ATP binding cassette transporter gene expression in rat liver progenitor cells

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.

Figure 1

mRNA levels of ATP binding cassette (ABC) transporter genes in rat liver after hepatic progenitor cell activation by 2-acetylaminofluorene/partial hepatectomy (2- AAF/PHx) treatment. mRNA expression levels of various ABC transporter genes were determined by real time detection reverse transcription-polymerase chain reaction, as described in materials and methods. Plac/sham, placebo pellet followed by sham operation; Plac/PHx: placebo pellet followed by PHx; 2-AAF/sham, 2-AAF pellet followed by sham operation; 2-AAF/PHx, 2-AAF pellet followed by PHx. Data are means (SD), n=3–5 per group. *p<0.05.

Figure 2

Immunohistochemical staining of OV6 (A, B), C219 (C, D), Mrp1 (E, F), and Mrp3 (G, H) in control rat liver (C, E, F), 2-acetylaminofluorene (2-AAF) treated (A) and 2- acetylaminofluorene/partial hepatectomy (2-AAF/PHx) treated rat liver (day 9) (B, D, F, H). Frozen liver sections were stained with primary antibodies directed against a common epitope in cytokeratin 14 and 19 (OV6, dilution 1/200), all p-glycoproteins (C219, dilution 1/5), Mrp1 (SC-7774, dilution 1/5), or Mrp3 (anti-Mrp3, dilution 1/500). Typical staining patterns of n=3–5 per group. B, bile duct, P, portal tract, C, central vein; arrows indicate progenitor cell reaction. In rats exposed to 2-AAF, OV6 stained the bile duct and a number of ductuli (A). In 2-AAF/PHx treated rats, a massively increased number of ductuli were apparent from intensive OV6 staining (B). In normal liver, C219 weakly stained the canalicular membrane of hepatocytes (C). In 2-AAF/PHx treated rat liver, C219 staining was increased (D). SC- 7774 did not stain normal liver (E) but significantly stained 2-AAF/PHx treated liver (F). In normal liver, anti-Mrp3 stained pericentral hepatocytes (G) and bile ducts (insert in (G)). In treated rat liver there was additional staining of oval cells (H).

Figure 3

mRNA levels of ATP binding cassette (ABC) transporter genes in RLF φ 13 cells, Thy-1 positive cells (Thy-1⁺), hepatocytes (Hep), and cholangiocytes (Chol). Relative expression levels of various ABC transporter genes in the different cell fractions were determined by real time detection reverse transcription-polymerase chain reaction, as described in materials and methods.

Figure 4

Fluorescence histogram. Representative fluorescence histogram of RLF φ 13 cells showing carboxyfluorescein (CF) content after 60 minutes of incubation with or without the specific Mrp inhibitor MK571 (10 μM and 20 μM). Autofluorescence=control.