Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos) proteolysis

The molecular mode of action of arsenic, a therapeutic agent employed in the treatment of acute promyelocytic leukemia, has been elusive. Here we provide evidence that arsenic compounds may act on mitochondria to induce apoptosis. Arsenite induces apoptosis accompanied by a loss of the mitochondrial transmembrane potential (Delta Psim). Inhibition of caspases prevents the arsenite-induced nuclear DNA loss, but has no effect on the Delta Psim dissipation and cytolysis induced by arsenite. In contrast, Bcl-2 expression induced by gene transfer prevents all hallmarks of arsenite-induced cell death, including the Delta Psim collapse. PK11195, a ligand of the mitochondrial benzodiazepine receptor, neutralizes this Bcl-2 effect. Mitochondria are required in a cell-free system to mediate arsenite-induced nuclear apoptosis. Arsenite causes the release of an apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space. This effect is prevented by the permeability transition (PT) pore inhibitor cyclosporin A, as well as by Bcl-2, which is known to function as an endogenous PT pore antagonist. Arsenite also opens the purified, reconstituted PT pore in vitro in a cyclosporin A- and Bcl-2-inhibitible fashion. Altogether these data suggest that arsenite can induce apoptosis via a direct effect on the mitochondrial PT pore.     

Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L -cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O) and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation. J. Cell. Physiol. 177:324–333, 1998. © 1998 Wiley-Liss, Inc.     

Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells

Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents. The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure, and are associated with various human malignancies, including skin, lung and bladder cancers. In addition, arsenic could induce cell death, including apoptosis or autophagy in malignant cells. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes, in a dose-dependent manner. When the cells were pretreated with inhibitors 5-aza-CdR or U0126 for 24 h, the effect of arsenite on ERK1/2, LC3B, Beclin-1 and DAPK proteins expression is suppressed. Furthermore, our results support the notion that arsenite can induce the ERK1/2 signaling pathway to stimulate autophagy and DAPK promoter hypermethylation in human uroepithelial SV-HUC-1 cells. These findings may contribute to a better understanding of the carcinogenesis of arsenite.     

Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite.

It is well-known that p38 mitogen-activated protein kinase (p38MAPK) participates in cellular responses to mitogenic stimuli, environmental and genotoxic stresses, and apoptotic agents. Although there are several reports on p38MAPK in relation to cell growth and apoptosis, the exact mechanism of p38MAPK-mediated cell growth regulation remains obscure. Here, we examined possible roles of p38MAPK in the sodium arsenite-induced cell growth inhibition in NIH3T3 cells. Sodium arsenite induced transient cell growth delay with marked activation of p38MAPK. In addition, arsenite induced CDK inhibitor p21(CIP1/WAF1) and enhanced its binding to the CDK2, which resulted in inhibition of CDK2 activity. The levels of cyclin D1 expression and the CDK4 kinase activity were also significantly reduced. pRB was hypophosphorylated by sodium arsenite. SB203580, a specific inhibitor of p38MAPK, blocked arsenite-induced growth inhibition as well as the arsenite-induced p21(CIP1/WAF1) expression. Expression of dominant negative p38MAPK also blocked arsenite-induced p21(CIP1/WAF1) expression. Inhibited-CDK2 activity was also completely reversed by SB203580 or expression of dominant negative p38MAPK, while the decreased-cyclin D1 protein by the compound was not restored. These data demonstrate a possible link between the activation of p38MAPK and induction of p21(CIP1/WAF1), suggesting that the activation of p38MAPK is, at least in part, related to the cell growth inhibition by sodium arsenite.     

砷暴露诱导人肝细胞氧化应激过程中NADPH氧化酶作用机制

目的：探讨砷暴露诱导细胞氧化应激的分子机制。方法：采用人正常肝细胞进行亚砷酸钠和砷酸钠的暴露处理,并设相应对照组,采用SOD模拟物MnTMPyP和还原型谷胱甘肽（reducedglutathione,GSH）预处理,检测细胞超氧阴离子（02。）和细胞整体ROS的水平。WestemBlot方法检测细胞氧化／抗氧化重要酶微粒体谷胱甘肽硫转移酶（microsomalglutathioneS-transferase-l,Mgst．1）、半胱氨酸双加氧酶l（cysteinedioxygenasel,Cd01）和NADPH氧化酶的催化亚基NOX4的表达。针对NADPH氧化酶,采用特异性抑制剂（diphenyleneiodoniumchloride,DPI）进行预处理,观察对砷暴露引起的细胞ROS水平及细胞凋亡的影响。结果：砷暴露能够显著诱导细胞超氧阴离子的产生,提高细胞整体ROS水平,其中三价砷（亚砷酸钠,A矿）诱导氧化应激作用显著强于五价砷（砷酸钠,As5＋）。亚砷酸钠能够显著提高NOX4的表达。针对NADPH氧化酶的抑制剂DPI能够显著抑制砷暴露引起的细胞ROS水平升高以及细胞凋亡的增加。结论：NADPH氧化酶是砷暴露诱导人肝细胞的作用靶点,砷能够通过NADPH氧化酶产生大量超氧阴离子,提高ROS水平,造成氧化应激,诱导人正常肝细胞凋亡。     

Involvement of calcium-dependent protein kinase C in arsenite-induced genotoxicity in chinese hamster ovary cells

Arsenic is the first metal to be identified as a human carcinogen. Arsenite, one inorganic form of arsenic, has been found to induce sister chromatid exchange, chromosome aberrations, and gene amplification in a variety of in vitro systems. In this study of arsenite-induced genotoxicity represented as micronuclei production in Chinese hamster ovary cells (CHO-K1), we found that the calcium channel blocker, verapamil, can potentiate arsenite-induced micronuclei. And after arsenite treatment, the elevation of intracellular calcium was observed. When extracellular calcium was depleted during arsenite treatment, the arsenite-induced micronuclei formation was significantly suppressed. These data indicated that a calcium ion plays an essential role in arsenite-induced genotoxicity. Further, it was found that the cotreatment of arsenite and a calcium ionophore, A23187, can increase the micronuclei induction. In contrast, pretreatment of the intracellular calcium chelator, quin 2, significantly inhibited micronuclei production of arsenite administration. In addition, we measured the activity of calcium-and phospholipid-dependent protein kinase C (PKC) and found that arsenite can activate PKC activity in a dose-dependent manner. Subsequently, some PKC activators and inhibitors were applied to investigate the involvement of PKC on arsenite-induced micronuclei formation. It was found that H7, a PKC inhibitor, can depress but TPA, a PKC activator, can enhance arsenite-induced micronuclei significantly. These data indicated that arsenite exposure perturbs intracellular calcium homeostasis and activates PKC activity. As a result, the activation of PKC activity may play an important role in arsenite-induced genotoxicity. J. Cell. Biochem. 64:423–433. © 1997 Wiley-Liss, Inc.     

Involvement of reactive oxygen species in arsenite-induced downregulation of phospholipid hydroperoxide glutathione peroxidase in human epidermoid carcinoma A431 cells

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is unique in the substrate specificity among the glutathione peroxidase family because it can interact with lipophilic substrates, including the peroxidized phospholipids and cholesterol, and reduce these hydroperoxide to hydroxide compounds. However, what kinds of ligand can regulate the PHGPx expression is still unknown. In the present study, we found that sodium arsenite induced downregulation of mRNA, protein expression, and enzyme activity of PHGPx in time- and dose-dependent manners. At the same time, it upregulated mRNA and protein expression of p21(WAF1/CIP1). With the aid of agarose gel electrophoresis, and propidium iodide and annexin-V staining, we found that treatment of 30 microM sodium arsenite for 24 h induced apoptosis in human epidermoid carcinoma A431 cells and EA.hy926 cells. An increase of intracellular peroxide levels was measured by flow cytometry using 2',7'-dichlorofluorescin diacetate (DCFH-DA) after treatment of arsenite. Overexpression of PHGPx prevented arsenite-induced increase of intracellular peroxide levels, downregulation of PHGPx, upregulation of p21(WAF1/CIP1), and apoptosis in A431 cells. N-Acetyl-L-cysteine also significantly prevented arsenite-induced effects in A431 cells. Therefore, we concluded that reactive oxygen species were involved in arsenite-induced downregulation of PHGPx, upregulation of p21(WAF1/CIP1), and apoptosis in A431 cells.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Role of HO-1 in the Arsenite-Induced Neurotoxicity in Primary Cultured Cortical Neurons

In the present study, the role of heme oxygenase (HO)-1 in sodium arsenite (arsenite)-induced neurotoxicity was investigated using primary cultured cortical neurons. Incubation with arsenite was found to cause cell death of primary cultured cortical neurons in concentration- and time-dependent manners. Furthermore, arsenite induced caspase 3 activation and decreased procaspase 12 levels, indicating that apoptosis is involved in the arsenite-induced neurotoxicity. The oxidative mechanism underlying arsenite-induced neurotoxicity was investigated. Western blot assay showed that arsenite significantly increased HO-1 levels, a redox-regulated protein. Co-incubation with glutathione (10 mM) attenuated arsenite-induced HO-1 elevation and caspase 3 activation, suggesting that oxidative stress is involved in the arsenite-induced neurotoxicity. The neurotoxic effects of inorganic arsenics were compared; arsenite was more potent than arsenate in inducing HO-1 expression and caspase 3 activation. Moreover, the cell viabilities of arsenite and arsenate were 60?±?2 and 99?±?2 % of control, respectively. HO-1 siRNA transfection was employed to prevent arsenite-induced HO-1 elevation. At the same time, arsenite-induced caspase 3 activation and neuronal death were attenuated in the HO-1 siRNA-transfected cells. Taken together, HO-1 appears to be neuroprotective in the arsenite-induced neurotoxicity in primary cultured cortical neurons. In addition to antioxidants, HO-1 elevation may be a neuroprotective strategy for arsenite-induced neurotoxicity.     

Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27)

Exposure to ultraviolet (UV) light poses a health risk for eye disease, and solar ultraviolet in the B range (UVB, 280-320 nm) is known to be related to various corneal disorders. In this study, we investigated whether pre-conditioning of cells with arsenite (AsO2(-1)) can reduce UVB-induced apoptosis in human corneal epithelial cells, and whether the anti-apoptotic activity of 27 kDa heat shock protein (HSP27), a small heat shock protein, plays a role in this protection. UVB at levels comparable to physiologic solar exposure induces apoptosis of corneal epithelial cells in culture, demonstrated by activation of caspase 9 and caspase 3, and DNA fragmentation. When cells were pre-conditioned with arsenite prior to UVB exposure, the UVB-induced cell death was reduced, and UVB-induced activation of caspases and DNA fragmentation was inhibited. When cells were pre-treated with SB 203580, which inhibits HSP27 phosphorylation through inhibition of p38 MAP kinase activation, the arsenite-induced reduction of UVB-induced apoptosis was partially reversed. Arsenite pre-conditioning inhibited UVB-induced apoptosis in a two-phase pattern, which was temporally correlated with arsenite-induced HSP27 expression and phosphorylation. Neutralization of intracellular HSP27 with its antibody reduced arsenite's inhibition of UVB-induced caspase3 activation. Our results suggest that forms of stress that upregulate HSP27 and its phosphorylation may be useful as novel approaches to prevent adverse ocular effects arising from UV exposure in humans.     

Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. Here we demonstrate that treatment of promonocytic U937 cells with arsenic trioxide leads to G2/M arrest which was associated with a dramatic increase in the levels of cyclin B and cyclin B-dependent kinase and apoptosis. We further show that apoptosis occurs after bcl-2 phosphorylation and caspase-3 activation followed by cleavage of PARP and PLC-gamma1 degradation and DNA fragmentation. The arsenic trioxide-induced apoptosis could be blocked by the protein synthesis inhibitor cycloheximide. In addition, pretreatment of U937 cells with the DNA polymerase inhibitor aphidicolin also blocked apoptosis, but did not cause the arrest of cells in the G2/M phase. The findings suggest that arsenic trioxide exerts its growth-inhibitory effects by modulating expression and/or activity of several key G2/M regulatory proteins. Furthermore, arsenic trioxide-mediated G2/M arrest correlates with the onset of apoptosis.     

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Haloperidol-induced neuronal apoptosis: role of p38 and c-Jun-NH(2)-terminal protein kinase

We examined patterns and mechanisms of cell death induced by haloperidol. Cortical cell cultures exposed to 10-100 microM: haloperidol for 24 h underwent neuronal death without injuring glia. The degenerating neurons showed hallmarks of apoptosis, featuring cell body shrinkage, nuclear chromatin condensation and aggregation, nuclear membrane disintegration with intact plasma membrane, and prominent internucleosomal DNA fragmentation. Neither glutamate antagonists nor antioxidants prevented the haloperidol-induced neuronal apoptosis. The c-Jun-NH(2)-terminal protein kinase and p38 mitogen-activated protein kinase were activated within 1 h and were sustained over the next 3 h following exposure of cortical neurons to 30 microM haloperidol. Haloperidol-induced neuronal apoptosis was partially attenuated by 10-30 microM PD169316, a selective inhibitor of p38 mitogen-activated protein kinase. Inclusion of 1 microg/ml cycloheximide, a protein synthesis inhibitor, or 100 ng/ml insulin prevented activation of both kinases and subsequent neuronal death. The present study demonstrates that cortical neurons exposed to haloperidol undergo apoptosis depending on activation of p38 mitogen-activated protein kinase and c-Jun-NH(2)-terminal protein kinase sensitive to cycloheximide and insulin.     

Glutathione protects cells against arsenite-induced toxicity

To understand the role of glutathione (GSH) in the protection of cells from arsenite toxicity, we studied the mechanism of apoptotic cell death in cells genetically unable to synthesize GSH (GCS-2 cells). Arsenite stimulated an increase in protein ubiquitination in GCS-2 cells while the wild-type cells were unaffected. Arsenite treatment increased lipid peroxidation and induced ubiquitination of molecular chaperone Hsp90 and impaired its ability to bind cochaperone p50(Cdc-37) and client proteins Plk-1 and Cdk-4 in GCS-2 cells. Treatment with arsenite also partially inhibited proteasome activity in GCS-2 cells. In these cells stably transfected with GFP(u) (a reporter consisting of a short degron fused to the COOH-terminus of GFP), intracellular fluorescence increased, suggesting the accumulation of GFP aggregates. GCS-2 cells underwent apoptosis accompanied by release of cytochrome c into the cytoplasm. Taken together, these data suggest that a possible mechanism of arsenite-induced apoptosis is the accumulation of ubiquitinated proteins and impairment of the protein degradative pathway. Further, protection from arsenite-induced ubiquitination is mediated by GSH and to a lesser extent by available reducing equivalents in the cells.     

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.     

Coordination of JNK1 and JNK2 is critical for GADD45alpha induction and its mediated cell apoptosis in arsenite responses

Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45alpha (growth arrest and DNA damage 45alpha), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45alpha expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1-/-) or jnk2 (JNK2-/-). Knockdown of GADD45alpha by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45alpha in either JNK1-/- or JNK2-/- mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45alpha by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1-/- and JNK2-/- cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45alpha induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45alpha-mediated cellular apoptosis induced by arsenite.     

EGF receptor-dependent JNK activation is involved in arsenite-induced p21Cip1/Waf1 upregulation and endothelial apoptosis

Arsenic exposure is associated with an increased risk of atherosclerosis and vascular diseases. Although endothelial cells have long been considered to be the primary targets of arsenic toxicity, the underlying molecular mechanism remains largely unknown. In this study, we sought to explore the signaling pathway triggered by sodium arsenite and its implication for endothelial phenotype. We found that sodium arsenite produced time- and dose-dependent decreases in human umbilical vein endothelial cell viability. This effect correlated with the induction of p21Cip1/Waf1 (up to 10-fold), a regulatory protein of cell cycle and apoptosis. We also found that arsenite-stimulated EGF (ErbB1) and ErbB2 receptor transactivation, manifest as receptor tyrosine phosphorylation, appeared to be a proximal signaling event leading to p21Cip1/Waf1 induction, because both pharmacological inhibitors and knockdown of receptors by RNA interference blocked arsenite-induced p21Cip1/Waf1 upregulation. Arsenite-induced activation of JNK and p38 MAPK was distinct, with only JNK as a downstream target of the EGF receptor. Moreover, inhibition of JNK with SP-600125 or dominant negative MKK7 inhibited only p21Cip1/Waf1 induction, whereas the p38 MAPK inhibitor SB-203580 or dominant negative MKK4 inhibited both p21Cip1/Waf1 and p53 induction. Functionally, inhibition of p21Cip1/Waf1 induction prevented endothelial apoptosis due to arsenite treatment. Insofar as endothelial dysfunction promotes vascular disease, these data provide a mechanism for the increased incidence of cardiovascular disease due to arsenite exposure.