Ethanol-mediated regulation of cytochrome P450 2A6 expression in monocytes: role of oxidative stress-mediated PKC/MEK/Nrf2 pathway

Cytochrome P450 2A6 (CYP2A6) is known to metabolize nicotine, the major constituent of tobacco, leading to the production of toxic metabolites and induction of oxidative stress that result in liver damage and lung cancer. Recently, we have shown that CYP2A6 is induced by ethanol and metabolizes nicotine into cotinine and other metabolites leading to generation of reactive oxygen species (ROS) in U937 monocytes. However, the mechanism by which CYP2A6 is induced by ethanol is unknown. In this study, we have examined the role of the PKC/Nrf2 pathway (protein kinase C-mediated phosphorylation and translocation of nuclear erythroid 2-related factor 2 to the nucleus) in ethanol-mediated CYP2A6 induction. Our results showed that 100 mM ethanol significantly induced CYP2A6 mRNA and protein (~150%) and increased ROS formation, and induction of gene expression and ROS were both completely blocked by treatment with either a CYP2E1 inhibitor (diallyl sulfide) or an antioxidant (vitamin C). The results suggest the role of oxidative stress in the regulation of CYP2A6 expression. Subsequently, we investigated the role of Nrf2 pathway in oxidative stress-mediated regulation of CYP2A6 expression in U937 monocytes. Our results showed that butylated hydroxyanisole, a stabilizer of nuclear Nrf2, increased CYP2A6 levels >200%. Staurosporine, an inhibitor of PKC, completely abolished ethanol-induced CYP2A6 expression. Furthermore, our results showed that a specific inhibitor of mitogen-activated protein kinase kinase (MEK) (U0126) completely abolished ethanol-mediated CYP2A6 induction and Nrf2 translocation. Overall, these results suggest that CYP2E1-mediated oxidative stress produced as a result of ethanol metabolism translocates Nrf2 into the nucleus through PKC/MEK pathway, resulting in the induction of CYP2A6 in monocytes. An increased level of CYP2A6 in monocytes is expected to further increase oxidative stress in smokers through CYP2A6-mediated nicotine metabolism. Thus, this study has clinical relevance because of the high incidence of alcohol use among smokers, especially in HIV-infected individuals.     

Induction of cIAP-2 in human colon cancer cells through PKC delta/NF-kappa B

Activation of protein kinase C (PKC) prevents apoptosis in certain cells; however, the mechanisms are largely unknown. Inhibitors of apoptosis (IAP) family members, including NAIP, cIAP-1, cIAP-2, XIAP/hILP, survivin, and BRUCE, block apoptosis by binding and potently inhibiting caspases. Activation of NF-kappa B contributes to cIAP-2 induction; however, the cellular mechanisms regulating cIAP-2 expression have not been entirely defined. In this study, we examined the role of the PKC and NF-kappa B pathways in the regulation of cIAP-2 in human colon cancers. We found that cIAP-2 mRNA levels were markedly increased in human colon cancer cells by treatment with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), or bryostatin 1. Inhibitors of the Ca2+-independent, novel PKC isoforms, but not inhibitors of MAPK, PI3-kinase, or PKA, blocked PMA-stimulated cIAP-2 mRNA expression, suggesting a role of PKC in PMA-mediated cIAP-2 induction. Pretreatment with the PKC delta-selective inhibitor rottlerin or transfection with an antisense PKC delta oligonucleotide inhibited PMA-induced cIAP-2 expression, whereas cotransfection with a PKC delta plasmid induced cIAP-2 promoter activity, which, taken together, identifies a role for PKC delta in cIAP-2 induction. Treatment with the proteasome inhibitor, MG132 or inhibitors of NF-kappa B (e.g. PDTC and gliotoxin), decreased PMA-induced up-regulation of cIAP-2. PMA-induced NF-kappa B activation was blocked by either GF109203x, MG132, PDTC, or gliotoxin. Moreover, overexpression of PKC delta-induced cIAP-2 promoter activity and increased NF-kappa B transactivation, suggesting regulation of cIAP-2 expression by a PKC delta/NF-kappa B pathway. In conclusion, our findings demonstrate a role for a PKC/NF-kappa B-dependent pathway in the regulation of cIAP-2 expression in human colon cancer cells. These data suggest a novel mechanism for the anti-apoptotic function mediated by the PKC delta/NF-kappa B/cIAP-2 pathway in certain cancers.     

HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded on Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. We previously identified RtsA, an AraC-like protein similar to the known HilC and HilD regulatory proteins. Like HilC and HilD, RtsA activates expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 TTSS structural genes. Here we present evidence that hilA expression, and hence the SPI1 TTSS, is controlled by a feedforward regulatory loop. We demonstrate that HilC, HilD and RtsA are each capable of independently inducing expression of the hilC, hilD and rtsA genes, and that each can independently activate hilA. Using competition assays in vivo, we show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, HilD has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through HilD, thus inducing hilC, rtsA and hilA. However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, we show that 'SPI1 inducing conditions' cause independent activation of the rtsA, hilC and hilD genes in the absence of known regulators. Our model of SPI1 regulation provides a framework for future studies aimed at understanding this complicated regulatory network.     

Star-PAP control of BIK expression and apoptosis is regulated by nuclear PIPKIα and PKCδ signaling

BIK protein is an initiator of mitochondrial apoptosis, and BIK expression is induced by proapoptotic signals, including DNA damage. Here, we demonstrate that 3' end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P(2)-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCδ is a key mediator of apoptosis, and DNA damage stimulates PKCδ association with the Star-PAP complex where PKCδ is required for Star-PAP-dependent BIK expression. PKCδ binds the PI4,5P(2)-generating enzyme PIPKIα, which is essential for PKCδ interaction with the Star-PAP complex, and PKCδ activity is directly stimulated by PI4,5P(2). Features in the BIK 3' UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIα, PI4,5P(2), and PKCδ regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3' end processing.     

Exploring the five different genes associated with PKCα in bladder cancer based on gene expression microarray

Much progress has been made in understanding the mechanism of bladder cancer (BC) progression. Protein kinase C-α (PKCα) is overexpressed in many kinds of cancers. Additionally, PKCα is considered an oncogene that regulates proliferation, invasion, migration, apoptosis and cell cycle in multiple cancers. However, the mechanism underlying how these cellular processes are regulated by PKCα remains unknown. In the present study, we used PKCα siRNA to knock down PKCα gene expression and found that down-regulation of PKCα could significantly inhibit cell proliferation, migration and invasion and induce apoptosis and G1/S cell cycle arrest in vitro. Overexpression of PKCα promotes tumour growth in vivo. We applied cDNA microarray technology to detect the differential gene expression in J82 cells with PKCα knockdown and found that five key genes (BIRC2, BIRC3, CDK4, TRAF1 and BMP4) were involved in proliferation and apoptosis according to GO analysis and pathway analyses. Correlation analysis revealed a moderate positive correlation between PKCα expression and the expression of five downstream genes. BIRC2 and BIRC3 inhibit apoptosis, whereas CDK4, TRAF1 and BMP4 promote proliferation. Essentially, all five of these target genes participated in proliferation, and apoptosis was regulated by PKCα via the NF-kB signalling pathway.