Choon-Myung Lee

Regulation of Drug Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer

Under conditions of innate immune system activation (i.e., inflammation), the functions of specific cytochrome P450 enzymes, other drug metabolizing enzymes (DMEs), and drug transporters (DTs) are altered in the liver, small intestine, lung, kidney, and central nervous system (CNS). Many of these effects are primarily manifest at the transcriptional/RNA level, leading to corresponding changes in protein levels and function. This not only leads to altered drug and xenobiotic toxicity and action in diseased humans, but also has importance for disease therapy with biologic drugs that target inflammatory mediators or their receptors. Major roles for proinflammatory cytokines such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α(TNFα) are inferred from the abilities of these agents to affect DMEs and DTs in cultured cells and in vivo, but the in vivo contributions of cytokines to regulation of these proteins in different inflammatory disease states is still poorly understood. Keywords: inflammation; infection; cytokines; toll-like receptors; biologics

Nitric oxide-dependent proteasomal degradation of cytochrome P450 2B proteins.

Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes after exposure to bacterial endotoxin (lipopolysaccharide) in a nitric oxide (NO) -dependent manner. In this study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed >60% after 6 h of treatment with interleukin-1β (IL-1). This effect was NO-dependent, and treatment of cells with the NO donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione, and S-nitroso-N-acetylpenicillamine also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH4Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-Myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with S-nitrosoglutathione caused S-nitrosylation of CYP2B protein and enhanced the ubiquitination pattern of CYP2B compared with unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species.

Dual mechanisms of CYP3A protein regulation by proinflammatory cytokine stimulation in primary hepatocyte cultures.

Whereas many cytochrome P450 enzymes are transcriptionally suppressed by inflammatory stimuli, down-regulation of CYP2B protein by the inflammatory cytokine interleukin (IL)-1β is nitric oxide (NO)-dependent and occurs via polyubiquitination and proteasomal degradation. Here, we used iTRAQ proteomic analysis to search for other proteins that are potentially down-regulated by cellular NO in cultured rat hepatocytes, and we identified CYP3A1 as one such protein. Therefore, we examined whether CYP3A proteins, like CYP2B, undergo NO- and proteasome-dependent degradation in response to cytokine treatment of rat hepatocytes. In cultured rat hepatocytes treated with phenobarbital, IL-1β stimulation failed to down-regulate CYP3A1 mRNA within 24 h of treatment, whereas CYP3A protein was down-regulated to 40% of control within 6 h, showing the post-transcriptional down-regulation of CYP3A1 protein. The down-regulation of CYP3A after 9 h of stimulation by IL-1β was attenuated by inhibitors of NO synthase (NOS) and of the proteasome, showing NO- and proteasome-dependent down-regulation at earlier time points. However, the down-regulation of CYP3A evoked by IL-1β measured 24 h after stimulation was not affected by the inhibition of NOS or by proteasomal inhibitors, showing that CYP3A1 down-regulation at later time points is NO- and proteasome-independent. IL-6, which did not evoke NO production nor affect CYP3A1 mRNA within 24 h, produced a delayed proteasome-independent down-regulation as well. Taken together, these observations show a novel dual mode of post-transcriptional CYP3A down-regulation by cytokines: NO- and proteasome-dependent at earlier time points and NO- and proteasome-independent at later times.

Roles of nitric oxide in inflammatory downregulation of human cytochromes P450.

The purpose of this study was to determine the role of nitric oxide (NO) in the downregulation of human cytochrome P450 (CYP) enzymes and mRNAs by an inflammatory stimulus in cultured human hepatocytes. We focused on CYP2B6 because previous studies showed that rat CYP2B proteins undergo an NO-dependent degradation in response to inflammatory stimuli. To ensure high-level expression of CYP2B6, the inducer phenytoin was present at all times. Stimulation of cells with a mixture of tumor necrosis factor-alpha, interleukin-1, and interferon-gamma (ILmix) downregulated CYP2B6 mRNA and protein to 9 and 19% of control levels. The NO donor NOC-18 downregulated CYP2B6 protein to 30% of control, with only a small effect on CYP2B6 mRNA. Nitric oxide synthase inhibitors attenuated the downregulation of CYP2B6 protein but not mRNA by ILmix. These findings demonstrate that the posttranscriptional NO-dependent downregulation of CYP2B enzymes, observed previously in rat hepatocytes, is conserved in human CYP2B6. This mechanism is specific for CYP2B6 among the enzymes tested. No evidence was found for regulation of CYP2E1 mRNA or protein by NO. NOC-18 treatment downregulated CYP3A4 mRNA to 50% of control. However, NOS inhibitors failed to block the effects of ILmix on CYP3A4 expression.

Modulation of Hepatic Cytochrome P450s by Citrobacter rodentium Infection in Interleukin-6- and Interferon-γ-null Mice

After infection with Citrobacter rodentium, murine hepatic cytochrome P450 (P450) mRNAs are selectively regulated. Several serum proinflammatory cytokines are elevated, the most abundant being interleukin-6 (IL6). To elucidate the role of cytokines in the regulation of P450s during infection, we orally infected wild-type, IL6(−/−), or interferon-γ(−/−) [IFNγ(−/−)] female C57BL/6J mice with C. rodentium and analyzed hepatic P450 expression 7 days later. The majority of P450 mRNAs were equally affected by infection in each genotype, indicating that IL6 and IFNγ are not the primary mediators of P450 down-regulation in this disease model. The down-regulation of CYP3A11 and CYP3A13 and induction of CYP2D9 mRNAs were attenuated in the IL6(−/−) mice, suggesting a role of IL6 in the regulation of only these P450s. Similar evidence implicated IFNγ in the regulation of CYP2D9, CYP2D22, CYP3A11, CYP3A25, and CYP4F18 mRNAs in C. rodentium infection and CYP2B9, CYP2D22, and CYP2E1 in the bacterial lipopolysaccharide model of inflammation. This is the first indication of an in vivo role for IFNγ in hepatic P450 regulation in disease states. The deficiency of IL6 or IFNγ affected serum levels of the other cytokines. Moreover, experiments in cultured hepatocytes demonstrated that tumor necrosis factor α (TNFα) is the most potent and efficacious of the cytokines tested in the regulation of murine P450 expression. It is therefore possible that part of the IFNγ(−/−) and IL6(−/−) phenotypes could be attributed to the reduced levels of TNFα and part of the IFNγ(−/−) phenotype could be caused by reduced levels of IL6.

Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen

Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1-/-), interleukin-1 (IL1) receptor null mice (IL1R1-/-), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1-/- mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R1-/- mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection.

Metabolism and Action of Proteasome Inhibitors in Primary Human Hepatocytes

Proteasome inhibitors are important tools for studying the roles of the proteasome in cellular processes. In this study, we observed that the proteasome inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), epoxomicin, and lactacystin were ineffective and bortezomib was completely effective in inhibiting cytokine-stimulated nitric oxide production in primary cultures of human hepatocytes that had been treated with the cytochrome P450 inducer phenobarbital. The inefficacy of MG132 was due to its metabolism by CYP3A enzymes, as deduced from its rapid, ketoconazole-sensitive clearance by pooled human liver microsomes and cultured hepatocytes. The efficacy of MG132 was increased by inclusion of ketoconazole in the hepatocyte incubations and decreased by prior treatment of the cultures with the CYP3A inducers phenobarbital or rifampicin. Epoxomicin was also rapidly metabolized by CYP3A, whereas bortezomib and lactacystin were much more stable metabolically in human liver microsomes or hepatocyte cultures. Thus, bortezomib is a better choice than MG132, epoxomicin, or lactacystin in cells with high activities of CYP3A enzymes. The reason for the lack of efficacy of lactacystin in human hepatocytes has yet to be determined, but it too should not be used for studies of proteasome function in human hepatocytes.

Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system

Abstract We showed previously that rat cytochrome P450 CYP2B1 undergoes NO‐dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down‐regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 down‐regulation, we made several cell lines (HeLa and HuH7 cells) in which native CYP2B6 or CYP2B6 with a C‐terminal V5 tag (CYP2B6V5) are expressed from a lentiviral vector with a cytomegalovirus promoter. Native CYP2B6 protein was rapidly down‐regulated in HeLa cells within 3 h of treatment with the NO donor (Z)−1‐[2‐(2‐Aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2‐diolate, while its mRNA level was not down‐regulated. Treatment of the cells with the NO donor (Z)−1‐[N‐(3‐aminopropyl)‐N‐(3‐ammoniopropyl)amino]diazen‐1‐ium‐1,2‐diolate also resulted in rapid down‐regulation of CYP2B6 activity, measured as the formation of 7‐hydroxy‐4‐trifluoromethylcoumarin, as well as 2B6 protein in the CYP2B6 HeLa cell line. CYP2B6V5 was also down‐regulated by NO donors in HuH7 cells. Down‐regulation was observed in the presence of cycloheximide, demonstrating that this occurs via a post‐translational mechanism. We generated a HeLa cell line expressing both CYP2B6V5 and human nitric oxide synthase 2 (NOS2), the latter under positive control by tetracycline. The cellular NO produced by doxycycline treatment also effectively down‐regulated CYP2B6 protein, which was blocked by the co‐treatment with the NOS2 competitive inhibitor L‐NG‐nitroarginine methyl ester (L‐NAME). We next investigated the proteolytic enzymes responsible for NO‐dependent CYP2B6 degradation. Neither calpain inhibitors (N‐Acetyl‐L‐leucyl‐L‐leucyl‐L‐norleucinal, carbobenzoxy‐valinyl‐phenylalaninal), nor lysosomal protease inhibitors (3‐methyladenine and chloroquine) inhibited the NO dependent CYP2B6V5 down‐regulation. The proteasome inhibitors MG132 and bortezomib attenuated, but did not completely block the NO‐induced down‐regulation in the HuH7 cell line. However, when cells were co‐treated with NO donor and proteasome inhibitors, high molecular mass species could be detected on native CYP2B6 as well as CYP2B6V5 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by co‐treatment with NO donor and bortezomib. Taken together, our data demonstrate that CYP2B6 is down‐regulated in an NO‐dependent manner via ubiquitination and proteasomal degradation. Graphical abstract Figure. No Caption available. HighlightsCytochrome P450 2B6 is downregulated by nitric oxide from NOS2 or NO donors.Occurs in presence of cycloheximide, indicating NO‐mediated protein degradation.Down‐regulation is partially inhibited by proteasome inhibitors.Ubiquitin‐CYP2B6 conjugates accumulate in presence of NO and proteasome inhibition.

Hepatic cytochrome P450s, phase II enzymes and nuclear receptors are downregulated in a Th2 environment during Schistosoma mansoni infection.

Inflammation and infection downregulate the activity and expression of cytochrome P450s (P450s) and other drug metabolizing enzymes (DMEs) involved in hepatic drug clearance. Schistosoma mansoni infection was reported to cause a downregulation of hepatic P450-dependent activities in mouse liver, but little is known about the specific enzymes affected or whether phase II DMEs are also affected. Here we describe the effect of murine schistosomiasis on the expression of hepatic P450s, NADPH-cytochrome P450 reductase (Cpr), phase II drug metabolizing enzymes, and nuclear receptors at 30 and 45 days postinfection (dpi). Although the hepatic expression of some of these genes was altered at 30 dpi, we observed substantial changes in the expression of the majority of P450 mRNAs and proteins measured, Cpr protein, as well as many of the UDP-glucuronosyltransferases and sulfotransferases at 45 dpi. S. mansoni infection also altered nuclear receptor expression, inducing mRNA levels at 30 dpi and depressing levels at 45 dpi. S. mansoni evoked a T helper 2 (Th2) inflammatory response at 45 dpi, as indicated by the induction of hepatic Th2 cytokine mRNAs [interleukins 4, 5, and 13], whereas the hepatic proinflammatory response was relatively weak. Thus, chronic schistosomiasis markedly and selectively alters the expression of multiple DMEs, which may be associated with Th2 cytokine release. This would represent a novel mechanism of DME regulation in disease states. These findings have important implications for drug testing in infected mice, whereas the relevance to humans with schistosomiasis needs to be determined.

Nitric oxide-dependent CYP2B degradation is potentiated by a cytokine-regulated pathway and utilizes the immunoproteasome subunit LMP2.

CYP2B proteins in rat hepatocytes undergo NO-dependent proteolytic degradation, but the mechanisms and the reasons for the specificity towards only certain P450 (cytochrome P450) enzymes are yet unknown. In the present study we found that down-regulation of CYP2B proteins by the NO donor NOC-18 is accelerated by pretreatment of the hepatocytes with IL-1 (interleukin-1β) in the presence of an NO synthase inhibitor, suggesting that an NO-independent action of IL-1 contributes to the lability of CYP2B proteins. The immunoproteasome subunit LMP2 (large multifunctional peptidase 2) was significantly expressed in hepatocytes under basal conditions, and IL-1 induced LMP2 within 6–12 h of treatment. CYP2B protein degradation in response to IL-1 was attenuated by the selective LMP2 inhibitor UK-101, but not by the LMP7 inhibitor IPSI. The results show that LMP2 contributes to the NO-dependent degradation of CYP2B proteins, and suggest that induction of LMP2 may be involved in the potentiation of this degradation by IL-1.