Impact of Folic Acid on the Resistance of Non-small Cell Lung Cancer Cells to Osimertinib by Regulating Methylation of DUSP1 / 中国肺癌杂志

BACKGROUND@#Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1).@*METHODS@#The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group.@*RESULTS@#Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05).@*CONCLUSIONS@#FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.

Effects of Glucocorticoid-Induced Transcript 1 Gene Deficiency on Glucocorticoid Activation in Asthmatic Mice / 中华医学杂志(英文版)

Background@#Glucocorticoid (GC) is the first-line therapy for asthma, but some asthmatics are insensitive to it. Glucocorticoid-induced transcript 1 gene (GLCCI1) is reported to be associated with GCs efficiency in asthmatics, while its exact mechanism remains unknown.@*Methods@#A total of 30 asthmatic patients received fluticasone propionate for 12 weeks. Forced expiratory volume in 1 s (FEV) and GLCCI1 expression were detected. Asthma model was constructed in wild-type and GLCCI1 knockout (GLCCI1) mice. Glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase 1 (MKP-1) expression were detected by polymerase chain reaction and Western blotting (WB). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was also detected by WB.@*Results@#In asthmatic patients, the change of FEV was well positively correlated with change of GLCCI1 expression (r = 0.430, P = 0.022). In animal experiment, GR and MKP-1 mRNA levels were significantly decreased in asthmatic mice than in control mice (wild-type: GR: 0.769 vs. 1.000, P = 0.022; MKP-1: 0.493 vs. 1.000, P < 0.001. GLCCI1: GR: 0.629 vs. 1.645, P < 0.001; MKP-1: 0.377 vs. 2.146, P < 0.001). Hydroprednisone treatment significantly increased GR and MKP-1 mRNA expression levels than in asthmatic groups; however, GLCCI1 asthmatic mice had less improvement (wild-type: GR: 1.517 vs. 0.769, P = 0.023; MKP-1: 1.036 vs. 0.493, P = 0.003. GLCCI1: GR: 0.846 vs. 0.629, P = 0.116; MKP-1: 0.475 vs. 0.377, P = 0.388). GLCCI1 asthmatic mice had more obvious phosphorylation of p38 MAPK than wild-type asthmatic mice (9.060 vs. 3.484, P < 0.001). It was still higher even though after hydroprednisone treatment (6.440 vs. 2.630, P < 0.001).@*Conclusions@#GLCCI1 deficiency in asthmatic mice inhibits the activation of GR and MKP-1 and leads to more obvious phosphorylation of p38 MAPK, leading to a decremental sensitivity to GCs.@*Trial Registration@#ChiCTR.org.cn, ChiCTR-RCC-13003634; http://www.chictr.org.cn/showproj.aspx?proj=5926.

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.</p><p><b>METHODS</b>Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.</p><p><b>RESULTS</b>Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).</p><p><b>CONCLUSION</b>Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.</p>

The distribution of MAP kinase phosphatase-1 in the cerebrospinal fluid-contacting nucleus and its functional contribution to depressive behaviors / 生理学报

The purpose of this research is to explore the distribution and expression of MAP kinase phosphatase-1 (MKP-1) in cerebrospinal fluid (CSF)-contacting nucleus in depression, and provide experimental evidence to reveal the biological function and regulatory mechanisms of CSF-contacting nucleus in depression. Depression model was produced by chronic forced swimming stress (CFSS) in Sprague-Dawley (SD) rats. Intracerebroventricular injection of cholera toxin subunit B (CTb) labeled with horseradish peroxidase (CB-HRP) was used to specifically mark distal CSF-contacting nucleus. The rate of animal growth and behavioral tests including sucrose preference test (SPT) and open field test (OFT) were used to validate the model of depression. The expressions of MKP-1 and fos proteins in CSF-contacting nucleus were detected by immunofluorescence. Software Image-Pro Plus version 6.0 was used to count the positive neurons. The results showed that, the distributions of MKP-1 were found in the CSF-contacting nucleus. After 28 days of swimming, the rats in stress group had a lower growth rate, a less consumption of sucrose and lower scores of OFT compared to control group. The number of neurons double labeled with CB-HRP/fos or CB-HRP/MKP-1 in stress group was significantly higher than that in control group (P < 0.01). These results suggest that the CSF-contacting nucleus may be involved in the process of depression via the MKP-1.

The inhibitory effects of dexamethasone on cisplatin induced apoptosis of human lung adenocarcinoma cell SPC-A1 and its molecular mechanism / 生物医学工程学杂志

The aim of this study is to investigate the apoptotic inhibition and its molecular mechanism of dexamethasone (DEX) acting on cisplatin (CDDP)-induced apoptosis of human lung adenocarcinoma cell SPC-A1; SPC-A1 cells were pre-cultured in vitro for 24 hours with DEX in different concentrations and then CDDP was added in different concentrations for culturing for further 48 hours. The survival rates of the cells were determined by MTT. The expression of serum/glucocorticoid-induced kinase (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1) in SPC-A1 cells after being cultured by 1 micromol/l DEX at different time was detected by semi-quantitative RT-PCR technology. The expression of glucocorticoid receptor (GR) in SPC-A1 cells was measured by immunohistochemistry (IHC) with biotin-labeled anti-GR. The results of MTT showed that SPC-A1 cells had resistance to CDDP-induced apoptosis with pre-cultured DEX and the resistance intensity presented DEX concentration-dependent. The expressing quantity of SGK-1 in SPC-A1 cells stimulated by DEX could be elevated and increased with intention of time, but the express of MKP-1 was not detected. Up-regulated expression of GR in SPC-A1 cells stimulated by DEX was detected by IHC. The number of cells expressing GR in SPC-A1 cells was significantly higher than that in the control group. The results showed that DEX inhibited apoptosis of SPC-A1 cells induced by CDDP. The possible molecular mechanism is that elevated expression of GR induced by DEX up-regulates the expression of SGK-1 which locates at the downstream of anti-apoptosis pathway. The apoptosis resistance of SPC-A1 cells may account for all above the factors.

Post-transcriptional regulation of dual-specificity phosphatase-1 by RNA-binding protein HuR T118 in heat shock / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the post-transcriptional regulation of dual-specificity phosphatase-1 (DUSP1) by the RNA- binding protein HuR in heat shock.</p><p><b>METHODS</b>The recombinant plasmids carrying wild-type (WT) HuR or its mutants at threonine 118 were constructed and transiently transfected into NIH 3T3 cells via liposome, and the changes in the expressions of DUSP1 mRNA and protein were detected by quantitative real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Heat shock caused significantly enhanced phosphorylation of HuR at the residue T118. In 3T3 cells transfected with the plasmids carrying wild-type HuR for its over-expression showed significantly up-regulated DUSP1 mRNA and protein expressions at 24 h after transfection. Over-expression of HuR(T118A) down-regulated DUSP1 mRNA and protein expressions in cells challenged with heat shock, while HuR(T118E) over-expression significantly increased DISP1 expression at both mRNA and protein levels. After heat shock, HuR(WT) translocated from the cell nucleus to the cytoplasm to form particles. HuR(T118E) was diffusely distributed in the cytoplasm before heat shock and formed particles after heat shock. HuR(T118A) did not undergo such translocation in response to heat shock challenge.</p><p><b>CONCLUSION</b>HuR regulates DUSP1 mRNA and protein expression at the post-transcriptional level to increase its expression after heat shock by enhancing the phosphorylation HuR T118.</p>

Expression of mitogen activated protein kinase phosphatase-1 in pancreatic cancer / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the expression of mitogen activated protein kinase phosphatase-1(MKP-1)in pancreatic cancer.</p><p><b>METHODS</b>Totally 60 cases of normal pancreas, chronic pancreatitis(CP), and pancreatic cancer tissues were collected by operation in our hospital. Pancreatic tissues were analyzed by Northern blot analysis and Western blot analysis. Meanwhile, MKP-1 expression was detected in 6 pancreatic cancer cell lines by Western blot analysis.</p><p><b>RESULTS</b>Northern blot analysis of total RNA revealed relatively low MKP-1 mRNA expression in 7 of 20(35%)normal pancreatic samples. In the remaining 13 samples, the MKP-1 mRNA was absent to faint detectable. In 7 of the 20 CP samples, MKP-1 was demonstrated moderate to high expression. In contrast, 12 of 20(60%)pancreatic cancer samples MKP-1 mRNA was expressed at high levels, whereas in the remaining 8 cancer tissues this mRNA moiety was present at low to moderate levels. Densitometric analysis with normalization to 7S revealed that the median level of MKP-1 mRNA in CP and cancerous tissues was increased by 6.2 folds(P=0.035)and 8.1 folds(P=0.016)in comparison with the median level in the normal pancreatic samples, respectively. Overexpression of MKP-1 was also found in 6 pancreatic cancer cell lines, in which the expression of MKP-1 was slightly lower in one pancreatic cancer cell line but high in the remaining 5 cell lines.</p><p><b>CONCLUSIONS</b>MKP-1 is over-expressed in pancreatic cancer, CP tissues, and pancreatic cell lines. It is speculated that MKP-1 may play an important role in tumorigenesis of pancreatic cancer.</p>

Research progress on MKP-1 in tumor drug resistance / 浙江大学学报·医学版

The main obstacle for chemotherapy is tumor drug resistance. Studying the mechanisms of drug resistance and reversing drug resistance is the key to improve the effectiveness of chemotherapy. It has been reported that MKP-1 plays an important role in tumor drug resistance. MKP-1, as a negative regulator of MAPKs, is involved in the MAPKs mediated drug resistance and is regulated by ERK and p38 signaling pathways.However, the relationship between MKP-1 and other drug resistance-related signaling pathways is not clear and requires further investigation.

Constitutive Expression of MAP Kinase Phosphatase-1 Confers Multi-drug Resistance in Human Glioblastoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Current treatment of glioblastoma after surgery consists of a combination of fractionated radiotherapy and temozolomide. However, it is difficult to completely remove glioblastoma because it has uncertain boundaries with surrounding tissues. Moreover, combination therapy is not always successful because glioblastoma has diverse resistances. To overcome these limitations, we examined the combined effects of chemotherapy and knockdown of mitogen-activated protein kinase phosphatase-1 (MKP-1). MATERIALS AND METHODS: We used ten different anti-cancer drugs (cisplatin, cyclophosphoamide, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, mitomycin C, and vincristine) to treat glioblastoma multiforme (GBM) cells. Knockdown of MKP-1 was performed using siRNA and lipofectamine. The basal level of MKP-1 in GBM was analyzed based on cDNA microarray data obtained from the Gene Expression Omnibus (GEO) databases. RESULTS: Anti-cancer drug-induced cell death was significantly enhanced by knockdown of MKP-1, and this effect was most prominent in cells treated with irinotecan and etoposide. Treatment with these two drugs led to significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in a time-dependent manner, while pharmacological inhibition of JNK partially inhibited drug-induced cell death. Knockdown of MKP-1 also enhanced drug-induced phosphorylation of JNK. CONCLUSION: Increased MKP-1 expression levels could be the cause of the high resistance to conventional chemotherapeutics in human GBM. Therefore, MKP-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy.

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid suppresses UV-induced IL-6 synthesis in keratinocytes, exerting an anti-inflammatory activity

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is an enzymatic product of prostaglandin H2 (PGH2) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-kappaB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-kappaB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.

The mitogen-activated protein kinase phosphatase-1 (MKP-1) gene is a potential methylation biomarker for malignancy of breast cancer

The mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P < 0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P < 0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver-operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P < 0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.

Sildenafil potentiates the proliferative effect of porcine pulmonary artery smooth muscle cells induced by serotonin in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Sildenafil is one of the selective phosphodiesterase 5 inhibitors that has been proven by many investigators to suppress growth factor stimulated (e.g. platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)) proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMCs) via cGMP/cGKIa pathway. Serotonin promotes cell cycle progression leading to cell mitogenesis and plays a key role in the pathogenesis of pulmonary artery hypertension. The role of sildenafil in proliferation of PASMCs induced by serotonin has not been investigated so far. In this study we explored the underlying mechanism of the effect of sildenafil on serotonin induced proliferation of porcine PASMCs.</p><p><b>METHODS</b>PASMCs were cells from primary cultures by the explant method from the pulmonary artery of swine and cells at passage 3 - 5 were used in this study. MTT colorimetric assay and flow cytometry analysis were used to evaluate the cell proliferation and alterations in cell cycle progression respectively. Western blotting analysis was applied to determine the expression of phosphorylated extracellular signal-regulated kinase (ERK), proliferating cell nuclear antigen (PCNA) and mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1).</p><p><b>RESULTS</b>Serotonin (10 µmol/L) induced the upregulation of phosphorylation of ERK1/ERK2 and PCNA, an increase in the percentage of cells in S phase and subsequent cell proliferation. Pretreatment with 1 µmol/L sildenafil potentiated the phosphorylation of ERK1/ERK2, an increase in the percentage of cells in S phase and cell proliferation, compared with serotonin stimulation alone (P < 0.05). Furthermore, 30-minute pretreatment with 10 µmol/L U0126, specific antagonist for ERK kinase (MEK) prevented the increase in phosphorylation of ERK1/ERK2 and abolished cell cycle progression and the proliferation of PASMCs induced by sildenafil.</p><p><b>CONCLUSION</b>This study shows that sildenafil potentiated the proliferative effect of serotonin on PASMCs via phosphorylation of ERK1/ERK2.</p>

Down regulation of mitogen activated protein kinase phosphatase-1 mediate acquired multidrug resistance in pancreatic adenocarcinoma cell line SW1990/Fu / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the role of mitogen activated protein kinase phosphatase-1 (MKP-1) in mediating acquired multidrug resistance in pancreatic adenocarcinoma cell line SW1990/Fu.</p><p><b>METHODS</b>To detect MKP-1 mRNA expression, Northern blot analysis was carried out in well established drug resistant pancreatic adenocarcinoma cell line SW1990/Fu, SW1990 and MiaPaCa-2 cell lines. To further elucidate the exact role of MKP-1, Western blot hybridization was performed in these three cell lines.</p><p><b>RESULTS</b>Northern blot analysis of total RNA isolated from SW1990/Fu, SW1990 and MiaPaCa-2 cell lines revealed the presence of the 2400 bp MKP-1 transcript 7 at relatively high levels in pancreatic cancer cell lines SW1990 and MiaPaCa-2. In the SW1990/Fu, the MKP-1 transcript was detectable at very low level. Densitometric analysis with normalization to 7S indicated that MKP-1 mRNA expression level was significantly decreased in SW1990/Fu in comparison with the parental and MiaPaCa-2 cell lines. MKP-1 protein expression level in SW1990/Fu detected by Western blot was coincident with mRNA level.</p><p><b>CONCLUSIONS</b>MKP-1 may be involved in acquired multidrug resistance in pancreatic adenocarcinoma, and we could hypothesized that alterations of intra-cellular transduction signal system acts as an important role in multidrug resistance of tumor cells.</p>

Effects of angiotensin II on extracellular signal-regulated protein kinases signaling pathway in cultured vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats / 生理学报

The aim of this study was to investigate the effects of angiotensin II (Ang II) on extracellular signal-regulated protein kinase (ERK) signaling pathway in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. VSMCs from SHR and WKY rats were treated with 1x10(-7) mmol/L Ang II for 24 h in the absence or presence of 30 min of pre-treatment of valsartan (1x10(-5) mmol/L) or PD98059 (1x10(-5)mmol/L), selective inhibitor of ERKs- dependent pathways, when they were cultured in 20% calf serum medium. VSMCs of SHR and WKY cultured in serum-free medium were used as control groups. Among the different treatments, VSMCs from the SHR and WKY were devided into four groups: (1) control, (2) Ang II, (3) Ang II + valsartan, (4) Ang II + PD98059. ERK activity in VSMCs was measured by immuno-precipitation. Proteins of total ERK (t-ERK), phosphorylated-ERK (p-ERK) and mitogen-activated protein kinases phosphatase-1 (MKP-1) in VSMCs were detected by Western blot. MKP-1 mRNA in VSMCs was measured by RT-PCR. In VSMCs from WKY or SHR rats, ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in Ang II group were higher than those in control group (P<0.05). In both SHRs and WKYs, there were no significant differences in ERK activity, p-ERK, MKP-1 and MKP-1 mRNA among the control group, Ang II + valsartan group and Ang II + PD98059 group. ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in SHRs were significantly higher than those in WKYs with same treatments (P<0.01). There was no significant difference in t-ERK among different groups and no difference in t-ERK between SHRs and WKYs (P>0.05). Our results show that Ang II activates VSMCs ERK signaling pathways via Ang II type 1 (AT(1)) receptors. Ang II increased ERK activity and p-ERK, but not t-ERK, accompanied by an increase in MKP-1 mRNA expression and protein. Among the different treatments, ERK activity and p-ERK were higher in SHR than in WKY. Valsartan and PD98059 blocked Ang II-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of Ang II on SHR and WKY VSMCs' ERK signaling pathway may be mediated by AT(1) receptors, enhancing ERK activity and the amount of p-ERK, and then increasing MKP-1 mRNA and its expression.

Change of mitogen-activated protein kinase phosphatase-1 in heart and aorta of SHR and its effect on proliferation of vascular smooth muscle cells stimulated by angiotensin II / 中国应用生理学杂志

<p><b>AIM AND METHODS</b>To investigate the role of mitogen-activated protein kinase phosphatase-1 (MKP-1) in the regulation of cells proliferation, the expression of MKP-1 and extracellular signal-regulated kinase-1 (ERK-1) in heart and aorta of spontaneous hypertensive rat (SHR) and WKY were studied. We also investigated the effect of MKP-1 genes,which were transfected into vascular smooth muscle cells (VSMC) using the classical calcium phosphate coprecipitation technique, on the incorporation of 3H-TdR in VSMC stimulated by angiotensin II (Ang II).</p><p><b>RESULTS</b>(1) Compared with that of WKY, MKP-1 expression in heart and aorta were significantly decreased by 53% (P < 0.01) and 45% (P < 0.01) in SHR, respectively. While the expression of ERK-1 in heart and aorta of SHR were higher than that of WKY (P < 0.01). The ratio of ERK-1/MKP-1 in heart and aorta of SHR were significantly higher than that of WKY. (2) 3H-TdR incorporation in VSMC stimulated by Ang II (10(-7) mol/L) was increased by 207% (P < 0.01), compared with control group. In the transfected cells with wild MKP-1 gene, Ang II-induced incorporation of 3H-TdR lowered 63%, compared with untransfected cells (P < 0.05). There were no marked inhibitive role between mutant MKP-1-transfected cells and blank vector-transfected cells in response to Ang II, compared with Ang II group (P > 0.05).</p><p><b>CONCLUSION</b>These results showed that the expression of ERK-1 in heart and aorta isolated from SHR, which stimulated proliferation and hypertrophy of cells, is higher than that of MKP-1 which dephosphorylates and inactivated ERK-1. In addition, MKP-1 significantly inhibits Ang II-stimulated proliferation of VSMC.</p>