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Differential activation of AP-1 in human bladder epithelial cells by inorganic and methylated arsenicals¹

ZUZANA DROBNÁ^*, ILONA JASPERS^*,, DAVID J. THOMAS and MIROSLAV STBLO^*,,¶2

^* Department of Pediatrics,
Center for Environmental Medicine and Lung Biology, and
^¶ Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina, USA; and
Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

²Correspondence: Department of Pediatrics, CB# 7220, 535 Burnett-Womack Clinical Sciences Bldg., University of North Carolina, Chapel Hill, NC 27599-7220, USA. E-mail: styblo{at}med.unc.edu

SPECIFIC AIMS

Inorganic arsenic (iAs), an environmental contaminant and potent carcinogen, is metabolized in human body to yield mono- and dimethylated trivalent and pentavalent arsenicals. Chronic exposure to iAs has been linked to increased incidences of various cancers, including cancer of urinary bladder. To characterize the role of metabolism in the toxic and cancer-promoting effects of iAs, particularly in the induction of activator protein-1 (AP-1)-dependent gene transcription, we examined effects of inorganic and methylated arsenicals on AP-1 composition and DNA binding activity in UROtsa cells, an SV40-immortalized cell line derived from normal human urinary bladder epithelium.

PRINCIPAL FINDINGS

1. DNA binding activity and composition of AP-1 in cells exposed to arsenicals
The effects of trivalent arsenicals on DNA binding activity of AP-1 in UROtsa cells as determined by the electrophoretic mobility shift assay (EMSA) were species dependent and varied with concentration and exposure time. AP-1 DNA binding activity was suppressed in cells exposed for 1 h to 0.1 or 0.5 µM arsenite (iAs^III) or iododimethylarsine (DMAs^IIII) but was strongly induced by 5 µM DMAs^IIII. AP-1 DNA binding activity remained unchanged in cells exposed for 1 h to 1 or 5 µM iAs^III, but was slightly increased after 2 h exposures. Methylarsine oxide (MAs^IIIO) was a potent activator of AP-1 DNA binding at either exposure time. One hour exposure to MAs^IIIO activated AP-1 at concentrations as low as 0.1 µM; the largest effect occurred at 1 µM. Pentavalent inorganic or methylated arsenicals (1–100 µM) did not alter the AP-1 DNA binding activity in UROtsa cells. Immunochemical (EMSA supershift) analysis showed that phosphorylated c-Jun (p-c-Jun) and Fra-1 are the major constituents of the AP-1-DNA binding complex both in cells treated with trivalent arsenicals and untreated cells (Fig. 1 ). JunD but not c-Fos, FosB, or ATF-2 were detected in the complex. JunB was a significant component of the AP-1-DNA binding complex only in cells treated with MAs^IIIO.

View larger version (67K): [in this window] [in a new window]   Figure 1. EMSA supershift analysis of nuclear protein extracts from UROtsa cells treated for 1 h with 5 µM iAsIII, 1 µM MAsIIIO, or 5 µM DMAsIIII and from control (untreated) cells. For immunochemical characterization of the AP-1 constituents, the DNA protein binding assay was carried out in the presence of antibodies specific for a) p-c-Jun, c-Jun, or c-Fos (Santa Cruz Biotechnology); b) c-Jun, JunB, JunD, c-Fos, or FosB (Geneka Biotechnology) and Fra-1, p-ATF-2 or ATF-2 (Santa Cruz Biotechnology). Specificity of the assay was established using a 50-fold excess of a wild-type (wt) or mutated (mt) AP-1 probe. A nuclear protein extract from 3T3 SR cells was used as a positive control for analysis of the c-Fos subunit. To confirm the specificity of the c-Fos antibodies, 3T3 SR nuclear extract was incubated in the DNA binding assay mixture in the presence of an excess of c-Fos peptide. Each lane represents 6 µg of nuclear protein.

2. Expression and activation of AP-1 constituents in cells exposed to arsenicals
The effects of trivalent arsenicals on expression and activation (phosphorylation) of c-Jun and Fra-1, the major AP-1 constituents, were examined using immunoblot analysis. Low concentrations of iAs^III (0.1 and 0.5 µM) decreased p-c-Jun levels while higher concentrations of iAs^III (1 or 5 µM) increased p-c-Jun levels in a concentration-dependent manner. Methylated trivalent arsenicals, MAs^IIIO and DMAs^IIII, were both more potent inducers of p-c-Jun than iAs^III. A >20-fold increase in nuclear p-c-Jun levels was found in cells exposed to 5 µM MAs^IIIO vs. 13- and 4-fold increases in cells treated with 5 µM DMAs^IIII and 5 µM iAs^III, respectively. No changes in nuclear levels of c-Jun or c-Fos were detected in exposed cells regardless of treatment conditions. Increased nuclear levels of Fra-1 were found in UROtsa cells exposed to MAs^IIIO and DMAs^IIII, but not iAs^III. An electrophoretic shift of the immunoreactive fractions of Fra-1 to a 43 kDa band has been detected in exposed cells, indicating that besides the induction of Fra-1, production of its phosphorylated form (p-Fra-1) also increased.

3. Induction of AP-1-dependent gene expression by trivalent arsenicals
Effects of trivalent arsenicals on AP-1-dependent gene transcription were examined in UROtsa cells transiently cotransfected with pAP-1-luc (an AP-1-dependent promoter-reporter construct containing cDNA for firefly luciferase) and pSV-ß-gal (an SV-40 promoter-ß-galactosidase construct). The activity of constitutively expressed ß-galactosidase was used to normalize luciferase activity for well-to-well variations in transfection efficiency, cell number, and viability. In transfected cells, only treatment with iAs^III and MAs^IIIO (not DMAs^IIII) resulted in a significant increase in normalized AP-1-dependent luciferase activity. MAs^IIIO (1 µM) was a more potent inducer of the luciferase activity than was 5 µM iAs^III.

4. Effects of trivalent arsenicals on enzymes of the mitogen-activated protein kinase (MAPK) family
To identify the upstream mechanisms responsible for c-Jun and Fra-1 phosphorylation and AP-1 activation by trivalent arsenicals, we analyzed protein extracts from control and exposed cells for the presence of the parent and activated (phosphorylated) forms of enzymes of the MAPK family, JNK, p38, and ERK. No changes in JNK1, JNK2, and p38 levels or induction of the corresponding phosphorylated forms (p-JNK1, p-JNK2, and p-p38) were detected in cells treated with iAs^III, MAs^IIIO, or DMAs^IIII for up to 2 h. Similarly, no changes in ERK1 and ERK2 levels were found (Fig. 2 a–c). However, all three arsenicals induced production of the phosphorylated form of ERK-2 (p-EKR2). Increased p-ERK1 levels were found in cells exposed to DMAs^IIII but not to iAs^III or MAs^IIIO (Fig. 2c ).

View larger version (29K): [in this window] [in a new window]   Figure 2. Immunoblot analysis of ERK1,2 and p-ERK1,2 in UROtsa cells exposed to 5 µM iAsIII (a), 1 µM MAsIIIO (b), or 5 µM DMAsIIII (c) for up to 2 h. Each panel shows immunoblot images and results of the quantitative analysis of these images (bar charts). The bar charts show folds of ERK activation.

CONCLUSION AND SIGNIFICANCE

The metabolic pathway for iAs in humans includes reduction and oxidative methylation steps, during which arsenic cycles between +5 and +3 oxidation states. iAs^III has commonly been considered the most harmful metabolite responsible for toxic and carcinogenic effects associated with exposures to iAs. Therefore, biomethylation of iAs has been regarded as a mechanism for its detoxification. However, recent studies have shown that methylated trivalent arsenicals, methylarsonous acid (MAs^III), and dimethylarsenious acid (DMAs^III), are potent cytotoxins, genotoxins, and enzyme inhibitors. In light of these findings, biomethylation can be viewed as a process that potentiates toxicity and cancer-promoting effects of iAs. In this study, we show that methylated trivalent arsenicals, especially MAs^IIIO (a derivative of MAs^III), are significantly more potent than iAs^III as activators of AP-1 DNA binding activity in UROtsa cells. The activation of AP-1 coincides with a significant increase in nuclear levels of p-c-Jun and Fra-1 in exposed cells. The immunoblot patterns suggest that exposures to methylated trivalent arsenicals also induce phosphorylation of Fra-1. Fra-1 and p-c-Jun are the major components of the AP-1-DNA binding complex in UROtsa cells exposed to trivalent arsenicals. Thus, induction of c-Jun and Fra-1 phosphorylation by these arsenicals is likely to underlie the activation of AP-1 DNA binding in exposed cells. Unlike other arsenicals, MAs^IIIO increases the presence of JunB in the AP-1-DNA binding complex. These data suggest that a specific MAs^III-sensitive mechanism for AP-1 activation exists in UROtsa cells. In human cells, c-Jun phosphorylation is catalyzed mainly by activated (phosphorylated) JNK1 or 2 (p-JNK1,2); p-ERK1 and 2 are thought to be responsible for phosphorylation of Fra-1. JNK activity can be induced in cultured cells by exposures to high cytotoxic concentrations (40–400 µM) of iAs^III. The results of this study show that exposure of UROtsa cells to low concentrations of iAs^III, MAs^III or DMAs^III derivatives induces phosphorylation of ERK1 and/or 2, but not JNK1,2 or p38. Under these conditions, p-ERKs are involved at least in part in the activation of c-Jun and Fra-1 in UROtsa cells. The upstream mechanism of ERK activation by trivalent arsenicals remains to be examined.

Exposures to trivalent arsenicals, increase AP-1 DNA binding activity in UROtsa cells through the ERK-dependent induction of c-Jun and possibly Fra-1 phosphorylation (Fig. 3 ). Among these arsenicals, MAs^IIIO is the most potent inducer of c-Jun and Fra-1 phosphorylation and the activator of AP-1 DNA binding. Thus, methylated trivalent arsenicals chemically consistent with methylated metabolites of iAs, MAs^III, and DMAs^III, are considerably more potent AP-1 activators in human urinary bladder cells than is iAs^III. Activation of AP-1 DNA binding, and thereby induction of the AP-1-regulated gene transcription, by methylated trivalent metabolites of iAs may contribute to the toxic and/or cancer-promoting effects associated with exposures to iAs. These findings support a recently promoted hypothesis that the biomethylation is a process that activates iAs as a toxin and human carcinogen.

View larger version (33K): [in this window] [in a new window]   Figure 3. Scheme. Hypothetical signal transduction pathway for AP-1 activation in human bladder epithelial cells exposed to trivalent arsenicals. This pathway includes induction of ERK phosphorylation by trivalent arsenicals formed in the course of iAs metabolism and delivered to the cell via the bloodstream. Phosphorylation of ERKs likely occurs through a cellular receptor (R)-mediated activation of MAPK/ERK kinase (MEK). p-ERKs catalyze phosphorylation of Fra-1 and/or c-Jun, activating AP-1 DNA binding in the nucleus. The activated AP-1, which is represented in the exposed cells mainly by p-c-Jun/p-Fra1 heterodimers or p-c-Jun/p-c-Jun homodimers, binds to the TPA response element (TRE) in a promoter region, initiating the AP-1-dependent gene transcription.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0287fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0287fje

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