Elsevier

Biochemical Pharmacology

Volume 57, Issue 6, 15 March 1999, Pages 619-630
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Regulation of cytochrome P4501B1 (CYP1B1) in mouse embryo fibroblast (C3H10T1/2) cells by protein kinase C (PKC)

https://doi.org/10.1016/S0006-2952(98)00344-XGet rights and content

Abstract

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.

Section snippets

Materials

TPA and CC were bought from L. C. Services Inc.; TCDD was obtained from Accustandard, Inc. [α-32P]dCTP and [γ-32P]ATP were purchased from New England Nuclear. Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (DMEM/F12) and RPMI 1640 medium were obtained from GIBCO. FBS was bought from Gemini. Rabbit polyclonal antibody to CYP1B1 was generated in this laboratory. Rabbit antibodies to AhR and Arnt were gifts from Dr. Edward Glover of the McArdle Laboratory. Anti-active MAPK polyclonal antibody

Results

Other studies have demonstrated that CYP1A1 activity is down-regulated in mouse kidney [18], epidermis [19], and human keratinocytes [20] following phorbol ester (TPA) treatment. We therefore investigated if the constitutive and/or TCDD-induced activity of the novel CYP1B1 was affected by TPA treatment in the same manner. The assessment of CYP1B1-catalyzed DMBA metabolism in 10T1/2 cells has been established in this laboratory as a convenient measure of the functional expression of the CYP1B1

Discussion

The present study provides evidence for the possible participation of PKC in the regulation of the novel CYP1B1 in 10T1/2 cells. Our studies reveal that in TPA-exposed cells a considerable amount of PKC activity remained at 6 hr but was diminished dramatically almost to basal level at 12 hr. This diminished PKC activity paralleled CYP1B1 activity in cells that had been exposed for 2 hr to the PKC specific inhibitor CC.

Our observation that activation or inhibition of PKC suppresses basal and

Acknowledgements

This work was funded by a Minority Investigator Supplementary (FII) grant to National Cancer Institute Grant CA 16265 (C.R.J)

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