Molecular Aspects of Medicine 34 (2013) 646–654

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Molecular Aspects of Medicine

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Review

The choline transporter-like family SLC44: Properties and rolesin human diseases q

Elisabeth Traiffort a,⇑, Seana O’Regan b, Martial Ruat a

a CNRS, UPR-3294, Laboratory of Neurobiology and Development, Institute of Neurobiology Alfred Fessard IFR2118, Signal Transductionand Developmental Neuropharmacology Team, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, Franceb CNRS/Université Paris Descartes UMR 8192, Centre Universitaire des Saints-Pères, 45, rue des Saints-Pères, 75270 Paris cedex 06, France

Guest Editor Matthias A. HedigerTransporters in health and disease (SLC series)

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 November 2011Accepted 14 March 2012

Keywords:Membrane phospholipidsMitochondriaNeuronsOligodendrocytesAutoimmune hearing lossTransfusion-related acute lung injurySLC44

0098-2997/$ - see front matter � 2012 Elsevier Ltdhttp://dx.doi.org/10.1016/j.mam.2012.10.011

q Publication in part sponsored by the Swiss NatUniversity of Bern, Switzerland; Director Matthias A⇑ Corresponding author. Tel.: +33 1 6982 4301; fa

E-mail address: Elisabeth.Traiffort@inaf.cnrs-gif.f

The Na+-independent, high affinity choline carrier system proposed to supply choline forthe synthesis of cell membrane phospholipids was recently associated with SLC44 familymembers (SLC44A1-5) also called choline-like transporter family. SLC44A1 is widelyexpressed throughout the nervous system in both neurons and oligodendrocytes, whileSLC44A2-4 are mainly detected in peripheral tissues. The subcellular localization of theproteins was mainly addressed for SLC44A1 through the development of specific antibod-ies. SLC44A1 is detected in both the plasma and mitochondrial membranes where the pro-tein is able to transport choline at high affinity and in a Na+-independent manner. Thephysiological relevance of SLC44A1 as a choline carrier is indicated by its likely involve-ment in membrane synthesis for cell growth or repair, and also by its role in phospholipidproduction for the generation of lung surfactant. Moreover, an autoimmune disease hasbeen related to the blockade of SLC44A2 function, which results in the alteration of haircells in the inner ear and leads to autoimmune hearing loss. In the alloimmune syndromecalled transfusion-related acute lung injury, antibodies to SLC44A2 cause a deleteriousaggregation of granulocytes. Therefore transporters of the SLC44 family represent attrac-tive and promising targets for therapeutic and diagnostic applications regarding bothimmune and degenerative diseases.

� 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6472. Molecular characterization of the SLC44 family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6483. Tissue distribution and choline transport activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648

3.1. The transcripts of SLC44 family members are widely expressed in tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6483.2. SLC44 proteins and choline transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6493.3. SLC44 proteins are plasma membrane and mitochondrial transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650

. All rights reserved.

ional Science Foundation through the National Center of Competence in Research (NCCR) TransCure,. Hediger; Web: http://www.transcure.ch.x: +33 1 6982 3639.r (E. Traiffort).

FCNh

E. Traiffort et al. / Molecular Aspects of Medicine 34 (2013) 646–654 647

4. SLC44 family members in physiopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651

ig. 1.ommi- andearing

4.1. Involvement of SLC44A1 in membrane synthesis for cell growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6514.2. SLC44 family proteins in the neonatal respiratory distress syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6514.3. SLC44A2, a target for autoimmune hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6524.4. Polymorphisms in SLC44A2 are responsible for TRALI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653

1. Introduction

The organic cation choline is an essential component of membrane phospholipids including phosphatidylcholine andsphingomyelin, both required for the synthesis of cell membranes. Choline plays an additional role in the brain as a precursorfor the synthesis of the neurotransmitter acetylcholine. Choline uptake is dependent upon carrier-mediated transport, sincea charged cation under physiological pH does not cross cell membranes readily by passive diffusion. During the last decades,two main transport systems have been characterized biochemically: a high-affinity, Na+-dependent system localized in pre-synaptic cholinergic nerve terminals and likely coupled with acetylcholine synthesis, and a low-affinity, Na+-independentsystem found throughout various tissues proposed to supply choline for the synthesis of phospholipids in the cellular mem-brane (for review, Lockman and Allen, 2002). More recently, proteins have been associated with each of these transport sys-tems. The high affinity (Km � 2 lM) choline transporter, CHT1 (SLC5A7), belongs to the Na+/glucose co-transporter family, issensitive to the choline analogue hemicholinium-3 (HC-3) and is thought to be part of the rate-limiting step in acetylcholinesynthesis (Apparsundaram et al., 2000, 2001; Okuda et al., 2000). Choline is also a substrate for the low affinity (Km -� 200 lM) organic cation transporters or OCTs (SLC22) widely expressed in peripheral tissues as well as in the CNS (for re-view, Michel et al., 2006) and which recognize multiple endogenous and exogenous organic cations with low specificity. Theconcentrations of free choline in plasma or in CSF (12 and 7 lM in rats, respectively; Klein et al., 1992) being low in com-parison to the affinities of these low affinity carriers, the physiological relevance of a general metabolic high affinity cholinetransporter that would supply choline for membrane phospholipid synthesis remained obvious. Ten years ago, candidateproteins were proposed to play such a role. They were called choline transporter-like proteins or CTL. Now, they constitutethe SLC44 family since their inclusion in the solute carrier series (see http://www.genenames.org/genefamilies/SLC). Amongthe five SLC44 gene family members found in the human genome (Fig. 1A), the first to be identified, CTL1/CDw92/SLC44A1,was proposed to encode a protein involved in supplying choline to several cell types including a specific subset of cholinergicneurons (O’Regan et al., 2000) and to be present on various cells of the hematopoietic system (Wille et al., 2001).

This review will focus on the molecular identification and tissue distribution of SLC44 family members, their activity incholine transport, their implication in the development of diverse immune diseases, their role in the maturation of the

Phylogenetic tree and structural model of SLC44 family members in human. (A) The dendrogram is derived from the HUGO Gene Nomenclaturettee (HGNC) at http://www.genenames.org/genefamilies/SLC). (B) Predicted topology for SLC44A2 with 10 helical transmembrane domains with theC-terminal portions residing in the cytoplasm. Potential N-linked carbohydrate site for binding of the antibody KHRI-3 promoting autoimmuneloss in humans (black triangles) and mutations associated with transfusion-related acute lung injury (yellow stars).

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respiratory system or in cell growth. We will also present the most recent data indicating that the members of this familyshould be considered both as potentially therapeutic targets themselves and for the development of new screening assays inauto- and allo-immune syndromes related to these proteins.

2. Molecular characterization of the SLC44 family

CTL1 was initially cloned from Torpedo marmorata and was first characterized as a suppressor for a yeast choline transportmutation derived from a Torpedo electric lobe yeast expression library (O’Regan et al., 2000; O’Regan and Meunier, 2003).The rat Slc44a1 (69% identity with its Torpedo counterpart) was then cloned from a brain cDNA library and its protein se-quence found to display 96% identity with its human ortholog itself isolated from a cDNA expression library derived frommyeloid KG1a cells using a monoclonal antibody for the human antigen CDw92 (Wille et al., 2001). Four other related pro-teins (SLC44A2-5) exist in human and mouse. SLC44 paralogs are found throughout the phylogenetic tree of all eukaryotes,while no prokaryote homologue has been found (O’Regan et al., 2000). In this family, SLC44A5 is closer to the SCL44A2/SLC44A4 than to the SLC44A1/SLC44A3 subgroup (Fig. 1A). Moreover, if we consider the transporter classification systemof Milton Saier (Saier et al., 2006), only SLC44A1 and SLC44A2 are present in this classification as 2.A.92.1.1 and2.A.92.1.2. Nevertheless they are not included in any transporter superfamily in agreement with their lack of homology withother transporters.

Computerized topology models initially proposed an even number of 10–12 transmembrane domains for rat and humanSLC44A1 possibly with intracellular N and C-terminal ends (O’Regan et al., 2000; Wille et al., 2001; Fig. 1B). According to thismodel, a first large and variable loop between transmembrane domains 1 and 2 is potentially extracellular and glycosylated.A highly conserved region covers the last four transmembrane domains and includes the fourth extracellular loop that con-tains 3 of the 11 conserved cysteines. However more recent analyses have questioned this model. SLC44A1 might display anintracellular N-terminus and an extracellular C-terminus as suggested by the ability of two newly generated SLC44A1 anti-bodies to bind extracellularly to peptides located in the first extracellular loop and the C-terminal end, respectively (Micheland Bakovic, 2009). Moreover, an extracellular N-terminus has been proposed for SLC44A2 despite a greater entropy cost(Nair et al., 2004). All five human SLC44 proteins share a common domain structure (EMBL-EBI accession: IPR007603) withthe other eukaryotic CTL-like proteins, but the structure of this domain has not yet been resolved.

The diversity and complexity of the SLC44 family is further increased by alternative splicing mechanisms. The SLC44A1gene displays 17 exons spanning about 200 kb and contains an alternative splicing site located at the 30 end of exon 15(O’Regan et al., 2000) leading to transcripts SLC44A1a (use of exon 16), SLC44A1b (exon 16 skipped, use of exon 17) andanother one located in exon 16 giving rise to SLC44A1c (Wille et al., 2001). Rat Slc44a1a and Slc44a1b encode differentC-terminal peptides corresponding in the rat sequence to the amino acids ASGASSA and LRKR, respectively. Though theRXR motif occurring in SLC44A1b has been classically identified as a potential endoplasmic reticulum retention/retrievalmotif in ion channels or G protein-coupled receptors, the functionality of this motif remains to be demonstrated in SLC44A1b(Traiffort et al., 2005). Interestingly, AS⁄GAS⁄SA contains two phosphorylated (⁄) serines (Dephoure et al., 2008) suggestingthe splicing must also change the phosphorylation state of the corresponding protein.

Likewise, the genomic sequences for SLC44A2 and SLC44A5 contain the corresponding exons from which the alternativelyspliced transcripts can be generated. In addition, SLC44A2, SLC44A3 and SLC44A4 also show alternate splicing at the N-ter-minal end.

3. Tissue distribution and choline transport activity

3.1. The transcripts of SLC44 family members are widely expressed in tissues

The distribution of Slc44a1-4 transcripts has been determined by Northern blot analysis in rat tissues. Slc44a1 was de-tected as a major 3.5 kb transcript in all brain regions studied including the striatum, cerebral cortex, hippocampus, hypo-thalamus, cerebellum, midbrain, thalamus and olfactory bulbs and also in the spinal cord. A minor 5.0 kb form wasprominently detected in the colon and in a notable manner in the lung and spinal cord (O’Regan et al., 2000; Traiffortet al., 2005). Interestingly, Slc44a1a and Slc44a1b probes detected selectively the 5.0 and 3.5-kb transcripts, respectively. Un-like SLC44A1, the other members are poorly expressed (SLC44A2) or barely detectable (SLC44A3, SLC44A4) in the nervous sys-tem. However SLC44A2-4 are present in several peripheral tissues as a single transcript. SLC44A2 is easily observed in tongue,muscle, kidney, heart and lung, but is fainter in testis, intestine and stomach. In addition, age-related changes in expressionof SLC44A2 N-terminal isoforms were reported in the murine inner ear (Beyer et al., 2011). A moderate SLC44A3 expression ispresent in kidney, ileum and colon, while a very strong SLC44A4 expression can be detected in intestine, stomach and kidney.Much less data are available for SLC44A5 expression which was found quite low in the brain, higher in the spinal cord, but toa lower extent than SLC44A1. In agreement with these data, other studies have shown notable levels of Slc44a1/SLC44A1mRNA in the mouse (Yuan et al., 2004) and human brain (Yuan et al., 2006).

A detailed analysis of Slc44a1-expressing cells was performed using in situ hybridization in rat tissues which displayed astrong signal in Northern analysis (O’Regan et al., 2000; Traiffort et al., 2005). In the CNS, Slc44a1 is expressed by both neu-rons and oligodendrocytes. Its transcripts were clearly visualized in brainstem and spinal cord motor neurons and at a lower

Table 1Human and rodent cell lines or primary cell cultures characterized for choline transport exhibiting features of SLC44 transporters. The asterisk indicates thatthe identity of the SLC44 member has been investigated using siRNA approaches. In the absence of the asterisk, the identity of the SLC44 transporter which ismentioned is derived from biochemical and pharmacological approaches.

Name Description SLC44 family member Km (lM) References

C2C12 Mouse muscle SLC44A1⁄ n.d. Michel and Bakovic (2009)MCF7 Human breast cancer SLC44A1⁄ n.d. Michel and Bakovic (2009)FL83B Mouse liver SLC44A1⁄ n.d. Michel and Bakovic (2009)NG108–15 Rodent cholinergic neuroblastoma x glioma SLC44A1⁄ 4.9 Machova et al. (2009)C6 Rat glioma SLC44A1⁄ 7.7 Machova et al. (2009)A549 Human lung adenocarcinoma SLC44A1-2⁄ 15.0 Ishiguro et al. (2008) and Nakamura et al. (2010)THP1 Human monocyte-derived macrophages SLC44A1 56.0 Fullerton et al. (2006)HT-29 Human colon carcinoma SLC44A1 12.1 Kouji et al. (2009)NRK-52E Rat renal tubule epithelial cells SLC44A1 16.5 Yabuki et al. (2009)TR-TBT Rat syncytiotrophoblast SLC44A1 68.3 Lee et al. (2009)SH-SY5Y Human neuroblastoma non cholinergic SLC44A1 6.2 Yamada et al. (2011)LA-N-2 Human neuroblastoma cholinergic SLC44A1 4.8 Yamada et al. (2011)MDA-MB-

231Human cancerous mammary cells SLC44A1 17.0 Eliyahu et al. (2007)

MCF7 Human cancerous mammary cells SLC44A1 20.0 Eliyahu et al. (2007)ZR-75-1 Human cancerous mammary cells SLC44A1 11.0 Eliyahu et al. (2007)SKBR-3 Human cancerous mammary cells SLC44A1 6.0 Eliyahu et al. (2007)T47D Human cancerous mammary cells SLC44A1 6.5 Eliyahu et al. (2007)ATII Rat alveolar type II epithelial cellsa SLC44A1 10–100 Ishiguro et al. (2008)

Rat astrocytesa SLC44A1⁄ 35.7 Inazu et al. (2005)Human keratinocytesa SLC44A1 12.3 Uchida et al. (2009)Mouse cerebral cortex neuronsa SLC44A1 26.7 Fujita et al. (2006)Human mammary epithelial cellsa SLC44A1 14.0 Eliyahu et al. (2007)

a Indicates primary cell cultures.

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level in other neuronal populations throughout the brain, but surprisingly not in other neurons expressing choline acetyl-transferase mRNA such as those found in the septal areas or in the nucleus basalis which constitute the brain cholinergicnuclei. This observation thus indicated that Slc44a1 distribution clearly differs from that expected for mRNA encoding thepresynaptic cholinergic protein.

The strong expression of Slc44a1 observed in the major myelinated fiber tracts corresponds to myelin basic protein-po-sitive oligodendrocytes. Moreover, Slc44a1 expression is regulated in the fiber tracts with a distribution profile reminiscentof that for MBP indicating that it occurs in oligodendrocytes during the period of myelination and remains in mature oligo-dendrocytes. Interestingly, Slc44a1 splice variants, Slc44a1a and Slc44a1b, display overlapping but not identical distributionsin the developing rat cerebellum. Both are strongly expressed in chains of cells in the fibre tracts indicating labeling of oli-godendrocytes. However, only Slc44a1a is also found in the granule cells and/or their precursors within the internal andexternal granule cell layers of the cerebellum indicating that Slc44a1b labeling is restricted to oligodendrocytes, which isconsistent with its expression in fibre tracts in other brain areas. Outside the central nervous system, especially in the colon,high levels of Slc44a1 expression are observed in the mucosal cell layer both lining the crypts and exposed to the lumen.

Many cell lines have been investigated for the expression of SLC44 family members (Table 1). Slc44a1 is also expressed invarious cell primary cultures including astrocytes (Inazu et al., 2005) and neurons (Fujita et al., 2006) from rodent cerebralcortex, alveolar epithelial type II (ATII) cells from rat lung (Ishiguro et al., 2008) and human keratinocytes from individualneonatal donor foreskins (Uchida et al., 2009).

3.2. SLC44 proteins and choline transport

SLC44A1 was initially identified as a protein able to restore choline transport in a triple yeast mutant strain deficient inboth choline transport and neosynthesis under selective conditions (O’Regan et al., 2000). In agreement with these data, Tor-pedo Slc44a1 overexpressed in Xenopus oocytes was found to increase Na+-independent high affinity choline uptake (O’Reganand Meunier, 2003). Analysis of the kinetic parameters of this choline transport led to a Km value for choline close to 1 lMand a high sensitivity to the competitive inhibitor HC-3 (Ki = 0.5 lM) (O’Regan et al., 2000). Choline uptake by mutant yeastexpressing Torpedo slc44a1 thus closely resembled the choline uptake in non mutant yeast and only partly resembled theneuronal choline transporter in cholinergic nerve terminals, known to have a high affinity for choline and sensitivity toHC-3, but to be Na+-dependent (Haga and Noda, 1973; Yamamura and Snyder, 1973). The capacity of SLC44A1 to transportcholine was confirmed upon transient transfection of rat Slc44a1a or Slc44a1b in the neuroblastoma N18 cell line identified asa cell line expressing only low endogenous levels of those transcripts. A [3H]choline uptake both saturable (Km � 33 lM) andinhibited by HC-3 was found increased compared with the basal level of choline uptake constitutively present in N18 cells(Traiffort et al., 2005). Moreover, a Na+-independent high affinity choline transport system was proposed to be mediatedby SLC44 members, in most cases SLC44A1, as shown by the use of specific siRNA or through the biochemical and

Table 2SLC44 – Choline-like transporter family. For detailed information about the SLC gene tables, please visit: http://www.bioparadigms.org.

Humangenename

Proteinname

Aliases Predominantsubstrates

Transporttype/couplingionsa

Tissue distribution andcellular/subcellularexpression

Link to disease Humangenelocus

Sequenceaccession ID

Splicevariantsandtheirfeatures

SLC44A1 CTL1 CDw92,CHTL1

Choline E Brain and spinal cordneurons andoligodendrocytes, colon(mucosal cells), lung,numerous tumoral ornon-tumoral cell lines/plasma membrane andmitochondria

Neonatalrespiratorydistresssyndrome

9q31.2 NM_080546.3 3 Splicevariants

SLC44A2 CTL2 PP1292 Choline O Tongue, muscle, kidney,heart, lung, testis,intestine stomach,inner ear (vestibularsupporting cells)

Auto-immunehearing loss,neonatalrespiratorydistresssyndrome,transfusion-related acutelung injury

19p13.1 NM_020428.3 5 Splicevariants

SLC44A3 CTL3 MGC45474 O Kidney, ileum, colon 1p21.3 NM_152369.3 2 Splicevariants

SLC44A4 CTL4 NG22,FLJ14491

O Intestine, stomach,kidney

6p21.3 NM_025257 3 Splicevariants

SLC44A5 CTL5 MGC34032 O Brain, spinal cord 1p31.1 NM_152697 2 Splicevariants

a E: Exchanger; O: Orphan transporter.

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pharmacological characterization of the response (Table 1). The capacity of the other members of SLC44 family to transportcholine has been much less studied, but it seems that not all SLC44 proteins are associated with choline uptake since neitherthe yeast (Zufferey et al., 2004) nor the nematode (Mullen et al., 2007) paralogs transport choline. However, uptake of [3-

H]choline by oocytes expressing human SLC44A2 led to suggest that this protein is functional as a choline transporter as well(Kommareddi et al., 2010). This was confirmed in the human lung adenocarcinoma cell line A549 where specific siRNA treat-ment led to a significant decrease of [3H]choline uptake (Nakamura et al., 2010).

Together with their wide expression pattern throughout tissues and cell types (see Table 2), the pharmacological param-eters reported for SLC44 family members, are consistent with the hypothesis that these proteins may generally provide cho-line for cell membrane phospholipid synthesis.

3.3. SLC44 proteins are plasma membrane and mitochondrial transporters

Several antibodies have been developed for characterizing the biochemical properties and visualizing SLC44 family mem-bers in tissues. Most of them have been raised against SLC44A1. They target alternatively the C-terminal domain of TorpedoSlc44a1b (G047; Meunier and O’Regan, 2002) or human SLC44A1a (G103; Machova et al., 2009), and the first extracellularloop or a C-terminal peptide of mouse SLC44A1a (LV-58 and EN-627, respectively Michel and Bakovic, 2009). In addition, themonoclonal antibody VIM15 generated by immunization of BALB/c mice with cells of a myeloid cell line, allowed recognitionof a surface epitope from CDw92/SLC44A1 in human leucocytes and endothelial cells (Wille et al., 2001). In western blotanalyses, G047 detects an immunoreactive signal almost exclusively in the CNS and PNS tissues from Torpedo (Meunierand O’Regan, 2002). VIM15 was successfully used to detect an immunoreactive signal in mouse skeletal muscle (Yuanet al., 2004) and in several human tissues despite a low signal in the brain (Yuan et al., 2006). G103 recognized nativeSLC44A1b and SLC44A1a in rat cerebral cortex and colon membranes, respectively (Machova et al., 2009).

The predominant signal detected by G047 and G103 antibodies was found at a lower apparent molecular weight (55–60 kDa) than the predicted size based on cDNA expression (O’Regan and Meunier, 2003; Machova et al., 2009). This shiftwas proposed to be related to interhelical hydrogen bonding and tissue specific post-translational modifications rather thanto proteolysis or disulphide bridges. In contrast, LV-58 and EN-627 both recognize a 72-kDa protein under non-denaturingconditions in preparations from human breast cancer (MCF-7), mouse skeletal muscle (C2C12) cells and mouse liver which isconsistent with the predicted size. Interestingly, in those preparations as well as in mouse kidney, but not in the brain,SLC44A1-immunoreactive signal was present not only in the plasma membrane, but also in the mitochondria fraction. Suchmitochondrial expression was confirmed by confocal analysis using mitochondria-specific dye in both untransfected and

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SLC44A1-transfected C2C12 cells. Mitochondrial preparations from these cells allowed identification of mitochondrialSLC44A1 as a choline transporter too (Michel and Bakovic, 2009).

In tissue sections from human normal CNS, G103 antibody immunostains the cell bodies of subpopulations of neurons inbrain cortical grey matter and motor neurons in the spinal cord grey matter. In contrast, G047 antibody stains mainly endo-thelial cells in blood vessels of the grey matter. G047 immunostains endothelial cells and glia identified as oligodendrocytesin brain white matter as well as Schwann cells in the PNS. In contrast, indistinct glial cells were observed in the brain whitematter using G103. The mutually exclusive immunolabelling of neuronal subpopulations, oligodendrocyte perikarya andendothelial cells by G103 and G047 was thus proposed to reflect the finding that while both SLC44A1a and SLC44A1b areexpressed in oligodendrocytes, only SLC44A1a is expressed by neuronal cell populations in the cerebellum (Machovaet al., 2009).

Less antibodies are available for the characterization of SLC44A2. One of the major works regarding this protein is thediscovery that SLC44A2 is the target of a guinea pig antibody (KHRI-3 or Kresge Hearing Research Institute-3) able to pro-mote hearing loss (Nair et al., 2004). KHRI-3 and two rabbit antisera raised against a phylogenetically conserved antigenicsegment in the N-terminal domain of SLC44A2 can identify 68–72 kDa glycoproteins in preparations from guinea pig innerear. SLC44A2 protein was prominently detected by immunofluorescence on vestibular supporting cells in both guinea pigvestibular system and human inner ear derived from vestibular tissues removed from patients undergoing therapeutic trans-labyrinthic surgical procedures (Nair et al., 2004). Some, but not all, patients with autoimmune hearing loss have circulatingantibodies for SLC44A2 (Kommareddi et al., 2009).

4. SLC44 family members in physiopathology

4.1. Involvement of SLC44A1 in membrane synthesis for cell growth

The physiological relevance of SLC44A1 as a choline transporter was addressed in several studies. The suppression ofSLC44A1 via siRNA results in the impaired growth of the cholinergic hybrid neuroblastoma cell line NG108-15 cells. Thesedata are consistent with a role for SLC44A1 in providing choline for incorporation into membrane phospholipids. However,such a role might be not true for all cerebral cells. In the same study, no growth reduction was observed in the glioma cellline C6, despite the significant reduction of choline uptake also induced by siRNA treatment. This discrepancy was proposedto be related to a higher resistance of non-neuronal cells to choline deprivation (Machova et al., 2009).

The regulation of SLC44A1 expression has been reported in various models. Following facial nerve transection, SLC44A1transcripts are up-regulated suggesting that the protein may be involved in activation of choline uptake for membrane syn-thesis in motor neurons following nerve injury (Che et al., 2002). In the same line, phorbol esters treatment which is knownto induce THP-1 monocyte differentiation to macrophages promotes a decrease in choline uptake associated with an im-paired SLC44A1 trafficking at the cell membrane (Fullerton et al., 2006). Interestingly, SLC44A1 expression appears to be alsoregulated by the availability of extracellular choline as shown by the down-regulation of both mRNA and protein during cho-line deficiency in C2C12 mouse muscle cells arguing for the importance of the transporter for choline homeostasis (Micheland Bakovic, 2009).

As previously mentioned, SLC44A1 can transport choline not only at the plasma membrane but also at the mitochondrialmembrane. This effect is consistent with the mitochondrial localization of one of the two major metabolic pathways of cho-line present in hepatic tissues and corresponding to the oxidation of choline to betaine (Fig. 2). Moreover, SLC44A1 could beinvolved in the transport of choline both into and out of the mitochondria, since a complex series of reactions contribute alsoto a substantial release of free choline and phosphocholine from mitochondrial phospholipids which likely become availablefor phospholipid synthesis in the endoplasmic reticulum (Michel and Bakovic, 2009). As SLC44A1 is up-regulated in variouscancer cell lines (Wang et al., 2007) and functionally expressed in mitochondria of MCF7 human breast cancer cells (Micheland Bakovic, 2009), a link has been established between the increase of mitochondrial total phospholipids reported in tumorcells and the levels of SLC44A1 expression. One hypothesis is that SLC44A1 may be involved in the decrease of ceramide pro-duction and apoptotic signaling related to the abnormal mitochondrial membrane metabolism detected in tumoral cells (Bir-bes et al., 2001; Michel and Bakovic, 2009).

4.2. SLC44 family proteins in the neonatal respiratory distress syndrome

Pulmonary surfactant is a complex mixture of lipids and proteins produced by type II alveolar ATII cells that serves tolower the surface tension at the alveolar air/water interface, increases alveolar stability during the respiratory cycle and pre-vents atelectasia. Surfactant is composed of approximately 10% proteins and 90% lipids in which the lipid component ismainly phospholipids, with phosphatidylcholine being the most abundant. Developmental insufficiency of pulmonary sur-factant production in premature children is called neonatal respiratory distress syndrome and can be prevented by gluco-corticoid administration which likely stimulates the biosynthesis of all four surfactant associated proteins andphosphatidylcholine as shown in ATII cells (Bolt et al., 2001).

Recently, SLC44A1 and SLC44A2 expression and choline uptake were found increased by dexamethasone treatment in thehuman lung adenocarcinoma cell line A549 (Nakamura et al., 2010). The proposed involvement of both transporters in the

Fig. 2. Choline metabolism. The intracellular pathways of choline metabolism include oxidation phosphorylation and acetylation which are cell and tissuespecific and lead respectively to the production of betaine, phosphatidylcholine and acetylcholine. The carrier implicated in choline transport through thecytoplasmic or mitochondrial membrane (grey bar) is indicated for each of the three main metabolic pathways.

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synthesis of phosphatidylcholine led to suggest that insufficient activity of SLC44A1 and SLC44A2 could result in a decreaseof lung surfactant synthesis leading to neonatal respiratory distress syndrome (Nakamura et al., 2010). In the same line, theanticancer agent, Gefitinib that acts as a selective inhibitor of Epidermal Growth factor receptor Tyrosine Kinase, has beenassociated with the onset of severe interstitial lung disease, whose molecular mechanisms have long remained largely un-known. Investigation of this mechanism led to demonstrate that Gefitinib is able to reduce phosphatidylcholine biosynthesisin A549 and primary-cultured rat ATII cells via the inhibition of choline uptake mainly mediated by SLC44A1. These dataconfer on the transporter a role in this mechanism of drug-induced lung toxicity (Ishiguro et al., 2008).

4.3. SLC44A2, a target for autoimmune hearing loss

Autoimmune damage to the inner ear is suspected to be a cause of rapidly progressing hearing loss. The monoclonal anti-body KHRI-3 was developed against cochlear epithelium and characterized as recognizing a specific epitope in the guinea pigorgan of Corti and promoting hearing loss in mice. The epitope was recently identified as SLC44A2 expressed on inner-earsupporting cells and the antibody to cause hair-cell death and hearing loss in guinea pigs (Nair et al., 2004). This hypothesiswas strengthened by the observation that autoimmune hearing loss patients have antibodies able to bind to inner-ear sup-porting cells with the same distribution pattern as anti-SLC44A2 antibodies. The KHRI-3 antibody would bind to an N-linkedcarbohydrate moiety which may be associated with asparagine residues N187 and N200 on extracellular loop 1 and/or N417on extracellular loop 3 in SLC44A2 amino acid sequence (Fig. 1B). Such binding should lead to hair-cell loss in the organ ofCorti and to hearing loss likely through blocking of the protein function. Despite the fact that the reasons for sensory celldeath consecutive to this insult are unknown, these data suggest that SLC44A2 may be necessary for hair-cell survival. Asa consequence, genetic defects in SLC44A2 could lead to hearing loss (Nair et al., 2004). More recently, Cochlin, an abundantprotein in the inner ear, postulated to be an extracellular matrix protein, was shown to co-immunoprecipitate with SLC44A2.Because of the implication of Cochlin in autoimmune hearing loss in humans and its ability to interact with SLC44A2, Cochlincould contribute to SLC44A2 function, though the functional consequences of this interaction remain to be elucidated (Kom-mareddi et al., 2007). While SLC44A2 is likely not the only supporting cell antigen target in autoimmune hearing loss, it couldbe one of the most important. Therefore, the development of an assay that can quickly and specifically identify patients withanti-SLC44A2 antibodies was undertaken. The recombinant human SLC44A2 protein was efficiently produced in insect cellsand used as a substrate for testing human sera. This assay led to detect specific antibodies in 50% of inner-ear–reactive auto-immune hearing loss sera. Additionally, circulating antibodies to the recombinant protein was associated with response tocorticosteroids suggesting that this assay may be also predictive of response to steroid treatment (Kommareddi et al., 2009).

4.4. Polymorphisms in SLC44A2 are responsible for TRALI

Transfusion-related acute lung injury (TRALI) is a frequent cause of transfusion-associated morbidity and mortality. Thisacute respiratory distress occurs 6 h after blood transfusion. Patients present namely with bilateral infiltrates in the chestX-ray and signs of hypoxemia. Severe TRALI is often due to antibodies in blood components directed against the humanneutrophil alloantigen-3a (HNA-3a). Recently, HNA-3a antigen was identified as a nucleotide polymorphism in SLC44A2gene, with the resulting variation at amino acid position 154 (Fig. 1B) determining the reactivity of the protein with

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HNA-3a-specific antibodies. The variant with an arginine at this position, rather than a glutamine (Q154R), constitutes theHNA-3a antigen. HNA-3a-specific antibodies cause active aggregation of granulocytes which may release molecules that in-crease permeability of the pulmonary vessels, which together with injury caused by occlusion of pulmonary vessels by cellaggregates may lead to the clinical symptoms of TRALI (Curtis et al., 2010; Greinacher et al., 2010). More recently, new allelicforms of SLC44A2 were identified (Fig. 1B). They include a C > T mutation at position 457 resulting in amino acid substitutionleucine to phenylalanine at position 153 (L153F) and a C > T mutation at position 988T involving a 301 threonine to methi-onine substitution, which presumably is located at the intersection between the membrane compartment and the cytoplas-mic domain (Bayat et al., 2011; Flesch et al., 2011).

To reduce the risk of TRALI caused by HNA-3a-specific antibody transmission by blood transfusion, preventive screeningsof blood donors with alloantibodies reactive to HNA-3a are under development. Improvement of assays allowing reliabledetermination of the HNA-3a genotype by polymerase chain reaction with sequence-specific primers (PCR-SSP) or HNA-3a phenotype by granulocyte immunofluorescence and agglutination tests are underway (Curtis et al., 2010; Bayat et al.,2011; Berthold et al., 2011; Reil et al., 2011). Because of the difficulty to express the recombinant full-length SLC44A2 ina configuration recognized by alloantibodies, owing to the complex structure of the protein, synthetic SLC44A2 peptides con-taining arginine or glutamine at position 154 have been investigated and characterized by their reactions against a panel ofanti-HNA-3a antibodies. The most recent data indicate that peptide-based assays for detection of HNA-3a antibodies riskbeing insensitive and thus require SLC44A2 peptides to be long enough to create a target suitable for detecting all examplesof anti-HNA-3a in blood donors. The development of stable human cell lines expressing stable recombinant HNA-3a was alsoreported for the detection of antibodies in flow cytometry and antibody capture assays (Bayat et al., 2011). Finally, the re-cently described PCR-SSP approaches have yet allowed the conclusion that 5% of the population is at risk for developingHNA-3a antibodies (Reil et al., 2011).

5. Concluding remarks

Despite its recent discovery, the SLC44 family of choline transporters has yet been the focus of many studies which alto-gether make SLC44A1 and to a lesser extent SLC44A2, major players of the mechanisms regulating choline metabolismthroughout the organism. These data open the way to further investigations of the role of choline transport in health anddisease, since both proteins are implicated in physiological and pathological mechanisms. If their activity in choline trans-port appears to be consistent with the production of phosphatidylcholine for lung surfactant biosynthesis or for membranesynthesis aimed at cell growth or repair, this activity is less obvious in the development of autoimmune diseases which havebeen related to the blockade of SLC44A2 function. The latter finding suggests the potential existence of yet non identifiedfunctions, the blockade of which could result in the damage of specific cell types such as hair cells in the inner ear or inthe deleterious aggregation of granulocytes which may account for the occurrence of autoimmune hearing loss and trans-fusion-related acute lung injury, respectively. SLC44 family members should in the future represent attractive targets fortreating or detecting pathologies associated with their dysregulation.

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