Induction Of CYP1A1 Expression Research Articles

Activation of the Aryl Hydrocarbon Receptor by the Calcium/Calmodulin-Dependent Protein Kinase Kinase Inhibitor 7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic Acid (STO-609)

This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Ialpha, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 muM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+](i) and activation of CaMKIalpha, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIalpha/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

ジ（２－エチルヘキシル）フタレート（DEHP）によるCYP1A1の発現誘導

ジ（２－エチルヘキシル）フタレート（DEHP）によるCYP1A1の発現誘導

Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[ a]anthracene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo- p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

HDAC1 bound to the Cyp1a1 promoter blocks histone acetylation associated with Ah receptor-mediated trans-activation

Metabolic bioactivation of polycyclic aromatic hydrocarbons, such as the environmental procarcinogen benzo[ a]pyrene, is catalyzed by a cytochrome P450 monooxygenase encoded by the substrate-inducible Cyp1a1 gene. Cyp1a1 induction requires trans-activation by the heterodimeric transcriptional complex formed by the liganded Ah receptor (AHR) and its partner, ARNT. Previously, we showed that constitutively bound HDAC1 dissociates from Cyp1a1 promoter chromatin after ligand-mediated induction, concomitantly with the recruitment of AHR/ARNT complexes and p300. Here, we investigated the hypothesis that HDAC1 binding maintains the Cyp1a1 gene in a silenced state in uninduced cells. We find that Cyp1a1 induction by the AHR/ARNT is associated with modification of specific chromatin marks, including hyperacetylation of histone H3K14 and H4K16, trimethylation of histone H3K4, and phosphorylation of H3S10. HDAC1 and DNMT1 form complexes on the Cyp1a1 promoter of uninduced cells but HDAC1 inhibition alone is not sufficient to induce Cyp1a1 expression, although it allows for the hyperacetylation of H3K14 and H4K16 to levels similar to those found in B[ a]P-induced cells. These results show that by blocking the modification of histone marks, HDAC1 plays a central role in Cyp1a1 expression and that its removal is a necessary but not sufficient condition for Cyp1a1 induction, underscoring the requirement for a concerted series of chromatin-remodeling events to complete the initial steps of gene trans-activation by the Ah receptor.

The Arylhydrocarbon Receptor Sees the Light

When cells are exposed to ultraviolet light (UVB) the UV response is initiated. This signaling response has two main pathways. The first occurs in the nucleus where UV irradiation causes the formation of DNA photoproducts. A second, DNA-independent, cytoplasmic signaling pathway activates members of the family of mitogen-activated protein kinases (MAPKs). Activation of MAPKs and the resulting inflammation of the skin are important in the development of skin cancers. Although the identity of the initial signaling molecule of this pathway is unknown, some clues pointed to the involvement of the arylhydrocarbon receptor (AhR). AhR is a cytosolic receptor that mediates the toxic effects of polycyclic aromatic hydrocarbons. Upon binding to such compounds, it dissociates from its chaperones, Hsp90 (heat shock protein 90 kD) and c-src, and translocates to the nucleus, where it activates the transcription of cytochrome P450 (also known as CYP ), resulting in the synthesis of CYP protein, an enzyme important in the breakdown of toxic substances. Exposure of human skin to UVB results in the synthesis of CYP, and in vitro UVB irradiation of human cells increases expression of CYP . Fritsche et al . exposed a human keratinocyte cell line (HaCaT), transfected with a plasmid encoding an AhR-green fluorescent protein (GFP) fusion protein, to UVB radiation and monitored (by fluorescent microscopy) translocation of AhR-GFP to the nucleus. Real-time polymerase chain reaction assays showed that UVB irradiation of HaCaT cells (which have endogenous AhR) resulted in increased transcription of CYP1A1 compared with that in cells that had been treated with either an AhR-specific inhibitor or an AhR-specific siRNA (AhR KO) before UVB irradiation. UVB irradiation causes internalization of epidermal growth factor receptors (EGFRs), and the authors used fluorescence microscopy and Western blotting to show that UVB irradiation of HaCaT cells, but not AhR KO cells, resulted in EGFR internalization and the phosphorylation of MAPKs. The internalization of growth factor receptors has been suggested as a crucial early step in the cytosolic UV response. AhR nuclear translocation and CYP1A1 expression were blocked in cells cultured in media depleted of tryptophan. Tryptophan is converted to the AhR ligand FICZ (6-formylindolo[3,2-b]carbazole) after exposure to UVB, but the authors are the first to show this process occurring in UVB-treated human cells. Both CYP1A1 expression and EGFR internalization were measurable in FICZ-treated HaCaT cells, but not in AhR KO cells. FICZ-mediated internalization of EGFR was blocked by inhibition of c-src activity. Finally, the authors showed that UVB irradiation of wild-type, but not AhR-deficient, mice resulted in the induction of CYP1A1 expression, establishing a role for AhR in mediating the UVB stress response in vivo. E. Fritsche, C. Schäfer, C. Calles, T. Bernsmann, T. Bernshausen, M. Wurm, U. Hübenthal, J. E. Cline, H. Hajimiragha, P. Schroeder, L.-O. Klotz, A. Rannug, P. Fürst, H. Hanenberg, J. Abel, J. Krutmann, Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmic target for ultraviolet B radiation. Proc. Natl. Acad. Sci. U.S.A. 104 , 8851-8856 (2007). [Abstract] [Full Text]

The induction of CYP1A1 by oltipraz is mediated through calcium-dependent-calpain

The induction of CYP1A1 expression by oltipraz, a synthetic chemo-preventive agent, which increases intracellular calcium concentration, has previously been shown to result from transcriptional activation of CYP1A1 gene mediated by the Ah receptor (AhR), although oltipraz does not bind the receptor. The present study investigated the possible mechanisms of oltipraz-induced activation of AhR and the subsequent induction of CYP1A1 transcription. Treatment of the human metastatic breast cancer cell line MT-2 with oltipraz results in a concentration-dependent increase in the activity of the calcium-dependent calpain, as measured towards the BOC-LM-CMAC fluorescent substrate. This increase in calpain activity was coupled with the AhR activation, as evidenced by its nuclear localization and increased transcription of CYP1A1 gene. Treatment of cells with calpain specific inhibitor MDL 28170 completely blocked the oltipraz-induced nuclear translocation of AhR and subsequent CYP1A1 expression. Furthermore, treatment with oltipraz resulted in the classical ligand-dependent down-regulation of AhR protein, in a concentration dependent manner. The presented data established for the first time a mechanism of activating AhR and its transcription of CYP1A1 by oltipraz through activation of calcium-dependent calpain.

Ketoconazole and Miconazole Are Antagonists of the Human Glucocorticoid Receptor: Consequences on the Expression and Function of the Constitutive Androstane Receptor and the Pregnane X Receptor

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) play a major part in the control of drug metabolism and transport. We have previously shown that PXR and CAR expression is controlled by the glucocorticoid receptor (GR) and proposed the existence of a signal transmission cascade GR-(PXR/CAR)-drug metabolizing and transporter systems. In the current study, we investigated the effect of ketoconazole and other azole-derived drugs, miconazole and fluconazole, on the transcriptional activity of the human GR (hGR) in HeLa and HepG2 cells, and in primary human hepatocytes. The data show that ketoconazole inhibits GR transcriptional activity and competes with dexamethasone for hGR binding. In primary human hepatocytes, ketoconazole inhibits the expression of 1) GR-responsive genes tyrosine aminotransferase and both PXR and CAR; 2) CAR and PXR target genes, including cytochromes P450 (P450) CYP2B6, CYP2C9, and CYP3A4; UDP-glucuronosyltransferase 1A1, glutathione S-transferases A1 and A2; and transporter proteins (phase III) solute carrier family 21 form A6 and multidrug resistance protein 2. In parallel experiments, ketoconazole affected neither the expression of GR, the expression of glyceraldehyde-3-phosphate dehydrogenase, nor the inducible expression of CYP1A1 and 1A2. Miconazole behaved like ketoconazole, whereas fluconazole had no effect. We conclude that, in addition to their well known inhibitory effect on P450 enzyme activities, ketoconazole and miconazole are antagonists of hGR. These results provide a novel molecular mechanism by which these compounds may exert adverse and toxic effects on drug metabolism and other functions in human.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Role of Microtubules Network in CYP Genes Expression

Superfamily of cytochrome P450 enzymes (CYPs), a distinctive enzyme system by which human body defends itself against toxic compounds, is the subject of a complex regulation process involving various mechanisms, on the levels of expression and activity. Apart from physiological factors, several patho-physiological ones such as inflammation, infection, and stress affect CYP expression. The aim of this review is to summarize the current knowledge on the role of microtubules network in the regulation of drug metabolizing CYPs. Experiments on human and animal cell models revealed that microtubules disruption severely impaired basal and inducible expression of human CYP 1A1, 2B6, 2C8, 2C9, 2C19, and 3A4, and rat CYP 1A2, 2B1, 2B2, and 3A23. Inhibition of aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) transcriptional activity by microtubules disarray was found to be responsible for the suppressed CYP enzymes expression. However, the mechanism by which microtubules interfering agents (MIAs) inhibit GR and AhR transcriptional activities is not fully understood yet. Several lines of evidence indicate that: i) the cell cycle, G2/M phase in particular, has an influence on AhR and GR transcriptional activity, and ii) MIAs negatively modulate GR transcriptional activity via the activation of c-Jun-N-terminal kinase. In conclusion, down-regulation of major CYP enzymes by microtubules disarray is intriguing from the mechanistic point of view and in relation to the cell differentiation.

Arsenite Inhibition of CYP1A1 Induction by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Is Independent of Cell Cycle Arrest

We show here that arsenite (As(3+)) elicits multiple effects on gene control, such as the interruption of cell cycle control by initiating G(2)/M arrest as well as inhibiting the aryl hydrocarbon (Ah) receptor-mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible expression of CYP1A1. This raises the question as to whether As(3+) is selectively inhibiting TCDD induction of CYP1A1 independent of cell cycle control. As(3+) stimulated a concentration-dependent increase in G(2)/M phase arrest that was detected at 12.5 microM As(3). However, cotreatment of HepG2 cells with TCDD and concentrations of As(3+) as low as 0.5 microM stimulated a pronounced decrease in the induction of CYP1A1-dependent ethoxyresorufin-O-deethylase activity and protein, indicating that the inhibition of CYP1A1 induction by As(3+) was considerably more sensitive than As(3+)-initiated cell cycle arrest. Low concentrations of As(3+) also initiate a dose-dependent reduction in TCDD-induced mouse Cyp1a1 as well as human CYP1A1 in primary hepatocytes cultured from transgenic CYP1A1N(+/-) mice. Because primary hepatocytes in culture are quiescent, these results indicate that the actions of As(3+) on TCDD-initiated induction of CYP1A1 are independent of cell cycle control. As(3+) does not impact on Ah receptor function as evaluated by nuclear transport and binding to xenobiotic responsive element sequences, but it does reduce TCDD-induced CYP1A1 mRNA, a property that is concordant with RNA polymerase II association to the gene and the reduction in transcriptional heteronuclear RNA. We conclude from these studies that interruption of CYP1A1-induced transcription by As(3+) is not dependent upon cell cycle arrest.

The experimental antitumor agents Phortress and doxorubicin are equiactive against human-derived breast carcinoma xenograft models.

Phortress (the dihydrochloride salt of the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203)) is an experimental antitumor agent with potent and selective activity against human-derived carcinomas of breast, ovarian and renal origin. UK clinical trials of Phortress are scheduled to begin in 2004. The mechanism of action of Phortress is distinct from all classes of chemotherapeutic agents currently in the clinic, and involves metabolic activation by cytochrome P450 (CYP) 1A1 to electrophilic species, which generate DNA adducts in sensitive tumors only. In the present study, the antitumor efficacy of Phortress has been compared with that of doxorubicin (Dox) in nine human-derived mammary carcinoma xenograft models, cultivated subcutaneously in the flanks of nude mice. In addition, cyp1a1 mRNA expression was measured in tumors of control and treated animals. Phortress compared favorably with Dox: significant activity, independent of estrogen receptor (ER) status, was established in 7/9 xenografts; in one xenograft model, Phortress elicited superior antitumor activity; no model demonstrated complete resistance to Phortress. In accordance with this observation, all xenografts available for examination (8) displayed clear induction of cyp1a1 expression upon treatment of mice with Phortress whereas Dox failed to induce cyp1a1 expression in all models. Prolonged viability of tumor fragments, recovered for treatment ex vivo could not be sustained; thus correlations between tumor cells' response to Phortress and cyp1a1 or cyp1b1 inducibility following 5F 203 treatment could not be determined with confidence.

Different Structural Requirements of the Ligand Binding Domain of the Aryl Hydrocarbon Receptor for High- and Low-Affinity Ligand Binding and Receptor Activation

The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is responsible for the regulation of several response genes, of which the best characterized is the CYP1A1 gene. The present study was undertaken to elucidate the mechanism of activation of the AhR by omeprazole (OME), 2-mercapto-5-methoxybenzimidazole (MMB), and primaquine (PRQ), compounds that have previously been reported to induce CYP1A1 expression but that are not typical AhR ligands. All compounds caused a significant increase in luciferase activity in rat H4IIE and human HepG2 hepatoma cells transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. Furthermore, MMB and PRQ, but not OME, were capable of transforming cytosolic AhR to a DNA-binding form and displacing AhR-bound [3H]TCDD in rat hepatic cytosol in vitro. By performing site-directed mutagenesis of residues in the ligand-binding domain of the Gal4-AhR, a construct containing a Y320F substitution was found to be resistant to activation by OME, MMB, and PRQ, but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparable affinities of [3H]TCDD-binding to the wild-type and the Y320F mutant Gal4-proteins, expressed in human embryonic kidney 293 cells, were obtained in the ligand-binding assay. In contrast, the competition of receptor-bound [3H]TCDD by PRQ was absent from Gal4-Y320F but not from Gal4-AhR cell extracts. The results of this study confirm that MMB and PRQ are low-affinity ligands for the AhR and suggest that high- and low-affinity ligands interact with different residues of the AhR ligand-binding pocket. In addition, the data presented here indicate that Tyr320 plays an important role in AhR activation.

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells.

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.

Smoking-Induced Vascular Disease

Polycyclic aromatic hydrocarbons (PAHs) are recognized widely as potential environmental procarcinogens and have attracted their share of attention from both funding agencies and scientists interested in biomedical research. Curiously, their potential relevance to cardiovascular disease, rather than cancer, has stirred less emotion. This imbalance of attention is reflected also in society’s response to that most intimate form of self-pollution with PAHs (and much else), cigarette smoking. While the States have chosen to allocate varying proportions of their Tobacco Settlements to research, cancer tends to dominate the agenda. Yet, tobacco-related cardiovascular disease yields an even more striking burden on the public health than cancer. It has been estimated that cardiovascular morbidity and mortality are increased 4-fold in smokers and by one third in passive smokers.1 One might think that such daunting figures would prompt advocacy groups, such as the American Heart Association, to lobby vigorously at the State level for an appropriate allocation of the Tobacco Settlements to cardiovascular research. Our understanding of the mechanisms by which smoking mediates cardiovascular injury and the factors that determine interindividual susceptibility to smoking-induced tissue injury is remarkably limited. In this issue of Circulation Research , Kerzee and Ramos2 draw our attention to a mechanism of potential relevance to acceleration of atherogenesis by PAHs, including those in cigarette smoke. The aryl hydrocarbon receptor (Ahr) is a member of the family of basic helix loop helix proteins (bHLHs) that contain PAS domains. This family includes proteins related to differentiation, such as MyoD, the response to hypoxia, such as Hif1α, and circadian rhythms, such as CLOCK, …

Characterization of CYP1A1 and CYP1A3 gene expression in rainbow trout ( Oncorhynchus mykiss)

The rainbow trout CYP1A1 and CYP1A3 genes share 96% amino acid identity and have similar enzymatic activity. The expression of CYP1A1 and 1A3 genes was investigated in 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-treated rainbow trout tissues, sac fry and cell lines. Both CYP1A1 and CYP1A3 were induced by TCDD in all the tissues examined. While CYP1A3 gene was expressed constitutively at higher levels than CYP1A1 in trout intestine, preferential expression of CYP1A1 occurred in trout liver, heart, kidney and trout sac fry. In rainbow trout gonad (RTG)-2 and rainbow trout hepatoma (RTH)-149 cell lines, CYP1A1 was constitutively expressed and induced by exposure to TCDD, but CYP1A3 message was not detected, even after TCDD treatment. Quantitative analysis of CYP1A genes expression in rainbow trout liver revealed that TCDD induced CYP1A1 expression more than 50-fold and CYP1A3 RNA levels increased at least 100-fold over untreated fish. The cell- and tissue-specific expression indicates that these closely related CYP1A genes are independently regulated and that negative regulation may play a role in CYP1A3 gene expression.

Differential effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin and benzo( a)pyrene on proliferation and growth factor gene expression in human choriocarcinoma BeWo cells

Summary Benzo( a )pyrene (BaP) is a major polycyclic hydrocarbon toxicant in cigarette smoke, while 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) is the reference compound for the toxicity of halogenated aromatics. Our laboratory has previously reported that EGFR expression and hCG secretion were significantly downregulated by BaP in association with the induction of CYP1A1 in BeWo cells. This study presents evidence that TCDD markedly induced CYP1A1 expression but did not alter total cellular EGFR protein level or EGF binding. Data further showed that both TCDD and BaP affected hCG secretion, whereas only BaP significantly decreased cell proliferation. TCDD significantly increased the steady state mRNA level of transforming growth factor (TGF)-α in BeWo cells, while the mRNA levels of TGF-β1 and plasminogen activator inhibitor-2 (PAI-2) were unchanged by TCDD. In contrast, BaP had no significant effect on the mRNA levels of TGF-α and PAI-2, while TGF-β1 mRNA level was significantly increased. Thus, evidence from this study does not support a direct relationship between CYP1A1 induction and EGFR downregulation in BeWo cells. In addition, placental cell proliferation and endocrine function may be disrupted by environmental chemical exposure, but data also indicate that TCDD and BaP may produce placental toxicity through different mechanisms involving both AhR-dependent and AhR-independent pathways.

Differential Effects of Interleukin-1β, Interleukin-2, and Interferon-γ on the Inducible Expression of CYP 1A1 and CYP 1A2 in Cultured Rabbit Hepatocytes

The effects of interleukin-1β, interleukin-2 and interferon-γ and their combinations were investigated on induced cytochrome P 4501A of cultured rabbit hepatocytes considered 72 h after plating. Without apparent cellular toxicity, these cytokines provoke a significant decrease in TBZ- and BNF-induced P4501A1/2 expression. However specific patterns of action are revealed : IL-1β is the most potent cytokine in regard to CYP1A1/2 mRNA suppression whereas IL-2 exerts repressive effects only on P4501A1 induced expression. Although being a strong inhibitor of induced enzymatic activities and protein contents, IFN-γ exhibits only a weak influence on CYP1A1/2 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1β and IL-2 promote mainly a transcriptional repression mechanism whereas IFN-γ would stimulate a post-transcriptional suppressive pathway.

CAMP-dependent Negative Regulation of Rat Aldehyde Dehydrogenase Class 3 Gene Expression

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.

Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture

We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-1 alpha (Il-1 alpha), and tumor necrosis factor-alpha (TNF-alpha), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to Il-6 and Il-1 alpha, TNF-alpha reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by beta-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: Il-6 had the most potent effect on CYP3A4, whereas TNF-alpha was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with beta-naphthoflavone or rifampicin, the decay with time of the levels of the CYP1A2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures.

Differential effects of cytokines on the inducible expression of cyp1a1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture

We have investigated the effect of cytokines, including interleukin-6 (11-6), interleukin-lα (II-lα), and tumor necrosis factor-alpha (TNF-α), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to 11-6 and II-lα, TNF-α reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by β-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: 11-6 had the most potent effect on CYP3A4, whereas TNF-α was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with β-naphthoflavone or rifampicin, the decay with time of the levels of the CYPIA2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures. (Hepatology 1995; 22: 1143–1153.)