ABSTRACT
BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. METHODS: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF phi 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF phi 13 cells by flow cytometry. RESULTS: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF phi 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF phi 13 cells. CONCLUSION: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Liver/cytology , Stem Cells/metabolism , ATP Binding Cassette Transporter, Subfamily B/analysis , ATP-Binding Cassette Transporters/analysis , ATP-Binding Cassette Transporters/metabolism , Animals , Cell Division/physiology , Cell Line , Flow Cytometry/methods , Gene Expression , Genes, MDR/genetics , Immunohistochemistry/methods , Male , Multidrug Resistance-Associated Proteins/genetics , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Rats , Rats, Inbred F344 , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Specific Pathogen-Free OrganismsABSTRACT
BACKGROUND & AIMS: Our previous studies have shown that Cryptosporidium parvum induces biliary epithelial cell apoptosis in vivo and causes apoptosis in bystander uninfected biliary epithelia in vitro. We analyzed C. parvum-induced nuclear factor kappa B (NF-kappaB) activation in human biliary epithelial cells and assessed its relevance to epithelial cell apoptosis. METHODS: In vitro models of cryptosporidial infection using a human biliary epithelial cell line were used to assay C. parvum- induced NF-kappaB activation and associated apoptosis. RESULTS: Degradation of I(kappa)B and nuclear translocation of the NF-kappaB family of proteins (p65 and p50) were observed in the biliary epithelial cell cultures directly exposed to the parasite. Activation of NF-kappaB was found only in directly infected cells (but not in bystander uninfected cells). A time-dependent secretion of a known NF-kappaB gene product, interleukin 8, from infected cell cultures was detected. C. parvum-induced biliary epithelial cell apoptosis was limited to bystander uninfected cells. In contrast, inhibition of NF-kappaB activation resulted in apoptosis in directly infected cells and significantly enhanced C. parvum-induced apoptosis in bystander uninfected cells. CONCLUSIONS: These observations support the concept that, while C. parvum triggers host cell apoptosis in bystander uninfected biliary epithelial cells, which may limit spread of the infection, it directly activates the NF-kappaB/I(kappa)B system in infected biliary epithelia thus protecting infected cells from death and facilitating parasite survival and propagation.
