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Background & Aims: The multidrug resistance protein (MRP) functions as an organic anion efflux carrier. Recent studies suggest that hepatocytes contain two mrp homologues, named mrp1 and mrp2, localized on the lateral and canalicular membrane, respectively. The aim of this study was to evaluate the role of MRP1. Protein levels and localization of MRP1 in human hepatocytes, HepG2 hepatoma cells, and SV40 large T antigen-immortalized human hepatocytes were studied. Methods: Using specific antibodies, MRP1 protein levels and cellular localization were examined by Western blotting and fluorescence confocal microscopy, respectively. In addition, a fluorescent substrate, glutathione-methylfluorescein, was used to measure plasma membrane transport activity and to observe intracellular transport activity. Results: The level of MRP1 in normal hepatocytes is very low. In contrast, MRP1 is highly increased in both HepG2 and immortalized hepatocytes. In these cells MRP1 is localized in lateral membranes of adjacent cells. Plasma membrane staining is absent in separate cells. Glutathione-methylfluorescein is transported in the medium and intracellular vesicles. Conclusions: MRP1 protein level is greatly increased in the lateral membrane of proliferating hepatocyte-derived cells. The presence of a lateral domain seems necessary for plasma membrane localization. These results suggest that MRP1-mediated organic anion transport is important in proliferating hepatocytes, but not in quiescent cells.

The chicken very low density apolipoprotein II (apoVLDLII) gene is estrogen-inducible and specifically expressed in liver. We examined the possible involvement of the retinoid X receptor (RXR) and its ligand 9-cis-retinoic acid (9-cis-RA) in the activation of the apoVLDLII promoter. We first concentrated on a potential RXR recognition site, which deviates at only one position from a perfect direct A/GGGTCA repeat spaced by one nucleotide (DR-1) and was earlier identified as a common HNF-4/COUP-TF recognition site. However, band shift analysis revealed that this imperfect DR-1 motif does not interact with RXR alpha-homodimers. In accordance with this observation we found that this regulatory element does not mediate transactivation through RXR alpha in the presence of 9-cis-RA. However, our experiments revealed another, unexpected, effect of 9-cis-RA. Instead of stimulating, 9-cis-RA attenuated estrogen-induced expression of transfected estrogen-responsive VLDL-CAT reporter plasmids. This repression appeared to take place through the main estrogen response element (ERE) of the gene. Importantly, 9-cis-RA also strongly repressed the estrogen-induced expression of the endogenous apoVLDLII gene in cultured chicken hepatoma cells.

The gene coding for chicken very low density apolipoprotein II (apoVLDLII) is expressed exclusively in liver in response to estrogen. Previous work in our laboratory identified several protein binding sites, identified by the letters A to F, and their cognate factors within the first 300 bp flanking the gene. Here we present an extensive functional analysis of the apoVLDLII promoter by gene transfer experiments using a chicken hepatoma cell line and cultured non-hepatic cells. Deletion analysis revealed that the -301 to -163-bp promoter region, comprising elements E1, E2 and F, is sufficient for strong estrogen-dependent expression. Mutation analysis demonstrated that efficient transcription requires the interplay of the major estrogen response element El with several other cis-acting elements. Analysis of individual protein binding sites showed that element El is sufficient by itself to confer weak estrogen-induced transcription from the apoVLDLII promoter, and that additional promoter elements are required for full estrogen-responsiveness. Elements F and B1 were capable of strongly potentiating the activity of element E1. In general, the activity of certain cis-acting elements appeared to be strongly promoter-context dependent. Cultured non-liver cells expressed transfected VLDL-CAT reporter plasmids in the presence of cotransfected estrogen receptor expression vector in a hormone-dependent way, indicating that for the control of tissue specificity the 5'-proximal promoter region is not sufficient.

The chicken Very-Low-Density Apolipoprotein ll (apoVLDL II) gene is specifically expressed in liver in response to estrogen. In this study, we performed a functional analysis of the 300-base pair region immediately 5' to the gene by gene transfer of chloramphenicol acetyl transferase (CAT) constructs into chicken embryonic hepatocytes (CEH). Two estrogen response elements (EREs) could be distinguished which together form a potent estrogen response unit. Stimulation of transient expression by co-transfection with a plasmid expressing rat C/EBP confirmed that a similar protein in chicken liver may be involved in apoVLDL ll transcription. In vitro DNasel footprinting and band-shift analysis with liver, oviduct and spleen nuclear extract revealed the tissue distribution of the proteins binding to the promoter region. A liver-specific protein bound to multiple sites of which some resembled the recognition sequence of the CCAAT/Enhancer binding protein, C/EBP. Of the other proteins binding to the apoVLDL 11 promoter, one was identified as the liver-specific LF-A1 by mobility shift analysis, using purified bovine LF-A1, and another as the general COUP-transcription factor, using an antiserum against the human COUP-TF.