Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes.

2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car(-/-)) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car(-/-) livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor.

Ceramide promotes apoptosis in lung cancer-derived A549 cells by a mechanism involving c-Jun NH2-terminal kinase.

Ceramide regulates diverse signaling pathways involving cell senescence, the cell cycle, and apoptosis. Ceramide is known to potently activate a number of stress-regulated enzymes, including the c-Jun NH(2)-terminal kinase (JNK). Although ceramide promotes apoptosis in human lung cancer-derived A549 cells, a role for JNK in this process is unknown. Here, we report that ceramide promotes apoptosis in A549 cells by a mechanism involving JNK. The JNK inhibitor SP600125 proved effective at protecting cells from the lethal effects of ceramide. To understand which JNK-mediated pathway may be involved, a number of JNK target proteins were examined, including the transcription factor, c-Jun, and the apoptotic regulatory proteins Bcl-X(L) and Bim. A549 cells exhibited basal levels of phosphorylated c-Jun in nuclear fractions, revealing that active c-Jun is present in these cells. Ceramide was found to inhibit c-Jun phosphorylation, suggesting that JNK-mediated phosphorylation of c-Jun is not likely involved in ceramide-induced apoptosis. Ceramide did not promote Bcl-X(L) phosphorylation. On the other hand, ceramide promoted phosphorylation of Bim and induced translocation of active JNK from the nucleus to the cytoplasm and mitochondrial fraction. Ceramide-mediated changes in localization of JNK were consistent with the observed changes in phosphorylation status of c-Jun and Bim. Furthermore, ceramide promoted Bim translocation to the mitochondria. Mitochondrial localization of Bim has been shown recently to promote apoptosis. These results suggest that JNK may participate in ceramide-induced apoptosis in A549 cells by a mechanism involving Bim.

PKR regulates B56(alpha)-mediated BCL2 phosphatase activity in acute lymphoblastic leukemia-derived REH cells.

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56alpha is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56alpha at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56alpha in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56alpha-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56alpha phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56alpha proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56alpha protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2alpha activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56alpha, because wild-type but not mutant S28A B56alpha promoted mitochondrial PP2A activity. Cells expressing wild-type B56alpha but not S28A B56alpha were sensitized to etoposide. These results suggest that PKR regulates B56alpha-mediated PP2A signaling in REH cells.

Mitochondrial protein phosphatase 2A regulates cell death induced by simulated ischemia in kidney NRK-52E cells.

Acute renal failure can occur after an ischemic injury and results in significant mortality. The stress-signaling pathways that are activated during renal ischemia are unknown. PP2A has emerged as an important regulator of cell death. To study the role of PP2A in ischemia-induced cell death, we used an in vitro model of simulated ischemia. In the present study, simulated ischemia in rat renal tubule epithelial NRK-52E cells (A) results in cell death that involves both necrosis and apoptosis, (B) activates PP2A, and (C) up-regulates the PP2A B56 alpha regulatory subunit. Previous data have shown that PKC alpha negatively regulates B56 alpha protein expression. Consistent with this finding, simulated ischemia suppressed PKC alpha and up-regulated B56 alpha. Treatment of NRK-52E cells with ceramide suppressed PKC alpha and activated PP2A in a manner that mimicked simulated ischemia. A role for PP2A in simulated ischemia-induced cell death is likely since inhibition of PP2A protected NRK-52E cells. In addition, overexpression of exogenous B56 alpha but not B55 in NRK-52E cells enhanced simulated ischemia-induced cell death. These findings suggest that activation of a PP2A isoform that contains the B56 alpha regulatory subunit is required for ischemia-induced cell death in kidney epithelial proximal tubule cells.