Phorbol ester - mediated suppression of cytochrome P450 Cyp1a-1 induction in murine skin: Involvement of protein kinase C

doi:10.1016/0006-291X(92)90841-8

Biochemical and Biophysical Research Communications

Volume 186, Issue 2, 31 July 1992, Pages 970-976

https://doi.org/10.1016/0006-291X(92)90841-8 Get rights and content

Abstract

Epidermal 7-ethoxyresorufin O-deethylase (EROD) activity was elevated > 100-fold within 4 to 7 h of topical treatment of SENCAR mice with 100 nmol dibenz[a,c]anthracene (DB[a,c]A). Treatment of skin with 2 μg of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 2 to 8 h prior to DB[a,c]A application suppressed induction by 80%. Suppression was dose-dependent over the range of 0.01 to 5 μg TPA (ID₅₀ ∼ 0.6 nmol). EROD activities in normal and TPA-treated epidermis paralleled steady state P450 CYP1A1 mRNA content. Analogs of TPA incapable of activating or down-regulating protein kinase C (PKC) did not suppress induction. Pretreatment of skin with sn-1,2-didecanoylglycerol, an activator of PKC which causes translocation but no down-regulation, did not suppress EROD induction. However, induction was suppressed by chrysarobin, an anthralin analog that causes PKC down-regulation in the absence of prior activation. These studies suggest that PKC participates in the processes associated with Cyp1a-1 induction and that TPA effects Cyp1a-1 induction through its down-regulation of PKC.

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Suppression of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated CYP1A1 and CYP1B1 induction by 12-O-tetradecanoylphorbol-13-acetate: Role of transforming growth factor β and mitogen-activated protein kinases
2001, Biochemical Pharmacology
Citation Excerpt :
Collectively, these findings suggest that MEK/ERK activities neither mediate the suppressive effects of TPA on CYP1A1 induction, nor are required for AHR activation by TCDD. A number of studies document either the suppression [10–14] or enhancement [15–17] of AHR-mediated transcriptional processes by TPA. In our studies, a strong suppression occurred even if 12 hr elapsed between the time of TPA and TCDD exposures.
The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances or suppresses the transcriptional activation of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell/tissue-specific manner. The basis for these effects is not known. Exposure of the immortalized human breast epithelial cell line MCF10A-Neo to TPA at the time of, or up to 12 hr prior to, the addition of TCDD strongly suppressed the transcriptional activation of CYP1A1 and CYP1B1 (ic₅₀ ∼0.5 nM). A recent study (Carcinogenesis 2000;21:1303–12) demonstrated that TPA-treated MCF10A-Neo cells rapidly activate the latent transforming growth factor β (TGFβ) in the serum used to supplement the culture medium. The suppressive effects of TPA on CYP1A1 induction by TCDD in MCF10A-Neo cultures could be partially suppressed by: (a) co-incubation of TCDD + TPA-treated cultures with a neutralizing TGFβ pan antibody; (b) prior removal of latent TGFβ from the culture medium; or (c) switching cultures to serum- and growth factor-free medium immediately before the addition of TPA and TCDD. Exposure of cultures to TPA 24–48 hr prior to subsequent TPA + TCDD treatment not only inhibited the suppressive effects of TPA, but markedly enhanced CYP1A1 mRNA accumulation. TPA caused a rapid and protracted activation of extracellular signal-regulated kinases (ERKs). Pretreatment of cultures with the mitogen-activated protein kinase kinase (MEK) inhibitor PD184352 [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropyl-methoxy-3,4-difluoro-benzamide] completely inhibited ERK activation by TPA. However, PD184352 did not prevent the suppressive effects of TPA on CYP1A1 activation by TCDD. These studies demonstrate that TPA initiates protein kinase C-dependent, ERK-independent processes that suppress CYP1A1 activation by TCDD in MCF10A-Neo cells. Furthermore, TGFβ mediates a small portion of this suppressive activity.
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The effects of the mycotoxin fumonisin B₁ (FB₁) on the hepatic cytochrome P450 system were investigated in male rats dosed daily by oral gavage with 3 mg FB₁ per kg body weight for 9 consecutive days. FB₁ treatment resulted in a reduced weight gain. At the same time, CYP2E activity was increased, which is considered to mark the metabolic changes inherent to growth retardation in young rats. Treatment with FB₁ also resulted in a selective inhibition of CYP2C11 and to a lesser extent, CYP1A2 in liver microsomes obtained from treated animals, whereas it did not affect significantly the activity of CYP2A1/2A2, CYP2B1/2B2, CYP3A1/3A2 and CYP4A. The significant inhibition of CYP2C11 is considered to reflect a suppressed activity of protein kinase activity resulting from the inhibition of sphingolipid biosynthesis caused by FB₁.
Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinase C-mediated modulation of the AhR pathway
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Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the “PMA effect.” A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR · ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR · ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRΔCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRΔCVP · ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR · ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.
Suppressive effects of α-Hederin on 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated murine Cyp1a-1 expression in the mouse hepatoma Hepa-1c1c7 cells
1999, Cancer Letters
Cultured mouse hepatoma cell line Hepa-1c1c7 cells were treated with α-Hederin to assess the role of α-Hederin in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as indicated by analysis of 7-ethoxyresorufin O-deethylation (EROD) activity and Cyp1a-1 protein. When α-Hederin and TCDD were both added to cultures, TCDD-inducible EROD activity was greatly suppressed by α-Hederin in a dose-dependent manner. TCDD-induced Cyp1a-1 protein and mRNA levels were markedly reduced in the concomitant treatment of TCDD and α-Hederin consistent with EROD activity. Electrophoretic mobility shift assay using nuclear extraction of cells revealed that α-Hederin reduced transformation of the Ah receptor to a form capable of specifically binding to an oligonucleotide containing a dioxin-response element (DRE) sequence of the Cyp1a-1 gene. These results suggest that the suppressive effect of α-Hederin on TCDD-induced Cyp1a-1 gene expression in Hepa-1c1c7 cells might be an antagonist of the DNA binding potential of a nuclear Ah receptor.
Regulation of cytochrome P4501B1 (CYP1B1) in mouse embryo fibroblast (C3H10T1/2) cells by protein kinase C (PKC)
1999, Biochemical Pharmacology
The effects of co-treatment of C3H10T1/2 (10T1/2) cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of the novel cytochrome P4501B1 (CYP1B1) were investigated. As monitored by CYP1B1-catalyzed 7,12-dimethylbenzanthracene (DMBA) metabolism, TPA suppressed basal and TCDD-induced DMBA metabolism in a concentration-dependent manner, with a maximum inhibitory concentration of 100 nM. The suppression of CYP1B1 catalytic activity occurred at two time points during which protein kinase C (PKC) was activated and down-regulated in these cells as judged by analyses of cellular PKC content and PKC-inhibitor (chelerythrine chloride)-influenced suppression of CYP1B1 catalytic activity. Experiments in which TCDD and benzanthracene (BA)-induced DMBA metabolism were monitored in PKCβ1-overexpressing 10T1/2 cells revealed that the suppression of CYP1B1 activity is a consequence of cellular PKC elevation. This suppression phenomenon could be accounted for by PKC-mediated suppression of TCDD-induced CYP1B1 mRNA and apoprotein and of nuclear translocation of the Ah-receptor. In contrast, the mitogen-activated protein kinase (MAPK) proteins ERKs 1 and 2 were stimulated by TCDD under conditions in which PKC was activated. Collectively, our results suggest that PKC participates in the regulation of CYP1B1 in 10T1/2 cells, positively by directly suppressing the Ah-receptor signaling pathway, followed by an indirect or negative activation of the MAPK signaling pathway.
Tumor promoter benzoyl peroxide induces sulfhydryl oxidation in protein kinase C: Its reversibility is related to the cellular resistance to peroxide-induced cytotoxicity
1999, Archives of Biochemistry and Biophysics
Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excluding thiol agents present in the purified PKC preparation, low concentrations of BPO modified PKC, resulting in the loss of both kinase activity and phorbol ester binding (IC₅₀= 0.2 to 0.5 μM). This modification, which was not dependent on transition metals, was totally blocked by a variety of thiol agents including GSH, which directly reacted with BPO. Substoichiometric amounts of BPO (0.4 mol/mol of PKC) oxidized two sulfhydryls in PKC and inactivated the enzyme which was readily reversed by dithiothreitol. The regulatory domain having zinc thiolate structures supporting the membrane-inserting region provided the specificity for PKC reaction with BPO, which partitioned into the membrane. Unlike H₂O₂, BPO did not induce the generation of the Ca²⁺/lipid-independent activated form of PKC. Other redox-sensitive enzymes such as protein kinase A, phosphorylase kinase, and protein phosphatase 2A required nearly 25- to 100-fold higher concentrations of BPO for inactivation. BPO also inactivated PKC in a variety of cell types. In the JB6 (30 P⁻) nonpromotable cell line and other normal cell lines, where BPO was more cytotoxic, it readily inactivated PKC due to a slow reversibility of this inactivation by the cell. However, in the JB6 (41 P⁺) promotable cell line, C3H10T1/2 and B16 melanoma cells, where BPO was less cytotoxic, it did not readily inactivate PKC due to a rapid reversibility of this inactivation by an endogenous mechanism. Nevertheless, BPO inactivated PKC at an equal rate in the homogenates prepared from all these cell types. Inclusion of NADPH reversed this inactivation in the homogenates to a different extent, presumably due to a difference in distribution of a protein disulfide reductase, which reverses this oxidative modification. BPO-induced modification of PKC occurred independent of the cellular status of GSH. However, externally added GSH and cell-impermeable thiol agents prevented the BPO-induced modification of PKC. Since BPO readily partitions into membranes, its reaction with redox-cycling thiols of membrane proteins such as PKC may trigger epigenetic events to prevent cytotoxicity, but favor tumor promotion.

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^∗: Current address: Institute of Chemical Toxicology, Wayne State University, 2727 Second Ave., Detroit, MI 48201.

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Phorbol ester - mediated suppression of cytochrome P450 Cyp1a-1 induction in murine skin: Involvement of protein kinase C

Abstract

Pharmac. Ther

J. Biol. Chem

Anal. Biochem

J. Invest. Dermatol

Anal. Biochem

Biochem. Pharmacol

Biochem. Biophys. Res. Commun

Cancer Res

Cancer Res

Drug Met. Dispos

Cancer Res

Mol. Carcinogenesis