The role of prostaglandin E receptor-dependent signaling via cAMP in Mdr1b gene activation in primary rat hepatocyte cultures.

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 E(2) release. In the present study, the prostaglandin E (EP) receptor agonist misoprostol (1-10 microg/ml) further enhanced intrinsic mdr1b mRNA expression in primary rat hepatocytes. On the other hand, [1alpha(z),2beta,5alpha]-(+)-7-[5-[1,1'-(biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848B) (30 microM), 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 microM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 microM) 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.

Transcriptional regulation of CYP2B1 induction in primary rat hepatocyte cultures: repression by epidermal growth factor is mediated via a distal enhancer region.

Phenobarbital (PB) alters expression of numerous hepatic genes, including genes involved in xenobiotic metabolism. Phenobarbital-dependent induction of cytochrome P-450 2B1 (CYP2B1) is subject to regulation by cytokines [e.g., by epidermal growth factor (EGF)], hormones [e.g., by growth hormone (GH)], or the cellular redox status. To investigate mechanisms involved in regulation of CYP2B1 transcription, we performed promoter activation studies using primary rat hepatocyte cultures transiently transfected with individual CYP2B1 promoter-luciferase reporter gene constructs. The 2679-bp native 5'-flanking region of the CYP2B1 gene conferred reporter gene activation by PB and the potent PB-like inducer permethrin (PM). Furthermore, this region mediated EGF- and GH-dependent repression of gene activation by PB-like inducers. A wide promoter mapping strategy with constructs bearing internal CYP2B1 promoter deletions led to identification of a distal responsive CYP2B1 enhancer region at -2230 to -2170, encompassing the section equivalent to the 51-bp PB-responsive enhancer module situated in the distal mouse Cyp2b10-5'-flanking region. The distal CYP2B1 enhancer region conferred gene activation by PM, repression of PM-dependent activation by EGF, and enhancement of activation by the antioxidant N-acetylcysteine (NAC). Mutational analyses of the region at -2230 to -2170 suggested that the mechanisms of PB-dependent induction of CYP2B1 and the modulating effects by EGF or NAC are closely related.

The cyclooxygenase system participates in functional mdr1b overexpression in primary rat hepatocyte cultures.

Overexpression of mdr1-type P-glycoproteins (P-gps) is thought to contribute to primary chemotherapy resistance of untreated hepatocellular carcinoma. However, mechanisms of endogenous multidrug resistance 1 (mdr1) gene activation still remain unclear. Because recent studies have demonstrated overexpression of cyclooxygenase-2 (COX-2) in hepatocytes during early stages of hepatocarcinogenesis, we investigated whether the COX system, which catalyzes the rate-limiting step in prostaglandin synthesis, participates in mdr1 gene regulation. In the present study, primary rat hepatocyte cultures, exhibiting time-dependent mdr1b overexpression, demonstrated basal COX-2 and COX-1 mRNA expression and liberation of prostaglandin E(2) (PGE(2)), indicative of an active COX-dependent arachidonic acid metabolism. PGE(2) accumulation in culture supernatants was further enhanced by arachidonic acid (1mumol/L) and epidermal growth factor (EGF) (16 nmol/L). PGE(2) and prostaglandin F(2alpha) (PGF(2)alpha) (3-6mug/mL), added directly to the culture medium, significantly up-regulated intrinsic mdr1b mRNA overexpression and mdr1-dependent transport activity. Up-regulation was maximal after 3 days of culture. Like prostaglandins, the COX substrate, arachidonic acid, also induced mdr1b gene expression. Apart from this, structurally different COX inhibitors (indomethacin, meloxicam, NS-398) mediated significant inhibition of time-dependent and EGF-induced mdr1b mRNA overexpression, resulting in enhanced intracellular accumulation of the mdr1 substrate, rhodamine 123 (Rho123). Thus, the present data support the conclusion that the release of prostaglandins through activation of the COX system participates in endogenous mdr1b gene regulation. COX-2 inhibition might constitute a new strategy to counteract primary mdr1-dependent chemotherapy resistance.