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Am J Physiol Heart Circ Physiol 289: H99-H107, 2005. First published February 25, 2005; doi:10.1152/ajpheart.00901.2004
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EGF receptor-dependent JNK activation is involved in arsenite-induced p21Cip1/Waf1 upregulation and endothelial apoptosis

Nopparat Nuntharatanapong,1,2 Kai Chen,1 Palarp Sinhaseni,2 and John F. Keaney, Jr.1

1Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts; and 2Department of Pharmacology, Faculty of Pharmaceutical Sciences and Institute of Health Research, Chulalongkorn University, Bangkok, Thailand

Submitted 31 August 2004 ; accepted in final form 22 February 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

epidermal growth factor; mitogen-activated protein kinases; c-Jun NH2-terminal kinase

ARSENIC IS A NATURALLY OCCURRING ELEMENT in the earth's crust, and environmental exposure to arsenic results primarily from consumption of contaminated drinking water and food in certain regions of the world, including areas of the southwestern United States and Asia. On the basis of numerous epidemiological studies, arsenic has been classified as a potent human carcinogen (34), and chronic arsenic intake has been associated with other adverse effects such as cardiovascular disease, peripheral vascular disease, hypertension, diabetes mellitus, and neurological effects (32).

Arsenite has been shown to cause DNA damage evident as strand breaks and DNA-protein cross-links that subsequently invoke cell cycle arrest and apoptosis (11). In association with DNA damage, the tumor suppressor protein p53 is induced, and its activation plays important roles in the regulation of cell progression, particularly in G1/S-phase transition. Furthermore, p53 protein transactivates several checkpoint key proteins regulating the cell cycle, such as p21Cip1/Waf1, which is able to silence cyclin-dependent kinases essential for S-phase entry and thus inhibit cell cycle progression (30). Even though mitogen-activated protein kinases (MAPK) such as c-Jun NH2-terminal kinase (JNK) and p38 are well known to coordinate the cellular stress response, the signaling pathway leading to p21Cip1/Waf1 activation and cell apoptosis is not fully understood.

Emerging evidence indicates that endothelial cell dysfunction is an early, perhaps causal, component of both chronic vascular (26) and neurological diseases (10). In this regard, endothelial cells long have been suspected as primary targets of arsenic toxicity (12), although the precise molecular mechanisms for these observations are not well understood. Endothelial cells exposed to arsenite (trivalent arsenic) may exhibit diverse responses ranging from increased DNA synthesis (1) to apoptosis (37) and frank toxicity (4). Sublethal concentrations of arsenite induce endothelial cell reactive oxygen species (ROS) formation and the activation of transcription factors such as NF-{kappa}B and AP-1 (1). This latter action of arsenite may protect endothelial cells against the proapoptotic action of arsenite (37). The goal of this study was to examine the implications of arsenite for endothelial cell phenotype and to identify the underlying signaling mechanisms involved.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex. The COS-7 cell line was from American Type Culture Collection (ATCC, Manassas, VA). Endothelial cell culture medium including endothelial growth medium-2 (EGM-2) kit was obtained from Cambrex, and Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen (Carlsbad, CA). Protein kinase inhibitors, including AG1478, AG1295, AG825, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), SB-203580, and SP-600125, were obtained from Calbiochem (San Diego, CA). Primary antibodies directed against the phosphorylated (activated) forms of JNK, extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and phosphotyrosine were obtained from Cell Signaling Technology (Beverly, MA). Antibody against p21Cip1/Waf1 was obtained from Pharmingen (San Diego, CA). Agarose-conjugated anti-ErbB1 (the EGF receptor, EGFR), anti-p53, actin antibodies, and rabbit anti-goat secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFR and anti-ErbB2 were obtained from Neomaker. Small interfering RNA (siRNA) for EGFR, ErbB2, and negative control were from Upstate Biotechnology (Waltham, MA). Lipofectamine Plus reagent was purchased from Invitrogen. The RNeasy mini kit and TransMessenger transfection reagent were obtained from Qiagen (Valencia, CA). Dominant negative mutants for the MAP kinase kinases MKK4 (MKK4-KR) (28) and MKK7 (MKK7-KE) (41) were kindly provided by Dr. Leonard I. Zon (Children's Hospital, Boston, MA) and Dr. Tse-Hua Tan (Baylor College of Medicine, Houston, TX), respectively. All other reagents were obtained from Sigma (St. Louis, MO).

Cell culture. HUVECs were grown in EGM-2 and used between passages 3 and 6. COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/ml of penicillin G and streptomycin. Cells were grown at 37°C in a humidified, 5% CO2 atmosphere.

Cell viability assay. HUVECs were seeded at a density of 2 x 105 cells/ml into 96-well plates or 6-well plates, as appropriate, and treated with the indicated concentrations of sodium arsenite. Cell viability was determined by methylthiazoletetrazolium (MTT) assay or Trypan blue exclusion as described (6).

Immunoprecipitation and Western blotting. Immunoprecipitation and Western blotting procedures were performed essentially as described previously (7, 36). Briefly, after incubation, cells were washed twice with ice-cold PBS and incubated in lysis buffer containing 50 mM Tris·HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, and 1 mM EDTA for 30 min on ice, followed by brief sonication. Cell lysates were then cleared by centrifugation (13,600 g) for 10 min and subjected to immunoprecipitation as described previously (5, 36). Precipitated proteins or cell lysates were resolved by SDS-PAGE, transferred to membranes, and subjected to immunoblotting as described previously (5, 36). Densitometric analysis of immunoblots was performed using commercially available software (PDI Imageware System, Huntington Station, NY).

Cell transfection. COS-7 cells were seeded at a density of 2 x 105 cells/ml into six-well plates. Transfections were carried out using Lipofectamine Plus reagent (Invitrogen) with cells at 70% confluence according to the manufacturer's instructions. Typically, 2 µg plasmid/well were used in transfections, and the cells were then cultured for an additional 24 h before being used for the experiments. Transfection of HUVECs with siRNA (EGFR and ErbB2) and negative control siRNA was performed using the TransMessenger transfection reagent (Qiagen). Cells (~1x106) were seeded in 60-mm dishes and allowed to reach 50–70% confluence. For transfection, siRNA (1 µg) was mixed with enhancer reagent and incubated at room temperature for 5 min before the addition of TransMessenger transfection reagent (10 µl). The resulting mixture was incubated for an additional 10 min at room temperature to allow transfection-complex formation. Complexes of siRNA were then mixed with 1 ml OPTI-MEM (Invitrogen) and immediately incubated with cells for 3 h. After incubation, medium was replaced and experiments were performed after an additional 48 h of culturing.

RNA extraction and RT-PCR. Total RNA from the cells grown in six-well plates was isolated using RNeasy mini kit (Qiagen, Valencia, CA). The forward and reverse primers corresponding to human p53 and p21Cip1/Waf1 were 5'-ACAGCCAAGTCTGTGACTT-3' and 5'-CACGCACCTCAAAGCTGTT-3', and 5'-GCTGGGGATGTCCGTCAGAA-3' and 5'-GAGCGAGGCACAAGGGTACAA-3', respectively. Constitutively expressed glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified with forward (5'-ACAGTCCATGCCATCACTGCC-3') and reverse (5'-AGGAAATGAGCTTGACAAAGT-3') primers in a similar fashion. The RT-PCR products were resolved by electrophoresis on 1.5% agarose gels.

Fluorescence microscopy. After treatments, HUVECs were washed twice with PBS, fixed in 5% formaldehyde for 30 min, and washed further in PBS. Morphology was examined using phase-contrast microscopy. Cells were also stained with PI (10 µg/ml) in the dark for 20 min and visualized using fluorescence microscopy. Apoptotic cells were identified by the findings of condensation and fragmentation of chromatin.

Flow cytometric analysis of apoptosis. An annexin V-FITC kit (Oncogene, San Diego, CA) was used. Briefly, nonadherent and adherent cells, harvested by trypsinization, were pooled, washed in cold PBS, and resuspended in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, and 4% BSA). Annexin V-FITC (0.5 µg/ml) and PI (0.6 µg/ml) were then added to a 250-µl aliquot (~5 x 106 cells) of this cell suspension. After 15-min incubation in the dark at room temperature, stained cells were immediately analyzed using flow cytometry (MoFlo; Dako Cytomation, Fort Collins, CO).

Statistical analysis. All numerical data are presented as means ± SE. Western blots shown are representative of three or more independent experiments. Comparisons among treatment groups were performed with one-way analysis of variance and an appropriate post hoc comparison. Statistical significance was accepted if the null hypothesis was rejected with a P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Arsenite decreases endothelial cell viability. We observed both dose- and time-dependent decreases in endothelial cell viability as a function of sodium arsenite exposure (Fig. 1, A and B). These findings were evident using both the MTT assay and Trypan blue dye exclusion methods, consistent with previous reports (15, 42). Trends for an effect of arsenite were observed with concentrations as low as 5 µM, and significant effects were evident at 10 µM arsenite.



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  		Fig. 1. Sodium arsenite-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 50 µM sodium arsenite for the indicated time (A) or with increasing concentrations of sodium arsenite for 24 h (B). After treatment, cell viability was determined by methylthiazoletetrazolium (MTT) assay and Trypan blue exclusion as described in EXPERIMENTAL PROCEDURES. Values are means ± SE from 3–5 independent experiments. *P < 0.05 compared with respective untreated control.

 
Arsenite induces both p21Cip1/Waf1 and p53 in HUVECs. Arsenite has been linked to DNA damage and p53 induction in various cell types (19, 42, 43). To probe the mechanism(s) involved in arsenite-induced endothelial cell toxicity, we examined its effect on p53 and its downstream target, p21Cip1/Waf1. We found that arsenite produced a potent induction of both p53 and p21Cip1/Waf1, and this effect was noted at arsenite concentrations as low as 1–5 µM with prolonged incubations (Fig. 2, A and B). Induction of p53 and p21Cip1/Waf1 in response to arsenite required 4–6 h, suggesting a mechanism involving gene transcription (Fig. 2C). Consistent with this notion, we found that arsenite produced an increase in the steady-state mRNA levels of p53 and p21Cip1/Waf1 (Fig. 2D). To determine whether p21Cip1/Waf1 was also regulated at the posttranslational level, we examined the half-life of the protein level of p21Cip1/Waf1 in the presence of cycloheximide. As shown in Fig. 2E, there was no difference in p21Cip1/Waf1 stability between cells with and without arsenite treatment. Thus these data indicate that arsenite-induced upregulation of p53 and p21Cip1/Waf1 is the result of increased transcription.



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  		Fig. 2. Sodium arsenite-induced expression of both p21Cip1/Waf1 and p53 in HUVECs. HUVECs were treated with increasing concentrations of sodium arsenite for 4 (A) or 16 h (B) or with 50 µM sodium arsenite for the indicated time (C). After treatment, total cell lysates were subjected to immunoblot analysis for the levels of p21Cip1/Waf1 and p53. D: HUVECs were treated with 50 µM sodium arsenite for 4 h. Total cell RNA was obtained, and the mRNA levels of p21Cip1/Waf1, p53, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) were measured using RT-PCR. E: half-life of p21Cip1/Waf1 in HUVECs was measured by cycloheximide chase. Cells were treated with or without 50 µM sodium arsenite for 4 h, and then 5 µM cycloheximide was added to all cell cultures (As+CHX or CHX alone). p21Cip1/Waf1 and actin protein levels were determined at the indicated time points after administration of cycloheximide. The p21Cip1/Waf1 protein levels were quantified as the relative (fold) change in induction after normalization to the actin level and are presented as percent changes compared with time 0 of the cycloheximide chase.

 
Arsenite-induced p21Cip1/Waf1 and p53 upregulation involve ErbB receptor activation. The activation of p53 by oxidative stress involves the platelet-derived growth factor-{beta} (PDGF-{beta}) receptor (5) and the EGFR (ErbB1), and the latter has been implicated in arsenite-induced MAPK activation (8). We used pharmacological probes to test the involvement of these two growth factor receptors in our system. We found that both AG1478 and AG825, inhibitors of ErbB receptors, attenuated arsenite-mediated p21Cip1/Waf1 induction, whereas inhibition of the PDGF receptor with AG1295 was ineffective (Fig. 3A). Consistent with this finding, AG1478 produced dose-dependent inhibition of both p53 and p21Cip1/Waf1 induction with arsenite. We also found evidence for ErbB2 involvement, because selective inhibition of this growth factor receptor impaired arsenite-mediated p53 and p21Cip1/Waf1 induction (Fig. 3C). We were able to confirm activation of both the EGFR (Fig. 4A) and ErbB2 (Fig. 4B) in response to arsenite by tyrosine phosphorylation. In addition, each ErbB isoform receptor tyrosine phosphorylation was dependent on its intrinsic receptor tyrosine kinase activity and the nonreceptor Src-family kinases (Fig. 4, A and B). To substantiate these results, we utilized siRNA directed against the EGFR and ErbB2. We found that EGFR siRNA produced effective gene silencing (Fig. 5A) that significantly attenuated p21Cip1/Waf1 induction in response to arsenite (Fig. 5B). Similarly, siRNA against ErbB2 abrogated ErbB2 expression (Fig. 5C) and abolished arsenite-induced p21Cip1/Waf1 induction in HUVECs (Fig. 5D). Thus arsenite-induced p21Cip1/Waf1 induction requires the tyrosine kinase activity of both the EGFR and ErbB2.



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  		Fig. 3. Involvement of ErbB receptors but not platelet-derived growth factor (PDGF) receptor in sodium arsenite-induced p21Cip1/Waf1 and p53 expression in HUVECs. A: HUVECs were preincubated with PDGF receptor tyrosine kinase inhibitor AG1295 (40 µM) or ErbB receptor tyrosine kinase inhibitors AG1478 (40 µM) and AG 825 (40 µM) for 30 min before exposure to 50 µM sodium arsenite for 4 h. B and C: HUVECs were pretreated with increasing concentrations of the ErbB1 (EGF receptor, EGFR)-specific inhibitor AG1478 and ErbB2-specific inhibitor AG825, respectively. After treatments, cells were lysed and cell lysates were subjected to immunoblot analysis for p21Cip1/Waf1 and p53. Data are representative of 3 independent experiments.

 


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  		Fig. 4. Phosphorylation of EGFR (ErbB1) and ErbB2 by sodium arsenite. HUVECs were pretreated with AG1478 (40 µM), AG825 (40 µM), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2; 20 µM) for 30 min before exposure to 100 µM sodium arsenite for 30 min. Cells were then lysed, and EGFR (A) and ErbB2 (B) tyrosine phosphorylation was assessed using immunoprecipitation (anti-EGFR and anti-ErbB2 antibodies, respectively) and immunoblotting (IB; phosphotyrosine, PY-100) analysis as outlined in EXPERIMENTAL PROCEDURES. The blots were then reprobed with anti-EGFR and anti-ErbB2 antibodies, respectively. The blots shown are representative of 3 independent experiments.

 


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  		Fig. 5. Requirement of ErbB receptors for sodium arsenite-induced p21Cip1/Waf1 induction. HUVECs were transfected with EGFR (A) or ErbB2 (C) small interfering RNA (siRNA) in combination with negative control siRNA. After 48 h, EGFR and ErbB2 expression levels were determined using immunoprecipitation (IP) and immunoblot (IB) analysis as in Fig. 4. B and D: HUVECs after siRNA transfection were treated with 50 µM sodium arsenite for 4 h, and cell lysates were assessed for expression of p21Cip1/Waf1 and actin, with bar graphs representing composite data. Values are means ± SE. *P < 0.05 compared with respective controls. The blots shown are representative of 3 independent experiments.

 
Arsenite-induced p21Cip1/Waf1 and p53 upregulation involve distinct MAPKs. Because growth factor receptors often are upstream components of MAPK activation, we probed p38 MAPK, JNK, and ERK1/2 in HUVECs exposed to arsenite. We found that arsenite induced activation of p38 MAPK and JNK, but not ERK1/2, in our system (Fig. 6A). Moreover, inhibition of p38 MAPK with SB-203580 inhibited both p21Cip1/Waf1 and p53 induction (Fig. 6B), whereas the selective JNK inhibitor SP-600125 did not inhibit arsenite-mediated p53 induction (Fig. 6C). These data are most consistent with a p53-independent signaling pathway directly between JNK and p21Cip1/Waf1 that is distinct from p38 MAPK-mediated activation of p53. To assess this hypothesis directly, we transfected cells with dominant negative isoforms of MKK4 or MKK7 and examined the implications for arsenite-induced p21Cip1/Waf1 upregulation. It is known that MKK4 activates both JNK and p38 MAPK, whereas MKK7 serves as a specific activator of JNK. As shown in Fig. 6D, inhibition of MKK4 or MKK7 significantly attenuated arsenite-induced p21Cip1/Waf1 induction with distinct effect on p53. Inhibition of MKK4 inhibited both p21Cip1/Waf1 and p53 induction, whereas inhibition of MKK7 significantly attenuated arsenite-mediated p21Cip1/Waf1 induction without any material affect on p53. Thus arsenite-induced p21Cip1/Waf1 upregulation involves a unique role for EGFR/JNK that is independent of p53 (Fig. 6E).



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  		Fig. 6. Differential involvement of JNK and p38 kinases in sodium arsenite-induced p21Cip1/Waf1 and p53 expression. A: HUVECs were treated with 50 µM sodium arsenite for the indicated time. Cell lysates were subjected to immunoblotting with phospho-specific antibodies for p38, JNK, and ERK1/2. B and C: HUVECs were pretreated with increasing concentrations of SB-203580 and SP-600125, respectively, and p21Cip1/Waf1 and p53 induction was determined as described in A. D: COS-7 cells were transfected with the dominant negative MAPK kinase MKK7 or the dominant negative MKK4 before exposure to 50 µM sodium arsenite for 14 h. A green fluorescent protein (GFP) vector transfection served as a control. All blots were reprobed for actin to ensure equal protein loading. DN, dominant negative. E: HUVECs were pretreated with inhibitors for EGFR (AG1478, 40 µM), ErbB2 (AG825, 40 µM), p38 (SB-203580, 20 µM), and JNK (SP-600125, 40 µM) for 30 min before sodium arsenite treatment (50 µM, 4 h). Immunoblot analysis was performed as described above. Data are representative of 3 independent experiments.

 
Arsenite-induced endothelial cell apoptosis requires JNK-mediated p21Cip1/Waf1 upregulation. To determine the functional consequences of this pathway, we manipulated arsenite-induced p21Cip1/Waf1 upregulation and determined the implications for cell survival and apoptosis. Arsenite exposure produced a significant decrease in cell viability, an effect that was substantially reversed with inhibition of p21Cip1/Waf1 upregulation by interruption of arsenite signaling using AG1478, SP-600125, and SB-203580 (Fig. 7, A and B). PI staining of pyknotic nuclei suggested apoptosis as a mechanism of arsenite-induced cell death (Fig. 7C). We also used flow cytometric analysis of externalized phosphatidylserine to quantify early apoptotic cells and measured PI uptake to assess cells in the later stages of apoptosis or cells that sustained direct plasma membrane damage (necrosis). As shown in Fig. 7, D and E, arsenite treatment of HUVECs (50 µM) for 16 h increased the number of both early apoptotic cells and late apoptotic/necrotic cells. Moreover, these phenomena were significantly attenuated by inhibitors of p21Cip1/Waf1 induction. Similar results were observed in cells using EGFR gene silencing by siRNA (Fig. 7F). Together, these data are consistent with the notion that arsenite upregulates p21Cip1/Waf1 through multiple pathways resulting in cell apoptosis.



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  		Fig. 7. Role of sodium arsenite-induced p21Cip1/Waf1 in apoptosis. HUVECs were incubated with 100 µM sodium arsenite (As) for 8 h in the presence or absence of AG1478 (40 µM), SB-203580 (20 µM), or SP-600125 (40 µM). After treatment, cell morphology was observed using phase-contrast microscopy (A) and cell viability was measured using MTT assay (B). Ctl, control. *P < 0.05 compared with other groups. C: cells were fixed and stained with 10 µg/ml propidium iodide (PI) to detect pyknotic nuclei (arrows) with the use of a fluorescent microscope. D and E: HUVECs were treated with 50 µM sodium arsenite for 16 h in the presence or absence of AG1478 (20 µM), SB-203580 (10 µM), or SP-600125 (20 µM), followed by flow cytometric analysis of apoptosis: R3, viable or undamaged cells (annexin V–, PI–); R2, cells undergoing early apoptosis (annexin V+, PI–); R4, necrotic or late apoptotic cells (annexin V+, PI+). F: HUVECs were transfected with EGFR siRNA or control siRNA. After 48 h, cells were treated with arsenite and stained with PI as in C.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present report, we provide evidence that arsenite triggers membrane receptor-dependent signaling pathways that ultimately lead to endothelial cell toxicity. As an initial step of our study, we observed an evident toxicity in HUVECs at 10 µM arsenite after 24 h of treatment. This toxic effect is in agreement with that previously reported by Kao et al. (15) in HUVECs and appears to be cell type dependent with more resistance in porcine endothelial cells (42). The discrepancies with regard to species could be explained by differences in the genetic background. Although this article focuses on elucidating the signaling pathways by arsenite at toxic levels, it is important to note that the proliferative effect of arsenite at lower levels (<1 µM) (15) may involve distinct mechanisms that remain elusive.

We found that arsenite produced transactivation of the EGF (ErbB1) and ErbB2 receptors leading to induction of both p53 and p21Cip1/Waf1 in endothelial cells, although each process appears distinct (Fig. 8). In particular, we found that EGFR activation was required for JNK stimulation that facilitated p21Cip1/Waf1 induction and that this pathway was independent of p53. In contrast, we also found that arsenite induced ErbB2 and p38 MAPK activation independently, leading to increased p53 transcription that also contributed, in part, to p21Cip1/Waf1 expression. Finally, we have established the importance of these pathways in arsenite-mediated endothelial cell apoptosis, because we were able to attenuate both p21Cip1/Waf1 induction and apoptosis by inhibiting ErbB receptors, JNK, or p38 MAPK. Thus ErbB receptors are involved in coordinated MAPK activation that determines endothelial cell fate in response to arsenite-mediated injury. These data provide further information about the mechanisms of arsenite-mediated endothelial cell toxicity.



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  		Fig. 8. Schematic model depicting the signaling pathways involved in arsenite-induced p21Cip1/Waf1. See RESULTS and DISCUSSION for details.

 
Arsenic, a pervasive carcinogen in the environment, is known to promote DNA damage. Indeed, very low physiologically relevant doses of arsenite induce DNA strand breaks and DNA-protein cross-links in a variety of cell types (2, 29). Increasing evidence now links oncogenes and tumor suppressors such as Ras and p53, typically associated with carcinogenesis, with vascular disease (13, 23). We recently demonstrated that oxidative DNA damage induces p53 expression through protein stabilization via a PDGF-{beta} receptor-mediated process (5). Despite the similar propensity for DNA damage, however, it appears that arsenite and oxidative stress act on p53 via distinct mechanisms. In particular, we found in the present study that arsenite-induced p53 upregulation was largely a consequence of increased transcription (Fig. 2) that was independent of the PDGF-{beta} receptor (Fig. 3A) and appeared to involve ErbB receptors instead. Thus arsenite and oxidative stress facilitate increases in cellular p53 levels via different mechanisms.

Although distinct with regard to p53 activation, arsenite and oxidative stress do share some common features pertaining to ErbB receptors. In this study, we found that arsenite induced EGF and ErbB2 receptor activation in a manner dependent on both receptor kinase activity and Src-family kinases (Fig. 4). This finding is reminiscent of previously reported data on H2O2-mediated EGFR transactivation (7). Investigators in our laboratory (7) previously demonstrated that endothelial cells treated with H2O2 exhibit activation of the EGFR in a Src-dependent manner that is distinct from EGFR autophosphorylation. This "transactivation" of the EGFR has been described with respect to a number of diverse stimuli, including G protein-coupled receptors, cytokines, and cellular stress (45). This similarity between the response to H2O2 and arsenite is consistent with other data showing that arsenite exposure leads to the intracellular generation of ROS (39) and participation of the EGFR in redox-sensitive signal transduction (8, 21). Thus arsenite and H2O2 share common features with regard to the EGFR transactivation reported presently.

The similarities between the responses to arsenite and H2O2 may be consistent with putative mechanisms of signal transduction. For example, arsenite is a well-recognized sulfhydryl reactant that modifies cysteinyl residues of many cellular proteins, including the EGFR and protein tyrosine phosphatase (PTPase) (3). The EGFR contains an extracellular cysteine-rich domain that has proven important with regard to receptor dimerization (14). A similar scenario has been proposed in which arsenite, via reaction with vicinal dithiols, alters the conformation of the EGFR and produces an increase in its intrinsic tyrosine kinase activity (35). Upstream kinases also are targets of arsenite, as demonstrated by Simeonova et al. (31), who found that activation of Src was induced by arsenite in epithelial cells and that this process produced EGFR transactivation and stimulation of ERK. In that study, PP2 significantly inhibited arsenite-induced EGFR phosphorylation, consistent with both the present results reported and those of a previous study (7) demonstrating H2O2 transactivation of the EGFR in a Src-dependent manner. Finally, PTPase activity is subject to modulation by arsenite, and inhibition of PTPases has received considerable attention as a mechanism for signal transduction with ligand-induced growth factor activation (20, 25, 27, 33).

The link between arsenite and activation of the MAPK pathway has been studied in some detail. Arsenite has been shown to induce ERK, JNK, and p38 kinase components of the MAPK cascade in a number of human cell lines (24, 33, 35). For example, arsenite-treated human bronchial epithelial cells exhibit EGFR tyrosine phosphorylation, MEK1/2 activation, ERK1/2 phosphorylation, and enhanced transcriptional activity of Elk-1 (40). With regard to the current study, the p38 kinase and JNK components of the MAPK pathway have been linked to cell cycle regulation, particularly in response to external cellular stress (9, 18, 38). Treatment of NIH/3T3 cells with sodium arsenite induced growth inhibition through a mechanism that involved p38 kinase-mediated induction of p21Cip1/Waf1 (17). Our data are in general agreement with this report, given that we found both JNK and p38 kinase activation in response to arsenite and that inhibition of p38 kinase attenuated arsenite-induced p21Cip1/Waf1 induction (Fig. 6). However, our data add new information in that we found distinctions between p38 kinase and JNK activation. For example, activation of JNK required arsenite-induced ErbB receptor activation, whereas p38 MAPK did not. In addition, we found no role for p53 in the linear pathway of p21Cip1/Waf1 induction mediated by EGFR and JNK in response to arsenite. This latter finding is consistent with published reports that growth factors such as PDGF-{alpha} and basic fibroblast growth factor also have been shown to induce p53-independent transcription of p21Cip1/Waf1 (22, 44). With regard to PDGF-{alpha}, the mechanism of p21Cip1/Waf1 induction appears to involve JNK-1-responsive cis-acting regulatory elements residing in the p21 promoter (44). There is precedent for this mechanism, because c-Jun, a substrate of JNK1, mediates p53-independent p21Cip1/Waf1 promoter activation via physical interaction with the Sp1 protein (16). Thus our results are consistent with the model depicted in Fig. 8, which indicates that arsenite induces p21Cip1/Waf1 through a pathway that involves EGFR-mediated JNK activation. Our data also are consistent with the pathways involving ErbB2 and p38 kinase-mediated p53 induction that contributes to the p21Cip1/Waf1 upregulation in response to arsenite. The precise nature of the coordination between these pathways is not yet known. Given the important role of both p53 and p21 in regulating cell growth and apoptosis, however, one might submit that such coordination would be expected.

In summary, the data presented here indicate that arsenite induces endothelial cell death via a mechanism that involves p21Cip1/Waf1 induction. These data are in keeping with a large body of literature indicating that arsenite produces abnormalities in endothelial function. In this study, we have linked the mechanism of arsenite-induced injury, in part, to ErbB receptor activation and coordinated activation of the MAPK cascade. The data presented here suggest that p21Cip1/Waf1 may represent an attractive target to ameliorate arsenite-induced endothelial cell dysfunction.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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Dr. Chen is the recipient of a Scientist Development Grant from the American Heart Association (AHA) and a Boston University Department of Medicine Pilot Project Award. This work was partially supported by grant from the Thailand Research Fund through the Faculty of Pharmaceutical Sciences and Institute of Health Research, Chulalongkorn University. Work in the laboratory of J. F. Keaney, Jr., is supported by National Institutes of Health Grants DK-55656, HL-60886, HL-67206, and HL-68758 and a Juvenile Diabetes Research Foundation Complications Center Grant. This work was performed while J. F. Keaney, Jr., was an Established Investigator of the AHA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Chen, Boston Univ. School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St., Rm. W507, Boston, MA 02118 (E-mail: kaichen{at}bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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