RESUMO
The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively. The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação da Expressão Gênica , Proteínas Mitocondriais , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae , 2-Acetilaminofluoreno/farmacologia , Sequência de Bases , Clonagem Molecular , DNA Complementar , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Ácidos Hidroxâmicos/farmacologia , Immunoblotting , Dados de Sequência Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Nocodazol/farmacologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: The Dubin-Johnson syndrome is characterized by conjugated hyperbilirubinemia and by impaired secretion of anionic conjugates from hepatocytes into bile. Absence of the multidrug-resistance protein 2 (MRP2; symbol ABCC2), an adenosine triphosphate-dependent conjugate export pump, from the hepatocyte canalicular membrane is the molecular basis of this syndrome. The aim of this study was the elucidation of all exon-intron boundaries of the MRP2 gene as a prerequisite for the analysis of mutations in patients with Dubin-Johnson syndrome. METHODS: Exon-intron boundaries of MRP2 were determined, and the amplified exons were screened for mutations. Immunofluorescence microscopy served to localize the MRP2 protein in human liver. RESULTS: The human MRP2 gene is approximately 45 kilobases long; it contains 32 exons and a high proportion of class 0 introns. In 2 patients with Dubin-Johnson syndrome, we detected a nonsense mutation at codon 1066 and a 6-nucleotide deletion mutation affecting codons 1392-1394. The MRP2 protein was absent from the canalicular membrane of both patients. CONCLUSIONS: The mutations detected so far show that various mutations in the MRP2 gene can lead to the Dubin-Johnson syndrome. The exon-intron boundaries established in this article will facilitate the analysis of additional mutations in the MRP2 gene.
