Role of ATP5G3 in sodium nitroprusside-induced cell death in cervical carcinoma cells

We identified a gene, subunit C3 (ATP5G3) of mitochondrial ATP synthase, that displayed changes in gene expression under oxidative stress. We examined the role of ATP5G3 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using ATP5G3 small interfering RNA (siATP5G3)-transfected HeLa cells. A significant increase in cytotoxicity was observed in the transfected cells treated with SNP, which suggests a protective role of ATP5G3 in SNP-induced cytotoxicity in the cells. The transfected cells treated with photodegraded SNP showed equal cytotoxicity to SNP, and pretreatment with deferoxamine (DFO) completely inhibited this cytotoxicity. Further, cytotoxicity was significantly inhibited by pretreatment with a p38 inhibitor and was accentuated by the p38 activator in cells. Pretreatment with the Bcl-xL inhibitor also significantly accentuated cytotoxicity. The increase in p38 phosphorylation was significantly higher in siATP5G3-transfected cells treated with SNP in immunoblotting, which was inhibited by pretreatment with DFO. The increase in cytotoxicity with siATP5G3 transfection was completely blocked by cotransfection with sip38, and the blocking effect disappeared by cotransfection with additional siBcl-xL, which suggests that the protective role of ATP5G3 is mediated by Bcl-xL via the inhibition of p38 activity. Cytotoxicity was completely blocked by the cotransfection of siATP5G3 with siBax. No change in apoptotic parameters was observed during cytotoxicity. However, pretreatment with lysosomal inhibitors significantly inhibited cytotoxicity and increased p62 protein levels. These findings suggest that ATP5G3 plays a protective role in autophagic cell death/lysosome-associated cell death induced by SNP via the sequential signaling of ROS/p38/Bcl-xL/Bax in HeLa cells.     

Vacuolar H+ -ATPase c protects glial cell death induced by sodium nitroprusside under glutathione-depleted condition

We examined the role of the c subunit (ATP6L) of vacuolar H(+) -ATPase and its molecular mechanisms in glial cell death induced by sodium nitroprusside (SNP). ATP6L siRNA-transfected cells treated with SNP showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, but reduction of ATP6L did not affect the regulation of lysosomal pH in analyses with lysosomal pH-dependent fluorescence probes. Photodegraded SNP and ferrous sulfate induced cytotoxicity with the same pattern as that of SNP, but SNAP and potassium cyanide did not show activity. Pretreatment of the transfected cells with deferoxamine (DFO) reduced ROS production and significantly inhibited the cytotoxicity, which indicates that primarily iron rather than nitric oxide or cyanide from SNP contributes to cell death. Involvement of apoptotic processes in the cells was not shown. Pretreatment with JNK or p38 chemical inhibitor significantly inhibited the cytotoxicity, and we also confirmed that the MAPKs were activated in the cells by immunoblot analysis. Significant increase of LC3-II conversion was observed in the cells, and the conversions were inhibited by cotransfection of the MAPK siRNAs and pretreatment with DFO. Introduction of Atg5 siRNA inhibited the cytotoxicity and inhibited the activation of MAPKs and the conversion of LC3. We finally confirmed autophagic cell death and involvement of MAPKs by observation of autophagic vacuoles via electron microscopy. These data suggest that ATP6L has a protective role against SNP-induced autophagic cell death via inhibition of JNK and p38 in GSH-depleted glial cells.     

ERK‐1 MAP kinase prevents TNF‐induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells

Extracellular signal‐regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl‐2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK‐1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase‐deficient form of ERK‐1 (K71R) were more sensitive to TNF and CHX. In the ERK‐1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK‐1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK‐1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK‐1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase‐8 inhibitor IETD‐FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c‐Jun N‐terminal kinases activator, increased TNF‐killing. The ERK‐1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK‐1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death. J. Cell. Biochem. 108: 1166–1174, 2009. © 2009 Wiley‐Liss, Inc.     

Sodium nitroprusside induces autophagic cell death in glutathione‐depleted osteoblasts

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)‐depleted and control U2‐OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH‐depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP‐induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH‐depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD‐fmk, a pan‐specific caspase inhibitor. Large increases of LC3‐II were shown by immunoblot analysis of the SNP‐treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH‐depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP‐treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH‐depleted osteoblasts. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:313–322, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20340     

Unbalanced activation of ERK1/2 and MEK1/2 in apigenin-induced HeLa cell death

Apigenin, a dietary bioflavonoid with anticarcinogenic properties, was highly cytotoxic for HeLa cells (incubated with 0.5% FBS). This effect was accompanied with a marked increase in ERK1/2 but not MEK1/2 phosphorylation. The cytotoxic effects of apigenin were attenuated by the stimulation of these cells with 10% FBS, which provoked an increase in the phosphorylation levels of MEK1/2 and ERK1/2. The steps in the ERK1/2 pathway relevant to the cytotoxic effects of apigenin, as well as the contribution of other signaling pathways, were investigated. The activation of the pathway by transfection with the constitutively active Ras mutant (RasV12) conferred protection to serum-starved HeLa cells against apigenin, whereas the constitutively active MEK(E) mutant did not. MEK inhibitors (PD098059 or U0126) blocked ERK1/2 phosphorylation induced by apigenin and conferred partial protection against this flavonoid. The effects of apigenin did not involve p38-MAPK or JNK1/2, and were not simply due to inhibition of PI3kinase or protein kinase CK2. These data suggest that the deregulation of the ERK1/2 pathway, due to the potentiation of ERK1/2 phosphorylation without increasing MEK1/2 phosphorylation, is involved in apigenin-induced HeLa cell death.     

PARP-1-induced cell death through inhibition of the MEK/ERK pathway in MNNG-treated HeLa cells

Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N′-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.     

Dihydromyricetin enhances glucose uptake by inhibition of MEK/ERK pathway and consequent down‐regulation of phosphorylation of PPARγ in 3T3‐L1 cells

Accumulating evidence suggests that inhibition of mitogen‐activated protein kinase signalling can reduce phosphorylation of peroxisome proliferator‐activated receptor γ (PPARγ) at serine 273, which mitigates obesity‐associated insulin resistance and might be a promising treatment for type 2 diabetes. Dihydromyricetin (DHM) is a flavonoid that has many beneficial pharmacological properties. In this study, mouse fibroblast 3T3‐L1 cells were used to investigate whether DHM alleviates insulin resistance by inhibiting PPARγ phosphorylation at serine 273 via the MEK/ERK pathway. 3T3‐L1 pre‐adipocytes were differentiated, and the effects of DHM on adipogenesis and glucose uptake in the resulting adipocytes were examined. DHM was found to dose dependently increase glucose uptake and decrease adipogenesis. Insulin resistance was then induced in adipocytes using dexamethasone, and DHM was shown to dose and time dependently promote glucose uptake in the dexamethasone‐treated adipocytes. DHM also inhibited phosphorylation of PPARγ and ERK. Inhibition of PPARγ activity with GW9662 potently blocked DHM‐induced glucose uptake and adiponectin secretion. Interestingly, DHM showed similar effects to PD98059, an inhibitor of the MEK/ERK pathway. DHM acted synergistically with PD98059 to improve glucose uptake and adiponectin secretion in dexamethasone‐treated adipocytes. In conclusion, our findings indicate that DHM improves glucose uptake in adipocytes by inhibiting ERK‐induced phosphorylation of PPARγ at serine 273.     

Bacterial GroEL-like heat shock protein 60 protects epithelial cells from stress-induced death through activation of ERK and inhibition of caspase 3

Bacterial heat shock proteins (hsps) can have various effects on human cells. We investigated whether bacterial hsp60s can protect epithelial cells from cell death by affecting the mitogen-activated protein kinase (MAPK) signal pathways. Cell protection was studied by adding bacterial hsp60s to skin keratinocyte cultures (HaCaT cell line) before UV radiation. The results show that hsp60 significantly protected against UV radiation-induced cell death. Effects of UV radiation and exogenous hsp60 on phosphorylation of MAPKs and on activation of caspase 3 were examined by Western blot analysis. UV radiation strongly induced phosphorylation of p38 MAPK and formation of active caspase 3. A p38 inhibitor, SB 203580, totally blocked UV radiation-mediated activation of caspase 3. Preincubation with hsp60 strongly induced phosphorylation of ERK1/2 and inhibited UV radiation-mediated activation of caspase 3. PD 98059, a specific inhibitor of the ERK1/2 pathway, blocked this inhibitory effect of exogenous hsp60. Studies on the association between activity of MAPKs or caspase 3 and cell death showed that the ERK1/2 pathway inhibitor reversed protective effect of hsp60 while specific inhibition of p38 and caspase 3 reduced cell death. These results indicate that in HaCaT cells UV radiation mediates cell death through activation of p38 followed by caspase 3 activation. Exogenous hsp60 partially protects against UV radiation-mediated epithelial cell death through activation of ERK1/2, which inhibits caspase 3 activation.     

Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.     

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.     

Differential functions of PKC-delta and PKC-zeta in cisplatin response of normal and transformed thyroid cells

We investigated the effects of cisplatin (cisPt) in normal PC Cl3 and in transformed and tumourigenic PC E1Araf cells. cisPt cytotoxicity was higher in PC Cl3 than in PC E1Araf cells. In both cell lines, cisPt provoked the ERK1/2 phosphorylation; this was unaltered by G?6976, a conventional PKC inhibitor, whilst it was blocked by low doses (0.1 microM) or high doses (10 microM) of GF109203X, an inhibitor of all PKC isozymes, in PC Cl3 and in PC E1Araf cells, respectively. In PC E1Araf, the cisPt-provoked ERK phosphorylation was also blocked by the use of a myristoylated PKC-zeta pseudosubstrate peptide. Conversely, in PC Cl3 the cisPt-provoked ERK phosphorylation was blocked by the use of rottlerin, a PKC-delta inhibitor. Results show that cisPt activates both PKC (the -delta and the -zeta isozymes in PC Cl3 and in PC E1Araf cells, respectively) and ERK in association with prolonged survival of thyroid cell lines.     

A predominant role for inhibition of the adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid receptor 1 in N1E-115 neuroblastoma cells

Cannabinoids activate several members of the mitogen-activated protein kinase superfamily including p44 and p42 extracellular signal-regulated kinase (ERK). We used N1E-115 neuroblastoma cells and the cannabinoid receptor agonist WIN 55,212-2 (WIN) to examine the signal transduction pathways leading to the activation of ERK. ERK phosphorylation (activation) was measured by Western blot. The EC50 for stimulation of ERK phosphorylation was 10 nm, and this effect was blocked by pertussis toxin and the CB1 (cannabinoid) receptor antagonist SR141716A. The MEK inhibitors PD 98059 and U0126 blocked ERK phosphorylation, as did the adenylate cyclase activator forskolin. The phosphatidylinositol (PI) 3-kinase inhibitor LY 294002 and the Src kinase inhibitor PP2 partially occluded the response but also decreased basal levels of phospho-ERK. The PI 3-kinase and Src pathways are known to promote cell survival in many systems; therefore, MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) conversion was used to examine the effects of these inhibitors on cellular viability. LY 294002 decreased the number of viable cells after 18 h of treatment; therefore, the inhibition of ERK by this inhibitor is probably because of cytotoxicity. Forskolin blocked ERK phosphorylation with an EC50 of <3 microm, and the protein kinase A (PKA) inhibitor H-89 enhanced ERK phosphorylation. c-Raf phosphorylation at an inhibitory PKA-regulated site (Ser259) was also reduced by WIN. This is probably due to constitutive phosphatase activity because WIN did not directly stimulate PP1 or PP2A activity when measured using 6,8-difluoro-4-methylumbelliferyl phosphate as a fluorogenic substrate. These data implicate the inhibition of PKA as the predominant pathway for ERK activation by CB1 receptors in N1E-115 cells. PI 3-kinase and Src appear to contribute to ERK activation by maintaining activation of kinases, which prime the pathway and maintain cellular viability.     

Activation of ERK during DNA damage-induced apoptosis involves protein kinase Cdelta

We have previously shown that protein kinase C (PKC) acts upstream of caspases to regulate cisplatin-induced apoptosis. Since extracellular signal-regulated kinases (ERKs) have also been implicated in DNA damage-induced apoptosis, we have examined if ERK signaling pathway acts downstream of PKC in the regulation of cisplatin-induced apoptosis. PKC activator PDBu induced ERK1/2 phosphorylation which was inhibited by general PKC inhibitor bisindolylmaleimide and G? 6983 as well as the MEK inhibitor U0126 but not by the PKCdelta inhibitor rottlerin. Cisplatin caused a concentration-dependent activation of ERK1/2 in HeLa cells. The level of ERK2 was decreased in HeLa cells that acquired resistance to cisplatin (HeLa/CP). The MEK inhibitor U0126 inhibited cisplatin-induced ERK activation and attenuated cisplatin-induced cell death. Inhibition of PKCdelta by rottlerin or depletion of PKCdelta by siRNA inhibited cisplatin-induced ERK activation. These results suggest that cisplatin-induced DNA damage results in activation of ERK1/2 via PKCdelta.     

Leptin potentiates antiproliferative action of cAMP elevation via protein kinase A down‐regulation in breast cancer cells

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT‐5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA‐MB‐231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT‐5720 blocked the forskolin‐induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin‐induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down‐regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin‐dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA‐MB‐231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed. J. Cell. Physiol. 225: 801–809, 2010. © 2010 Wiley‐Liss, Inc.     

NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

Transmembrane signaling pathway mediates oxidized low-density lipoprotein-induced expression of plasminogen activator inhibitor-1 in vascular endothelial cells

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.     

Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK,RhoA, PI3K/Akt,and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [³H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010. © 2010 Wiley‐Liss, Inc.     

Delayed and persistent ERK1/2 activation is required for 4-hydroxytamoxifen-induced cell death

Tamoxifen (Tam), and its active metabolite, 4-hydroxytamoxifen (OHT), compete with estrogens for binding to the estrogen receptor (ER). Tam and OHT can also induce ER-dependent apoptosis of cancer cells. 10-100nM OHT induces ER-dependent apoptosis in approximately 3 days. Using HeLaER6 cells, we examined the role of OHT activation of signal transduction pathways in OHT-ER-mediated apoptosis. OHT-ER activated the p38, JNK and ERK1/2 pathways. Inhibition of p38 activation with SB203580, or RNAi-knockdown of p38alpha, moderately reduced OHT-ER mediated cell death. A JNK inhibitor partly reduced cell death. Surprisingly, the MEK1/2 inhibitor, PD98059, completely blocked OHT-ER induced apoptosis. EGF, an ERK1/2 activator, enhanced OHT-induced apoptosis. OHT induced a delayed and persistent phosphorylation of ERK1/2 that persisted for >80h. Addition of PD98059 as late as 24h after OHT largely blocked OHT-ER mediated apoptosis. The antagonist, ICI 182,780, blocked both the long-term OHT-mediated phosphorylation of ERK1/2 and OHT-induced apoptosis. Our data suggests that the p38 and JNK pathways, which often play a central role in apoptosis, have only a limited role in OHT-ER-mediated cell death. Although rapid activation of the ERK1/2 pathway is often associated with cell growth, persistent activation of the ERK1/2 pathway is essential for OHT-ER induced cell death.     

The specific Na(+)/Ca(2+) exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells

The Na⁺/Ca²⁺ exchanger (NCX) plays a role in the regulation of intracellular Ca²⁺ levels, and nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured glial cells via NCX-mediated pathways and the mechanism for NO-induced cytotoxicity is cell type-dependent. The present study examined using the specific NCX inhibitor 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX is involved in NO-induced injury in cultured neuronal cells. The treatment of neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity was blocked by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase (MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP60012. SNP increased Ca²⁺ influx and intracellular Ca²⁺ levels. In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of reactive oxygen species (ROS) in an extracellular Ca²⁺-dependent manner. These effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not affected by inhibitors of the Ca²⁺, Na⁺ and store-operated/capacitative channels. Moreover, SNP-induced increase in intracellular Ca²⁺ levels, ROS production and decrease in cell viability were blocked by a cGMP-dependent protein kinase (PKG) inhibitor. These results suggest that Ca²⁺ influx via the reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and NO activates the NCX through guanylate cyclase/PKG pathway.