Abstract
Multidrug resistance (mdr) proteins of the mdr1 type function as multispecific xenobiotic transporters in hepatocytes. In the liver, mdr1 overexpression occurs during regeneration, cirrhosis, and hepatocarcinogenesis and may contribute to primary chemotherapy resistance. Cultured rat hepatocytes exhibit a time-dependent “intrinsic” increase in functional mdr1b expression, which depends on cyclooxygenase-catalyzed prostaglandin E2 release. In the present study, the prostaglandin E (EP) receptor agonist misoprostol (1–10 μg/ml) further enhanced intrinsic mdr1b mRNA expression in primary rat hepatocytes. On the other hand, [1α(z),2β,5α]-(+)-7-[5-[1,1′-(biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848B) (30 μM), an antagonist of the cAMP-coupled EP4 receptor, and the protein kinase A (PKA) inhibitor, N-(2-[bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H89) (10 nM), repressed intrinsic mdr1b mRNA up-regulation, whereas the stable cAMP analog 8-bromo-cAMP (10 μM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 μM) further enhanced intrinsic mdr1b expression. Primary rat hepatocytes, transiently transfected with reporter gene constructs controlled by mdr1b 5′-gene-flanking regions [–1074 to +154 base pairs (bp) or –250 to +154 bp], demonstrated pronounced mdr1b promoter activity, already without the addition of exogenous modulators. Nevertheless, activity was further stimulated by misoprostol, 8-bromo-cAMP, or IBMX. Cotransfection with expression vectors for PKI, an inhibitor protein of cAMP-dependent PKA, or KCREB, a dominant-negative mutant of the cAMP-responsive element-binding protein (CREB), decreased high-intrinsic mdr1b promoter activity. KCREB also counteracted misoprostol-induced mdr1b promoter activation. In conclusion, these data provide evidence for a pivotal role of EP receptor-stimulated, cAMP-dependent activation of PKA and CREB or CREB-related proteins in mdr1b gene activation in primary rat hepatocytes. Thus, these data might offer potential new target structures for the reversal of primary drug resistance, for example, of liver tumors.
Footnotes
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This work was supported in part by a grant from the “Forschungsförder-programm 2000,” provided by the Faculty of Medicine, University of Göttingen (to C.Z.) and by the Deutsche Forschungsgemeinschaft (SFB 402).
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This work was presented in abstract form at the 43rd Spring Meeting of the German Society of Experimental and Clinical Pharmacology and Toxicology; 2002 March 12–14, Mainz, Germany, at the 44th Spring Meeting of the German Society of Experimental and Clinical Pharmacology and Toxicology; 2003 March 17–22, Mainz, Germany, and at the 15th International Symposium on Microsomes and Drug Oxidations; 2004 July 4–9, Mainz, Germany.
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doi:10.1124/jpet.105.094193.
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ABBREVIATIONS: P-gp, P-glycoprotein; mdr1, multidrug resistance transporter 1; COX, cyclooxygenase; PGE2, prostaglandin E2; EP receptor, prostaglandin E receptor; MDR, multidrug resistance; AH6809, 6-isopropoxy-9-oxoxanthene-2-carboxylic acid; AH23848B, [1α(z),2β,5α]-(+)-7-[5-[1,1′-(biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid; H89, N-(2-[bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide); IBMX, 3-isobutyl-1-methylxanthine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Rho123, rhodamine 123 [(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid methyl ester]; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); DMSO, dimethyl sulfoxide; PKA, protein kinase A; PKI, PKA inhibitor protein; CREB, cAMP-responsive element-binding protein; CRE, cAMP-responsive element.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received August 12, 2005.
- Accepted January 12, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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