International Journal of Molecular Sciences Review Structural and Functional Characterization of the ABCC6 Transporter in Hepatic Cells: Role on PXE, Cancer Therapy and Drug Resistance Faustino Bisaccia, Prashant Koshal, Vittorio Abruzzese, Maria Antonietta Castiglione Morelli and Angela Ostuni * Department of Sciences, University of Basilicata, 85100 Potenza, Italy; [email protected] (F.B.); [email protected] (P.K.); [email protected] (V.A.); [email protected] (M.A.C.M.) * Correspondence: [email protected] Abstract: Pseudoxanthoma elasticum (PXE) is a complex autosomal recessive disease caused by mutations of ABCC6 transporter and characterized by ectopic mineralization of soft connective tissues. Compared to the other ABC transporters, very few studies are available to explain the structural components and working of a full ABCC6 transporter, which may provide some idea about its physiological role in humans. Some studies suggest that mutations of ABCC6 in the liver lead to a decrease in some circulating factor and indicate that PXE is a metabolic disease. It has been reported that ABCC6 mediates the efflux of ATP, which is hydrolyzed in PPi and AMP; in the extracellular milieu, PPi gives potent anti-mineralization effect, whereas AMP is hydrolyzed to Pi and adenosine which affects some cellular properties by modulating the purinergic pathway. Structural and functional studies have demonstrated that silencing or inhibition of ABCC6 with Citation: Bisaccia, F.; Koshal, P.; probenecid changed the expression of several genes and proteins such as NT5E and TNAP, as well Abruzzese, V.; Castiglione Morelli, M.A.; Ostuni, A. Structural and as Lamin, and CDK1, which are involved in cell motility and cell cycle. Furthermore, a change in Functional Characterization of the cytoskeleton rearrangement and decreased motility of HepG2 cells makes ABCC6 a potential target ABCC6 Transporter in Hepatic Cells: for anti-cancer therapy. Collectively, these findings suggested that ABCC6 transporter performs Role on PXE, Cancer Therapy and functions that modify both the external and internal compartments of the cells. Drug Resistance. Int. J. Mol. Sci. 2021, 22, 2858. https://doi.org/10.3390/ Keywords: ABCC6; TNAP; NT5E; Pseudoxanthoma elasticum (PXE); cancer ijms22062858 Academic Editor: Thomas Falguières 1. Introduction Received: 20 February 2021 Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease, which was de- Accepted: 9 March 2021 scribed in 1881 by a French dermatologist. In 2000, it was first recognized that mutation Published: 11 March 2021 in ABCC6 is responsible for PXE [1]. It is affecting approximately 1:50,000 people world- wide, with the prominent characteristic feature of ectopic mineralization of soft tissues Publisher’s Note: MDPI stays neutral like skin, eyes, and arteries (Figure1), for which no effective curative treatment is avail- with regard to jurisdictional claims in published maps and institutional affil- able [2,3]. Moreover, PXE shows similar phenotypic characteristics with other common iations. health problems like kidney diseases (chronic kidney disease (CKD) and nephrocalcinosis) and cardiovascular diseases (coronary heart disease, cardiomyopathy, and dyslipidemia), which makes PXE a complex disorder [4,5]. Different hypotheses were proposed for the factors pathologically involved with PXE. The “Metabolic Hypothesis” stated that decrease or loss of ABCC6 functionality Copyright: © 2021 by the authors. especially in the liver may lead to a decrease in some circulating factors in the blood Licensee MDPI, Basel, Switzerland. stream, which should be responsible for preventing ectopic mineralization of soft tissues. This article is an open access article distributed under the terms and The “PXE Cell Hypothesis” stated that absence of ABCC6 in PXE tissues leads to an conditions of the Creative Commons alteration in cell proliferation due to changes in the biosynthetic pathway and alters cells Attribution (CC BY) license (https:// to extracellular matrix interactions. The most recent “ATP Release Hypothesis” stated that creativecommons.org/licenses/by/ ABCC6 mediates the efflux of ATP in extracellular milieu, where it is hydrolyzed into AMP 4.0/). and pyrophosphate and prevents the mineralization of soft tissues [1]. Int. J. Mol. Sci. 2021, 22, 2858. https://doi.org/10.3390/ijms22062858 https://www.mdpi.com/journal/ijms Int.Int. J. J. Mol. Mol. Sci.Sci.2021 2021,,22 22,, 2858 x FOR PEER REVIEW 22 of of 12 12 PreviousPrevious studiesstudies of of serum serum analysis analysis either either from from the ABCC6 the ABCC6knock-down knock-down mouse modelmouse ormodel from or PXE from patients PXE patients showed anshowed inability an toinab preventility to calcium prevent and calcium phosphate and phosphate deposition dep- and suggestedosition and that suggested PXE is a metabolicthat PXE is disease a metabolic with very disease slow onsetwith very [1,6,7 slow]. It should onset be[1,6,7]. noted It thatshould the be tissues, noted which that the mostly tissues, express which ABCC6, mostly are express the liver ABCC6, and, toare some the liver lesser and, extent, to some the kidneylesser extent, and differ the fromkidney those and in differ which from ectopic thos mineralizatione in which ectopic is mostly mineralization evident, namely is mostly soft tissuesevident, of namely skin, eyes, soft and tissues the cardiovascularof skin, eyes, and system. the cardiovascular The origin of this system. apparent The paradoxorigin of hasthisnot apparent been explained paradox has yet (Figurenot been1)[ explained8]. yet (Figure 1) [8]. OnOn thethe basisbasis ofof ourour previousprevious studiesstudies ofof thethe ABCC6ABCC6 transportertransporter inin hepatichepatic cells,cells, thethe presentpresent review review is focusedis focused on lighteningon lightening changes changes in cellular in cellular function function associated associated with ABCC6 with transporterABCC6 transporter activity. activity. Figure 1. Pictorial presentation of ABCC6 expression and affected tissues in Pseudoxanthoma Figure 1. Pictorial presentation of ABCC6 expression and affected tissues in Pseudoxanthoma elasticum (PXE). The protein is expressed mainly in liver and kidney cells but the main areas in- elasticumvolved in ectopic (PXE). Thecalcification protein are is expressed the elastic mainlytissues of in heart, liver andblood kidney vessels, cells skin, but and the eyes. main areas involved in ectopic calcification are the elastic tissues of heart, blood vessels, skin, and eyes. 2.2. StructuralStructural PropertiesProperties ofof ABCC6ABCC6 TransporterTransporter TheThe ATP-bindingATP-binding cassettecassette (ABC)(ABC) transporterstransporters areare aa well-knownwell-known familyfamily andand ubiqui-ubiqui- touslytously foundfound inin allall livingliving organisms.organisms. AboutAbout 5050 differentdifferent typestypes ofof ABCABC transporters transporters havehave beenbeen recognizedrecognized inin humans,humans, divideddivided intointo sevenseven subfamiliessubfamilies (ABCA(ABCA toto ABCG)ABCG) basedbased onon theirtheir structurestructure andand geneticgenetic sequences.sequences. AmongAmong themthem wewe cancan findfind bothboth halfhalf transporters,transporters, withwith onlyonly one one transmembrane transmembrane domain domain (TMD) (TMD and) and nucleotide-binding nucleotide-binding domain domain (NBD), (NBD), and fulland transporters, full transporters, with two with TMDs two and TMDs NBDs and [9,10 NBDs]. ABCC6 [9,10]. belongs ABCC6 to the belongs ATP-binding to the cassetteATP-binding (ABC) cassette transporter (ABC) subfamily transporter C andsubfam hasily been C and found has to been be highly found expressed to be highly in basolateralexpressed in plasma basolateral membrane plasma of membrane hepatocytes of and, hepatocytes to some lesserand, to extent, some inlesser the proximalextent, in tubulesthe proximal of the tubules kidney [of11 the]. kidney [11]. TheThe ABCC6ABCC6 genegene hashas 3131 exonsexons andand encodesencodes aa proteinprotein ofof 15031503 aminoamino acidacid residues,residues, MRP6.MRP6. It is made-upmade-up ofof twotwo nucleotide-bindingnucleotide-binding domainsdomains (NBD1(NBD1 andand NBD2)NBD2) andand twotwo transmembranetransmembrane domainsdomains (TMD1(TMD1 andand TMD2)TMD2) withwith anan additionaladditional auxiliary auxiliary NH2-terminal NH2-terminal transmembranetransmembrane domaindomain knownknown as as TMD0, TMD0, which which is is connected connected with with the the canonical canonical compo- com- nentsponents through through the the cytoplasmic cytoplasmic L0 L0 loop loop [12 [12,,1313].]. The The NBDs NBDs of of ABC ABC transporters transporters consistconsist ofof differentdifferent conservedconserved motifsmotifs whichwhich bindbind andand hydrolyzehydrolyze ATP. LikeLike inin otherother transporters,transporters, ABCC6ABCC6 NBDs domains have have Walker Walker A A motif motif (P (P loop), loop), Walker Walker B motif B motif (Mg (Mg2+ binding2+ binding site), site),histidine histidine loop loop(Switch (Switch region), region), signature signature moti motiff (C loop), (C loop), Q loop Q loop (between (between Walker Walker A and A Int. J. Mol. Sci. 2021, 22, 2858 3 of 12 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 12 andC-loop), C-loop), and and D loop D loop (involved (involved in inthe the interm intermolecularolecular interactions interactions at atthe the NBD NBD dimmer dimmer in- interface).terface). In In the the general general