MRP6 (ABCC6) Detection in Normal Human Tissues and Tumors

Abstract

M RP6 (ABCC6) is a member of the subfamily of the multidrug resistance proteins (MRPs, reviewed by Borst et al, 2000), but its putative role in multidrug resistance (MDR, reviewed by Moscow et al, 1997) is still under investigation. Closely related proteins such as MDR1 P-glycoprotein (P-gp, ABCB1, reviewed by Ambudkar et al, 1999), breast cancer resistance protein (BCRP, ABCG2;Doyle et al, 1998), and MRP1, -2, and -3 (ABCC1-3) are established MDR transporters. The exact range of substrates for MRP6 has not yet been determined, but a preliminary report suggested that MRP6 may be involved in the transport of certain anticancer agents, including anthracyclines and epipodophyllotoxins (M.G. Belinsky et al, Proceedings of the AACR, abstract 1510, 2001). Recently it was found that mutations in the MRP6 gene cause pseudoxanthoma elasticum (PXE), an inheritable disorder of the connective tissue involving impaired visual acuity, skin lesions, and cardiovascular complications (Bergen et al, 2000). The expression of MRP6 in normal human tissues has only been studied at the mRNA level. High MRP6 mRNA levels were reported in liver and kidney, whereas low expression was found in a range of other tissues, including lung, intestines, retina, skin, and vessel walls (Bergen et al, 2000;Kool et al, 1999).To study MRP6 at the protein level, three rat Mabs (M 6 II-7, M 6 II-21, and M 6 II-31) were generated from rats immunized with a fusion protein containing amino acids 764 to 964 of human MRP6 (FP M 6 II), according to described methods (Scheffer et al, 2000 Detection of the approximately 65 kD MRP6-MBP fusion protein and full length MRP6 from protein preparations of MRP6-transfected HEK 293 cells, using anti-MRP6 Mab M 6 II-7. The approximately 180,000 kD MRP6 migrates slightly faster than MRP2, as shown by control lanes stained for MRP2, using M 2 III-6 and MRP2-transfected cells. Total cell lysates were made as previously described (Scheffer et al, 2000). Ten to forty g of cell lysates or fusion proteins were fractionated on a 7% polyacrylamide slab gel and transferred onto a nitrocellulose membrane by electroblotting. After blocking, the membrane was incubated for 2 hours with primary antibody in the appropriate dilution. Horseradish peroxidase (HRP)-labeled-anti-rat or -mouse serum (1:1000; Dako, Copenhagen, Denmark) was used as a secondary antibody. Enhanced chemoluminescence (Amersham, Buckinghamshire, United Kingdom) was used to detect Mab binding.

The MRP family of drug efflux pumps

“…A similar pattern of transcript expression was reported for the rat MRP6 homologue except that its expression in kidney and gut appeared to be lower and higher, respectively, that the case in humans Madon et al, 2000). In addition, an immunohistochemical analysis in which a monoclonal MRP6 antibody was employed showed that the human protein was abundant in liver and kidney -where it was localized to basolateral surfaces of hepatocytes and proximal tubules, but not expressed in many other tissues including those affected in PXE patients (ie, skin and retina) (Scheffer et al, 2002a). These findings raised the possibility that PXE might result from the absence of a substance that is normally extruded from liver or kidney into blood, and which is involved in connective tissue homeostasis (Uitto et al, 2001).…”

ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE)

“…It consists of 31 exons spanning approximately 73kb and encodes for a protein (MRP6, Multidrug Resistance associated Protein 6) belonging to the ATP-binding cassette sub-family C of membrane transporters (Bergen et al, 2000;Le Saux et al, 2000;Ringpfeil et al, 2000). The precise localization in human tissues and the physiological role of MRP6 are still unknown (Scheffer et al, 2002). Recent reports suggest its implication in the cell extrusion of glutathionated metabolites (Belinsky et al, 2002;Ilias et al, 2002).…”

HNF4α and NF-E2 are key transcriptional regulators of the murine Abcc6 gene expression

“…Based on homology with the well-characterized ABCC1 protein (~45% homology) and its localization on the basolateral membrane of hepatocytes, ABCC6 is thought to be a metabolite pump exporting compounds from these cells [3]. Ilias et al [4] demonstrated in vesicular studies that glutathione S-conjugates, including leukotriene-C4 and N-ethylmaleimide-Sglutathione, are actively transported by the human ABCC6 whereas the endothelin-1 receptor antagonist BQ123 is not efficiently transported in contrast to Abcc6 in rat [5].…”