Enhanced Expression of the Human Multidrug Resistance Protein 3 by Bile Salt in Human Enterocytes

Abstract

The enterohepatic circulation is essential for the maintenance of bile acids and cholesterol homeostasis. The ileal bile acid transporter on the apical membrane of enterocytes mediates the intestinal uptake of bile salts, but little is known about the bile salt secretion from the basolateral membrane of enterocytes into blood. In the basolateral membrane of enterocytes, an ATP-binding cassette transporter, multidrug resistance protein 3 (MRP3), is expressed, which has the ability to transport bile salts. We hypothesized that MRP3 might play a role in the enterohepatic circulation of bile salts by transporting them from enterocytes into circulating blood through the up-regulation of MRP3 expression, so we investigated the transcriptional control of MRP3 in response to bile salts. MRP3 mRNA levels were increased about 3-fold in human colon cells by chenodeoxycholic acid (CDCA), in a dose-and time-dependent manner. In the promoter assay, the promoter activity of MRP3 was increased about 3-fold over the basal promoter activity when treated with CDCA, and the putative bile salt-responsive elements exist in the region ؊229/؊138 including two ␣-1 fetoprotein transcription factor (FTF)-like elements. Constructs with a specific mutation in the consensus sequence of FTF elements showed no increase in basal transcriptional activity following CDCA treatment. In electrophoretic mobility shift assay with nuclear extracts, specific binding of FTF to FTF-like elements was observed when treated with CDCA. The expression of FTF mRNA levels were also markedly enhanced in response to CDCA, and overexpression of FTF specifically activated the MRP3 promoter activity about 4-fold over the basal promoter activity. FTF thus might play a key role not only in the bile salt synthetic pathway in hepatocytes but also in the bile salt excretion pathway in enterocytes through the regulation of MRP3 expression. MRP3 may contribute as a plausible bile salt-exporting transporter to the enterohepatic circulation of bile salts.

The Fetoprotein Transcription Factor (FTF) Gene Is Essential to Embryogenesis and Cholesterol Homeostasis and Is Regulated by a DR4 Element

“…profiling using the Affymetrix U74Av2 GeneChip array displayed reduced expression (4.3-fold) of FTF target gene mrp3 (20,34), but a strong increase in CYP7A1 mRNA levels (8.4-fold) and a slightly increased SHP mRNA level (array 1 in Table III). Northern blot analysis confirmed the CYP7A1 and SHP results (Fig.…”

“…In FTFϩ/Ϫ liver, FTF target genes mrp3 and CYP7A1 showed opposite changes in steady-state conditions (arrays 1-3, Table III), and CYP7A1, but not mrp3, was inhibited by cholic acid feeding (array CA, Table III). Likewise, in isolated intestinal cells, bile acids activate mrp3 and deactivate ASBT, via their FTF-regulatory domains (20,21). Thus, mrp3 and CYP7A1/ASBT respond to bile acids in opposite directions, as a coherent systemic way to reduce cellular toxicity.…”

“…FTF in the Adult-Converging evidence suggests that FTF basically operates the enterohepatic cholesterol/BA cycle by activating liver BA production and export via CYP7A1 and mrp3 (14 -17,34), intestinal BA absorption via ASBT (21), and BA reentry from intestinal cells into portal blood via mrp3 (20). Short term overexpression of hepatic FTF in transgenic TgF35 mice provides compelling in vivo support for the activating effect of FTF on CYP7A1 and SHP.…”

“…A model emerged whereby FTF would act as a "competence" factor driving the BA pathway, subject to negative feedback regulation by interaction of FTF with orphan receptor SHP, activated by the BA receptor FXR and by FTF itself (15,16,18,19). Additional reports associated further FTF action with membrane transporters of the enterohepatic BA recycling system (20,21) and with pancreatic cholesterol esterase (22), all of which pointing to an important systemic role for FTF in adult cholesterol/BA homeostasis.…”

The MRP family of drug efflux pumps

“…(A recent study showed that MRP2 is also able to transport glycocholate, but the absence of reported bile acid abnormalities in MRP2-deficient humans and rats suggests that the bile salt export pump (BSEP, SPGP) is predominately responsible for this apical activity under normal conditions (Gerloff et al, 1998;Bodo et al, 2003).) The determination that MRP3 can be induced by bile acids, at least in the context of an enterocyte cell line (Caco2), and the identification of MRP3 promoter elements that mediate bile acid induction suggest that the increased levels of bile acids consequent to cholestatic conditions may directly contribute to upregulation of the pump in hepatocytes (Inokuchi et al, 2001). It has also been speculated that MRP3 may be involved in the enterohepatic circulation of bile acids, in that the pump is localized to the basolateral surfaces of enterocytes (Rost et al, 2002), where it may mediate the transport into blood of bile acids carried into the gut by the bile and taken up across the apical surfaces of enterocytes by the ileal Nadependent bile salt transporter.…”