Article

Seminars in Liver Disease

	Jahrgang Volume 20 (Heft Number 03/2000), Seiten 323-338

Transcriptional Control of Hepatocanalicular Transporter Gene Expression
MICHAEL MÜLLER, Ph.D.
From the Division of Gastroenterology and Hepatology, University Hospital Groningen, The Netherlands

ABSTRACT

Transport processes for larger organic solutes at the canalicular membrane are mainly driven by members of the superfamily of ATP-binding cassette (ABC) transporters. The functions of these transporters range from bile component secretion to xenobiotica and phase II-conjugate export. The transcriptional control of the expression of their respective genes differs, and this may be to guarantee tissue specificity, effective response to stress, or changes in substrate concentrations. Inside the nucleus, the concentration of competing and specifically activated transcription factors determines the transcriptional activation in transporter gene expression. Some transcription factors function as sensors for metabolites (LXR, FXR, CAR, SREBP, PPARs), xenobiotics (PPARs, PXR), oxidative stress (NF-κB, AP-1), or DNA damage (p53). Changes in their nuclear concentrations and activity will influence the transcription rates of the respective target genes that contain specific responsive elements in their 5′-promoter/enhancer DNA sequences. Until now little was known about the transcriptional control of most ABC transporter proteins. However, due to the enormous progress in molecular biology, many tools have become recently available to study and understand the ``battle inside the nucleus'' with respect to hepatic transporter gene expression.

KEYWORD

bile secretion - nuclear hormone receptors - ABC-transporter proteins - tumor necrosis factor

Bile formation is a regulated ATP-dependent process that depends on the coordinated action of a number of transporter proteins in the sinusoidal and canalicular domains of the hepatocyte.[1][2][3] Transporter proteins located in the canalicular membrane are responsible for the rate-limiting and tightly coupled biliary secretion of bile salts, phosphatidylcholine (PC), unesterified cholesterol, and reduced glutathione (GSH) on the one hand and for the excretion of potentially toxic endogenous and exogenous compounds on the other hand (Fig. [1]). These transporter proteins such as the bile salt transporter BSEP, the PC translocator MDR3, the anionic conjugate transporter MRP2, and the multidrug transporter MDR1 all belong to the superfamily of ATP-binding cassette (ABC) transporter protein family.[2][4][5] Because of their different functions, it can be assumed that also the regulation of these transporters and the transcriptional control of the expression of their respective genes will be different. These control mechanisms should allow either hepatocyte-specific gene expression (MDR3, BSEP) or gene expression due to stress response (MDR1, MRP2, and its (baso)lateral homologues MRP1 + MRP3) and response to differences in substrate concentrations (MDR3, MDR1, MRP3). With identification of molecules involved in intracellular signaling and the cloning and characterization of transporter genes and their 5′-flanking DNA regions, insight into the molecular mechanisms of transcriptional regulation of transporter gene expression is progressing. This review updates transcriptional control mechanisms that are involved in the expression of hepatocyte ABC transporters or that have potential impact on transporter gene expression.

GENE TRANSCRIPTION

Gene expression by transcription of mRNA by RNA polymerase II can be regulated at least at five potential control points[6]: activation of the gene structure, initiation of transcription (for most genes the major control point), processing the transcript, transport to cytoplasm, and translation of RNA. Many factors act together with RNA polymerase II: 1) factors of the basal transcription apparatus; 2) not regulated upstream DNA-binding proteins recognizing specific short consensus elements located upstream of the startpoint; and 3) other factors that are inducible or that can be activated and have regulatory functions. These transcription factors bind to so-called responsive elements (RE).

Activity of transcription factors may be controlled by protein synthesis (C/EBP), covalent modification of the protein (c-JUN), ligand binding (nuclear hormone/orphan receptors such as FXR, LXRα, PPARs), cleavage to release the active factor (SREBPs), release after breakdown of an inhibitor (NF-κB), or change of partner (MYC). Because knowledge of molecular events in transporter gene expression is still poor, I first discuss transcription factors that play important roles for liver physiology by controlling gene expression in response to changes in metabolite load or during stress response. Most of these factors are also potential candidates to control ABC transporter gene expression. In fact, some of them are already known to bind to bona fide REs in transporter gene promoter sequences and modulate gene transcription activity (Table [1]).

TRANSCRIPTION FACTORS

Nuclear Ligand-activated Receptors

Nuclear hormone or orphan receptors (NHR or NOR) comprise a large superfamily of ligand-modulated transcription factors that, in part, mediate response to steroids, retinoids, and thyroid hormones and play key roles in development and body physiology (for reviews, see Refs. 7-11). Shortly after their isolation, the strategy of ``reversed endocrinology'' was used to identify orphan ligands of these NORs. This has led to the identification of ligands for RARs (9-cis retinoic acid receptors), RXRs (retinoid X receptors), PPARs (peroxisome proliferator-activated receptors), FXRs (farnesoid X receptors), and LXRs (liver X receptors). Many of the recently identified ``orphans'' turned out not to represent an (unidentified) hormone ligand. For FXR, the endogenous ligands appear to be bile salts[12][13][14] and PPARs bind for example eicosanoids and certain unsaturated fatty acids.[15] These NHRs and several other recently identified NORs such as PXR (pregnane X receptor) and CAR (constitutively activated receptor) require heterodimerization with RXR for high-affinity DNA binding (Table [1]). Furthermore, most factors possess the feature of activating target genes only when bound by specific ligands. In contrast, CAR[16] is deactivated after binding ligands such as androstane metabolites.[17] The preferred organization of the NHR/NOR RE are direct repeats (DR) of AGTTCA or AGGTCA separated by one (DR-1) to five nucleotides (DR-5) (Table [1]). For several NHRs it is known that they recruit transcriptional coactivators (e.g., steroid receptor coactivator-1) after ligand binding that destabilize chromatin by mechanisms that include histone acetylation and contacts with the basal transcription machinery.[18] In contrast, the recruitment of corepressors (e.g., NHR corepressor, silencing mediator of retinoid, and thyroid receptors) in the absence of ligand serves to stabilize chromatin by the targeting of histone deactylases.[18]

Pregnane-activated Receptor PXR Binds Steroids and Xenobiotics

PXR is predominantly expressed in the liver and is transcriptionally activated by pregnanes, synthetic steroids, or steroid antagonists.[19][20][21][22] One of the best activators of PXR is the synthetic glucocorticoid antagonist, pregnenolone-16-carbonitrile, that is also known to induce cytochrome P450 (CYP) 3A in rodent liver and intestine. These inducible members of the rodent Cyp3A gene family contain a DNA-response element that mediates this induction by PXR/RXR. The drug rifampicin is another excellent PXR activator that is also known to be involved in numerous drug-drug interactions as a consequence of drastically altering their metabolism by inducing CYP3A4. These PXR ligands are known substrates of MDR1 and likely also of other ABC transporter proteins, and this may result in ``cross-talks'' between transporters (as ``phase III-drug metabolizing systems'')[23] and phase I-drug metabolizing enzymes via PXR (see below).

Ligand ``Inactivated'' Receptor CAR

Almost all ligand-dependent nuclear receptors are activated by a ligand-induced association with coactivator proteins. Studies of CAR (also termed ``constitutive androstane receptor'') has introduced a new concept in NHR/NOR action.[17] When isolated, CAR was found to be constitutively active as heterodimer with RXR on candidate DR-5 response elements[24] (Table [1]). Further search for ligands resulted in the identification of androstanol and androstenol as selective transcriptional inhibitors.[17] Although the constitutive activity of CAR can be suppressed by androstanes, various phenobarbital-related compounds have been demonstrated to reverse this ``inverse agonist'' effect of androstanes on the CYP2B6 promoter. Phenobarbital is also known as one of the prototype xenobiotic inducers of the CYP2B genes.[25][26][27] Together with PXR and PPARα, CAR appears to control the transcription of CYP genes in response to hydrophobic xenobiotic or endogenous compounds such as drugs, lipids, or steroids. Again, these NHRs may play a role in transcriptional control of the gene expression of ABC transporter proteins that transport NHR ligands (see also below).

Liver X Receptors as Sensors for Dietary Cholesterol

LXRs are a family of transcription factors that were first identified as orphan members of the nuclear receptor superfamily.[28][29] In later studies, naturally occurring oxysterols were identified as physiologic ligands for LXRs.[30][31] By binding as heterodimers with RXR to DR-4-HREs (Table [1]), LXRs control the expression of genes that encode enzymes involved in metabolism of several important lipids, including cholesterol and bile salts (Fig. [2]). Mice lacking the oxysterol receptor, LXRα, lose their ability to respond normally to dietary cholesterol,[32] because in these mice the regulation of cholesterol 7α-hydroxylase (CYP7α) is altered.

Bile Acid Receptor FXR

FXR, originally shown to be activated by high concentrations farnesol, binds to DR-4 REs of target gene promoter in a complex with RXR[11] (Table [1]). FXR is expressed in tissues that are exposed to high concentrations of bile salts, including liver, intestine, and kidney. Recently, FXR has been identified to be activated by bile salts such as chenodeoxycholate, deoxycholate, and their glycine and taurine conjugates (in the low μM range).[12][13][14] Ursodeoxycholate, the 7β-epimer of chenodeoxycholate, was a poor activator indicating that the seven position is a crucial determinant of FXR activity.[14] One of the identified target genes of FXR/RXR is the gene encoding the intestinal bile acid binding protein (I-BABP) (Fig. [2]). The I-BABP promoter was further shown to contain a perfect inverted repeat 1 element (Table [1]). Further, low μM concentrations of bile acids induce I-BABP promoter and reduce CYP7α RNA and protein levels. Chenodeoxycholate appears to be the most potent suppressor of the CYP7α gene. This is likely due to an inhibition of LXRα transactivation of CYP7α by FXR (Fig. [2]). Thus, LXRα and FXR possess opposing functions. Although LXRα is a positive regulator of Cyp7α transcription and bile salt synthesis, FXR functions as an endogenous bile salt sensor that plays an important role in the regulation of cholesterol homeostasis. In view of these recent findings, it is fascinating that bile salts possess so many different functions: as detergents, as activators of protein kinase C isoforms or of phosphatidylinositol-3 kinase (see elsewhere in this issue), and as important gene regulators.

Peroxisome Proliferator Activated Receptor PPARα

Three PPAR genes have been identified in mammals: PPARα, PPARβ, and PPARγ (for reviews, see ref. 7, 33-35). In promoters of genes controlling the lipid and carbohydrate metabolism, PPARs bind to DR-1 HREs as a heterodimer with RXR (Table [1]). PPARα is highly expressed in tissues with high rates of β-oxidation such as the liver. Hepatic PPARα expression levels may vary widely in individual animals, likely due to hormonal variation, physical stress, or fasting.[33] A broad spectrum of endogenous and exogenous high-affinity ligands were identified such as fibrates, fatty acids, eicosanoids, leukotriene B₄, or nonsteroidal anti-inflammatory drugs indicating the importance of this NHR in controlling various different pathways in the liver (Fig. [2]) and other organs. Interestingly, a direct interaction between PPARα and LXRα suggests a role for LXRα in modulating PPAR-signaling pathways in cells.[36]

Sterol-responsive Element Binding Proteins SREBPs

Identification of a sterol-responsive element (SRE-1) in the promoter of the low density lipoprotein (LDL) receptor gene has resulted in the purification and cloning of a family of transcription factors, termed sterol-responsive element binding proteins (SREBPs) (Table [1]).[37][38][39] Two SREBP genes (SREBP1 and SREBP2) have been identified, which produce three proteins (SREBP1a, SREBP1c, and SREBP2). SREBPs are sequestered in the endoplasmic reticulum (ER) by two membrane spanning domains in cells cultured in medium containing sufficient cholesterol. When cellular sterol levels drop, the amino-terminal portions of the SREBPs are released from the ER by two ordered proteolytic events.[38] Statins (e.g., simvastatin, atorvastatin, lovastatin) are cholesterol-lowering drugs that cause a transient state of cellular free cholesterol deprivation, resulting in enhanced transcription of sterol-regulated genes mediated by SREBPs. SREBP1 is downregulated and SREBP2 upregulated in livers of hamsters and mice during treatment with statins.[37] The liberated mature SREBPs enter the nucleus where they activate transcription of various genes in the fatty acid and cholesterol metabolic pathways (Fig. [2]). Additionally, fatty acids have also been shown to affect the regulated processing of the SREBPs from their membrane-bound precursor state[40]; thus, the SREBPs are important regulators of both cholesterol and fatty acid metabolism. SREBPs are weak activators of transcription by themselves, and for efficient promoter activation they require coregulatory transcription factors that bind nearby DNA sequences such as stimulating protein 1 (SP1) or CCAAT-binding factor/nuclear factor-Y (NF-Y).

Transcription Factors Involved in Stress Response

Nuclear Factor κB (NF-κB) as Early Control Factor in Cellular Stress Response

Members of the Rel/NF-κB family of transcription factors play a central role in the regulation of inflammatory and immune responses (for recent reviews, see Refs. 41-46). NF-κB consists of homo- or heterodimers of Rel (c-Rel), p65 (RelA), RelB, p50, and p52, all of which have a conserved N-terminal Rel homology domain that has the DNA-binding and dimerization domains and contains the nuclear localization signal. In most unstimulated cells, a large portion of NF-κB is retained in the cytoplasm as inactive complexes by a family of inhibitory proteins called IκB (IκBα, IκBβ, IκBε, IκBγ, and bcl-3) that bind to the Rel homology domain and mask the nuclear localization signal. NF-κB is activated by a variety of stimuli ranging from cytokines, radiation, drugs, or oxidative stress.[47][48] Upon cell stimulation by a broad variety of stimuli, IκB kinases are activated and phosphorylate IκB proteins (Fig. [3]). The phosphorylated IκBs are subsequently ubiquitinated and targeted for degradation by the 26S proteasome, releasing the NF-κB dimers that then translocate to the nucleus to activate the transcription of genes containing the so-called κB-binding site (Table [1]). Among the NF-κB-inducible genes are IκB members (Fig. [3]), leading to autoregulation of the NF-κB system. Many genes induced by NF-κB encode for proteins that function in ``anti-apoptotic'' pathways of cells. In fact, inhibition of NF-κB translocation after activation of the tumor necrosis factor (TNF) receptor 1 by TNF-α results in programmed cell death (apoptosis) (Fig. [3]).

Gatekeeper p53

The p53 gene encodes for a 393 amino acid protein that has functional domains for transactivation, DNA binding, nuclear localization, and oligomerization. p53 is involved in many cellular processes such as transcription, DNA repair, cell cycle control, senescence, and apoptosis (for recent reviews, see Refs. 49-52). p53 is an important tumor suppressor whose inactivation is considered to be a critical step in tumorigenesis. The p53 phosphoprotein functions as a transcription factor for genes containing the consensus DNA sequence 5′-RRRCWWGYYY-3′ (Table [1]). However, p53 can also repress the activity of promoters that lack p53-consensus sites (see below). Activation of p53 after exposure of cells to DNA damaging agents results in cell cycle arrest. This process allows the DNA repair machinery to restore the DNA damage and prevents mutations and genetic alterations, which can ultimately lead to malignancies. p53 can also mediate apoptosis after DNA damage. Expression of the ``death receptor'' CD95/APO-1/FAS is under control of p53 by the presence of a p53-responsive element within the first intron of the CD95/APO-1/FAS gene and three putative elements within the promoter.[53] A cross-talk between the ``anti-apoptotic'' and the ``pro-apoptotic'' pathways has been demonstrated by showing that NF-κB, mainly considered as a ``anti-apoptotic'' transcription factor (see above), can activate p53, and this activation is inducible by TNF-α[53] (Fig. [3]). Because NF-κB induction occurs as a response to stress and p53 arrests cells in G1/S, where repair may be initiated, activation of p53 by NF-κB could be a mechanism by which cells can recover from stress.[53] Sustained stress, however, will result in apoptosis illustrated by the fact that the CD95/APO-1/FAS promoter contains a NF-κB-responsive sequence[54] and also the FAS ligand is upregulated in an NF-κB-dependent manner.[55]

REGULATION OF HEPATIC ABC TRANSPORTER GENE TRANSCRIPTION

ABC Transporter Proteins Involved in Bile Formation

PC Translocase MDR3

Secretion of PC, cholesterol, and bile salts are closely coupled and regulated processes that are mainly controlled by MDR3 (ABCB4; in rodents Mdr2) and BSEP (ABCB11) activities (Fig. [1]). Expression of the PC translocase Mdr2 in rodent liver appears to be unaltered under most conditions of cellular stress (Table [2], Fig. [4]). Mdr2 expression was not affected after endotoxin treatment[56] and was only slightly enhanced after partial hepatectomy.[57] Recent studies with fibrates,[58][59] bile salts,[60][61] and statins,[62][63] however, provide evidence that Mdr2 expression is ``controlled'' by its substrates cholesterol, PC, and bile salts (as ``cosubstrates'' in PC secretion) (Table [2], Fig. [2]).

When mice were fed with a diet supplemented with the peroxisome proliferators ciprofibrate or clofibrate, increased Mdr2 mRNA and protein levels and increased PC secretion were observed,[58][59] suggesting a potential involvement of PPARα (Fig. [2]) in Mdr2 gene expression. In mice fed a diet supplemented with the hydrophobic bile salt cholate, Mdr2 mRNA levels were found to be induced, which was functionally reflected in a concomitant increase of the maximal PC secretion capacity.[60] Feeding the (relatively) hydrophilic bile salt ursodeoxycholate did not influence the Mdr2 mRNA levels nor the maximal PC output capacity.[60] These latter findings imply that the type of bile salt in plasma may influence the expression level of Mdr2 and therefore the rate of PC secretion. The finding that plasma bile salt concentrations may influence Mdr2 expression may also explain increased Mdr2 levels found in regenerating rat livers after 70% partial hepatectomy.[57] In these animals, a 10-fold increased plasma bile salt level was found. Recent studies with isolated rat hepatocytes provided further evidence for regulatory functions of hydrophobic bile salts and cholesterol on Mdr2 expression[61] (Fig. [2]). Taurocholate and taurodeoxycholate both increased Mdr2 mRNA levels in a time- and concentration-dependent manner. Squalestatin, an inhibitor of cholesterol biosynthesis, increased Mdr2 mRNA levels by sevenfold in primary hepatocyte cultures. In contrast, cholesterol feeding and chronic bile diversion decreased Mdr2 mRNA significantly.[61]

Continuous exposure of rats to the statins simvastatin or pravastatin, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, resulted in decreased levels of liver cholesterol and increased biliary PC output.[62][63] This was accompanied by increased levels of Mdr2 mRNA[62][63] and protein.[63] Our study[63] shows further that statins also increase the expression of Mdr1b in rat liver (Table [2]). For an effect on Mdr2 expression, a continuous exposure to statins was necessary, because Mdr2 mRNA levels returned to control levels within 9-12 hours after drug withdrawal. However, during this rebound phase, Mdr2 protein levels remained elevated; and accordingly, biliary phospholipid secretion was increased in both continuously fed and in rebound rats. In contrast, Mdr1b mRNA levels remained increased in the rebound group, indicating different mechanisms of induction of Mdr2 and Mdr1b gene expression or differences in mRNA stability. In this model, NF-κB may be activated by statins as demonstrated for other xenobiotics. Alternatively, NF-κB activation may be the result of ER stress caused by overexpression of HMG-CoA reductase.[64]

The finding that biliary cholesterol/PC ratios in continuously fed and control animals were identical, despite suppression of cholesterol synthesis in the first group, suggests that PC secretion per se is an important regulatory factor for cholesterol secretion[63] (Fig. [1]). Data from diosgenin-treated rats demonstrate that hypersecretion of cholesterol can occur independently of Mdr2 induction and that cholesterol hypersecretion per se does not cause induction of Mdr2.[63]

Mdr2 is localized in periportal hepatocytes in control and in statin-treated livers. This zonal distribution is very similar to the reported distribution of HMG-CoA reductase and HMG-CoA synthase before and after statin treatment, which suggests that the factors controlling the expression of these proteins may be similar.[63].

Until now, SP1 is the only transcription factor that is identified to functionally interact with the promoter of the MDR3/Mdr2 gene (Fig. [5]), and it seems necessary for basal expression.[65] We have hypothesized from our study with statin-induced Mdr2 expression that transcriptional control of Mdr2 gene expression might, at least partially, be mediated via SREBPs. The 5′-flanking region of the Mdr2 gene contains elements that are possibly recognized by SREBPs.[63][66] We have recently tested this hypothesis. Exposure of freshly isolated rat hepatocytes to statins (simvastatin, lovastatin, or atorvastatin [0.1-100 μM] for 24 or 48 hours) caused a strong increase in mRNA levels of the gene coding for HMG-CoA reductase and Srebp2, whereas Mdr2 mRNA levels were moderately increased. Srebp1 mRNA levels were not significantly affected by statin treatment. Transient transfection studies with HepG2 cells revealed that statins stimulated Mdr2 promoter activity up to 10-fold, whereas cotransfection with a nuclear-SREBP1 expression plasmid enhanced Mdr2 promoter activity >10-fold. We conclude from these preliminary studies that Mdr2 gene expression is, at least partially, under control of Srebps.[66] These findings further demonstrate the importance of the hepatic PC translocator Mdr2 in the regulation of cholesterol homeostasis (Fig. [1]).

Bile Salt Transporter BSEP

The rat, mouse, and human BSEP genes have been cloned recently[67][68][69][70]; however, little is known yet on the regulation of BSEP expression. From animal models, some information is present on the behavior of rat Bsep under conditions of endotoxin treatment,[56][71] bile-duct ligation,[71] and ethinyl estradiol-induced cholestasis.[71] In the above-mentioned cholestatic and stress models, Bsep mRNA and protein expression levels only slightly decrease (Table [2]) compared with levels of the basolateral bile salt carriers Ntcp,[72] Oatp1 and Oatp2, or the canalicular transporter Mrp2.[57][71][73] Thus, Bsep may continue to secrete bile salts, although at impaired rates (Fig. [4]). Remarkably, after partial hepatectomy, the mRNA level of Bsep is only somewhat decreased and the proteins level of Bsep were unaffected (Table [2]) in contrast to the bile salt uptake transporter Ntcp.[57][74] This may explain the fact that after partial hepatectomy the remnant liver is not cholestatic. In the regenerating liver other basolateral transport systems such as Oatp1 and Oatp2 will still take up bile salts into the cells. Furthermore, due to the 10-fold increase of serum bile salts,[57] hepatocytes of the whole acinus will contribute to bile salt secretion (see above).

Modulation of liver cholesterol content does not have any effect on Bsep expression.[63] Administration of statins did not affect Bsep mRNA and protein expression and did not alter biliary bile salt output in animals continuously fed with the statin and in animals where treatment was withdrawn 9-12 hours before the end of the experiment.[63]

Recently, we cloned the promoter sequence of the human BSEP gene. It contains potential REs for CCAAT enhancer binding protein (C/EBP) β hepatocyte nuclear factor (HNF) 3β and FXR/RXR (Table [1]) (J. Plass, O. Mol, P.L.M. Jansen, and M. Müller, unpublished data, 2000) that may explain expression of BSEP almost exclusively in the liver. However, the importance of these liver-enriched transcription factors for the regulation of BSEP gene transcription needs to be further evaluated.

Transporters Involved in Detoxification, Phase II Conjugate Transport, and Stress Response

Multidrug Resistance Protein MRP2

The anionic conjugate transporter MRP2 (ABCC2) contributes to the bile formation by transporting GSH, a major driving force for bile salt-independent bile flow (Fig. [1]). However, because MRP2 has also a major role in canalicular anionic phase II conjugate transport, MRP2 regulation is discussed here.

A dose- and time-dependent induction of Mrp2 expression was observed in isolated rat hepatocytes cultured in the presence of xenobiotics, including vincristine, tamoxifen, or the PXR-ligand rifampicin[75] (Table [2]), indicating that Mrp2 gene transcription may respond to substrates of MRP2 itself or of phase I and II enzymes. This response to xenobiotics is also in line with the finding that MRP2 can confer drug resistance in vitro.[76]

The promoter regions of the human MRP2 and the rat Mrp2 genes have been isolated.[77][78] Sequence analysis of the human MRP2 promoter showed a number of putative consensus binding sites for both ubiquitous and liver-enriched transcription factors, including activating protein (AP) 1, SP1, HNF1, and HNF3β[77][79] (Fig. [5]). From studies with various deletion constructs, it appears that important elements are localized in the -431/-258 region that controls expression in HepG2 cells. This region contains a putative binding site for C/EBPβ and mutations in this site result in a 50% decrease of promoter activity. Thus, C/EBPβ likely has an important role in the transcriptional control of MRP2 gene expression, at least in HepG2 cells.[72]

A major question is still unanswered: Why is rat Mrp2 so rapidly downregulated under conditions of endotoxin-treatment? Recently, an important role of the NHRs RXR and RAR has been demonstrated.[80] Similar to the bile salt uptake transporter Ntcp, Mrp2 is rapidly downregulated via reduction in gene transcription. Ntcp suppression by endotoxin in vivo is caused by downregulation of transactivators, including the footprint B binding protein.[72] Both the Ntcp footprint B binding protein RE and the Mrp2 promoter contain RAR/RXR REs (for MRP2, see Fig. [5]). The RAR/RXR complex is downregulated by interleukin-1β in HepG2 cells. This mechanism likely contributes to the reduction of Mrp2 transcription during acute phase response.[80]

Multidrug Resistance Protein MRP1

The specificities of MRP1[81] (ABCC1) and MRP2[82] are very similar; however, the localization and the expression levels in hepatocytes are different. MRP1/Mrp1 expression levels in normal resting hepatocytes are very low.[83][84] However, levels of Mrp1 mRNA and protein are considerably increased after endotoxin administration[56] (Table [2]), whereas Mrp2 is strongly downregulated. Furthermore, MRP1 mRNA and protein levels were increased in HepG2 cells and SV40 large T antigen-immortalized human hepatocytes.[84] These results suggest that MRP1/Mrp1 expression and function may be associated with cell proliferation. Indeed, we recently reported that in isolated rat hepatocytes that have entered the cell cycle, Mrp1 expression is induced whereas expression of Mrp2 is decreased.[83] This switch in expression occurred in the mid-G1 phase of the cell cycle and appeared to be associated with a decrease in cell polarity.

Mrp1 is induced when rat hepatoma H4IIE cells are exposed to compounds that generate reactive oxygen species (ROS)[85] (Table [2]). This is coupled to an increased expression of γ-glutamylcysteine synthetase (γGCS), a rate-limiting enzyme in the biosynthesis of GSH. GSH is an important factor in Mrp1 function and in the defense against metabolites generated by oxidative stress.[85] Based on these results, it is proposed that the expression of Mrp1 and γGCS is, at least partially, mediated by the intracellular reduction-oxidation (redox) status.[85] A parallel expression pattern of MRP1 and γGCS has been reported for many drug-resistant cell lines, colon tumors from patients, and normal mouse tissues.[86] Analysis of the promoter region of the MRP1 gene has identified consensus binding sites for numerous transcription factors, including activator proteins (AP1 and AP2) REs, SP1 RE, AMP RE, estrogen RE, and glucocorticoid REs[87] (Fig. [5]). At present, the mechanisms underlying redox-mediated regulation of MRP1 expression are unknown. Several oxidative stress responsive-like sequences located upstream from the promoter of MRP1 have been noted; however, whether these sites can function as authentic oxidative stress RE (ORE) remains to be demonstrated.[85]

As for other genes, the SP1 binding site in the MRP1 promoter is essential for optimal transcriptional activity[88] (Table [1]). Interestingly, the tumor suppressor gene p53 suppressed MRP1 promoter activity, whereas no bona fide p53 binding site could be found in the MRP1 promoter.[89] Wild-type p53 downregulates several genes containing a TATA box by forming a complex with the TATA box-binding protein. However, this is not the case for the MRP1 promoter, which does not contain a TATA box but multiple start sites.[87] The explanation for the MRP1 suppression by p53 is that there may be competitive binding between SP1 and p53. SP1 transfection of Drosophila SL2 cells that do not contain SP1 stimulated MRP1 promoter activity up to approximately 200-fold; the latter was attenuated by coexpression of p53.[89] The effect was found with a minimal promoter (-91 and +103 bp) containing three Sp1 sites.[87][89]

A close correlation between the expression of MRP1 and the MYCN oncoprotein has been reported.[90][91] When MYCN expression was downregulated in antisense transfected cells, the level of MRP1 expression was decreased. Transfection of neuroblastoma cells with MYCN resulted in increased MRP1 expression and significantly increased resistance to MRP1 substrates. The MYC family members, such as MYCN, belong to the class of basic helix-loop-helix leucine-zipper transcription factors, and there is strong evidence that these oncoproteins are involved in the regulation of the cell cycle. Whether MYCN and other members of the MYC family interact directly with the MRP1 promoter (via, e.g., one or more of the three E-box motifs) needs to be investigated.

Basolateral Anionic Conjugate and Bile Salt Transporter MRP3

MRP3 (ABCC3) is another transporter protein that could provide basolateral export of organic anions, including bile salts from hepatocytes.[92][93] Interestingly, Mrp3 is upregulated in the Mrp2-deficient Eisai hyperbilirubinemic rat and in bile duct ligated cholestatic rats[82][94][95] (Table [2]). Also, increased amounts of MRP3 are detected in livers of Dubin-Johnson patients.[96] Considering the cellular localization of Mrp3, its upregulation during cholestatis (Fig. [4]), and its substrate specificity, it is hypothesized that Mrp3 may play a significant role in the basolateral export of organic anions under conditions in which Mrp2 (or Bsep) is downregulated. The inducible nature of the rat Mrp3 has recently been investigated.[92] An increase in Mrp3 expression was observed in Gunn rats exhibiting hyperbilirubinemia due to a defective UDP-glucuronosyl transferase. In addition, the elevated level of Mrp3 observed after bile duct ligation was associated with an elevated level of unconjugated bilirubin and bilirubin glucuronides.[92] These compounds were shown to induce the hepatic expression of Mrp3. Recently, also the human MRP3 promoter has been cloned and several putative binding sites for transcription factors, including AP1, AP2, and SP1, have been identified.[79][97] However, future experiments have to clarify which transcription factors, ligands, and REs are responsible for the interesting compensatory upregulation of Mrp3/MRP3 in hepatocytes under conditions where Mrp2/MRP2 (and BSEP) function is disturbed.

Multidrug Transporter MDR1 and Its Rodent Homologues Mdr1a/Mdr1b

In vitro studies reveal that expression of the human MDR1 (ABC B1) gene is induced by a variety of toxic agents, ultraviolet irradiation,[98] and heat shock,[99] implying that MDR1 promoter activation may be part of a general stress response in many cells. The human MDR1 promoter contains an inverted CCAAT box (-82 to -73), which is known as core sequence of the Y-box, a GC element (-56 to -42), and a number of putative recognition sites for transcription factors, including those for AP1, NF-Y, and Y-box binding protein (YB) 1.[98][99] Recently, an important role for both NF-Y and SP1 in the transcriptonal activation of the MDR1 gene after genotoxic stress was demonstrated. NF-Y and SP1 interact with the Y box and the GC-rich region, respectively. In contrast, YB-1, which has been identified by others as important for the ultraviolet-response in MDR1 upregulation,[100][101] was found not to be sufficient to mediate this activation.[98]

As in many other promoters, the SP1 site the MDR1 promoter (GGGGCGTGGG) significantly overlaps with binding sites for transcription factors of the early growth response (EGR) family (GCGTGGGGCG). Binding of SP1 or EGR1 to these GC-rich DNA sequences is often mutually exclusive and the result can be different depending on the cellular ``context.''[102] The rat Mdr1b promoter is positively regulated by SP1, whereas overexpression of EGR1 decreased Mdr1b expression.[103] p53 is another factor involved in the basal regulation of MDR1, rat Mdr1a, and Mdr1b[104] (Fig. [5]). Interestingly, whereas wild-type p53 repressed Mdr1a expression, overexpressing of mutant p53 resulted in markedly elevated levels of rat Mdr1a mRNA and protein levels. Similar regulation was reported for MDR1. In contrast, a functional p53 binding site has been identified in the rat Mdr1b promoter[105] (Table [1], Fig. [5]). In fact, wild-type p53 was shown to upregulate Mdr1b promoter activity and endogenous expression of rat Mdr1b.[105] These results and other studies indicate that the two rodent Mdr1 genes are differentially regulated.[99] The expression of Mdr1a in rat liver is, for example, not affected by endotoxin treatment and increases only slightly after bile duct ligation or partial hepatectomy[57] (Table [2]). In contrast, Mdr1b expression is markedly enhanced during endotoxin- and bile duct ligation-induced cholestasis and even more in the remnant liver after partial hepatectomy[56][57][99][106] (Table [2]).

The induction of rat Mdr1b expression after exposure to insulin is mediated by binding of the transcription factor NF-κB to a functional NF-κB-like binding site in the rat Mdr1b promoter[107] (Table [1], Fig. [5]). Upregulation of rat Mdr1 proteins has been shown recently under conditions of cadmium-induced oxidative stress.[108] Mdr1 upregulation correlated with a reduction of apoptosis, whereas MDR1 inhibitors such as PSC833, cyclosporine A, or verapamil increased cadmium-induced apoptosis. Both cadmium- and ROS-associated Mdr1 upregulation were linked to activation of the transcription factor NF-κB.[108] These data are in line with data from our laboratory where we found that TNF-α mediated upregulation of Mdr1b in rat hepatocytes is dependent on NF-κB activation[109] (Fig. [3]). Incubation of hepatocytes or H4IIE cells cultured on rat tail collagen with TNF-α resulted in increased levels of Mdr1b. This upregulation of Mdr1b could be blocked under conditions of inhibition of NF-κB activation: in the presence of the proteasome-inhibitor MG132 or after infection of the cells with the recombinant adenovirus construct Ad/IκBα.[109] In the presence of MG132 or the IκBα super-repressor, the basal level of Mdr1b in hepatocytes was also strongly decreased. Gel mobility shift assays showed an inhibition of NF-κB binding to the mdrκB promoter site under these conditions. We speculate that mdr1b upregulation, at least in part, provides anti-apoptotic protection for cells against oxidative-stress induced cell damage (Fig. [3]).

The impact of MDR1/Mdr1a expression on the drug-inducible expression of CYP3A has been tested in human and mice samples.[110] This has been demonstrated for the drug rifampicin that is an excellent inducer of CYP3A and a substrate for MDR1 and its rodent homologues. Consequently, cells with increased levels of MDR1 needed higher rifampicin concentrations to reach CYP3A induction. MDR1 and CYP3A (and other CYPs) are likely complementary systems to detoxify hydrophobic and potentially toxic compounds. Decreased MDR1 levels result in increased CYP expression; on the other hand, under conditions of suppressed CYP expression (e.g., under cytokine-induced stress), Mdr1b expression was found to be increased[56] (Fig. [4]).

More recently, the impact of hepatic Mdr1a/Mdr1b expression on CYP expression in the liver was studied.[111] Mdr1a(-/-) and Mdr1a/Mdr1b(-/-) mice[112][113] were used to demonstrate that these proteins have distinguishable functional roles in influencing expression of CYPs. Mdr1a appears to be the major regulator of hepatic CYP expression.[111] Somewhat surprisingly, the strong effects on CYP expression were almost exclusively seen in mice housed in the Amsterdam animal house and not in U.S.-housed animals.[111] Different contents of inducing agents in the diet such as pesticides, endogenous steroids, or phytoestrogens may be the cause for this. These compounds are efficient stimulators of the NHR PXR (see above)[19][114] and upregulate many CYPs, phase II enzymes, and possibly also drug efflux transporters. However, the cellular bioavailability of PXR ligands will be largely affected by MDR1 and functionally related transporter proteins. This concept will have consequences for human drug therapy because individual differences in expression of MDR1 (and other drug-transporting ABC-transporter proteins) will consequently result in individual differences in the expression of CYPs (phase I), phase II-conjugating enzymes, and ABC transporter proteins (phase III).

PERSPECTIVE

In the light of the recent data on NHRs as sensors for endogenous and exogenous metabolites and ABC transporter gene regulation, we have to reconsider the function of (some of) these transporters not as ``simple'' efflux pumps but as regulatory transporters that control the intracellular concentration of NHR ligands.

Many tools have become available to study the ``battle inside the nucleus'' with respect to hepatic transporter gene expression. Because of the enormous progress in molecular biology, we are able to unravel the basal mechanism in transcriptional control of the transporter gene expression. In the near future, we understand many of the mechanisms in transporter gene transcription that control tissue specificity of their expression, effective response to stress, or changes in substrate concentrations.

ACKNOWLEDGMENTS

The author thanks Dr. P.L.M. Jansen for his continuous support. These studies were supported by the Dutch Scientific Organization (NWO-902-23-207, NWO 902-23-208, NWO 902-23-191, NWO 902-23-253, NWO 902-23-257), Dutch Cancer Society (KWF/RUG 95-1007, KWF/RUG 96-1218), and material grants from the foundations Jan Kornelis De Cock (Groningen/Netherlands) and Mucoviscidose:ABCF-proteins (Paris/France).

ABBREVIATIONS

ABC, ATP-binding cassette
BSEP, bile salt transporter
CAR, constitutively activated receptor
C/EBP, CCAAT-enhancer binding protein
CYP, cytochrome P450
CYP7α, cholesterol 7α-hydroxylase
DR, direct repeats
EGR, early growth response
ER, endoplasmic reticulum
FXR, farnesoid X receptor
γGCS, γ-glutamylcysteine synthetase
GSH, reduced glutathione
HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A
HNF, hepatocyte nuclear factor
I-BABP, intestinal bile acid binding protein
LXR, liver X receptor
MDR1, multidrug resistance protein 1
Mdr2, rodent phosphatidylcholine transporter
MDR3, human phosphatidylcholine transporter
MRP, multidrug resistance-associated protein and its homologues
NF-Y, nuclear factor Y
NHR, nuclear hormone receptor
NOR, nuclear orphan receptor
PC, phosphatidylcholine
PPAR, peroxisome proliferator activated receptor
PXR, pregnane X receptor
RAR, 9-cis retinoic acid receptor
RE, responsive element
ROS, reactive oxygen species
RXR, retinoid X receptor
SP1, stimulating protein 1
SREBP, sterol responsive element binding protein
TNF-α, tumor necrosis factor α

Figure 1. Hepatic ABC transporter proteins in normal cells. Transporter proteins located in the canalicular membrane are responsible for the coupled biliary secretion of bile salts, PC, cholesterol, and GSH on the one hand and for the excretion of potentially toxic compounds on the other hand.[2][4][5] These transporter proteins comprise the bile salt transporter Bsep, the PC translocator Mdr2, the anionic conjugate transporter Mrp2, and the multidrug transporters Mdr1a and Mdr1b (in humans MDR1). Little is known on the function, localization, and regulation of the recently described hepatic ABC transporters ABCA1, ABCA2, ABCA3,[127] ABCG1,[128] or MRP6/ABCC6.[95][129][130] They are not discussed in this review. For a recent overview on human ABC transporter proteins and the official nomenclature, see http://www.med.rug.nl/mdl/humanabc.htm.

Figure 2. Putative role of Mdr2 and Bsep in the regulation in hepatic cholesterol homeostasis. In the liver, cholesterol stimulates its own conversion to bile salts by activation of LXR, which activates the rate-limiting enzyme CYP7α. Oxysterols[131] block the activation of SREBPs that are involved in many genes in cholesterol supply and fatty acid synthesis pathways. Oxysterols themselves are ``deactivated'' by the alternative bile salt synthesis pathway. Bile salts activate FXR that control CYP7α expression by inhibition of LXR-mediated transactivation of CYP7α expression. Activated FXR also stimulates bile salt recycling from intestine by inducing I-BABP synthesis. The hepatic bile salt concentration is also controlled by Bsep. Mdr2 plays a crucial role in hepatic PC and cholesterol secretion, and its transcription is controlled by its substrates. This scheme has been compiled and modified from different sources.[7][13][14][132][133]

Figure 3. Signaling pathways downstream the TNF-α receptor 1. TNF-α receptor 1 (TNFR1) initiates activation of NF-κB by recruiting adaptor proteins such as TNFR1-associated death domain protein (TRADD) or TNF-associated factors (TRAFs). Activated NF-κB inducing kinase (NIK) is released and sequestered by complexes containing IκB kinase (IKK) α- and β-heterodimers. To be active, these kinases must be associated with IKKγ. IKKs also interact with IKK complex-associated protein (IKAP). IKAP is bound by NIK, which activates IKKs. For more information, see text. The caspase-pathway[134] becomes activated via the Fas-associated death domain protein (FADD). This activation may result in apoptosis. However, NF-κB activation results in parallel in the synthesis of ``anti-apoptotic'' proteins, thereby counteracting the apoptotic stimulus. This scheme has been compiled and modified from.[44][135][136]

Figure 4. ABC transporter proteins in hepatocytes under cytokine-induced stress conditions. During endotoxin-induced cholestasis,[137] Mrp2 is downregulated, whereas the basolateral transporter proteins Mrp3 and Mrp1 are upregulated. Mrp1 mainly transports GSH and glutathione S-conjugates (e.g., the GSH conjugates of the oxidative strss product 4-hydroxy-2-nonenal). Mrp3 transports both phase II conjugates (glucuronides, sulfates) and bile salts. Pro-inflammatory cytokines such as TNF-α or interleukin-1 have been shown to downregulate most CYPs.[138] We have shown that during endotoxemia, Mdr1b is upregulated, indicating that both systems (CYPs, MDR1) are complementary in keeping concentrations of potential toxic hydrophobic compounds low (see text). In conclusion, under conditions of oxidative stress (induced by, e.g., cytokines) cells downregulate oxidative stress-producing systems such as CYPs, downregulate GSH ``wasting'' systems like Mrp2, and upregulate, for example, γGCS and certain ``compensatory'' ABC transporter proteins.