Matrix Metalloproteinase Gene Expression Research Articles

402 Abnormal glucocorticoid synthesis in the lesional skin of erythematotelangiectatic rosacea

Rosacea is a chronic inflammatory disorder usually affecting the central face. Chronic use of topical steroids on the face causes lesions similar to rosacea. Synthesis and function of glucocorticoid (GC) have been established in healthy skin and various cutaneous diseases but not in rosacea. In most skin disorders, increased inflammation is usually coupled with a decline in the production of cutaneous GC. We designed this study to investigate the de novo GC synthesis in the skin and GC receptor expression in the lesional and the nonlesional skin of patients with erythematotelangiectatic rosacea (ETR). The key steroidogenic enzymes, 11βHSD1, CYP11A1, 3βHSD2 and CYP21A2, were increased in the lesional skin at the gene and protein levels. CYP17A1 and steroidogenic acute regulatory protein (StAR) tended to be higher in the lesional skin. GC receptor was also significantly increased in the lesional skin. Simultaneously, p65/p50 subunits of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κb) and matrix metalloproteinases (MMP) gene expressions were significantly increased in the lesional skin. These concurrent increments demonstrate that crosstalk between GC receptor and NF-κb may be hampered in the lesional skin of ETR and may contribute to rosacea pathogenesis. We demonstrate that ETR is an inherently inflammatory disorder mediated by NF-κb and that lesional GC synthesis and GC receptor expression is increased in ETR unlike in other cutaneous inflammatory diseases.

Flavonoid quercetin-methotrexate combination inhibits inflammatory mediators and matrix metalloproteinase expression, providing protection to joints in collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial tissues in joints, leading to progressive destruction of cartilage and joints. The disease-modifying anti-rheumatic drugs currently in use have side-effects. Thus, there is an urgent need for safe anti-inflammatory therapies for RA. This study aimed to evaluate the therapeutic effect of the flavonoid quercetin on arthritis in mice immunized with type II collagen (CII). An arthritis model was established in C57/BL6 mice by intradermal administration of chicken CII mixed with Freund's complete adjuvant. Quercetin (30mg/kg orally) and methotrexate (0.75mg intraperitoneally twice a week) were administered to investigate their protective effects against collagen-induced arthritis (CIA). Levels of tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and the matrix metalloproteinases (MMP), 3, and 9 were detected to assess the anti-inflammatory effect of quercetin. The mRNA expression of MMP3, MMP9, CCL2, and TNF-α was also measured by quantitative real-time PCR. Quercetin significantly alleviated joint inflammation by reducing the levels of circulating cytokines and MMPs. There was a significant decrease in the expression of TNFα and MMP genes in the ankle joints of arthritic mice. A significant reduction in the levels of knee-joint inflammatory mediators were observed with combined quercetin and methotrexate treatment. Thus, quercetin has the potential to prevent joint inflammation and could be used as an adjunct therapy for RA patients who have an inadequate response to anti-rheumatic monotherapy.

∆9-tetrahydrocannabinol inhibits epithelial-mesenchymal transition and metastasis by targeting matrix metalloproteinase-9 in endometrial cancer.

Limited therapeutic interventions are clinically available for treating aggressive endometrial cancer (EC). Therefore, effective therapies are urgently required. Therefore, the present study investigated the role of ∆9-tetrahydrocannabinol (THC), which is reported to impact proliferative and migratory activities during impairment of cancer progression. In the present study, cell migration in response to THC was measured using transwell assays. Using western blot analysis, the levels of cannabinoid receptors in EC tissues were detected and pathways leading to the inhibition of cell migration by THC on human EC cells were determined. Results suggested that cannabinoid receptors were highly expressed in EC tissues. Furthermore, THC inhibited EC cell viability and motility by inhibiting epithelial-mesenchymal transition (EMT) and downregulating matrix metalloproteinase-9 (MMP-9) gene expression in aggressive human EC cells. The results have the potential to promote the development of novel compounds for the treatment of EC metastasis. The presnet findings suggest that THC may inhibit human EC cell migration through regulating EMT and MMP-9 pathways.

Effect of FSH‐SP on inhibition winkle formation, melaningenesis, and promoting skin hydration in UVB‐irradiated HRM‐2 hairless mice

Skin is important organ to protect against dangerous external factors including bacteria, chemicals, and UV irradiation. UVB is directly induced destruction in skin epidermis barrier by damaging DNA. Expose to UVB can lead to low skin hydration, winkle formation and melanogenesis. The aim of this study was to investigate the effect of FSH‐SP to protect skin barrier in HRM‐2 hairless mice induced UVB irradiation 150 mJ/cm2 three times a week for 6 weeks. The groups divided PL (FSH‐SP 100 mg/kg b.w.) and PH (FSH‐SP 200 mg/kg b.w.). We examined histopathological experiment about skin damage including changes in skin thickness, melanin pigmentation and winkle formation. Also, we experimented SOD, catalase in plasma and expression of mRNA about procollagens, HAS2 (hyaluronan synthesis 2), MMPs (matrix metalloproteinase) and MITF. The serum SOD and catalase activity were significantly increased PH. The result of procollagen, HAS2 and elastin expression was significantly increased in PH. The gene expression of MMPs (1 and 9) and MITF which is a key factor to synthesis melanin were significantly reduced in PH. The skin hydration exposed to back skin was increased in PH. The winkles, skin thickness and melanin pigment looked like decreased in PL and PH. In conclusion, FSH‐SP decreased the expression of MMPs and MITF increasing the activity of antioxidant enzymes, thereby protecting the skin moisturizing factors and inhibiting melanogenesis. It is believed that effect of the FSH‐SP was reduced to suppress the wrinkle formation, whitening effect and moisturizing.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ovariectomy Exacerbates Apical Periodontitis in Rats with an Increase in Expression of Proinflammatory Cytokines and Matrix Metalloproteinases

IntroductionThe aim of this study was to evaluate the gene expression of proinflammatory cytokines, matrix metalloproteinases (MMPs), and cathepsin K in apical periodontitis (AP) and the volume of lesions in ovariectomized and sham-operated rats. MethodsTwenty 12-week-old female Wistar rats were subjected to ovariectomy (OVX) or sham surgery. After 9 weeks, access cavities were prepared in the maxillary and mandibular first molars, pulp tissue was removed, and canals were exposed to the oral environment during 21 days for the induction of AP. The groups were as follows: sham, OVX, sham+AP, and OVX+AP. Animals were euthanized, and blocks containing the maxillary first molar and the surrounding bone were removed for quantification of proinflammatory cytokines cathepsin K and MMP genes by real-time polymerase chain reaction. The hemimandibles containing the mandibular first molars were used for analysis of the AP lesion volume by micro–computed tomographic imaging. ResultsAP in OVX rats showed an increased expression of interleukin 1 beta, tumor necrosis factor alpha, interleukin 6, MMP-8, and MMP-13 (P < .05). OVX alone, without AP induction, did not affect the expression of the evaluated genes. Additionally, AP induced an increase in cathepsin K expression, without significant differences between AP in the sham and OVX groups (P > .05). Micro–computed tomographic imaging showed a significantly greater AP lesion mean volume in OVX compared with sham animals (P < .05). ConclusionsAP lesions in ovariectomized rats are larger and have an increased expression of proinflammatory cytokines and MMPs, indicating that the infection combined with ovariectomy has an important role in the regulation of these signaling molecules and enzymes during the development of AP. Based on that, it may be assumed that the hypoestrogenic condition aggravates inflammation and degradation of extracellular matrix components in AP, which may provide insight into understanding the development of AP in female postmenopausal patients.

Anti-metastatic potential of a proton beam is regulated by p38 MAPK/c-Fos signaling pathway in TPA-treated HepG2 human hepatocellular carcinoma

A proton beam therapy is considered the next generation radio-therapeutic tool for liver cancer treatments. However, its effect on metastasis has not been fully elucidated. Herein, we assessed the effects of a proton beam on the metastatic potential in HepG2, Hep3B and SK-Hep1 hepatocellular carcinomas. The result showed that a proton beam suppressed TPA-induced cell migration and invasion in HepG2 cells, but not in Hep3B and SK-Hep1 cells. In addition, matrix metalloproteinase-9 (MMP-9) activity and mRNA expressions were reversed by proton beam irradiation in TPA-treated HepG2 cells only. Furthermore, a proton beam suppressed TPA-induced gene expressions of urokinase plasminogen activator (uPA), uPA receptor (uPAR), Snail-1 and vascular endothelial growth factor (VEGF) in HepG2 cells in a dose-dependent manner. Moreover, we found that proton beam irradiation restrained p38 MAPK phosphorylation and c-Fos expression. Therefore, the result demonstrates that the anti-metastatic effects of a proton beam in TPA-treated HepG2 cells are associated with the inhibition of MMP-9 activity and the down-regulations of MMP-9, uPA, uPAR, Snail-1 and VEGF gene expression via the p38 MAPK/c-Fos signaling pathway. Taken together, this investigation suggests that the establishment of a customized proton beam therapeutic strategy for each liver cancer type is necessary to improve therapeutic efficiency.

The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and β-catenin, were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of β-catenin while PKF inhibited β-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.

Cordycepin rescues lidocaine-induced neurotoxicity in dorsal root ganglion by interacting with inflammatory signaling pathway MMP3

Application of local anesthetic reagents, such as lidocaine (Lid), could cause significant neurotoxicity in spinal cord dorsal root ganglia neurons (DRGNs). In this study, we investigated the potential rescuing effect of cordycepin (CDC) in an in vitro explant model of lidocaine-induced apoptosis in DRGNs. Explant of rat neonatal DRGNs was prepared, and treated with Lid in vitro to induce neuronal apoptosis. Prior to Lid treatment, DRGN explant was pre-incubated with various concentrations of CDC to evaluate its rescuing effect on Lid-induced apoptosis. We found that, in cultured DRGNs, Lid caused significant neuronal apoptosis whereas pre-incubation of CDC rescued Lid-induced neurotoxicity. In addition, gene and protein expressions of Caspase-9 and Matrix metalloproteinase 3 (MMP3), a key component of inflammatory signal pathway, were both upregulated by Lid but downregulated by CDC pre-incubation. We further overexpressed MMP3 in cultured DRGNs. And discovered that forced MMP3 overexpression upregulated endogenous MMP3 and caspase-9, and reversed the rescuing effect of CDC on Lid-induced neurotoxicity in DRGNs. Therefore, we concluded that CDC has protective effect on local anesthesia induced spinal cord neurotoxicity, possibly through the inverse regulation on inflammatory signaling pathway.

Interleukin‐17 regulates matrix metalloproteinase activity in human pulmonary tuberculosis

Tuberculosis (TB) is characterized by extensive pulmonary matrix breakdown. Interleukin‐17 (IL‐17) is key in host defence in TB but its role in TB‐driven tissue damage is unknown. We investigated the hypothesis that respiratory stromal cell matrix metalloproteinase (MMP) production in TB is regulated by T‐helper 17 (TH‐17) cytokines. Biopsies of patients with pulmonary TB were analysed by immunohistochemistry (IHC), and patient bronchoalveolar lavage fluid (BALF) MMP and cytokine concentrations were measured by Luminex assays. Primary human airway epithelial cells were stimulated with conditioned medium from human monocytes infected with Mycobacterium tuberculosis (Mtb) and TH‐17 cytokines. MMP secretion, activity, and gene expression were determined by ELISA, Luminex assay, zymography, RT‐qPCR, and dual luciferase reporter assays. Signalling pathways were examined using phospho‐western analysis and siRNA. IL‐17 is expressed in TB patient granulomas and MMP‐3 is expressed in adjacent pulmonary epithelial cells. IL‐17 had a divergent, concentration‐dependent effect on MMP secretion, increasing epithelial secretion of MMP‐3 (p < 0.001) over 72 h, whilst decreasing that of MMP‐9 (p < 0.0001); mRNA levels were similarly affected. Both IL‐17 and IL‐22 increased fibroblast Mtb‐dependent MMP‐3 secretion but IL‐22 did not modulate epithelial MMP‐3 expression. Both IL‐17 and IL‐22, but not IL‐23, were significantly up‐regulated in BALF from TB patients. IL‐17‐driven MMP‐3 was dependent on p38 MAP kinase and the PI3K p110α subunit. In summary, IL‐17 drives airway stromal cell‐derived MMP‐3, a mediator of tissue destruction in TB, alone and with monocyte‐dependent networks in TB. This is regulated by p38 MAP kinase and PI3K pathways. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

In Vitro Analysis of Bacosine as Novel Therapeutic Agent for Murine Breast Cancer

The anti-metastatic activity of bacosine (Bacopa monnieri plant) was studied using 4T1 murine breast cancer cell line by targeting matrix metalloproteinases (MMPs) which plays important role in invasion and metastasis. The inhibitory concentration (IC50) of bacosine for 4T1 cell line was 21 μM, which indicates that it is toxic to cancer cell line. The effect of different concentrations (10, 21 and 50 μM) of bacosine on metastatic matrix metalloproteinase-9 (MMP-9) gene expression of 4T1 murine breast cancer cells was studied by real time PCR. At high concentrations of bacosine (21 and 50 μM) MMP-9 gene expression was down-regulated after 24 h of treatment in a concentration dependent manner. The effect of bacosine on apoptosis was evaluated by annexin binding assay which showed anti-apoptotic effect in early and late apoptotic stages of cancer cells. These results suggest that bacosine plays a role in regulating cell metastasis by inhibiting MMP-9 and showing anti-apoptotic effect in breast cancer cells.

WITHDRAWN: DNA methylation and gene expression of matrix metalloproteinase-9 gene in Deficit and Non-Deficit Schizophrenia

// Ju Gao 1, 2, * , Hongwei Yi 3, * , Xiaowei Tang 4, * , Xiaotang Feng 5 , Miao Yu 1 , Weiwei Sha 4 , Xiang Wang 6 , Xiaobin Zhang 4 and Xiangrong Zhang 1, 7 1 Department of Geriatric Psychiatry, Nanjing Brain Hospital, Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210029, China 2 Centers of Disease Prevention and Control for Mental Disorders, Shanghai Changning Mental Health Center, Shanghai 200335, China 3 Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China 4 Department of Psychiatry, Affiliated WuTaiShan Hospital of Medical College of Yangzhou University, Yangzhou, Jiangsu 225003, China 5 Department of Psychiatry, Nanjing Qing Long Mountain Psychiatric Hospital, Nanjing, Jiangsu 211123, China 6 Medical Psychological Institute of the Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China 7 Department of Neurology, Affiliated ZhongDa Hospital, Neuropsychiatric Institute and Medical School of Southeast University, Nanjing, Jiangsu 210009, China * These authors contributed equally to this work Correspondence to: Xiangrong Zhang, email: drxrz@hotmail.com Keywords: deficit schizophrenia; matrix metalloproteinase-9; DNA methylation; gene expression; negative symptoms Received: July 31, 2017 Accepted: December 05, 2017 Published: January 02, 2018 ABSTRACT Background: The biological pathology of deficit schizophrenia (DS) remains unclear. Matrix metalloproteinase-9 (MMP-9) might be associated with neural plasticity and glutamate regulation, involved in schizophrenia pathogenesis. This study explored gene expression and DNA methylation of MMP-9 in peripheral blood mononuclear cells (PBMCs) and their relationship with clinical symptoms in DS and non-deficit schizophrenia (NDS). Materials And Methods: Pyrosequencing was used to determine DNA methylation at CpG sites in exon 4 and exon 5 of MMP-9 gene in 51 DS patients, 53 NDS patients and 50 healthy subjects (HC). RT-qPCR was used to detect MMP-9 gene expression. Clinical symptoms were assessed by BPRS, SANS and SAPS scales. Results: MMP-9 gene expression in PBMCs was significantly higher in DS than NDS and HC subjects. Compared to NDS patients, DS patients had significantly lower DNA methylation at individual CpG sites in exon 4 and exon 5 of MMP-9 gene. Correlation analysis showed that DNA methylation in exon 4 was negatively correlated with gene expression in DS group. Positive correlation was found between MMP-9 gene expression and negative symptoms in total schizophrenic patients. The social amotivation factor of SANS and negative syndrome of BPRS was negatively correlated with DNA methylation of CpG5-1 in DS patients but not in NDS patients. Conclusions: DS patients showed a specific abnormality of peripheral MMP-9 gene expression and DNA methylation, indicating a pathological mechanism underlying DS as a specific subgroup of schizophrenia.

Effects of abomasal supplementation of quercetin on performance, inflammatory cytokines, and matrix metalloproteinase genes expression in goats fed a high-grain diet

Ten abomasally fistulated male goats fed twice daily at a constant rate of 4% of body weight were used to investigate the effects of abomasal supplementation of quercetin on rumen fermentation and the mRNA expression of inflammatory cytokines in the rumen epithelium, liver and hoof tissues, and the expression of matrix metalloproteinase genes in hoof tissues. The goats were placed in individual pens (1.2 × 1.2m) with free access to water and randomly divided to two groups: one group fed an high grain diet (HG) without quercetin (control, n = 5) and the other group fed HG and treated with quercetin (treatment, n = 5) for 4 weeks. Results showed that abomasal supplementation of quercetin increased the average daily feed intake (P < 0.001) and body weight (P < 0.01). The concentration of free lipopolysaccharide (LPS) (P < 0.05) in rumen fluid was higher in goats supplemented with quercetin compared with the control group. Quercetin supplementation did not affect the mRNA expression of IL-1β, IL-6, TNF-α, IL-10, IL-12, and IFN-γ in the ruminal epithelial tissue (P > 0.05). Quercetin supplementation decreased the IL-1β mRNA expression (P < 0.05) and increased IL-10 mRNA expression (P < 0.05) in the liver tissues. Quercetin supplementation downregulated the mRNA expression of IL-1β (P < 0.01) and tended to downregulate the TNF-α mRNA expression (P > 0.05) in the hoof. Quercetin supplementation also lowered the mRNA expression levels of MMP-2 (P < 0.05) and MMP-9 (P < 0.01) in the hoof tissue. In conclusion, abomasal supplementation of quercetin improved the performance, decreased the mRNA expressions of inflammatory cytokines in the liver and hoof tissues, and reduced the risk of laminitis in goats.

Research on Roles of Mongolian Medical Warm Acupuncture in Inhibiting p38 MAPK Activation and Apoptosis of Nucleus Pulposus Cells.

Background Mongolian medical warm acupuncture has a desirable therapeutic effect on sciatica. Apoptosis of the nucleus pulposus cells is considered to play an important role in sciatica. Evidence has demonstrated that oxidative stress and its induced activation of the signaling pathways play important roles in sciatica. However, further research is expected to reveal whether Mongolian medical warm acupuncture can inhibit the apoptosis of nucleus pulposus cells and oxidative stress. Objective To study the effect of the p38 MAPK pathway activated by the generated ROS on apoptosis and the expression of the genes related to the balance of the extracellular matrix metabolism during treatment of sciatica with Mongolian medical warm acupuncture. Method The volume of the active oxygen generated in the nucleus pulposus cells was detected following intervention of Mongolian medical warm acupuncture. The p38 MAPK phosphorylation level was detected with Western blot. The genes are related to the metabolism of the nucleus pulposus extracellular matrix. Result Mongolian medical warm acupuncture reduced the active oxygen within the nucleus pulposus cells and inhibited the activation of the p38 MAPK pathway (P=0.013). Meanwhile, it upregulated the gene expression of Type II collagen, aggrecan, Sox-9, and tissue matrix metalloproteinase reagent 1 (P-0.015; P=0.025; P=0.031; P=0.045) and downregulated the gene expression of matrix metalloproteinase 3 (P=0.015). Conclusion Mongolian medical warm acupuncture may inhibit apoptosis of nucleus pulposus cells and activation of the extracellular matrix decomposition metabolism pathway and promote its anabolism. This process may rely on the oxidative stress matrix of the p38 MAPK pathway.

MicroRNA-378 suppresses myocardial fibrosis through a paracrine mechanism at the early stage of cardiac hypertrophy following mechanical stress.

Rationale: Excessive myocardial fibrosis is the main pathological process in the development of cardiac remodeling and heart failure; therefore, it is important to prevent excessive myocardial fibrosis. We determined that microRNA-378 (miR-378) is cardiac-enriched and highly repressed during cardiac remodeling. We therefore proposed that miR-378 has a critical role in regulation of cardiac fibrosis, and examined the effects of miR-378 on cardiac fibrosis after mechanical stress.Methods: Mechanical stress was respectively imposed on mice through a transverse aortic constriction (TAC) procedure and on cardiac fibroblasts by stretching silicon dishes. A chemically modified miR-378 mimic (Agomir) or an inhibitor (Antagomir) was administrated to mice by intravenous injection and to cells by direct addition to the culture medium. MiR-378 knockout mouse was constructed. Cardiac fibroblasts were cultured in the conditioned media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869. Quantitative real-time polymerase chain reaction analysis of gene and miRNA expression, Western blot analysis, immunochemistry and electron microscopy were performed to elucidate the mechanisms.Results: Mechanical stress induced significant increases in fibrotic responses, including myocardial fibrosis, fibroblast hyperplasia, and protein and gene expression of collagen and matrix metalloproteinases (MMPs) both in vivo and in vitro. All these fibrotic responses were attenuated by treatment with a chemically modified miR-378 mimic (Agomir) but were exaggerated by treatment with an inhibitor (Antagomir). MiR-378 knockout mouse models exhibited aggravated cardiac fibrosis after TAC. Media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869 enhanced the fibrotic responses of stimulated cardiac fibroblasts, confirming that miR-378 inhibits fibrosis in an extracellular vesicles-dependent secretory manner. Mechanistically, the miR-378-induced anti-fibrotic effects manifested partially through the suppression of p38 MAP kinase phosphorylation by targeting MKK6 in cardiac fibroblasts.Conclusions: miR-378 is secreted from cardiomyocytes following mechanical stress and acts as an inhibitor of excessive cardiac fibrosis through a paracrine mechanism.

Regulation of matrix metalloproteinase-3 gene expression in inflammation: A molecular study.

Introduction:Matrix metalloproteinases (MMPs) play a significant role in the efficient tissue turnover and remodeling. This study focuses on the regulation of the MMPs by the protein kinases at the level of gene expression and their signaling pathways.Materials and Methods:Lipopolysaccharide-induced murine macrophage-like RAW 264.7 cell lines were obtained and maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum under optimal temperatures. Primers used were MMP-3 forward primer, MMP-3 reverse primer, and glyceraldehyde-3-phosphate dehydrogenase forward primer and glyceraldehyde-3-phosphate reverse primer. Total RNA was isolated, the sample was prepared, and electrophoresis was performed. The first strand of cDNA was synthesized and amplification of specific isolated gene using polymerase chain reactor (PCR). The amplified products were then separated on a 1.0% agarose gel in 1XTBE at 75 V for 3 h. The gel was stained with ethidium bromide, and the amplified product was visualized and photographed on Gel Doc system.Results:Real-time PCR showed only bands at expected size of 595 bp for internal control amplification of glyceraldehyde-3-dehydrogenase gene. Analysis was done with densitometry, and these values are compared with the negative control. Results showed a statistically significant rise in the relative levels of MMP-3-mRNA when compared with negative control at 1, 2, and 3 h.Conclusion:This study proved the significantly increased levels of MMP gene at different period, thereby it can be concluded that MMP-3 levels are higher in inflammatory conditions.

Interferon regulatory factor 5 (IRF5) regulates the expression of matrix metalloproteinase-3 (MMP-3) in human chondrocytes

Matrix metalloproteinase-3 (MMP-3) plays a pivotal role in the destruction of articular cartilage in osteoarthritis (OA). The regulation of gene expression of MMP-3 is complicated. Interferon regulatory factor 5 (IRF5) is a member of the interferon regulatory factor family of transcription factors. Little information regarding the biological function of IRF5 on chondrocytes and the pathogenesis of OA has been reported. In the current study, for the first time, we report that IRF5 is expressed in human primary chondrocytes and human chondrosarcoma cell line SW1353 cells. In addition, IRF5 is upregulated in response to TNF-α treatment in a dose dependent manner. Interestingly, IRF5 is significantly higher in chondrocytes from OA patients compared to those from normal subjects. Notably, IRF5 mediates TNF-α- induced expression of MMP-3 in chondrocytes. Overexpression of IRF5 promotes the expression of MMP-3, however, knockdown of IRF5 reduces the expression of MMP-3. Mechanistically, IRF5 is able to enhance the transcription of MMP-3 by binding to its promoter. Also, we found that NF-κB was involved in the effects of IRF-5 on MMP-3 expression. These findings suggest that IRF5 might be a novel pharmacological target for the treatment of OA and RA.

The DNA methylation status of genes encoding Matrix metalloproteinases and tissue inhibitors of Matrix metalloproteinases in endometriosis.

Endometriosis is a benign disease, with malignant properties. A necessary step in the progression of endometriosis is tissue remodeling, which is coordinated by the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). This study evaluated the regulation of abnormal MMP and TIMP gene expression during endometriosis. Among the two genes families, promoter regions of MMP2, MMP3, MMP7, TIMP3, and TIMP4 were significantly altered in proliferative-phase endometriotic lesions compared to menstrual cycle-matched eutopic tissue from endometriosis-free women. In addition, a negative correlation was found between the DNA methylation status of the promoter region and transcript abundance of MMP2. Our findings suggest that changes in DNA methylation at the promoter region of MMP2 could underlie the changes in its expression in the ectopic endometria from patients with endometriosis.

Effects of transforming growth factor-β and interleukin-1β on inflammatory markers of osteoarthritis in cultured canine chondrocytes

OBJECTIVE To determine effects of transforming growth factor (TGF)-β and interleukin (IL)-1β on inflammatory markers in cultured canine chondrocytes to clarify the role of these cytokines in osteoarthritis of dogs. SAMPLE Pooled chondrocytes isolated from the stifle joints of 4 adult dogs. PROCEDURES Chondrocytes were isolated, cultured, and frozen at -80°C. Pooled cells were incubated in medium with or without TGF-β (1 or 10 ng/mL) and subsequently stimulated with IL-1β (10 ng/mL). Concentrations of nitric oxide (NO) and prostaglandin (PG) E were measured in culture supernatants. Gene expression of matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-2, inducible NO synthase (iNOS), and cyclooxygenase (COX)-2 was quantified by use of real-time quantitative PCR assays. RESULTS Stimulation with IL-1β increased gene expression of all inflammatory markers, except for TIMP-2. Incubation with TGF-β resulted in a significant decrease in MMP-3 and TIMP-2 mRNA concentrations but had no effect on PGE and NO concentrations. For cells treated with TGF-β followed by IL-1β, concentrations of PGE and NO were lower, compared with concentrations for IL-1β control cells. Furthermore, IL-1β-induced gene expression of iNOS, MMP-3, and COX-2 was downregulated. However, the IL-1β-induced downregulation of TIMP-2 gene expression was partially restored by pretreatment with TGF-β. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that IL-1β increased the expression of inflammatory genes and mediators, and TGF-β largely attenuated the IL-1β-mediated inflammatory response. Therefore, TGF-β might be a novel target for use in the prevention and treatment of cartilage breakdown in dogs with osteoarthritis.

Periostin promotes malignant potential by induction of epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma.

Periostin, a secreted matricellular protein, has been reported to induce epithelial‐mesenchymal transition (EMT), which increases motility and invasiveness in various epithelial cancer cells. Periostin is also overexpressed in intrahepatic cholangiocarcinoma (ICC) and suggested to be a biomarker for tumor progression and poor prognosis; however, its functional role in ICC is not fully understood. Here, we investigated whether periostin influences malignant potential through the induction of EMT in ICC. Analyses of surgical resected ICC specimens revealed that the gene expression of periostin was significantly higher in ICC tumors than in adjacent nontumor liver tissues and was closely correlated with the expression of mesenchymal markers, including N‐cadherin, vimentin, and fibronectin. However, the expression level of periostin varied in each case. Consistently, the expression of periostin in HuH28 (an undifferentiated ICC cell) was markedly higher than in HuCCT‐1 (a moderately differentiated ICC cell). In addition, high‐level secretion of periostin into culture media was observed in HuH28 but not in HuCCT‐1. To identify the biological significance of periostin in EMT, gene silencing of periostin by small interfering RNA was performed in HuH28 cells. Periostin knockdown in HuH28 cells significantly down‐regulated mesenchymal markers and up‐regulated epithelial markers, suggesting the reversal of EMT, namely mesenchymal‐epithelial transition. Along with these changes, cell proliferation was significantly suppressed by 52%. In addition, cell migration and invasion were significantly suppressed by 62% and 61%, respectively, with reduced gene expression of matrix metalloproteinase 2. Interestingly, chemosensitivity to gemcitabine was also significantly improved by periostin depletion. Conclusion: Periostin plays an important role in the regulation of malignant potential through EMT and is suggested to be a novel target for the treatment of ICC. (Hepatology Communications 2017;1:1099–1109)

Polyunsaturated fatty acids influence inflammatory markers in a cellular model for canine osteoarthritis.

Although it is well recognized that dietary supplementation with fish oil improves clinical symptoms in dogs suffering from osteoarthritis, the molecular basis for the dietary benefit is not yet completely resolved in dogs. This study was designed to further clarify how polyunsaturated fatty acids (PUFA) affect key factors of cartilage degeneration in a canine cell culture system mimicking osteoarthritis. Canine chondrocytes were incubated either without or with 10μm of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA) or 3.6μm ibuprofen (Ibu) as positive control for 6days. After the supplementation, cells were stimulated with 10ng/ml interleukin-1β (IL-1β) for another 48hr to induce osteoarthritic changes, or left unstimulated. We analysed fatty acid uptake via gas-liquid chromatography, nitric oxide (NO) production via Griess assay, prostaglandin E (PGE) production via ELISA and relative gene expression of several cartilage matrix proteinases, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 via RT-qPCR. After supplementation, the chondrocytes rapidly incorporated the PUFA into their fatty acid pools. The stimulation with IL-1β caused a marked increase of most of the inflammatory markers measured. N-3 PUFA EPA reduced IL-induced gene expression of iNOS and corresponding production of NO. N-6 PUFA AA also decreased iNOS and NO, but furthermore lowered gene expression of matrix metalloproteinase-3. On the other hand, AA upregulated the aggrecanase ADAMTS-5 and augmented the release of PGE. The effect of n-3 PUFA DHA turned out to be negligible. Our results reveal molecular mechanisms by which PUFA affect degenerative joint disease in dogs. Of particular importance is that not only EPA but also AA decreased several inflammatory markers in our model. Thus, we conclude that an appropriate balance of both n-3 and n-6 fatty acids deserves more attention in dietary interventions.