Matrix Metalloproteinase-9 mRNA Expression Research Articles

Molecular Pathology of Pulmonary Edema in Forensic Autopsy Cases with Special Regard to Fatal Methamphetamine Intoxication.

Pulmonary edema is a common finding in fatal methamphetamine intoxication. However, the underlying mechanism remains poorly understood. This study investigated the molecular pathology of alveolar damage involving pulmonary edema in forensic autopsy cases. Seven candidate reference genes (RPL13A, YWHAZ, GUSB, SDHA, GAPDH, B2M, and ACTB) were evaluated in the lung by the geNorm module in qBaseplus software. RPL13A, YWHAZ, and GUSB were identified as the most stable reference genes. Using these validated reference genes, intrapulmonary mRNA expressions of matrix metalloproteinases (MMPs), intercellular adhesion molecule-1 (ICAM-1), claudin-5 (CLDN-5), and aquaporins (AQPs) were examined. Relative mRNA quantification using TaqMan real-time PCR assay demonstrated higher expressions of all markers except for AQP-5 in fatal METH intoxication cases. These findings suggested alveolar damage and compensatory response in fatal METH intoxication cases. Systematic analysis of gene expressions using real-time qPCR is a useful tool in forensic death investigation.

Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid.

Increased receptor activator of nuclear factor κβ ligand/osteoprotegerin ratio exacerbates cartilage destruction in osteoarthritis in vitro.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage destruction, matrix degradation and bony changes. Subchondral bone alterations in osteoarthritis are associated with cartilage destruction. It has previously been demonstrated that osteoprotegerin (OPG) and receptor activator of nuclear factor κβ ligand (RANKL) mediate this process. The RANKL/OPG ratio is altered in OA chondrocytes compared with normal chondrocytes. In the pathogenesis of OA, abnormal expression levels of matrix metalloproteinase-13 (MMP-13) are secreted by chondrocytes has a vital role in the progression of cartilage erosion. In the present study, the effect of various RANKL/OPG ratios on MMP-13 expression levels was investigated in interleukin-1β-stimulated SW1353 human chondrosarcoma cells. Cell viability was assessed by MTT assay and MMP-13 mRNA and protein expression levels were analyzed by quantitative reverse-transcription-quantitative polymerase chain reaction, ELISA and western blot analyses, respectively. The results demonstrated that an increase in MMP-13 mRNA and protein expression levels was observed with increasing RANKL/OPG ratio. These findings suggest that this mechanism may be used as a novel therapeutic strategy against OA.

High-fat diet plus carbon tetrachloride-induced liver fibrosis is alleviated by betaine treatment in rats

Steatosis, the first lesion in non-alcoholic fatty liver disease (NAFLD), may progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Steatosis predisposes the liver to oxidative stress, inflammation, and cytokines. Betaine (BET) has antioxidant, antiinflammatory and hepatoprotective effects. However, the effects of BET on liver fibrosis development are unknown. Rats were treated with high-fat diet (60% of total calories from fat) for 14weeks. Carbon tetrachloride (0.2mL/kg; two times per week; i.p.) was administered to rats in the last 6weeks with/without commercial food containing BET (2%; w/w). Serum liver function tests and tumor necrosis factor-α, insulin resistance, hepatic triglyceride (TG) and hydroxyproline (HYP) levels and oxidative stress parameters were determined along with histopathologic observations. Alpha-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions and mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were evaluated. BET decreased TG and HYP levels, prooxidant status and fibrotic changes in the liver. α-SMA, COL1A1 and TGF-β1 protein expressions, MMP-2, TIMP-1, and TIMP-2 mRNA expressions diminished due to BET treatment. BET has an antifibrotic effect and this effect may be related to its antioxidant and antiinflammatory actions together with suppression on HSC activation.

Relationship between fluoride exposure and osteoclast markers during RANKL-induced osteoclast differentiation.

Skeletal fluorosis is a metabolic bone disease caused by excessive accumulation of fluoride. Although the cause of this disease is known, the mechanism by which fluoride accumulates on the bone has not been clearly defined, thus there are no markers that can be used for screening skeletal fluorosis in epidemiology. In this study, osteoclasts were formed from bone marrow cells of C57BL/6 mice-treated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand. The mRNA expression of tartrate-resistant acid phosphatase 5b (TRAP5b), osteoclast-associated receptor (OSCAR), calcitonin receptor (CTR), matrix metalloproteinase 9 (MMP9) and cathepsin K (CK) were detected using real-time PCR (RT-PCR). Results showed that fluoride between 0.5 and 8mg/l had no effect on osteoclast formation. However fluoride at 0.5mg/l level significantly decreased the activity of osteoclast bone resorption. Fluoride concentration was negatively correlated with the activity of osteoclast bone resorption. On day 5 of osteoclast differentiation maturity, MMP9 and CK mRNA expression were not only negatively correlated with fluoride concentration, but directly correlated with the activity of osteoclast bone resorption. TRAP5b, CTR and OSCAR mRNA expression were positively correlated with the number of osteoclast and they had no correlation with the activity of osteoclast bone resorption. Thus, it can be seen that MMP9 and CK may reflect the change of activity of bone resorption as well the degree of fluoride exposure. TRAP5b, CTR and OSCAR can represent the change of number of osteoclast formed.

Sclareol isolated from Salvia officinalis improves facial wrinkles via an antiphotoaging mechanism.

Ultraviolet (UV) irradiation triggers skin photoaging processes, which disrupt the normal three-dimensional integrity of skin. UV-induced oxidative stress, both directly and indirectly, stimulates complex signaling pathways. UV radiation activates skin cell surface receptors on a molecular level and triggers severe changes in extracellular matrix (ECM) proteins, resulting in skin photoaging. Sclareol isolated from Salvia officinalis is widely used as a fragrance material. Sclareol is known to exert various biological activities, but its antiphotoaging effect has not been elucidated to date. Therefore, we evaluated wrinkle improvement efficacy of sclareol. Human dermal fibroblast cell line (Hs68) and a reconstructed human epidermis (RHE) model were used to evaluate the antiphotoaging effect of sclareol in vitro. A clinical study treated with 0.02% sclareol-containing cream was conducted to identify the ability of sclareol to improve wrinkles. First, sclareol enhanced cellular proliferation and blocked UVB-induced cell death. Sclareol inhibited the UVB-induced mRNA expression of matrix metalloproteinases (MMPs) by regulating the protein expression of AP-1 constituents. In RHE model, sclareol recovered the UVB-induced decrease in epidermal thickness and the expression of proliferating cell nuclear antigen (PCNA). In clinical trial, visually assessed changes and several wrinkle parameters were considered to be statistically different between the test and control groups at 12 weeks. In this study, sclareol inhibited various photoaging phenomena in human fibroblasts and RHE model. In addition, sclareol-containing cream improved wrinkles in a clinical trial. Taken together, sclareol alleviates facial wrinkle formation via an antiphotoaging mechanism and may be an effective candidate ingredient.

Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

Expression of MMP and TIMP mRNA in peripheral blood leukocytes of patients with invasive ductal carcinoma of the breast.

An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) appears critical for tumor progression and metastasis. This study aimed to determine whether gene expression of MMP1, MMP2, MMP9, TIMP1 and TIMP3 and the MMP/TIMP expression ratio in peripheral blood leukocytes (PBLs) and the MMP1 and TIMP1 contents or MMP1/TIMP1 ratio in plasma were associated with clinicopathological characteristics in invasive ductal carcinoma (IDC) of the breast. Blood samples were collected from women newly diagnosed with IDC who had not received prior treatment (n = 102). Gene expression in PBLs was analyzed by quantitative real-time polymerase chain reaction. Concentrations of MMP1 and TIMP1 in plasma were measured using ELISA. In univariate analysis the expression levels of MMP2 and TIMP1 mRNA were significantly higher in premenopausal compared to postmenopausal patients (p<0.001 and p = 0.014, respectively). MMP2 mRNA expression negatively correlated with age (p<0.001, r = -0.43). We found that the MMP2/TIMP3 expression ratio was significantly higher in women after menopause (p = 0.007). The MMP2/TIMP1 expression ratio was higher in human epidermal growth factor receptor 2 (HER2)-positive patients (p = 0.022). Low-grade tumors had significantly lower MMP1/TIMP1 and MMP2/TIMP1 expression ratios (p = 0.047 and p = 0.048, respectively). TIMP1 plasma concentration was significantly higher in small tumors compared with T2-T3 tumors (p = 0.013). These findings reveal an important association between tumor characteristics and expression ratios of MMP1/TIMP1 and MMP2/TIMP1 in PBLs and TIMP1 concentration in plasma. Menopausal status may influence the mRNA expression levels of MMP2 and TIMP1 as well as the MMP2/TIMP3 expression ratio in IDC of the breast.

The effect of captopril on the expression of MMP-9 and the prognosis of neurological function in herpes simplex encephalitis mice

Background and purpose: Early increased matrix metalloproteinase-9 (MMP-9) expression is involved in the evolution of herpes simplex encephalitis (HSE) by facilitating the development of cerebrovascular complications. However, the molecular mechanism underlying the detrimental effects of MMP-9 in HSE has not been elucidated. Recent research finds angiotensin II plays an important role in regulation of MMP-9 activity. The aim of this work was to identify the influence of angiotensin-converting enzyme inhibitor (ACEI) captopril on MMP-9 activation after herpes simplex virus 1 (HSV-1) infection.Methods: Animal models of HSE were established by intracerebral inoculation of HSV-1 into mice. Brain tissue ROS levels were measured by staining with dihydroethidium. MMP-9 protein expression was detected by immunofluorescence and brain water content was measured with dry-wet weight method. Neurological function score was quantified 5 d after HSV-1 infection. Microglial cells were treated with various concentrations of captopril. MMP-9 gelatinolytic activity in the supematant of the cell cultures was assessed by zymography. RT-PCR was used to detect the mRNA expressions of p47phox and MMP-9.Results: Immunofluorescence showed that expression of MMP-9 in brain tissue was mainly presented in OX-42 positive microglia. Quantification of gelatinolytic activity by densitometry showed that expression of MMP-9 in microglia was significantly increased after HSV-1 infection and inhibited by captopril treatment. NADPH oxidase subunit p47phox and MMP-9 mRNA expression were significantly increased 6 h after HSV-1 infection, and were seen reduced after captopril treatment in dose dependence. Captopril also downregulated ROS and MMP-9 protein expression following encephalitis in vivo, and attenuated brain edema, and improved neurological function.Discussion: This compelling evidence suggests that MMP-9 is a key pathogenic factor within HSE. ACEI captopril could reduce the expression of MMP-9 mediated by ROS, then relieve cerebral edema and improve neurological function, which may lay a foundation for further basic research and clinical application.

Influence of puerarin on proliferative activity of human fetal scleral fibroblasts and expression of collagen type I and matrix metalloproteinase-2 in power frequency electromagnetic field

To investigate the influence of puerarin on the proliferative activity of human fetal scleral fibroblasts (HFSFs) and the expression of collagen type I and matrix metalloproteinase-2 (MMP-2) in HFSFs in the power frequency electromagnetic field. HFSFs were cultured and divided into four radiation groups (0.2 mT, 50 Hz) and control group. Puerarin at concentrations of 0.0, 1.0, 5.0, and 10.0 μmol/L was added to the four radiation groups, respectively. The methyl thiazolyl tetrazolium colorimetry was used to measure the influence on the proliferative activity of HFSFs, Western blot and RT-qPCR were used to measure the protein and mRNA expression of collagen type I and MMP-2 induced by puerarin in the power frequency electromagnetic field. The radiation groups and control group showed significant increases in the proliferative activity of HFSFs over the culture time (F= 959.472 and 279.468, both P<0.01). At 24 and 48 hours, the radiation groups showed significantly lower proliferative activity than the control group (0.432±0.038/0.591±0.017 vs 0.536±0.034/0.801±0.020, both P<0.01). With the increasing concentration of puerarin (0.0, 1.0, 5.0, and 10.0 μmol/L) , the proliferative activity of HFSFs significantly increased, with A values of 0.598±0.031, 0.809±0.041, 0.910±0.037, and 0.983±0.054, respectively (P<0.05). After exposure for 24 hours, the radiation groups showed significantly lower protein expression of collagen type I and significantly higher protein expression of MMP-2 (t=7.917 and 7.831, both P<0.01) ; compared with the 0.0 μmol/L puerarin group, the 1.0, 5.0, and 10.0 μmol/L puerarin groups showed significant increases in the protein expression of collagen type I and significant reductions in the protein expression of MMP-2 (all P<0.01). Compared with the control group, the radiation groups showed significant reductions in the mRNA expression of collagen type I and MMP-2 (t=17.293 and 16.378, both P<0.01) ; compared with the 0.0 μmol/L puerarin group, the 1.0, 5.0, and 10.0 μmol/L puerarin groups showed significant increases in the mRNA expression of collagen type I and significant reductions in the mRNA expression of MMP-2 (P<0.01). Puerarin can inhibit the reduction in the proliferative activity of HFSFs in the power frequency electromagnetic field, upregulate the expression of collagen type I, downregulate the expression of MMP-2, and thus exert its protective effect.

Enhancement of Matrix Metalloproteinase-2 (MMP-2) as a Potential Chondrogenic Marker during Chondrogenic Differentiation of Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (hASCs) have a capacity to undergo adipogenic, chondrogenic, and osteogenic differentiation. Recently, hASCs were applied to various fields including cell therapy for tissue regeneration. However, it is hard to predict the direction of differentiation of hASCs in real-time. Matrix metalloproteinases (MMPs) are one family of proteolytic enzymes that plays a pivotal role in regulating the biology of stem cells. MMPs secreted by hASCs are expected to show different expression patterns depending on the differentiation state of hASCs because biological functions exhibit different patterns during the differentiation of stem cells. Here, we investigated proteolytic enzyme activity, especially MMP-2 activity, in hASCs during their differentiation. The activities of proteolytic enzymes and MMP-2 were higher during chondrogenic differentiation than during adipogenic and osteogenic differentiation. During chondrogenic differentiation, mRNA expression of MMP-2 and the level of the active form of MMP-2 were increased, which also correlated with Col II. It is concluded that proteolytic enzyme activity and the level of the active form of MMP-2 were increased during chondrogenic differentiation, which was accelerated in the presence of Col II protein. According to our findings, MMP-2 could be a candidate maker for real-time detection of chondrogenic differentiation of hASCs.

Betaine treatment decreased oxidative stress, inflammation, and stellate cell activation in rats with alcoholic liver fibrosis.

The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH+CCl4) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl4 was administered intraperitoneally (i.p.) 0.2mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis.

Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts.

To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon's capsule, both in vitro and in vivo. Human primary Tenon's capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT) method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-1,2) and proliferating cell nuclear antigen (PCNA). Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d). A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson's trichrome to compare the neovascularization in the subconjunctiva area. In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3(rd), 7(th), 14(th) and 21(st) days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28(th) day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson's trichrome observation. By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

Abstract 167: Doxycycline Inhibits Revascularization After Hindlimb Ischemia

Objectives: p27 kip1 , a gene affecting human response to arterial injury, affects collateralization after hindlimb ischemia (femoral artery ligation), possibly by regulating matrix metalloproteinase 2 (MMP2). We hypothesized that MMP2 mRNA expression would increase significantly more in ko mice after hindlimb ischemia and that MMP inhibition would affect p27 -/- (ko) collateralization less than p27 +/+ (wt). Methods: Ko and wt mice had their femoral and collateral arteries harvested seven days after hindlimb ischemia for RNA isolation and qRT-PCR. Ko and wt mice were also fed chow without or with doxycycline (a broad spectrum MMP inhibitor) and subjected to hindlimb ischemia. The mice were followed with weekly footpad laser Doppler perfusion imaging for 28 days, and then sacrificed for microCT scanning. Vascular smooth muscle cells (VSMC) isolated from either wt or ko aortae were used in Boyden chamber migration assays without and with an MMP2 specific inhibitor. Results: MMP2 mRNA expression increased significantly more in ko collaterals than wt collaterals (312% vs. 50%, respectively). However, blood flow recovery in ko and wt mice treated with doxycycline decreased to a similar extent (27±2.5% and 33±1% decrease respectively), although ko mice still revascularized better than wt mice ( p &lt; 0.01). Gracilis collateral diameters increased less after doxycycline treatment in both groups compared to mice fed regular chow (78.5±12 and 80±7 vs 98±18 and 106±17 μm, respectively). The bridge collateral diameters increased less in ko mice fed doxycycline (66±32 vs 158±18.3 μm), and no bridge collaterals were detected in wt mice fed doxycycline. Wt mice fed regular chow had rare bridge collaterals. Ko VSMC migration was less affected by MMP2 inhibition than wt (29% less migration vs 48% less migration, p &lt;0.01). Conclusions: MMP2 mRNA expression increased significantly more in p27 ko collaterals after hindlimb ischemia. The non-specific MMP inhibitor doxycycline inhibited revascularization after hindlimb ischemia, independent of p27 expression. p27 ko mice still revascularized better than wt mice after doxycycline treatment, suggesting that p27 has additional effects other than regulating MMP expression in the collateralization response.

Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression.

Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma.

Bisphenol A Increases the Migration and Invasion of Triple‐Negative Breast Cancer Cells via Oestrogen‐related Receptor Gamma

Triple-negative breast cancer (TNBC) is characterized by great metastasis and invasion capability. Our study revealed that nanomolar bisphenol A (BPA), one of the most ubiquitous endocrine disruptors, can increase wound closure and invasion of both MDA-MB-231 and BT-549 cells. BPA treatment can increase protein and mRNA expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, while had no effect on the expression of vimentin (Vim) and fibronectin (FN) in TNBC cells. The expression of G-protein-coupled receptor (GPER), which has been suggested to mediate rapid oestrogenic signals, was not varied in BPA-treated MDA-MB-231 and BT-549 cells. Its inhibitor G15 also had no effect on BPA-induced MMPs expression and cell invasion. Interestingly, BPA treatment can significantly increase the mRNA and protein expressions of oestrogen-related receptor γ (ERRγ), but not ERRα or ERRβ, in both MDA-MB-231 and BT-549 cells. The knock-down of ERRγ can markedly attenuate BPA-induced expression of MMP-2 and MMP-9 in TNBC cells. BPA treatment can activate both ERK1/2 and Akt in TNBC cells. Both inhibitors of ERK1/2 (PD98059) and Akt (LY294002) can attenuate BPA-induced ERRγ expression and cell invasion of MDA-MB-231 cells. Collectively, our data revealed that BPA can increase the expression of MMPs and in vitro motility of TNBC cells via ERRγ. Both activation of ERK1/2 and Akt participated in this process. Our study suggests that more attention should be paid to the roles of xenoestrogens such as BPA in the development and progression of TNBC.

High molecular weight hyaluronic acid regulates MMP13 expression in chondrocytes via DUSP10/MKP5.

To determine the effect of high molecular weight hyaluronic acid (HA) on matrix metalloproteinase 13 (MMP13) expression induced by tumor necrosis factor α (TNF-α) in chondrocytes. Human chondrocytic C28/I2 cells were incubated with TNF-α and HA. In some experiments, the cells were pre-incubated with a CD44 function-blocking monoclonal antibody (CD44 mAb) prior to addition of TNF-α and HA. The expression of MMP13 was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay, while the phosphorylation of signaling molecules was measured by western blot analysis. The transcriptional activity of activator protein 1 (AP-1) was analyzed by a reporter assay. To further clarify the molecular mechanisms of HA in MMP13 regulation, the expression level of dual-specificity protein phosphatase 10 (DUSP10)/mitogen-activated protein kinases phosphatase 5 (MKP5) in HA-treated chondrocytes was assessed by real-time RT-PCR, western blotting, and immunofluorescence microscopy. HA decreased MMP13 mRNA and protein expression induced by TNF-α. Blockage of HA-CD44 binding by CD44 mAb suppressed HA-mediated inhibition of MMP13. HA inhibited transient phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun NH2 -terminal kinase (JNK) induced by TNF-α. Reporter assay findings also revealed that pre-treatment with HA inhibited the transcriptional activity of AP-1 mediated by TNF-α. Moreover, HA induced the expression of DUSP10/MKP5, a negative regulator of p38 MAPK and JNK pathways. These results indicate that HA-CD44 interactions downregulate TNF-α-induced MMP13 expression via regulation of DUSP10/MKP5, suggesting that HA plays an important role as a regulatory factor in cartilage degradation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:331-339, 2017.

The Development of Diabetic Retinopathy in Goto-Kakizaki Rat and the Expression of Angiogenesis-Related Signals.

The Goto-Kakizaki (GK) rat is a genetic model of type 2 diabetes. Diabetic retinopathy (DR) is a common complication of diabetes. In this study, we observed the development of DR in GK rats and the expression of some angiogenesis-related signals. GK rats were housed for 5, 6 and 7 months. Results of retinal vessels stained by cluster of differentiation 31 (CD31) showed that the number of retinal vessels was increased in GK rats at both 6 and 7 months. Retinal histological observation also evidenced such increase. Retinal mRNA expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), VEGFB and its receptors (VEGFR1/2), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) A/B was increased in GK rats at both 6 and 7 months. Retinal mRNA expressions of matrix metalloproteinase (MMP) 2/9 and insulin-like growth factor 1 (IGF-1) were increased at 7 months. Retinal mRNA expression of pigment epithelium-derived factor (PEDF) was increased in GK rats at 6 months. Serum contents of VEGF, bFGF, PDGFA, MMP2/9, IGF-1, PEDF were increased in GK rats at both 6 and 7 months, while PDGFB was increased at 7 months. In summary, our results indicate that retinal angiogenesis occurred in GK rats at 6 and 7 months, and the expressions of some angiogenesis related factors were increased during the development of DR in GK rats.

Whitening and Anti-Wrinkle Effects of Tremella fuciformis Extracts

Background : The white jelly mushroom (Tremella fuciformis), one of the most popular edible fungi, has medicinal properties. However, the effects of T. fuciformis in skin whitening or anti-wrinkle efficacy has not been defined to date. The aim of the present study was to investigate the effects of T. fuciformis extracts on whitening and anti-wrinkle efficacy in skin cells. Methods and Results :We prepared T. fuciformis extracts with water. The extracts (80℃) contained 12.11 ㎎/g polyphenol and 8.54 ㎎/g flavonoid concentration. T. fuciformis extracts markedly decreased melanin contents and tyrosinase activity in α-MSHstimulated melanocytes (B16F10 cells). In addition, the mRNA expression of melanin formation factors, such as microphthalmiaassociated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) were significantly down-regulated in α-MSH-stimulated melanocyte. Furthermore, T. fuciformis extracts increased the synthesis of type I procollagen and reduced mRNA expression of matrix metalloproteinase 1 (MMP-1) in the human dermal fibroblast (HDFn cells). These data indicated that T. fuciformis extracts induce repression of cellular melanogenesis and protect against wrinkles caused by UVB-stimulated damage. Conclusions : Thus T. fuciformis extracts could be a cosmetic candidate for skin whitening and anti-wrinkle effects.

A novel role of MMP-13 for murine DC function: its inhibition dampens T-cell activation

Dendritic cells (DCs) have been shown to express matrix metalloproteinase 13 (MMP-13), but little is known about its specific function in DCs and its role in inflammatory conditions. In the present study, we describe a novel role of MMP-13 in regulating the immunostimulatory function of murine DCs through moderating MHC-I surface presentation, endocytosis and cytokine/chemokine secretion. MMP-13 expression was confirmed in bone marrow-derived DCs at both the mRNA and the protein level and, furthermore, at the activity level. Remarkably, LPS treatment strongly enhanced MMP-13 mRNA expression as well as MMP-13 activity, indicating an important role of MMP-13 in inflammatory processes. Functionally, MMP-13 inhibition did not influence the DC migratory capacity, while endocytosis of ovalbumin was significantly decreased. Inhibition of MMP-13 lowered the capability of murine DCs to activate CD8+ T cells, apparently through reducing MHC-I surface presentation. Decreased surface expression of CD11c on DCs, as well as changes in the DC cytokine/chemokine profile after MMP-13 inhibition, emphasizes the influence of MMP-13 on DC function. Moreover, T-cell-targeting cytokines such as IL-12, IL-23 and IL-6 were significantly reduced. Collectively, our data reveal a novel involvement of MMP-13 in regulating DC immunobiology through moderating MHC-I surface presentation, endocytosis and cytokine/chemokine secretion. Furthermore, the reduced MHC-I surface presentation by DCs resulted in a poor CD8+ T-cell response in vitro This novel finding indicates that MMP-13 might be a promising target for therapeutic intervention in inflammatory diseases.