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. 1998 Mar 1;330(Pt 2):889–895. doi: 10.1042/bj3300889

Protein serine/threonine phosphatase inhibitors suppress phenobarbital-induced Cyp2b10 gene transcription in mouse primary hepatocytes.

P Honkakoski 1, M Negishi 1
PMCID: PMC1219221  PMID: 9480906

Abstract

Using a primary hepatocyte culture in which the mouse Cyp2b10 gene transcription is activated by phenobarbital (PB)-type inducers, we examined the cellular signalling mechanisms associated with PB induction. Low nanomolar concentrations of protein serine/threonine phosphatase inhibitors okadaic acid (OA) and calyculin A blocked the induction of CYP2B10 mRNA. Nuclear run-on assays indicated that OA suppressed Cyp2b10 gene transcription. Pretreatment of the cells with an inhibitor of Ca2+/calmodulin-dependent protein kinases ¿1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62)¿ or with a flavonoid, naringin, were completely or partly protective respectively against the OA-mediated suppression of CYP2B10 mRNA. Several other established modulators of protein kinase activities did not greatly affect the induction of CYP2B10 mRNA, nor could they prevent the suppressive effect of OA. Our results indicate that specific protein phosphorylation-dephosphorylation is required for the induction of Cyp2b10 gene expression, which is modulated through multiple endogenous and exogenous signals.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aubrecht J., Hirsch-Ernst K. I., Becker-Rabbenstein V., Kahl G. F., Taniguchi H., Höhne M. W. Induction of cytochrome P-4502B1-related mouse cytochrome P-450 and regulation of its expression by epidermal growth factor/transforming growth factor alpha in primary hepatocyte culture. Biochem Pharmacol. 1995 Sep 7;50(6):781–785. doi: 10.1016/0006-2952(95)00200-j. [DOI] [PubMed] [Google Scholar]
  2. Barker C. W., Fagan J. B., Pasco D. S. Interleukin-1 beta suppresses the induction of P4501A1 and P4501A2 mRNAs in isolated hepatocytes. J Biol Chem. 1992 Apr 25;267(12):8050–8055. [PubMed] [Google Scholar]
  3. Blankson H., Holen I., Seglen P. O. Disruption of the cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid. Exp Cell Res. 1995 Jun;218(2):522–530. doi: 10.1006/excr.1995.1187. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Caron J. M. Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol. 1990 Mar;10(3):1239–1243. doi: 10.1128/mcb.10.3.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carrier F., Owens R. A., Nebert D. W., Puga A. Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation. Mol Cell Biol. 1992 Apr;12(4):1856–1863. doi: 10.1128/mcb.12.4.1856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen J. Q., Ström A., Gustafsson J. A., Morgan E. T. Suppression of the constitutive expression of cytochrome P-450 2C11 by cytokines and interferons in primary cultures of rat hepatocytes: comparison with induction of acute-phase genes and demonstration that CYP2C11 promoter sequences are involved in the suppressive response to interleukins 1 and 6. Mol Pharmacol. 1995 May;47(5):940–947. [PubMed] [Google Scholar]
  8. Cooney A. J., Tsai S. Y., O'Malley B. W., Tsai M. J. Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors. Mol Cell Biol. 1992 Sep;12(9):4153–4163. doi: 10.1128/mcb.12.9.4153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Denison M. S., Whitlock J. P., Jr Xenobiotic-inducible transcription of cytochrome P450 genes. J Biol Chem. 1995 Aug 4;270(31):18175–18178. doi: 10.1074/jbc.270.31.18175. [DOI] [PubMed] [Google Scholar]
  10. Enslen H., Sun P., Brickey D., Soderling S. H., Klamo E., Soderling T. R. Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation. J Biol Chem. 1994 Jun 3;269(22):15520–15527. [PubMed] [Google Scholar]
  11. Fraslin J. M., Kneip B., Vaulont S., Glaise D., Munnich A., Guguen-Guillouzo C. Dependence of hepatocyte-specific gene expression on cell-cell interactions in primary culture. EMBO J. 1985 Oct;4(10):2487–2491. doi: 10.1002/j.1460-2075.1985.tb03960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gordon P. B., Holen I., Seglen P. O. Protection by naringin and some other flavonoids of hepatocytic autophagy and endocytosis against inhibition by okadaic acid. J Biol Chem. 1995 Mar 17;270(11):5830–5838. doi: 10.1074/jbc.270.11.5830. [DOI] [PubMed] [Google Scholar]
  13. Gotoh Y., Nishida E., Sakai H. Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells. Eur J Biochem. 1990 Nov 13;193(3):671–674. doi: 10.1111/j.1432-1033.1990.tb19385.x. [DOI] [PubMed] [Google Scholar]
  14. Gurland G., Gundersen G. G. Protein phosphatase inhibitors induce the selective breakdown of stable microtubules in fibroblasts and epithelial cells. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8827–8831. doi: 10.1073/pnas.90.19.8827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holen I., Gordon P. B., Seglen P. O. Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors. Eur J Biochem. 1993 Jul 1;215(1):113–122. doi: 10.1111/j.1432-1033.1993.tb18013.x. [DOI] [PubMed] [Google Scholar]
  16. Honkakoski P., Moore R., Gynther J., Negishi M. Characterization of phenobarbital-inducible mouse Cyp2b10 gene transcription in primary hepatocytes. J Biol Chem. 1996 Apr 19;271(16):9746–9753. doi: 10.1074/jbc.271.16.9746. [DOI] [PubMed] [Google Scholar]
  17. Honkakoski P., Negishi M. Characterization of a phenobarbital-responsive enhancer module in mouse P450 Cyp2b10 gene. J Biol Chem. 1997 Jun 6;272(23):14943–14949. doi: 10.1074/jbc.272.23.14943. [DOI] [PubMed] [Google Scholar]
  18. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 1995 Jan 27;80(2):225–236. doi: 10.1016/0092-8674(95)90405-0. [DOI] [PubMed] [Google Scholar]
  19. Iynedjian P. B., Jotterand D., Nouspikel T., Asfari M., Pilot P. R. Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system. J Biol Chem. 1989 Dec 25;264(36):21824–21829. [PubMed] [Google Scholar]
  20. Jirtle R. L., Meyer S. A., Brockenbrough J. S. Liver tumor promoter phenobarbital: a biphasic modulator of hepatocyte proliferation. Prog Clin Biol Res. 1991;369:209–216. [PubMed] [Google Scholar]
  21. Klaunig J. E., Ruch R. J. Role of cyclic AMP in the inhibition of mouse hepatocyte intercellular communication by liver tumor promoters. Toxicol Appl Pharmacol. 1987 Nov;91(2):159–170. doi: 10.1016/0041-008x(87)90097-4. [DOI] [PubMed] [Google Scholar]
  22. Le Cam A., Pantescu V., Paquereau L., Legraverend C., Fauconnier G., Asins G. cis-Acting elements controlling transcription from rat serine protease inhibitor 2.1 gene promoter. Characterization of two growth hormone response sites and a dominant purine-rich element. J Biol Chem. 1994 Aug 26;269(34):21532–21539. [PubMed] [Google Scholar]
  23. Lindblad W. J., Schuetz E. G., Redford K. S., Guzelian P. S. Hepatocellular phenotype in vitro is influenced by biophysical features of the collagenous substratum. Hepatology. 1991 Feb;13(2):282–288. [PubMed] [Google Scholar]
  24. Meehan R. R., Forrester L. M., Stevenson K., Hastie N. D., Buchmann A., Kunz H. W., Wolf C. R. Regulation of phenobarbital-inducible cytochrome P-450s in rat and mouse liver following dexamethasone administration and hypophysectomy. Biochem J. 1988 Sep 15;254(3):789–797. doi: 10.1042/bj2540789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mumby M. C., Walter G. Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol Rev. 1993 Oct;73(4):673–699. doi: 10.1152/physrev.1993.73.4.673. [DOI] [PubMed] [Google Scholar]
  26. Nemoto N., Sakurai J. Glucocorticoid and sex hormones as activating or modulating factors for expression of Cyp2b-9 and Cyp2b-10 in the mouse liver and hepatocytes. Arch Biochem Biophys. 1995 May 10;319(1):286–292. doi: 10.1006/abbi.1995.1294. [DOI] [PubMed] [Google Scholar]
  27. Nirodi C. S., Sultana S., Ram N., Prabhu L., Padmanaban G. Involvement of synthesis and phosphorylation of nuclear protein factors that bind to the positive cis-acting element in the transcriptional activation of the CYP2B1/B2 gene by phenobarbitone in vivo. Arch Biochem Biophys. 1996 Jul 1;331(1):79–86. doi: 10.1006/abbi.1996.0285. [DOI] [PubMed] [Google Scholar]
  28. Porter T. D., Coon M. J. Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem. 1991 Jul 25;266(21):13469–13472. [PubMed] [Google Scholar]
  29. Ray A., Ray B. K. Serum amyloid A gene expression under acute-phase conditions involves participation of inducible C/EBP-beta and C/EBP-delta and their activation by phosphorylation. Mol Cell Biol. 1994 Jun;14(6):4324–4332. doi: 10.1128/mcb.14.6.4324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Salonpä P., Pelkonen O., Kojo A., Pasanen M., Negishi M., Raunio H. Cytochrome P4502A5 expression and inducibility by phenobarbital is modulated by cAMP in mouse primary hepatocytes. Biochem Biophys Res Commun. 1994 Nov 30;205(1):631–637. doi: 10.1006/bbrc.1994.2712. [DOI] [PubMed] [Google Scholar]
  31. Schuetz E. G., Schuetz J. D., May B., Guzelian P. S. Regulation of cytochrome P-450b/e and P-450p gene expression by growth hormone in adult rat hepatocytes cultured on a reconstituted basement membrane. J Biol Chem. 1990 Jan 15;265(2):1188–1192. [PubMed] [Google Scholar]
  32. Sidhu J. S., Omiecinski C. J. Modulation of xenobiotic-inducible cytochrome P450 gene expression by dexamethasone in primary rat hepatocytes. Pharmacogenetics. 1995 Feb;5(1):24–36. doi: 10.1097/00008571-199502000-00003. [DOI] [PubMed] [Google Scholar]
  33. Sidhu J. S., Omiecinski C. J. cAMP-associated inhibition of phenobarbital-inducible cytochrome P450 gene expression in primary rat hepatocyte cultures. J Biol Chem. 1995 May 26;270(21):12762–12773. doi: 10.1074/jbc.270.21.12762. [DOI] [PubMed] [Google Scholar]
  34. Sudo Y., Mariash C. N. Two glucose-signaling pathways in S14 gene transcription in primary hepatocytes: a common role of protein phosphorylation. Endocrinology. 1994 Jun;134(6):2532–2540. doi: 10.1210/endo.134.6.8194479. [DOI] [PubMed] [Google Scholar]
  35. Uchida C., Funai T., Oda T., Ohbayashi K., Ichiyama A. Regulation by glucagon of serine: pyruvate/alanine: glyoxylate aminotransferase gene expression in cultured rat hepatocytes. J Biol Chem. 1994 Mar 25;269(12):8849–8856. [PubMed] [Google Scholar]
  36. Varone C. L., Canépa E. T., Llambías E. B., Grinstein M. cAMP regulation of phenobarbital-mediated induction of delta-aminolevulinate synthase mRNA in hepatocytes from normal and experimental diabetic rats. Biochem Cell Biol. 1994 Sep-Oct;72(9-10):381–390. doi: 10.1139/o94-051. [DOI] [PubMed] [Google Scholar]
  37. Vintermyr O. K., Mellgren G., Bøe R., Døskeland S. O. Cyclic adenosine monophosphate acts synergistically with dexamethasone to inhibit the entrance of cultured adult rat hepatocytes into S-phase: with a note on the use of nucleolar and extranucleolar [3H]-thymidine labelling patterns to determine rapid changes in the rate of onset of DNA replication. J Cell Physiol. 1989 Nov;141(2):371–382. doi: 10.1002/jcp.1041410219. [DOI] [PubMed] [Google Scholar]
  38. Wadzinski B. E., Wheat W. H., Jaspers S., Peruski L. F., Jr, Lickteig R. L., Johnson G. L., Klemm D. J. Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol. 1993 May;13(5):2822–2834. doi: 10.1128/mcb.13.5.2822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Waxman D. J., Azaroff L. Phenobarbital induction of cytochrome P-450 gene expression. Biochem J. 1992 Feb 1;281(Pt 3):577–592. doi: 10.1042/bj2810577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Waxman D. J., Morrissey J. J., Naik S., Jauregui H. O. Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response. Biochem J. 1990 Oct 1;271(1):113–119. doi: 10.1042/bj2710113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yamazoe Y., Shimada M., Murayama N., Kato R. Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone. J Biol Chem. 1987 May 25;262(15):7423–7428. [PubMed] [Google Scholar]

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