J.J.M. Smit

Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs

We have generated mice homozygous for a disruption of the mdr1a (also called mdr3) gene, encoding a drug-transporting P-glycoprotein. The mice were viable and fertile and appeared phenotypically normal, but they displayed an increased sensitivity to the centrally neurotoxic pesticide ivermectin (100-fold) and to the carcinostatic drug vinblastine (3-fold). By comparison of mdr1a (+/+) and (-/-) mice, we found that the mdr1a P-glycoprotein is the major P-glycoprotein in the blood-brain barrier and that its absence results in elevated drug levels in many tissues (especially in brain) and in decreased drug elimination. Our findings explain some of the side effects in patients treated with a combination of carcinostatics and P-glycoprotein inhibitors and indicate that these inhibitors might be useful in selectively enhancing the access of a range of drugs to the brain.

Homozygous disruption of the murine MDR2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease

Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.

The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice.

The mouse mdr2 P‐glycoprotein (P‐gp) and its human MDR3 homologue are present in high concentrations in the canalicular membrane of hepatocytes. Mice lacking this protein are unable to secrete phosphatidylcholine (PC) into bile, suggesting that this P‐gp is a PC translocator. We have tested this in fibroblasts from transgenic mice expressing the MDR3 gene under a vimentin promoter. Transgenic and control fibroblasts were incubated with [14C]choline to label PC. When the labeled cells were incubated with a PC transfer protein and acceptor liposomes, transfer of radioactive PC was enhanced in transgenic cells relative to the wild type controls. We conclude that the MDR3 P‐glycoprotein is able to promote the transfer of PC from the inner to the outer leaflet of the plasma membrane, supporting the idea that this protein functions as a PC flippase.

Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.

Disruption of the mdr2 gene in mice leads to a complete absence of phospholipid from bile (Smit, J. J. M., et al. 1993. Cell. 75:451-462). We have investigated the control of both mdr2 P-glycoprotein (Pgp) expression and bile salt secretion on biliary lipid secretion in the mouse. Lipid secretion was monitored at various bile salt output rates in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. In (-/-) mice, phospholipid secretion was negligible at all bile salt output rates. In (+/-) mice, a curvilinear relation between bile salt and phospholipid secretion was observed similar to that in (+/+) mice; however, at all bile salt secretion rates phospholipid secretion was reduced compared to (+/+) mice, indicating that mdr2 Pgp exerts a strong control over secretion. Infusion of increasing amounts of taurocholate up to maximal secretory rate led to a decline in the phospholipid and cholesterol secretion in both (+/+) and (+/-) mice in accordance to what has been observed in other species. In contrast, in (-/-) mice cholesterol secretion increased under these conditions while phospholipid output remained extremely low. The increased cholesterol secretion may represent extraction of cholesterol from the canalicular plasma membrane by taurocholate micelles as opposed to the concomitant secretion of both phospholipid and cholesterol in the presence of a functional mdr2 Pgp. Increased bile flow in (-/-) mice could be attributed completely to an increase in the bile salt-independent fraction and may therefore be caused by the bile duct proliferation in these mice.

Multidrug resistance and the role of P-glycoprotein knockout mice

Drug resistance, be it intrinsic or acquired, is a major problem in cancer chemotherapy. In vitro, one well characterised form of resistance against many different cytotoxic drugs is caused by the MDR1 P-glycoprotein, a large plasma membrane protein that protects the cell by actively pumping substrate drugs out. Available evidence suggests that this protein may cause drug resistance in at least some clinical tumours. Drugs inhibiting the MDR1 P-glycoprotein activity are, therefore, co-administered during chemotherapy of these tumours. To predict the biological and pharmacological effects of the blocking of this protein, we have generated mice with a genetic disruption of the drug-transporting mdr1a P-glycoprotein. These mice are overall healthy, but they accumulate much higher levels of substrate drugs in the brain, and have markedly slower elimination of these drugs from the circulation. For some drugs, this leads to dramatically increased toxicity, indicating that P-glycoprotein inhibitors should be used with caution in patients.

Classical and novel forms of multidrug-resistance and the physiological functions of p-glycoproteins in mammals

In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism. Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile. This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located. These and other results show the importance of the genetic approach for studying drug metabolism. MDR is not only caused by increased activity of Pgps. When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity. Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur. This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms. The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.

Regulation of protein secretion into bile: Studies in mice with a disrupted mdr2 p-glycoprotein gene☆

BACKGROUND & AIMS Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. METHODS Protein secretion and biliary lipid output were studied in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). RESULTS Exocytosis and transcytosis were unaltered in (-/-) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (-/-) mice but had little effect in (+/-) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (-/-) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. CONCLUSIONS The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.

Characterization of the promoter region of the human MDR3 P-glycoprotein gene.

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.

Structure and Function of P-Glycoproteins

Overexpression of P-glycoprotein genes is one established cause of multidrug resistance in mammalian cells. We are studying P-glycoproteins and their genes in man, in the mouse and in the nematode worm Caenorhabditis elegans in order to understand the normal, physiological role of P-glycoproteins, and the mechanistics of P-glycoprotein function. Our data suggest that one common, and evolutionarily well-conserved function of P-glycoproteins is protection of the organism against naturally occurring xenotoxins present in ingested food. We consider it likely, however, that some P-glycoprotein variants perform a more specialized function in the organism.