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Contribution of the EP4-receptor, PKA and CREB to intrinsic mdr1b overexpression in rat hepatocyte cultures

Der EP4-Rezeptor, PKA und CREB sind an der intrinsischen mdr1b Überexpression von Rattenhepatozytenkulturen beteiligt
: Ziemann, C.; Rüdell, G.; Riecke, A.; Steinfelder, H.J.; Oetjen, E.; Hirsch-Ernst, K.I.

Naunyn-Schmiedebergs archives of pharmacology 369 (2004), Supplement 1, pp.R155
ISSN: 0028-1298
ISSN: 1432-1912
Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (Spring Meeting) <45, 2004, Mainz>
Journal Article, Conference Paper
Fraunhofer ITEM ()

Mdrl-type P-glycoproteins mediate ATP-dependent elimination of various amphi-philic cations. Since intrinsic mdrl overexpression can contribute to chemotherapy resistance in tumour cells, it is important to elucidate endogenous mechanisms of mdr1 gene regulation. In previous studies, primary rat hepatocyte cultures have been characterized as a model for intrinsic mdrl overexpression, as they reveal intrinsic, time-dependent mdrlb up-regulation, which is due to transcriptional activation of the mdr1b gene. Using rat hepatocytes cultures, we have formerly demonstrated that cyclooxygenase-2-dependent liberation of prostaglandin E2 (PGE2) contributes to intrinsic mdrlb overexpression and that overexpression is further enhanced by the stable cAMP analogue 8-Br-cAMP or the phosphodiesterase inhibitor IBMX. Since stimulation of the PGE2-receptors EP2 and EP4 is associated with an increase in intracellular cAMP levels, we addressed the question whether these PGE2-receptors participate in intrinsic mdr1b gene activation. In the present study, rat hepatocytes cultured with the specific EP4-receptor antagonist AH23848B indeed demonstrated a concentration-dependent decrease in intrinsic mdrlb mRNA overexpression (in Northern blot analyses). This decrease was maximal (~62% inhibition) at 15-30µM after 24h of incubation. The potent EPl/EP2-receptor antagonist AH6809 was less effective (~25% inhibition at 5-10µM). To further elucidate the signal transduction cascade leading to intrinsic, PGE2-receptor-mediated mdr1b gene activation, we focussed on cAMP-dependent protein kinase A (PKA) and PKA-associated transcription factors. Transient transfection assays with rat hepatocyte cultures were performed, using a luciferase reporter gene, controlled by 1264 bp of the mdr1b gene 5'-flanking region (-1074 to +150bp). The high intrinsic mdrlb promoter activity in this system could be markedly decreased by overexpression (via transfection of expression vectors) of a PKA-inhibitor protein (pPKI) or of a dominant negative mutant of the cAMP responsive element (CRE)-binding protein (KCREB). KCREB co-transfected cells exhibited luciferase activities of ~45% of control cells. In conclusion, these data suggest that stimulation of the EP4-receptor, cAMP-dependent activation of PKA, and CREB or factors which are able to dimerize with CREB contribute to intrinsic mdr1b gene overexpression in primary rat hepatocyte cultures.