced infiltration of M2 macrophages (Saline vs Mont; 7.51 % vs 14.7 %, P<.05). Conclusion Montelukast induces M2 macrophage polarization and prevents AAA formation in apoE−/− mice.

CNBP controls tumor cell biology by regulating tumor-promoting gene expression.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factorstimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology.

IL-1β Damages Fibrocartilage and Upregulates MMP-13 Expression in Fibrochondrocytes in the Condyle of the Temporomandibular Joint.

The temporomandibular joint (TMJ), which differs anatomically and biochemically from hyaline cartilage-covered joints, is an under-recognized joint in arthritic disease, even though TMJ damage can have deleterious effects on physical appearance, pain and function. Here, we analyzed the effect of IL-1β, a cytokine highly expressed in arthritic joints, on TMJ fibrocartilage-derived cells, and we investigated the modulatory effect of mechanical loading on IL-1β-induced expression of catabolic enzymes. TMJ cartilage degradation was analyzed in 8–11-week-old mice deficient for IL-1 receptor antagonist (IL-1RA−/−) and wild-type controls. Cells were isolated from the juvenile porcine condyle, fossa, and disc, grown in agarose gels, and subjected to IL-1β (0.1–10 ng/mL) for 6 or 24 h. Expression of catabolic enzymes (ADAMTS and MMPs) was quantified by RT-qPCR and immunohistochemistry. Porcine condylar cells were stimulated with IL-1β for 12 h with IL-1β, followed by 8 h of 6% dynamic mechanical (tensile) strain, and gene expression of MMPs was quantified. Early signs of condylar cartilage damage were apparent in IL-1RA−/− mice. In porcine cells, IL-1β strongly increased expression of the aggrecanases ADAMTS4 and ADAMTS5 by fibrochondrocytes from the fossa (13-fold and 7-fold) and enhanced the number of MMP-13 protein-expressing condylar cells (8-fold). Mechanical loading significantly lowered (3-fold) IL-1β-induced MMP-13 gene expression by condylar fibrochondrocytes. IL-1β induces TMJ condylar cartilage damage, possibly by enhancing MMP-13 production. Mechanical loading reduces IL-1β-induced MMP-13 gene expression, suggesting that mechanical stimuli may prevent cartilage damage of the TMJ in arthritic patients.