Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Effects of ERK1/2 signal pathway on cardiomyocyte during glucose lowering

Objective: This study investigated whether the extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway affects cardiomyocyte apoptosis and the expression of tumor necrosis factor (TNF-α) at different glucose-lowering rates.

Methods: Cardiomyocytes of Wistar neonate rats were maintained in a medium supplemented with 25?mmol/L glucosamine for 72?h. Then the medium was changed to different concentrations of glucosamine, and all cells were divided into five groups. The survival rate of cardiomyocyte was measured using the Cell Counting Kit-8; cardiomyocyte apoptosis was measured using the flow cytometry instrument and laser confocal microscope; TNF-α was measured using the enzyme-linked immunosorbent assay; and ERK1/2 protein and phosphorylation were measured using the Western blot. Cardiomyocyte apoptosis and TNF-α were measured again after adding U0126.

Results: As the glucose-lowering rate increased, the survival rate of cardiomyocytes increased in group B and decreased in groups C, D, and E. The TNF-α concentration increased in groups B, C, and D and decreased in group E. After 24?h, the apoptosis rate decreased in group B and increased in groups C, D, and E. The expression of p-ERK1/2 increased in groups B, D, and E, and was the lowest in group C. After adding U0126, the survival rate of cardiomyocyte in all groups increased and TNF-α concentration decreased.

Conclusions: The influence of glucose-lowering rate on cardiomyocyte apoptosis and TNF-α was caused by the p-ERK1/2 pathway. During the glucose-lowering course, the p-ERK1/2 pathway promoted cardiomyocyte apoptosis, and TNF-α secretion was related to not only osmotic pressure but also ERK1/2 signal pathway activation.     

ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5

During restenosis following arterial injury, vascular smooth muscle cells (VSMCs) form a neointimal layer in arteries by changing from a differentiated, contractile phenotype to a dedifferentiated, migratory, and proliferative phenotype. Several growth factors, cytokines, and extracellular matrix components released following injury have been implicated in these phenotypic changes. We have recently detected the expression of laminin-5, an ECM protein found predominantly in epithelial tissues, in the arterial vasculature. Here we report that ln-5 expression by VSMC is upregulated by platelet-derived growth factor (PDGF-BB), epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-beta1. Adhesion to ln-5 specifically enhances PDGF-BB-stimulated VSMC proliferation and migration. PD98059, a specific inhibitor of the ERK1/2 members of the Mitogen Activated Protein kinase family, increases both VSMC adhesion to ln-5 and blocks PDGF-BB-stimulated VSMC migration on ln-5. These results suggest that adhesion to ln-5 mediates a PDGF-BB-stimulated VSMC response to vascular injury via an ERK1/2 signaling pathway.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells

We have shown earlier a requirement for Ca²⁺ and calmodulin (CaM) in the H₂O₂-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H₂O₂-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H₂O₂-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H₂O₂, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H₂O₂-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H₂O₂-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H₂O₂-induced IGF-1R phosphorylation. H₂O₂ treatment also induced Thr²⁸⁶ phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H₂O₂ on ERK1/2, PKB, and IGF-1R phosphorylation.     

Human lactoferrin exerts bi-directional actions on PC12 cell survival via ERK1/2 pathway

Human lactoferrin (hLF) is a member of the transferrin family and is found in most body fluids of human. Recent study showed that hLF played some roles in the regulation of cell growth. However, the biological function of hLF in the central nervous system and neuronal cells is still unclear. The MTT was used to assay cell viability, ELISA tests were used to assay caspase activities, and TUNEL staining was used to test the cytotoxicity of hLF to the cells. Our result showed that 700 microg/ml hLF significantly reduced the cell viability and increased the caspase 3 and 8 activities in PC12 neuronal cells. TUNEL staining further showed that 700 microg/ml hLF was cytotoxic to the PC12 through apoptosis-mediated pathway. In addition, 700 microg/ml hLF significantly decreased the protein expressions of phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) and Bcl-2 in PC12 cells, whereas 50 microg/ml hLF significantly increased the phosphorylation of ERK1/2 which could be specifically inhibited by PD98059. Furthermore, 50 microg/ml hLF could not only up-regulate the Bcl-2 expression but also protect PC12 cells from FasL-induced apoptosis. In conclusion, hLF plays a crucial role in the regulation of apoptosis and anti-apoptosis in PC12 neuronal cells via ERK1/2 phosphorylation pathway.     

ERK1／2信号通路对黄芪苷Ⅳ抗H2O2诱导H9c2细胞氧化损伤的作用

目的：研究黄芪苷Ⅳ（AST）是否通过细胞外信号调节激酶1／2（ERK1／2）通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。方法：用200μmoL／L的H2O2处理细胞6h,采用MTT法检测细胞存活率,建立H2O2诱导的H9c2细胞氧化损伤模型;比色法测定细胞培养液中乳酸脱氢酶（LDH）活性、总超氧化物歧化酶（T—SOD）和锰超氧化物歧化酶（Mn—SOD）活力以及丙二醛（MDA）含量;Western blot检测H9c2细胞ERK1／2蛋白的磷酸化水平。结果：在H2O2浓度为200μmol／L作用6h条件下,细胞存活率降低程度适中,实验结果重复性好,确定后续实验采用200μmol／L H2O2作用6h建立模型。与H2O2组比较,10mg／L及20mg／L AST均显著提高细胞存活率（P〈0．01）,使细胞培养液中LDH活性显著降低（P〈0．01）,T—SOD及Mn—SOD活力显著提高（P〈0．01）,MDA含量显著降低（P〈0．01）。10mg/L及20mg/L AST均显著增加H2O2损伤的H9c2细胞p—ERK1／2蛋白的表达（P〈0．01）,当用PD98059（ERK1／2的抑制剂）预处理后,AST的作用则被取消。结论：黄芪苷Ⅳ可以通过ERK1／2通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。     

Down-regulation of matrix metalloproteinase-9 expression by nitric oxide in lipopolysaccharide-stimulated rat primary astrocytes

Immunologically activated astrocytes over-express matrix metalloproteinase-9 (MMP-9) and nitric oxide (NO). Because they have both beneficial and detrimental effects on the pathophyiological outcomes of several neurological diseases, their expression should be tightly regulated in the CNS. NO can modify the activity of other proteins either by directly modifying protein structure or regulating the expression of target proteins. In this study, we investigated the role of NO on the expression of MMPs in rat primary astrocytes. Rat primary astrocytes were stimulated with lipopolysaccharide (LPS), resulting in the over-expression of both MMP-9 and NO. Inhibition of NO production using nitric oxide synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), further increased MMP-9 expression, suggesting NO inhibits MMP-9 expression. In line with this observation, exogenous addition of NO donor, sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP), inhibited MMP-9 expression in astrocytes. The inhibitory effect of NO was mediated by the down-regulation of mRNA and protein levels of MMP-9 but not by the direct modification of the enzymatic activity of MMP-9. The effect of NO on MMP-9 expression was mimicked by dibutyryl-cGMP and inhibited by PKG inhibitor KT5823, suggesting NO regulates MMP-9 expression via guanylate cyclase-PKG pathway. Finally, SNP or dibutyryl-cGMP inhibited the activation of ERK1/2 in LPS-stimulated astrocytes, which is an essential regulator of MMP-9 expression in astrocytes. The regulation of MMP-9 expression by NO may confer additional levels of fine-tuning of the level of MMP-9 during brain inflammatory conditions.     

Effects of E-cadherin on mouse embryo implantation and expression of matrix metalloproteinase-2 and -9

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.