Nobuhito Ono

Ingrid Van Der Heijden

George L Scheffer

Koen Van De Wetering

Elizabeth Van Deemter

Marcel De Haas

Arjan Boerke

Bart M Gadella §

Dirk G De Rooij

Jacques J Neefjes ¶

Tom A M Groothuis ¶

Lauran Oomen ¶

Lenny Brocks ¶

Toshihisa Ishikawa

Piet Borst
email: p.borst@nki.nl

Multidrug Resistance-associated Protein 9 (ABCC12) is present in mouse and boar sperm

Keywords: ABC-transporters, drug transport, MRP, testis, mammary cancer, sperm. Biochemical

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INTRODUCTION

The largest family of mammalian primary active drug transporters is the Multidrug Resistance-associated Protein (MRP) family, belonging to the ABCC sub-group of ATP Binding Cassette (ABC) proteins. MRP1 (ABCC1) was discovered by Cole and Deeley and their co-workers [START_REF] Cole | Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line[END_REF] in 1992 and eight other MRPs have since followed one by one [START_REF] Büchler | cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats[END_REF][START_REF] Paulusma | Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene[END_REF][START_REF] Kool | Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologs of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines[END_REF], as reviewed by us [START_REF] Raggers | The human multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma membrane[END_REF][START_REF] Borst | Mammalian ABC transporters in health and disease[END_REF][START_REF] Borst | The multidrug resistance proteins 3-7. In ABC proteins: from bacteria to man[END_REF][START_REF] Borst | The potential impact of drug transporters on nucleoside-analog-based antiviral chemotherapy[END_REF][START_REF] Borst | On the putative cotransport of drugs by multidrug resistance proteins[END_REF][START_REF] Borst | Multidrug Resistance associated Proteins 3, 4 and 5[END_REF][START_REF] Ishikawa | The GS-X pump in plant, yeast, and animal cells: structure, function, and gene expression[END_REF][START_REF] Ishikawa | The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design[END_REF] and others [START_REF] Adachi | Therapeutic and biological importance of getting nucleotides out of cells: a case for the ABC transporters, MRP4 and 5[END_REF][START_REF] Bodo | The role of multidrug transporters in drug availability, metabolism and toxicity[END_REF][START_REF] Haimeur | The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation[END_REF][START_REF] König | MRP2, the apical export pump for anionic conjugates[END_REF][17][START_REF] Renes | The (patho)physiological functions of the MRP family[END_REF][START_REF] Deeley | Transmembrane Transport of Endoand Xenobiotics by Mammalian ATP-Binding Cassette Multidrug Resistance Proteins[END_REF]. The 8 human MRPs that have been characterized are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRPs may transport a variety of endogenous compounds, such as leukotriene C4 (MRP1), bilirubin glucuronides (MRP2, MRP3), prostaglandins E1 and E2 (MRP4), cGMP (MRP4, MRP5, MRP8), and several glucuronosyl-, or sulfatidyl steroids. The absence of two of these transporters is associated with human disease. Defective MRP2 causes the Dubin-Johnson syndrome; defective MRP6 pseudoxanthoma elasticum. A defect in MRP8 was recently associated with the inability to produce wet, brownish, sticky ear wax [START_REF] Yoshiura | A SNP in the ABCC11 gene is the determinant of human earwax type[END_REF].

The most recent addition to the MRP family is MRP9 (ABCC12), independently described in 2001 by Yabuuchi et al. [START_REF] Yabuuchi | Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12[END_REF], Tammur et al. [START_REF] Tammur | Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12[END_REF] and Bera et al. [START_REF] Bera | MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer[END_REF]. MRP9 is most closely related to MRP5 and MRP8. Human MRP9 is located next to MRP8 on chromosome 16q12.1 [START_REF] Yabuuchi | Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12[END_REF], presumably the result of a recent duplication [START_REF] Tammur | Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12[END_REF]. Whereas no mouse homolog of MRP8 (ABCC11) has been found [START_REF] Shimizu | Characterization of the mouse ABcc12 gene and its transcript encoding an ATPbinding transporter, an orthologue of human ABCC12[END_REF], the cDNA of murine Mrp9 (Abcc12) was cloned and characterized by Shimizu et al. [START_REF] Shimizu | Characterization of the mouse ABcc12 gene and its transcript encoding an ATPbinding transporter, an orthologue of human ABCC12[END_REF]. Both the human MRP9 and the mouse Mrp9 genes yield a plethora of alternatively spliced RNAs and this has led to uncertainty about the true nature of the protein encoded by MRP9. The gene structure looks unambiguous. It predicts 29 exons both in human and mouse, encoding a classical "short" MRP, lacking the N-terminal domain present in MRP1, 2, 3, 6, and 7, but absent in MRP4, 5, and 8. The predicted amino acid sequences of murine Mrp9 and human MRP9 are 84.5% identical and their hydropathy profiles are nearly identical [START_REF] Shimizu | Characterization of the mouse ABcc12 gene and its transcript encoding an ATPbinding transporter, an orthologue of human ABCC12[END_REF]. With MRP5 the human MRP9 shares 42% identity and all conserved elements in MRPs are also present in MRP9 [START_REF] Tammur | Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12[END_REF].

A transcript encoding full-length human MRP9 has been hard to find, however.

Tammur et al. [START_REF] Tammur | Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12[END_REF] (2001) identified transcripts of about 5000 bp on blots of human testis, ovary and prostate RNA, but were unable to clone a full length cDNA containing all MRP9 exons. [START_REF] Yabuuchi | Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12[END_REF] showed that the predominant MRP9 splice variants present in human tissues do not encode a canonical MRP, but they detected a small amount of RNA containing all putative exons in testis RNA [START_REF] Yabuuchi | Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12[END_REF]. The relevance of this RNA is supported by the results obtained with RNA from mouse testis [START_REF] Shimizu | Characterization of the mouse ABcc12 gene and its transcript encoding an ATPbinding transporter, an orthologue of human ABCC12[END_REF]. The predominant transcript in this Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society tissue is a 5.4 kb RNA, which encodes the complete Mrp9 protein sequence. Two minor variants were cloned as well, both encoding truncated proteins [START_REF] Shimizu | Characterization of the mouse ABcc12 gene and its transcript encoding an ATPbinding transporter, an orthologue of human ABCC12[END_REF].

In contrast, [START_REF] Bera | MRP9, an unusual truncated member of the ABC transporter superfamily, is highly expressed in breast cancer[END_REF] concentrated on the main splice products of the human MRP9 gene [START_REF] Bera | MRP9, an unusual truncated member of the ABC transporter superfamily, is highly expressed in breast cancer[END_REF]. They cloned two major RNAs: a 4.5 kb RNA lacking exons 5, 16, and 26, found only in testis and encoding a truncated protein of 930 amino acids; and a 1.3 kb RNA present at high levels in brain and encoding a putative protein of 233 amino acids.

Interestingly, substantial amounts of the 4.5 kb RNA were also found in some breast cancer cell lines. Moreover, tumor cells in some samples of breast cancer showed strong RNA in situ hybridization with the MRP9 probe. A band of about 100 kDA, presumably the 930 amino acid translation product of the 4.5 kb RNA, was detected in testis extracts and in a breast cancer cell line extract by an IgG fraction purified from rabbit antisera raised against MRP9 synthetic peptides [START_REF] Bera | MRP9, an unusual truncated member of the ABC transporter superfamily, is highly expressed in breast cancer[END_REF].

We have focussed on the full-length canonical versions of murine Mrp9 as well as human MRP9. We have tried to determine whether these proteins are actually made in vivo and what their transport function could be. Here we report that murine Mrp9 is present in murine sperm and sperm cell precursors.

MATERIALS AND METHODS

Chemicals and reagents

DMM (1-deoxymannojirimycin) was made by Broxterman et al. [START_REF] Broxterman | Synthesis of the antibiotic 1,5-dideoxy-1,5-imino-D-mannitol[END_REF] and was supplied by J.N. A polyclonal antibody against the human mitochondrial outer membrane protein Sam50 [START_REF] Kozjak | An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane[END_REF] was generated by T.G. and J.N. Sam50 fused to glutathione-S-transferase was made in Escherichia coli (E. coli) BL 21, purified over glutathione beads, and eluted with thrombin.

The thrombin was removed with a heparin column (Amersham) and the purified Sam50 was injected into rabbits according to standard protocols. The rabbit antisera recognized a mitochondrial protein in confocal microscopy and stained a 50 kDa band on Western blots of human, mouse and pig tissues.

Protein immunoblot (Western) analysis

Western blot analysis was done essentially as described before [28;29]. Protein was quantified using the Bio-Rad protein assay (Bio-Rad), fractionated on an 8% polyacrylamide slab gel and transferred to a nitrocellulose membrane by electroblotting. After blocking for 1 hour in PBS containing 1% nonfat milk powder, 1% bovine serum albumin, and 0.05% Tween 20, the blots were incubated with the MRP9 rat monoclonal antibodies, with subsequent use of a horseradish peroxidase-labeled goat anti-rat antibody (Santa Cruz Technology) and enhanced chemiluminescence for detection.

Transfected cell lines

HEK293 cells were grown in DMEM (GIBCO/BRFL) containing 10% FCS and penicillin/streptomycin. The cloning of the full-length human MRP9 cDNA has been described [START_REF] Yabuuchi | Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12[END_REF]. To make MRP9-expressing cell lines, the MRP9 cDNA was inserted into the EcoRI and Hind III sites of the pcDNA3.1(-)vector (Invitrogen). By using calcium phosphate precipitation, HEK293 cells were transfected with the pcDNA3.1(-)-MRP9 vector. After selection with G418 (1 mg/ml), positive colonies were picked and separately expanded in DMEM containing 10% FCS and penicillin/streptomycin for further studies. Although details are not described in this manuscript, several other transfected cell lines were also generated to characterize human MRP9. Briefly, to generate pEGFP-N2-MRP9, the 5' part of an EcoRI and PstI MRP9 cDNA fragment of PcDNA3.1 containing fulllength MRP9 cDNA was inserted into the corresponding restriction sites of the pEGFP-N2 vector. To replace the stop codon, 1327bp was amplified by PCR from an MRP9 cDNA template. The PCR fragment was digested with PstI and SmaI and inserted into pEGFP-N2 containing the 5' part of MRP9. The orientation and fidelity of the fragment was verified by sequence analysis. By analogous procedures, we constructed a MscV-MRP9-IRES-EGFP construct and expressed this in HEK293 cells. Finally, we expressed MRP9 in insect Sf9 cells using a baculovirus construct: The EcoRI MRP9 cDNA fragment from plasmid pcDNA3.1 (-) was inserted into the corresponding restriction sites of the pFastBac-1 vector. After the orientation of the MRP9 cDNA insert in the vector was verified, DH10Bac cells were transformed with the pFastBac-1-MRP9 construct to generate the recombinant bacmid DNA.

The sequence of the resulting recombinant bacmid DNA was verified by PCR analysis. Sf9 cells were transfected with the bacmid DNA to produce recombinant baculovirus. The MRP9producing Sf9 cells were used to prepare inside-out membrane vesicles for transport studies. The presence of MRP9 in these vesicles was checked with our new anti-MRP9 Mab M 9 I-27, 1µl of vesicle protein producing a clear 150-kDa band on a Western blot.

Cloning of rat Mrp9 (Abcc12) cDNA

The rat Mrp9 gene that is homologous to mouse Mrp9 or human MRP9 genes was identified in the NCBI mouse database as well as the EMBL/UCSC database. By using the GENSCAN program [START_REF] Burge | Prediction of complete gene structures in human genomic DNA[END_REF] (http://genes.mit.edu/GENSCAN.html), we have predicted plausible exons in the rat Mrp9 gene. Based on the predicted exons, rat expressed sequence tag (EST) clones were extracted from the EST database. We have screened the multiple tissue cDNA panels (MTC TM , Clontech, Palo Alto, CA, USA) by means of PCR using the following primers: the forward primer 5'-GACTATCGGATGAGATACAGAGACAACAC -3' the backward primer 5'-CAAAGCAGCTGGCGTTCTCCTACTGAGAAG -3'. Among the tissues screened, the highest expression of rat Mrp9 was detected in the testis.

To clone the rat Mrp9 cDNA, we have designed the following four sets of PCR primers: c12-1 (the forward primer: 5'-GTCCACAGAGGAGGAAGCTAGAGTGAAC -3' and the backward primer: 5'-GACTCACCTGCCCTAGAAGAGCAGAAATG-3'), c12-2 (the forward primer: 5'-GATACGTCCAAAGTGGGAACTCAGCCCTG-3' and the backward primer: 5'-CTGCATGGAGGAGGTGATGTGAGAGAAC-3'), c12-3 (the forward primer: 5'-CACATGTACCAGTTGGTTTACATAGCAAG-3' and the backward primer: 5'-CAAAGCAGCTGGCGTTCTCCTACTGAGAAG-3'), and c12-4 (the forward primer: 5'-GACTATCGGATGAGATACAGAGACAACAC-3' and the backward primer: 5'-GCGAGGAGCGCTGTTACCTCCAGCCTTG-3'). The PCR reaction was performed with rat testis cDNA (Clontech, Palo Alto, CA) and Ex Taq TM polymerase (TaKaRa, Japan). The PCR conditions consisted of 30 cycles of 95 O C for 30 sec, 58 O C for 30 sec, and 72 o C for 90 sec. After agarose gel electrophoresis, the PCR products were extracted from the gels and subsequently inserted into TA cloning vectors (Invitrogen USA) according to manufacturer's protocol. The sequences of the resulting inserts were analyzed with an automated DNA sequencer (TOYOBO Gene Analysis, Japan). Finally, the whole cDNA of rat Mrp9 was obtained by assembling those partial sequences.

Quantitative PCR analysis of human MRP9, mouse Mrp9 and rat Mrp9

The expression of human MRP9, mouse Mrp9 and rat Mrp9 in different organs was examined by quantitative PCR with human, mouse and rat Multiple Tissue cDNA (MTC) panel polymerase in a total volume of 25 µl. For this analysis, the following specific primers were used; for human (the forward primer 5'-GTAAGGTACAACTTGGATCCCT -3' and the backward primer: 5'-TGCTGCTAGTAACATCGCAA -3'), for mouse (the forward primer 5'-TATGGCCCGGG CACTTCTCCGTAA -3', the backward primer 5'-GACCTTTACAGTCCAACCTCTG CAGCTAGT -3') and for rat Mrp9 (the forward primer: 5'-GTAGGAGAACGCCAGCTGCTTTGTATGG -3' and the backward primer: 5'-GACCTTTACAGTCCAACTTCTGCAGCTAGT -3');. The PCR cycle consisted of four steps; i.e., denaturation (95˚C, 15 sec), annealing (60˚C, 15 sec), amplification (72˚C, 15 sec) and quantification (87˚C, 10 sec).

Rat monoclonal antibodies

For the generation of antibodies against MRP9, segments of MRP9 cDNA (Fig. 1) were subcloned into the pMal-c vector for production of a fusion protein of E. coli maltose binding protein with MRP9 segments. The fusion protein was produced in E. coli strain JM101 and purified by amylose affinity chromatography. The use of the fusion protein for the production of rat monoclonal antibodies was as described [31]. Briefly, the Mabs were selected by an ELISA test, and positive clones were cross-checked on cytospins of HEK293/MRP9 and HEK293 cells. The antibodies obtained were finally screened on Western blots of cellular lysates prepared from HEK293 cells transfected with MRP9 cDNA. Three mAbs of the M 9 I series against the N-terminal segment, and 4 of the M 9 II series against the linker segment were used for this study.

Immunohistochemistry of cells and tissues

For immunolocalization of the MRP9 protein in cells, HEK293 cells were grown for 2 days on glass slides. After fixation with acetone, we used the DAKO EnVision Plus HRP system (K4008; DAKO), which uses AEC as substrate, with minor modifications for staining.

Several rat monoclonal anti-MRP9 antibodies were used for MRP9 immunostaining. A rabbit anti-rat-IgG (Vector lab AI-4001, pre-absorbed with mouse IgG, 1:200 dilution) was used as the secondary antibody. Before mounting, slides were counterstained with hematoxylin.

Adult FVB mouse were used for the isolation of testis and epididymis.

Immunohistochemistry of testis cyto-sections fixed with acetone was performed as described for HEK293 cells.

Isolation and immunostaining of sperm

Mouse sperm were washed out of the caudal epididymis and centrifuged at 400 x g. The pellet was washed in PBS and a drop was placed on a glass slide, smeared out like a blood smear, and air dried. After fixation in acetone for 10 min. at room temperature, the slide was rinsed with PBS/0.05% Tween 20 for 5 min. and the smear was then stained using the DAKO EnVision Plus HRP system (DAKO). Preblocking was done with a mixture of PBS, 0.05% Tween and 5% normal donkey serum (Jackson ImmunoResearch). Rat anti-MRP9 Mabs were used in undiluted form (culture supernatant) for 2 hours at room temperature; after rinsing with PBS/0.05% Tween, the slide was pre-incubated with rabbit anti-rat IgG (Vector lab, preabsorbed with mouse IgG in 1:200 dilution), followed by the polymer-HRP incubation with AEC substrate, as specified by DAKO. The slides were counterstained for 5 sec with hematoxylin solution.

Boar semen was collected in the Cooperative Centre for Artificial Insemination in Pigs "Utrecht en de Hollanden" (Bunnik, the Netherlands). Semen was filtered through gauze to remove gelatinous material and diluted to 150 million cells/ml in Beltsville Thawing Solution (BTS: 0.2 M glucose, 20 mM sodium citrate, 15 mM NaHCO 3 , 3.36 mM Na 2 EDTA, 10 mM KCl, 20 mM Hepes, pH 7.4) and kept at 17 O C for transport or storage in air-tight 80 ml insemination tubes. Prior to further experimentation the sperm samples were first washed through BTS by centrifugation for 5 min at 400 x g and the resuspended sperm (1 billion sperm/ml) was then spun through a discontinuous (35%/70%) Percoll® (Pharmacia, Uppsala, Sweden) density gradient to remove epithelial cell contamination and immature sperm [START_REF] Gadella | Capacitation induces cyclic adenosine 3',5'monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells[END_REF].

After washing the pellet with PBS, sperm smears were stained as for the mouse sperm, but pre-blocking before adding anti-MRP Mabs was done with 4% BSA in PBS without the donkey serum.

Confocal microscopy of immunofluorescent cells

Confocal fluorescence images were obtained on a Leica TCS SP2-AOBS (Leica Microsystems, Heidelberg, Germany) confocal system, using the 488 nm line from an Argon The rat anti-MRP9 antibodies M 9 I-38 and M 9 II-19 were used undiluted on formalinfixed cells and 1:2 diluted on methanol-fixed cells. As secondary antibodies we used goat anti-rat-Alexa488 or anti-rabbit-Texas Red.

Analysis of protein-linked oligosaccharides

Total cellular lysates were incubated with an N-glycosidase cocktail (2.67mU/µl), a mixture of endoglycosidase F and peptide-N-glycosidase (Boehringer Mannheim, Mannheim, Germany), at 30 o C overnight, to remove N-linked oligosaccharides. High-mannose oligosaccharides were removed by incubation with endoglycosidase H (2 units/ml, Boehringer Mannheim, Mannheim, Germany) in 50mM sodium citrate (pH 5.5) and 0.2% SDS, at 37 o C overnight.

Cell fractionation studies

Mouse testis was dissected into small pieces in ice-cold mitobuffer (0.2 mM sodium-EDTA, 0.25 M sucrose, 10 mM Tris-chloride, pH 7.8) and homogenized by 15 strokes with a Wheaton Dounce Tissue Grinder with tight pestle clearance (0.05 mm). The homogenate was centrifuged for 10 min at 1000 g at 4 0 C. The supernatant (S1) was centrifuged for 15 min at 8000 g at 4 0 C (Sorvall, SS34 rotor), the pellet (P2) was washed twice with mitobuffer containing protease inhibitors (Roche Complete tablets) and stored frozen. (P2) was thawed, centrifuged and resuspended in a minimal volume of 0.8 M sucrose, containing 0.2 mM EDTA / Tris pH 7.8 and layered on a 5-ml 0.9 -2.0 M sucrose gradient (in 0.3 mM sodium EDTA, 10 mM Tris, pH 7.8). After centrifugation to equilibrium (2 hrs, 4 0 C, 80,000 g), about 15-20 fractions were collected through a hole punched in the bottom. The membranous material in each fraction was collected by centrifugation for 10 min at 20,000 g. The pellets were resuspended in mitobuffer.

RESULTS

Generation of antibodies against MRP9

Whereas transcripts of MRP9 have been detected in various human, rat and mouse tissues including brain, the expression levels are relatively low with the exception of rat and mouse testes (Fig. 2). Most of the human testis RNA consists of alternative splice products, but our previous analysis has also shown [START_REF] Yabuuchi | Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12[END_REF]. To test whether this protein is actually made, we generated a series of monoclonal antibodies (MAbs) and two polyclonal rabbit antibodies against human MRP9 segments with little sequence identity with other MRPs (Fig. 1). The MRP segments were fused to the E. coli maltose binding protein, and purified fusion proteins were used as immunogen (see Methods). The Mabs were raised in rats in the hope of getting some antibodies that would not only react with human MRP9, but also with murine Mrp9.

To characterize the antibodies, we used Human Embryonic Kidney 293 (HEK293) cells transfected with a MRP9 cDNA construct. Fig. 3A shows the results with 4 of the 7 different Mabs. Each of the Mabs recognizes a single 150 kDa band in the transfected cells, but not in the parental cells and the same result was obtained with the 3 Mabs not shown in Fig. 3A. Fig. 3B presents immunohistochemical results with 3 of the Mabs. In the HEK293 cells MRP9 does not localize to the plasma membrane, as other MRPs do, but remains intracellular. The two rabbit antisera raised against MRP9 segments also recognized a 150 kDa band, but as these sera gave more background than the MAbs, they were not used in most further experiments.

Detection of full-length Mrp9 in mouse tissues

With the antibodies generated we were unable to detect any form of MRP9 in human tissues or cell lines, or on Western blots of extracts from tissues or cell lines. These included testis and mammary tumor cell lines in which a short 100-kDa form of MRP9 has been detected by others. We therefore turned to murine tissues. Fig. 4A shows a Western blot of mouse tissue extracts, incubated with Mab M 9 II-19. Only testis extracts contained a 150-kDa band comigrating with the 150-kDa band in HEK293/MRP9 cell extract. More rapidly migrating bands were observed in mouse liver extracts, but these were non-specific as they were also present in the control with second antibody only (results not shown). The 150 kDa band in mouse testis reacted with 6 of our 7 anti-MRP9 Mabs; only M 9 II-26 was negative (Fig. 4B), although it reacted as intensely with human MRP9 as the other Mabs (Fig. 3A). Mab 9 II-26 therefore provided a convenient control for further mouse studies. Whereas the Mabs directed against the N-terminus of MRP9 (M 9 I series) only stained a 150-kDa band, the Mabs directed against the linker region (M 9 II series; see Fig. 1) also stained a band of about 105 kDa (Fig. 4 A and B, indicated by arrow). This is unlikely to be a protein corresponding to one of the known alternatively spliced RNAs of mouse Mrp9 [START_REF] Shimizu | Characterization of the mouse ABcc12 gene and its transcript encoding an ATPbinding transporter, an orthologue of human ABCC12[END_REF], as both RNAs encode the N-terminal sequence of Mrp9 and the corresponding proteins should therefore also react with the Mabs of the M 9 I series (Fig. 1A) It is therefore either a degradation product of Mrp9 or the result of an internal translation start. The variable amount of this band and its absence in other tissues than testis makes the degradation product alternative the more likely one. In a (failed) attempt to generate a mouse Mrp9 KO by oligonucleotide targeting [START_REF] Dekker | Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3[END_REF] we obtained mice producing MRP9 without the N-terminal segment used to get the M 9 I series of antibodies. Western blots of testis extracts of these mice contained a 150-kDa band that reacted with the M 9 II series of Mabs, but not the M 9 I series, confirming the specificity of the M 9 I series for MRP9 (see supplementary Fig. 1). We estimate that the missing segment in truncated Mrp9 is only 31 aa (see supplementary material), explaining why the truncated Mrp9 co-migrates with wild-type Mrp9.

We studied the distribution of Mrp9 in mouse testis by immunocytochemistry using 3 No significant staining was observed in Leydig cells, or other interstitial elements. Within the seminiferous tubules we found no staining of the Sertoli cells and Mrp9 staining appeared to be specific for testicular germ cells.

During mouse testis development, Mrp9 was first detected in mice three weeks of age (fig. 5A). Only a few cells were positive in 3-week testis (Fig. 5B), the pachytene spermatocytes. As the mice aged, more Mrp9-positive cells appeared (Fig. 5B). Ìn the adult seminiferous tubules, specific associations of subsequent generations of spermatogonia, spermatocytes and spermatids at particular development steps, occur in a cyclic manner referred to as stages of the cycle of the seminiferous epithelium [START_REF] Russel | Histological and histopathological evaluation of the testis[END_REF]. To study which types of cells during spermatogenesis were positive for Mrp9, we investigated sections of seminiferous tubules in the different epithelial stages, of which there are 12 in the mouse.

During germ cell differentiation in the adult mouse testis, pachytene spermatocytes in stage VI of the epithelial cycle were the first germ cells to show Mrp9 expression (Figure 6B). Subsequently, Mrp9 stayed present during further development of the spermatocytes, round spermatids and elongating spermatids (Figure 6A). During the maturation of the spermatids, Mrp9 accumulated in the tail of elongating spermatids (Figure 6B). There was no significant staining of spermatogonia or pachytene spermatocytes in stage I-IV (Figure 6A), of type B spermatogonia in stage VI (Figure 6B), in leptotene spermatocytes in stage VIII-IX (Figure 6C) or in leptotene/zygotene spermatocytes in stage X (Figure 6D).

Analysis of Mrp9 in mouse sperm

To study whether mature mouse sperm contain Mrp9, we analysed sperm from the epididymis. Fig. 7A shows a Western blot of mature sperm lysates analysed with 6 different antibodies. All three antibodies of the M 9 I series detect the characteristic 150 kDa Mrp9 band, M 9 II-3 and -19 detect this band and the additional 105 kDA band, whereas the control Mab M 9 II-26 yields an "empty" lane. The background with control Mab M 9 II-26 is somewhat higher in epididymis (Fig. 7B) than in testis (Fig. 4C). The upper part of panel B of To test whether any MRP9 glycosylation was detectable, we incubated cell lysates of HEK293/MRP9 and of mouse testis with endoglycosidases, prior to Western blot analysis. As shown in supplementary Fig. 2, no indication for glycosylation of MRP9/Mrp9 was obtained.

The lack of detectable N-linked glycosylation of MRP9 suggests that it is not routed through the Golgi cisternae to the plasma membrane or to a post-Golgi endosomal compartment. Indeed, Fig. 3B does not show MRP9 in the plasma membrane or in lysosomes, the usual final destinations of MRPs in transfected cells [START_REF] Borst | The multidrug resistance protein family[END_REF]. To analyse the distribution of MRP9 in the transfected HEK cells in more detail, we did co-localization studies with markers for intra-cellular membranous compartments. As shown in Fig. 8, MRP9 is clearly not located in mitochondria (represented by the mitochondrial outer membrane marker Sam50) and also not in lysosomes (analysed with the lysosomal marker LAMP-1; results not shown). Co-localization was most extensive with the ER markers protein disulfide isomerase (PDI) and calnexin, as also shown by the pixel analysis in supplementary Fig. 3. Lack of colocalization of MRP9 with mitochondrial markers was also observed in HEK293 cells transfected with other MRP9 constructs and with an EGFP-MRP9 fusion construct (see Methods).

Localization of Mrp9 in murine and boar sperm

Although the staining for Mrp9 of sperm midpiece shown in Fig. 7B is clear, we were unable to resolve the intracellular localization. The staining was too weak for reliable immunofluorescence staining; none of our 6 MAbs were suitable for (cryo) electron microscopy and neither were the two anti-MRP9 sera made. We therefore turned to cell fractionation. Sucrose gradients of sub-cellular fractions of mouse testis showed that Mrp9 clearly separated from the mitochondrial markers Sam50 and cytochrome c, confirming that Mrp9 is not in mitochondria (supplementary Fig. 4). In contrast, MRP9 did not separate Attempts to fractionate mouse sperm cells failed for lack of material and we therefore turned to boar sperm. Of our 7 MRP9-specific Mabs, 2 reacted well with the boar sperm midpiece, as shown in Fig. 9, and these antibodies also detected a band of approximately 150 kDa on Western (protein) blots (results not shown). Unfortunately, our attempts to separate a boar sperm homogenate into sub-cellular fractions failed, as the membranous fractions aggregated so strongly that we were unable to separate them in a sucrose gradient.

DISCUSSION

MRP9 was the last member of the MRP family cloned and no more will follow. The genome sequence is complete and the only other candidate MRP left, -MRP10 (ABCC13) -is clearly a pseudo-gene [36;37]. MRP9 is an unusual MRP in many respects; it contains no detectable N-linked glycosylation; the full-length protein is only made in detectable amounts in testicular germ cells and sperm; it appears to be localized mainly in the ER in transfected HEK293 cells and in a still poorly defined (non-mitochondrial) membrane fraction in sperm.

We have tried to find a substrate for MRP9 by testing the ability of MRP9 to induce cellular resistance to cytotoxic compounds and to transport known MRP substrates in vesicular transport, without any positive result thusfar (see supplementary materials).

We have been unable to detect any form of MRP9 in any human tissue or cell line.

Given the fact that MRP9 RNA was readily detected in many tissues and in cell lines from various tumors, this is puzzling. Each of the 7 Mabs that we raised against human MRP9 (Fig. 1A) should have sensitively detected both full-length MRP9 and all long splice variants described. This includes the 100-kDa band detected by Bera et al. [START_REF] Bera | MRP9, an unusual truncated member of the ABC transporter superfamily, is highly expressed in breast cancer[END_REF] in testis and in a breast cancer cell line. It is possible (but unlikely) that the single polyclonal antibody used by Bera et al. [START_REF] Bera | MRP9, an unusual truncated member of the ABC transporter superfamily, is highly expressed in breast cancer[END_REF] was a more sensitive tool than the 7 Mabs we generated. It is also possible that the Bera antiserum reacted with a 100-kDa cross-reacting band and that there is very little translation of the MRP9 RNA detected with PCR. It may be significant that there are hardly any published RNA blots containing MRP9 transcripts; most MRP9 transcripts were thusfar detected by PCR, indicating low expression of MRP9.

MRPs evolve rapidly and substantial differences have been observed between human MRPs and their murine counterparts. The most striking thus far is the absence of MRP8 in mice [START_REF] Yoshiura | A SNP in the ABCC11 gene is the determinant of human earwax type[END_REF]. Nevertheless, it seems unlikely that MRP9 is a pseudo-gene in humans, as the basic structure of MRPs is fully conserved in MRP9 and as it is 84.5 % identical in amino Our results suggest a role for MRP9 during the latter part of the male meiotic prophase, the development of spermatids and/or possibly in sperm function. MRP9 is clearly and exclusively localized in the sperm mid-piece, the anterior part of the flagellum characteristically containing all sperm's mitochondria. Nevertheless we are confident that MRP9 is not in mitochondria. Conventional wisdom has it that sperm does not contain ER, the sub-cellular fraction in which we find most of the MRP9 made in transfected HEK293 cells. However, Olson and Winfrey have described a mid-piece specific sub-mitochondrial reticulum in mammalian spermatozoa [START_REF] Olson | Mitochondria-cytoskeleton interactions in the sperm midpiece[END_REF], and this structure remains a possible location for MRP9. The generation of a Mrp9 KO mouse may shed more light on MRP9 function.

Legends to Figures

Figure 1 Segments of MRP9 used for raising antibodies

The figure shows the putative topology of MRP9 in the membrane [START_REF] Bera | MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer[END_REF]. Arrows above the membrane indicate the protein segments used to raise rabbit polyclonal antibodies against a fusion protein containing amino-acids 1-33 (9 FP-A) or amino acids 1317-1349 (9 FP-D).

Arrows below the membrane indicate the MRP9 segments used to generate rat monoclonal antibodies. Series I was raised against a mixture of fusion proteins containing amino acids 1-33 and 1-42; series II against amino acids 690-734. All series I antibodies react with the fusion protein containing amino acids 1-33. See Methods for details.

  Commercial antibodies came from the following sources: Polyclonal rabbit antihuman calreticulin (Upstate); polyclonal rabbit anti-human EEA1 (Upstate); polyclonal rabbit anti-bovine Catalase (Abcam); monoclonal rat anti-mouse CD107a (LAMP-1) (BD Pharmingen); monoclonal mouse anti pigeon Cytochrome c (B D Pharmingen); polyclonal rabbit anti-human Calnexin (Santa Cruz); polyclonal goat anti-human GRASP65 (Santa Cruz); rabbit anti-PDI rec 442 (a kind gift of Dr. Hidde Ploegh, Boston); goat anti-rabbit-HRP (Santa Cruz); goat anti-rat-HRP (Santa Cruz); rabbit anti-mouse-HRP (DAKO); rabbit anti-Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society goat-HRP (DAKO); rabbit anti-rat (Vector Labs); goat anti-rat-Alexa 488 (Molecular Probes); goat anti-rabbit-Texas Red (Molecular Probes).

(

  Clontech, Palo Alto, CA), respectively. The PCR was performed with TaKaRa Ex Taq TM R-PCR version in a TaKaRa Smart Cycler TM System (TaKaRa, Japan), where amplification of DNA was monitored with SYBR Green I. The reaction mixture contained 2 µl of cDNA (MTC panel), 2.5 µl of 10 x PCR buffer, 0.75 µl of 10 mM dNTP mix, 0.75 µl of 10 µM PCR Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society primers, 0.3 µl of 250 mM Mg 2+ solution, 0.25 µl of 1/300-dilluted SYBR Green I (Bio-Whittaker Molecular Applications, Rockland, ME, USA), and 0.25 µl of Ex Taq TM

Biochemical

  Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  laser and the 594 nm line from a HeNe laser as excitation source. Images were taken using a HCX PL APO CS 63x NA 1.4 oil objective. Possible crosstalk between the different fluorochromes, which could give rise to false-positive colocalisation of the signals, was totally avoided by careful selection of the imaging conditions. For the analysis of the intracellular location of MRP9 in transfected cells, the MRP9-HEK293 cells were grown overnight on poly-L-lysine-coated coverslips. After rinsing with PBS, the cells were fixed with either methanol (5 min icecold methanol, rehydration in PBS for 5 min) or formalin (20 min formalin at room temp., 3 PBS washes, 30 min PBS incubation, permeabilization for 5 min in a mixture of 1% BSA and 0.2% Tween X-100 in PBS). For antibody incubations, the Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society cells were pre-blocked with 1% BSA in PBS for 30 min, incubated with primary antibody for 1 hr, washed with 1% BSA/PBS, incubated with the secondary antibody for 1 hr, washed with 1% BSA/PBS and PBS, and mounted in vectashield.

  the presence of a minor fraction of an RNA able to encode Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society full-length MRP9

Biochemical

  Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Mabs. Fig 4C shows strong staining of the testicular tubules with the Mrp9-specific Mabs M 9 I-38 and M 9 II-3, but not with M 9 II-26, which does not react with murine Mrp9 (Fig 4B).

Fig. 7 Biochemical

 7 Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society shows specific staining in the lumen of the epididymal ducts and originates from the sperm that are migrating towards the ductus deferens, but not in the epithelium. That the sperm cells are responsible for the luminal staining is confirmed in the lower half of Fig. 7B and in Fig. 7C. The Mrp9-specific Mabs stain the midpiece of sperm; the control Mab M 9 II-26 gives no staining at all.Post-translational modification and intra-cellular location of MRP9 in transfected HEK cellsIn polyacrylamide gels human MRP9 and murine Mrp9 run faster than MRP5, although these proteins are of similar size on the basis of their amino acid sequences. The MRP9 band is also much sharper on Western blots than of the other MRPs, which are all heavily glycosylated.

Biochemical

  Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society unambiguously from the lysosomal marker LAMP1 or the ER markers calnexin (Supplementary Fig. 4) or calreticulin (not shown).

Biochemical

  Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20070292 © 2007 The Authors Journal compilation © 2007 Biochemical Society acid sequence to murine Mrp9, a high level of identity conserved over all the 29 putative exons of MRP9.

Figure 2 MRP

 2 Figure 2MRP RNA levels in rat, mouse and human tissues, determined by semiquantitative RT-PCR.Note differences in scale for human versus rodent tissues. See Methods for details.

Figure 3

 3 Figure 3 Detection of MRP9 in HEK 293 cells transfected with a MRP9 cDNA construct (A) Western blot of cell lysates from transfected and parental HEK 293 cells stained with 4 different rat anti-MRP9 MAbs. (B) Immunocytochemical staining of transfected and parental HEK 293 cells stained with 3 different rat MAbs. See Methods for details.

Figure 4

 4 Figure 4 Tissue distribution of MRP9 in mice (A) Western blot analysis of MRP9 in protein extracts of various mouse tissues, using rat Mab M 9 II-19. The asterisk indicates a non-specific band reacting with the second antibody. The arrowhead indicates the 105 kDa band. (B) Western blot analysis of MRP9 in mouse testis extracts, using three different rat anti-MRP9 MAbs, as indicated. The arrowhead indicates the 105 kDa band. (C) Sections of adult mouse testis, stained with two rat MAbs that detect MRP9 and a control MAb M 9 II-26 that does not react with mouse MRP9, as shown in panel B. See Methods for details.

Figure 5

 5 Figure 5 Appearance of Mrp9 during mouse development (A) Western blots of testis extracts from mice 2 (two different mice) or 3 weeks after birth with adult testis extract for comparison. (B) Immunocytochemistry of testis sections of mice of the age indicated. AEC was used instead of DAB to visualize the antibody signal. Mab 9 I-38 was used in both panels.

ACKNOWLEDGEMENTS

We are grateful to Elly Mesman and Dr. P. Peters (NKI-AVL) and to Dr. L. Looijinga and Dr. J.W. Oosterhuis (Erasmus Medical Centre) for advice and help with microscopy; to M. Dekker and Dr. H. te Riele (NKI-AVL) for help in the generation of mice with a truncated form of Mrp9; and to Dr. J. Wijnholds (Netherlands Institute for Neuroscience) for advice and assistance. Dr. N. Ono was supported by a postdoctoral fellowship from the Japan Research Foundation for Clinical Pharmacology. This work was also supported by grants NKI 2001-2473 and -2474 of the Dutch Cancer Society to P. Borst and J. Wijnholds.

The panel shows from top to bottom: boar sperm stained with Mab M 9 II-3, M 9 II-19, or M 9 II-26 (which does not react with pig MRP9). The presence of the anti-MRP9 Mab was detected by peroxidase staining as described in Methods, resulting in coarse granular precipitates of the peroxidase reaction product.


Emilio Lecona

Nieves Olmo

Javier Turnay

Angélica Santiago-Gómez

Isabel López De Silanes

Myriam Gorospe

Prof Antonia Mª

Lizarbe
email: lizarbe@bbm1.ucm.es

Kinetic analysis of butyrate transport in human colon adenocarcinona cells reveals two different carrier-mediated mechanisms

Keywords: anion exchanger, butyrate uptake, colon adenocarcinoma, monocarboxylate transporter, PKC activators and inhibitors, siRNA AE, anion exchanger, BSM, bisindolylmaleimide I, 4-CHC, α-cyano-4

Butyrate has anti-tumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration which is regulated by its transport. We have analyzed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT29), in butyrate resistant cells (BCS-TC2.BR2), and in normal colonic cells (FHC). The properties of transport were analyzed with structural analogues, specific inhibitors, and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K m =109±16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K m =17.9±4.0 μM in BCS-TC2 cells) protonmonocarboxylate cotransporter that was energy-dependent and activated via PKCδ. All adenocarcinoma cells analyzed express MCT1, MCT4, ancillary protein CD147, and AE2.

Silencing experiments show that MCT1, whose expression increases with butyrate-treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate-bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time-and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumor regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.

INTRODUCTION

Short-chain fatty acids (SCFAs) are produced by anaerobic bacterial fermentation of dietfiber within the large bowel. Among them, butyrate plays a key role in colonic epithelium homeostasis and represents the main fuel for colonocytes in vivo. Butyrate induces cell differentiation and apoptosis and regulates proliferation of mucosal epithelial cells, reducing the growth rate of colorectal cancer cells in culture [START_REF] Andoh | Role of dietary fiber and short-chain fatty acids in the colon[END_REF][START_REF] Miller | Cellular and physiological effects of short-chain fatty acids[END_REF]. It is currently accepted that butyrate effects are mainly due to the regulation of the expression of specific genes through its activity as histone-deacetylase inhibitor [START_REF] Myzak | Dietary agents as histone deacetylase inhibitors[END_REF]. SCFA uptake can be rate-limiting and deficiencies in the availability or utilization of butyrate have been implicated in the development of colitis and in colon carcinogenesis [START_REF] Soergel | Colonic fermentation: metabolic and clinical implications[END_REF].

Different studies using several model systems indicate that SCFAs are transported into colonic epithelial cells by two different mechanisms: passive nonionic diffusion (unspecific component) of the undissociated SCFA form, and specific carried-mediated transport of SCFA anions. Although butyrate should be ionized at the physiological pH of the colonic lumen, the nonionic form of butyrate can exist due to the luminal acidic microclimate generated in the apical region of colonocytes by H + -secreting systems, as the Na + -H + exchanger (isoforms 2&3) [START_REF] Krishnan | Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon[END_REF]. Earlier work suggests that this is the main uptake mechanism [START_REF] Chu | Non-ionic diffusion and carrier-mediated transport drive extracellullar pH regulation of mouse colonic crypts[END_REF], but later evidence supports a greater participation of carrier-mediated transport [START_REF] Rajendran | Characterization and molecular localization of anion transporters in colonic epithelial cells[END_REF].

Carrier-mediated transport of the ionized form of butyrate has been described so far as: i) a nonelectrogenic SCFA --HCO 3 antiporter, ii) an electroneutral H + -coupled monocarboxylate transporter, and iii) an electrogenic Na + -coupled monocarboxylate transporter. The SLC4 family mediates Na + -independent anion exchange and is involved in the electroneutral and reversible exchange of Cl -and HCO 3 -across the plasma membrane.

The AE2 isoform (SLC4A2) has been described to mediate apical butyrate transport in HEK293 cells [8]. Experiments with membrane vesicles show an exchange butyrate-HCO 3 in apical and basolateral membranes from surface cells, but not from crypt cells, that is stimulated by a pH gradient [START_REF] Reynolds | Bicarbonate-stimulated [14C]butyrate uptake in basolateral membrane vesicles of rat distal colon[END_REF][START_REF] Tyagi | Mechanism of n-butyrate uptake in the human proximal colonic basolateral membranes[END_REF].

The proton-linked monocarboxylate transport is carried out by members of the SLC16 family. MCT1 (SLC16A1) and MCT4 (SLC16A3) require a monotopic ancillary protein, CD147, for proper cell surface expression and butyrate transport. MCTs may transport one monocarboxylate with either a proton or sodium [START_REF] Halestrap | The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation[END_REF][START_REF] Enerson | Molecular features, regulation, and function of monocarboxylate transporters: implications for drug delivery[END_REF], or exchange one monocarboxylate for another, via electroneutral transport, with a 1:1 stoichiometry. Monocarboxylate flux direction depends on the chemical gradients for H + and monocarboxylates across the Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society membrane. Heterologous MCT1 expression has revealed its specificity for monocarboxylic acids such as pyruvate, L-lactate and acetate [START_REF] Enerson | Molecular features, regulation, and function of monocarboxylate transporters: implications for drug delivery[END_REF][START_REF] Garcia | Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle[END_REF] in association with a Na + -bicarbonate cotransporter to compensate for the proton influx [START_REF] Becker | Facilitated lactate transport by MCT1 when coexpressed with the sodium bicarbonate cotransporter (NBC) in Xenopus oocytes[END_REF]. It is inhibited by structural analogues, phloretin, tiol and amino reagents and derivatives of α-cyanociannamate, with a lower sensitivity for 4,4´-diisothiocyanostilbene-2,2´-disulfonate (DIDS) and other stilbenes. MCT1

has been localized to the basolateral membranes of immature intestinal epithelial cells, whereas it appears in brush border membranes of absorptive cells. MCT4 is expressed in cells with a high glycolytic rate associated with hypoxic energy production [START_REF] Dimmer | The lowaffinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells[END_REF], exhibiting a much lower affinity for most substrates and inhibitors than MCT1 [START_REF] Halestrap | The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation[END_REF].

SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters with a Na + :monocarboxylate stoichiometry of 4:1 for SCFAs [START_REF] Srinivas | Cloning and functional identification of slc5a12 as a sodiumcoupled low-affinity transporter for monocarboxylates (SMCT2)[END_REF]. They are very similar to MCTs in substrate selectivity but they are Na + -dependent. SLC5A12 is expressed in the small intestine (but not in colon) whereas SLC5A8 is expressed in the apical membranes of the colonic epithelium. Due to its location and lower substrate affinity, SLC5A12 is unlikely to participate in physiological SCFA absorption.

Poorly differentiated colon tumor cells grow in multilayer without polarization, what

impairs the availability of oxygen and nutrients. Thus, transport mechanisms are likely to differ from those observed in colonocytes. There are only a few reports on butyrate transport in human colon adenocarcinoma cells, which mainly employ HT-29 cells [START_REF] Cuff | The human colonic monocarboxylate transporter Isoform 1: its potential importance to colonic tissue homeostasis[END_REF] or polarized Caco-2 cells [18, 19] as a model system. Nevertheless, the latter cells more closely resemble the properties of small intestine epithelium [20], underscoring the need to further analyze this process in colon adenocarcinoma cells. Here, we examine butyrate transport in different human colon adenocarcinoma cell lines (butyrate-sensitive BCS-TC2 cells [START_REF] Turnay | Establishment and characterization of a new human colon adenocarcinoma cell line: BCS-TC2[END_REF], butyrateresistant BCS-TC2.BR2 cells [START_REF] López De Silanes | Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells[END_REF], both established in our laboratory, and HT-29 and Caco-2 cells), and investigate its regulation by butyrate. In addition, a human fetal normal colonic cell line (FHC cells [START_REF] Siddiqui | Primary and long term epithelial cell cultures from human fetal normal colonic mucosa[END_REF]) has been included in the study. Data show that two carrier-mediated butyrate transport mechanisms coexist in colon carcinoma cells enabling uptake of butyrate at different concentrations.

EXPERIMENTAL Cell lines and cell culture

BCS-TC2 cells obtained from a poorly differentiated human colon adenocarcinoma show null tumorigenicity, low differentiation [START_REF] Turnay | Establishment and characterization of a new human colon adenocarcinoma cell line: BCS-TC2[END_REF], and are sensitive to the apoptotic effects of butyrate [START_REF] López De Silanes | Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells[END_REF]. The continuous growth of these cells in the presence of 2 mM butyrate allowed the establishment of the BCS-TC2.BR2 cell subline, resistant to the apoptotic effects of butyrate, more differentiated and with intrinsic tumorigenicity [START_REF] López De Silanes | Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells[END_REF]. Human colon adenocarcinoma cell lines HT-29 (HTB38) and Caco-2 (HTB37) and the fetal normal colonic cell line FHC (CRL-

[ 14 C]Butyrate uptake measurements and kinetics

Cells were seeded on 24-well plates at 10 5 cells/cm 2 , and the assays performed 2 days later, when cultures were confluent. In Caco-2 cells, uptake was studied at least 5 days after reaching confluence. Cell monolayers were washed twice with 0.5 ml of Hanks´ balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.5 (buffer A) and incubated in this buffer for 30 min at 37ºC or, alternatively, pre-equilibrated 15 min at 4ºC. Buffer A was replaced with 0.5 ml HBSS containing 10 mM PIPES, pH 6.5 (buffer B) and [1- (both from Calbiochem, Darmstadt, Germany) were also analyzed.

Gene silencing using interference RNA

MCT1 gene silencing was performed on BCS-TC2 cells using two different synthetic small interfering RNAs (siRNAs) [START_REF] Wang | Characterization of monocarboxylate transport in human kidney HK-2 cells[END_REF]: si1-sense GCAGUAUCCUGGUGAAUAAdTdT in a duplex with si1-antisense UUAUUCACCAGGAUACUGCdTdG, and si2-sense CGAAUAAAGAUAGGAUUGGdTdT in a duplex with si2-antisense CCAAUCCUAUCUUUAUUCGdTdT, all purchased from Sigma-Proligo. A scrambled siRNA was also added as a control. Transfection of siRNAs was carried out using Escort IV (Sigma) according to the manufacturer's instructions. After transfection, the cells were cultured for 72 h under standard culture conditions and the change of MCT1 expression was analyzed using Western blot. The uptake of [ 14 C]butyrate in transfected cells was examined in parallel cultures prepared under identical conditions.

Other procedures

RT-PCR analysis was carried out using total RNA [isolated using the ToTALLY RNA kit from Ambion (Austin, TX)] and the SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA) using gene-specific primer pairs (Table 1). Western blot analysis was carried out as described [START_REF] López De Silanes | Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells[END_REF] densitometric analysis was carried out using Multi Gauge v3.0 software (Fujifilm). Alkaline phosphatase activity (a marker of colonic epithelium differentiation) was measured in cell extracts obtained as described [START_REF] Guzmán-Aránguez | Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5[END_REF] using the ALP 10 kit (Sigma).

Statistical analysis

Butyrate uptake data were obtained from at least three independent experiments with triplicate samples and represent mean values (±SD; 95% confidence interval). Differences between the mean values were analyzed using a two-tailed Student's t-test; statistical significance was considered to be achieved at the p<0.05 level.

RESULTS

In preliminary experiments, [ 14 C]butyrate transport was shown to be linear in 30-min incubation periods, in all cell lines at the range of concentrations employed (not shown). The effect of butyrate concentration on transport was analyzed at 4°C and 37ºC. At 4ºC a linear variation was observed whereas at 37ºC both saturable and non-saturable components were detected; the specific uptake was determined by subtracting the non-saturable component at 4°C from the global uptake at 37ºC or after direct non-linear regression of the data obtained at 37°C as described under Experimental.

[ 14 C]Butyrate uptake kinetics and characterization in BCS-TC2 cells

Uptake kinetics was studied in BCS-TC2 cells using 10 to 150 mM butyrate. Figure 1A shows the experimental transport data obtained both at 4°C and 37°C. In addition, the saturable (hyperbolic) and non-saturable (linear) uptake components at 37°C obtained after fitting are shown; the linear component was quite similar to experimental data obtained at 4°C. The saturable transport showed an apparent Michaelis constant of 109±16 mM with a maximum uptake velocity of 90±6 x 10 -4 nmol•μg -1 •min -1 . These parameters agree with a lowaffinity and high-capacity system. Butyrate transport was characterized at 20 mM butyrate (Figure 1B). The uptake of radiolabeled butyrate was almost completely abolished by 200 mM unlabeled butyrate or reduced by ~30% after addition of the structural analogue L-lactate , we analyzed the effect of their main substrate: bicarbonate. Forty and 120 mM bicarbonate inhibited ~75% of specific butyrate uptake (Figure 1B) and the presence of bicarbonate partly prevented the DIDS-stimulated butyrate uptake (not shown), suggesting a DIDS-insensitive butyrate-bicarbonate transport at these butyrate concentrations.

Additionally, the absence of sodium did not significantly modify butyrate transport.

A detailed analysis of butyrate transport at concentrations below 5 mM (Figure 2A;

Table 2) showed an additional hyperbolic specific component with an apparent K m of 17.9 μM and a maximum uptake velocity of 0.71 x 10 -4 nmol•μg -1 •min -1 . The linear component at 37°C was again quite similar to the uptake kinetics at 4°C (not shown). Characterization of this high-affinity/low-capacity transport is shown in Figure 2(B&C). The pH dependence and the effect of different agents were analyzed at 40 μM butyrate. A decrease in extracellular pH from 7.5 to 5.5 increased butyrate transport in BCS-TC2 cells by 61% (Figure 2B). Increased osmolarity (40 mM mannitol) had no effect on transport whereas treatment with the structural analogues acetate and L-lactate inhibited the uptake by 53 and 85%, respectively, and 20 mM unlabeled butyrate abolished (99% inhibition) transport. Treatment with 4-CHC and NaHS each inhibited uptake significantly (up to 90% for 5 mM 4-CHC, and 84% for 2 mM NaHS), while DIDS did not significantly change butyrate transport (Figure 2C). Bicarbonate inhibited butyrate uptake in a concentration-dependent manner (120 mM bicarbonate totally prevented the uptake; Figure 2C), indicating that a different sensitivity to bicarbonate was displayed at high or low butyrate concentrations. Additionally, the absence of sodium dramatically reduced butyrate uptake. The Na + -H + exchanger inhibitor amiloride (2 mM) did not affect butyrate uptake whereas a 16% inhibition was achieved using 4 mM acetazolamide, a carbonic anhydrase inhibitor that blocks the Na + -bicarbonate cotransporter. The incubation of the cells with both inhibitors at the indicated concentrations induced a synergic reduction (36%) of butyrate uptake.

Together, these results support the existence of two specific carrier-mediated transport mechanisms in BCS-TC2 cells kinetically characterized as low-affinity/high-capacity and high-affinity/low-capacity transporters, being the latter described and characterized here for

High-affinity/low-capacity butyrate transport is present in other human colon carcinoma cells and in normal colon cells

To extend the relevance of the analysis, we have also studied the presence of the highaffinity/low-capacity transport in the widely used HT-29 and Caco-2 human colon adenocarcinoma cells, as well as in butyrate-resistant BCS-TC2.BR2 cells. In addition to a low-affinity/high capacity transport (not shown), these cell lines displayed the highaffinity/low-capacity butyrate uptake at concentrations below 5 mM (Figure 3A). Figure 3B shows a detail of transport kinetics below 200 μM butyrate. The four cell lines presented apparent Michaelis constants (K m ) in the micromolar range without significant differences; in contrast, the maximum uptake velocitiy (V max ) differed among the cell lines (Table 2). The V max value was highest for confluent Caco-2 cells and lowest for BCS-TC2 cells. Butyrateresistant BCS-TC2.BR2 cells exhibited a 1.4-fold higher V max than the parental cells. HT29 cells showed an intermediate value between Caco-2 and BCS-TC2.BR2 cells.

Butyrate uptake was also measured in human normal colonic cells (FHC). Figure 3C shows uptake kinetics at 37°C at concentrations below 200 μM (5 mM in the insert) and the nonlinear regression with the hyperbolic and the non-saturable components described above in colon adenocarcinoma cells. K m and V max values are similar to those obtained for HT-29 cells (Table 2).

Even though Caco-2 cells show the highest alkaline phosphatase activity values and V max for butyrate transport among colon adenocarcinoma cells, no clear relationship between differentiation and high-affinity transport was found (Table 2). In addition, FHC cells showed the highest differentiation degree according to their alkaline phosphatase activity, in agreement with a normal colon cell phenotype. However, these cells presented kinetic parameters similar to those of low-differentiated adenocarcinoma cells.

Characterization of butyrate transport in butyrate-resistant BCS-TC2.BR2 cells

We have characterized butyrate transport in butyrate-resistant BCS-TC2.BR2 cells (Figure 4).

Regarding the low-affinity/high-capacity transport, the main differences with parental butyrate-sensitive cells were: i) higher inhibition by lactate, ii) inhibition of the transport by 5 mM 4-CHC (~50%), iii) increase in DIDS activation (~3-fold), and iv), lower inhibition by bicarbonate (Figure 4A). When the high-affinity/low-capacity butyrate transport was analyzed, the most significant differences with BCS-TC2 cells were found in the effect of bicarbonate, as 40 mM bicarbonate did not inhibit butyrate transport, and 120 mM only inhibited it by 30% (Figure 4B). A slightly stronger inhibition was detected with structural analogues.

In BCS-TC2.BR2 cells, butyrate transport also increased (47%) when the pH was lowered from 7.5 to 5.5; the uptake at all pH values was higher in the butyrate-resistant cells compared to BCS-TC2 cells, although this difference was not significant at pH 5.5 (not shown).

Energy requirements of butyrate transport and effect of PKC inhibitors and activators

The energy dependence of the high-affinity/low-capacity butyrate transport was analyzed in BCS-TC2 and BCS-TC2.BR2 cells by inhibiting the respiratory chain electron transfer complex III with antimycin A. Butyrate transport in both cell lines was deemed to be an energy-dependent process as it was significantly inhibited by this agent (Figure 5). Similarly, treatment with the specific PKCδ inhibitor rottlerin significantly decreased butyrate transport in both cell lines, although BCS-TC2.BR2 cells were more refractory to the inhibition (Figure 5). The general PKC competitive inhibitor BSM only reduced butyrate uptake when used at 250 nM (close to the Ki value of PKCδ). Activation of PKC had the opposite effect; an increase to around 150% butyrate uptake was achieved using the general PKC activator PMA at 1 μM or the specific PKCδ activator ingenol-3-angelate at 50 nM in both cell lines (Figure 5).

Effect of butyrate treatment on [ 14 C]butyrate uptake

We analyzed the time-and dose-dependent effects of butyrate treatment on the highaffinity/low-capacity [ 14 C]butyrate uptake (Figure 6A). A steady increase in uptake was detected in BCS-TC2 cells treated with 4 mM butyrate for different times (62% increase after 4 days treatment), while BCS-TC2.BR2 cells showed no increase over time. For HT29 cells, after a small decrease, an increase in transport is also observed (48%), while no significant modification takes place in Caco-2 cells (not shown).

After 4 days of treatment with different butyrate concentrations, a dose-dependent increase in butyrate uptake was seen in BCS-TC2 cells (90% increase at 10 mM butyrate).

BCS-TC2.BR2 cells showed no significant changes with up to 4 mM; but a 58% increase was observed at 10 mM (Figure 6A). HT29 and Caco-2 cells showed a small increase and decrease, respectively (not shown).

Detection of potential butyrate transporters by RT-PCR

Total RNA was obtained from the different cell lines and from butyrate-treated BCS-TC2 cells, and the expression levels of mRNAs encoding MCT1, MCT4, AE2, SLC5A8, SLC5A12, and CD147 were assessed using gene-specific primers (Table 1). No differences were detected in the expression of AE2 or CD147 among the cells lines. On the other hand, SLC5A8 was only expressed in Caco-2 cells, and SLC5A12 in Caco-2 and butyrate-treated BCS-TC2 cells. MCT1 was expressed in all the cell lines studied; the lowest expression was observed in BCS-TC2 cells followed by BCS-TC2.BR2. MCT4 was also expressed in all of the cell lines with maximal expression in BCS-TC2 cells and very low in Caco-2 cells (Figure 7A).

Role of MCT1 in high-affinity/low-capacity butyrate uptake

The characteristics of high-affinity/low-capacity butyrate uptake (effect of structural analogues, inhibitors and pH) suggest the potential involvement of MCT1 in this transport.

To verify this hypothesis, MCT1 expression was silenced using two different specific siRNAs. BCS-TC2 cells were transfected with increasing amounts of MCT1-directed siRNAs up to 500 nM to determine the optimal silencing conditions (data not shown). The addition of 250 or 500 nM siRNA #1 or #2, respectively, resulted in ~70% reduction of the amount of MCT1 expressed in the absence of siRNA or in the presence of an scrambled one (Figure 8A).

Butyrate uptake measured in parallel wells was decreased in MCT1-silenced cells by 33 and 45% after transfection with siRNA #1 and #2 respectively (Figure 8C).

In addition, MCT1 expression was analyzed in BCS-TC2 and BCS-TC2.BR2 cells under the experimental conditions described in Figure 6. MCT1 expression showed a similar behavior to butyrate uptake (Figure 6B&C). No variation in MCT1 levels were detected with incubation time in the presence of 4 mM butyrate in BCS-TC2.BR2 cells whereas BCS-TC2 cells showed a progressive increase (Figure 6C, left panel). Regarding the effect of 4 days incubation in the presence of increasing butyrate concentrations, BCS-TC2.BR2 cells showed no MCT1 variations up to 6 mM while BCS-TC2 showed a sharp increase in expression at 2 and 4 mM butyrate with a slower increase thereafter (Figure 6C, right panel).

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DISCUSSION

Intestinal health and function are strongly dependent on appropriate levels of butyrate and other SCFAs in the colon lumen; concentration of SCFAs may reach around 100 mM with a 20-30% butyrate [START_REF] Cummings | Short chain fatty acids in the human colon[END_REF]. Butyrate is taken up by colon epithelial cells and not only serves as a major source of energy for the colonic mucosa, but it also induces differentiation and apoptosis which may contribute to cancer prevention [START_REF] Miller | Cellular and physiological effects of short-chain fatty acids[END_REF]. Butyrate prevents colonic mucosa inflammation [START_REF] Andoh | Role of dietary fiber and short-chain fatty acids in the colon[END_REF] and infection by certain pathological bacteria as Shigella by induction of antimicrobial peptides as LL-37 [START_REF] Raqib | Improved outcome in shigellosis associated with butyrate induction of an endogenous peptide antibiotic[END_REF]. The absence of butyrate in colonic tissue is associated with mucosal atrophy and colonocyte death. Many tumor cells are highly dependent on glycolysis, but glucose availability in colon tumor cells is significantly reduced and these cells are more dependent on butyrate as a fuel nutrient. In this context, understanding the mechanisms that govern butyrate transport acquires a remarkable role in cancer studies.

The study of butyrate transport in the colonocyte is complex due to: i) differences between apical and basolateral membranes of cells at the surface or at the colonic crypt (absent in transformed unpolarized cells), ii) marked differences between enterocytes from the proximal and distal colon, iii) species-specific differences in the transport of the ionized and protonated forms of SCFAs, and iv) the coexistence of multiple transport systems showing different substrate specificity or affinity. This scenario is even more complex in colorectal tumor cells.

Besides a diffusive unspecific monocarboxylate transport in cultured cells, a saturable transport with low affinity for butyrate (millimolar range) has been reported [START_REF] Ogihara | Structural characterization of substrates for the anion exchange transporter in Caco-2 cells[END_REF]. Here we describe for the first time that human colon adenocarcinoma cells present at least two different types of carrier-mediated butyrate transport mechanisms according to the kinetic parameters (affinity/capacity): a low-affinity/high-capacity transport system (K m around 100 mM), and a high-affinity/low-capacity transport system (K m in the micromolar range). The existence of these two types of specific transport may have physiological and pathophysiological relevance. When butyrate concentration is high in the colonic lumen (e.g.

after digestion of dietary fiber) the low-affinity/high-capacity system could become active.

On the other hand, when butyrate concentration is low (e.g. in the intermeal interval, at the bottom of colonic crypts or in the inner layers of tumors) the high-affinity carrier-mediated transport could play an important role in cell survival. Previous reports in human colon adenoma (AA/C1) [START_REF] Cuff | Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and carrier mediated transport mechanism[END_REF] and carcinoma (Caco-2) [19, 30] cells showed affinity constants in the millimolar range; however, here we demonstrate the existence of a high-affinity transport Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society system that was not previously detected in these reports as they analyze butyrate transport at millimolar concentrations. Differences among the analyzed cell lines or the regulation of this transport mainly affected the V max, as described for the uptake regulation by butyrate in AA/C1 cells [START_REF] Cuff | Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and carrier mediated transport mechanism[END_REF] or by PMA in Caco-2 cells [START_REF] Alrefai | Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1[END_REF]. This high-affinity transport is not only present in transformed cells, but we have also detected it in FHC cells that maintain the characteristics of normal human colonic cells [START_REF] Siddiqui | Primary and long term epithelial cell cultures from human fetal normal colonic mucosa[END_REF][START_REF] Hofmanova | The effects of parenteral lipid emulsions on cancer and normal human colon epithelial cells in vitro[END_REF].

Which transporters might elicit the kinetically different carrier-mediated butyrate uptake mechanisms? According to data gathered from intestinal brush-border membrane vesicles [START_REF] Tamai | Anion antiport mechanism is involved in transport of lactic acid across intestinal epithelial brush-border membrane[END_REF] and Caco-2 cells [START_REF] Ogihara | Structural characterization of substrates for the anion exchange transporter in Caco-2 cells[END_REF], L-lactate and other monocarboxylates are transported through either proton-cotransporter or anion-antiporter mechanisms. We therefore studied the differential characteristics of the low-and the high-affinity components detected in the human colon adenocarcinoma cells. is weakly expressed in Caco-2 cells. Thus, an additional butyrate-bicarbonate antiporter whose molecular identity is yet unknown must participate in the low-affinity butyrate uptake, as described in other systems [START_REF] Tyagi | Mechanism of n-butyrate uptake in the human proximal colonic basolateral membranes[END_REF][START_REF] Ogihara | Structural characterization of substrates for the anion exchange transporter in Caco-2 cells[END_REF][START_REF] Vidyasagar | Role of short-chain fatty acids in colonic HCO(3) secretion[END_REF].

The high-affinity component is inhibited by butyrate structural analogues, by 4-CHC and thiol group modifiers; it is insensitive to DIDS and is stimulated at acidic pH. These characteristics point to MCT1 as a promising candidate for this type of transport. Expression of MCT1 in Caco-2 cells has been reported, but associated to a low-affinity transport [START_REF] Garcia | Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle[END_REF]19,[START_REF] Ogihara | Structural characterization of substrates for the anion exchange transporter in Caco-2 cells[END_REF]30]. We confirmed the expression of MCT1 in all of the cell lines, with the lowest expression seen in BCS-TC2 cells, which also showed the lowest transport. However, differences in uptake among the other cell lines only partially correlate with the levels of expression of this transporter or its ancillary protein CD147 [START_REF] Kirk | CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression[END_REF] suggesting the involvement of additional factors. Butyrate treatment enhanced MCT1 expression in non-tumorigenic human colon adenoma cells (AA/C1) [START_REF] Cuff | The human colonic monocarboxylate transporter Isoform 1: its potential importance to colonic tissue homeostasis[END_REF][START_REF] Cuff | Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and carrier mediated transport mechanism[END_REF]. Here we have verified a time-and concentration-dependent up-regulation of MCT1 in BCS-TC2 cells concomitant with an increase in high-affinity transport, further supporting the role of MCT1. In BCS-TC2.BR2 cells, only concentrations higher than 6 mM butyrate induce an increase in high-affinity Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society transport along with higher MCT1 levels. The absence of effects at lower concentrations reflects the resistance of these cells to butyrate [START_REF] López De Silanes | Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells[END_REF]. MCT1 involvement in high-affinity transport has been further confirmed by gene silencing using siRNAs. The reduction of MCT1 expression is accompanied by a significant decrease in uptake at low butyrate concentrations although not to the same extent. This effect is similar to that reported for human kidney HK-2 cells [START_REF] Wang | Characterization of monocarboxylate transport in human kidney HK-2 cells[END_REF] reinforcing the notion that MCT1 plays a key role in highaffinity butyrate uptake, but additional factors may modulate this transport.

The high-affinity butyrate transport is Na + -dependent, but SLC5A8 and SLC5A12

Na + -monocarboxylate transporters are only expressed in Caco-2 cells. Thus, inhibition of this transport may be related to the disappearance of the extracellular acidic microclimate adjacent to the membrane, maintained either by Na + -bicarbonate cotransporters [START_REF] Becker | Facilitated lactate transport by MCT1 when coexpressed with the sodium bicarbonate cotransporter (NBC) in Xenopus oocytes[END_REF] or Na + -H + exchangers [START_REF] Martín-Venegas | Monocarboxylate transporter 1 mediates DL-2-Hydroxy-(4-methylthio)butanoic acid transport across the apical membrane of Caco-2 cell monolayers[END_REF]. In fact, simultaneous inhibition of both systems partially impairs butyrate uptake. A proton gradient is required by MCT1 for its H + -monocarboxylate cotransport, which is maintained by an energy-dependent mechanism, emphasizing the influence of the cellular metabolic activity [START_REF] Gabel | Influence of isoform and DNP on butyrate transport across the sheep ruminal epithelium[END_REF]. In addition, high-affinity butyrate transport is dependent on PKCδ activity as specific activation or inhibition of this PKC isoform results in enhancement or reduction of uptake. In good agreement with these data, it has been described that butyrate modulates PKCδ in colon cancer cells [START_REF] Mcmillan | Opposing effects of butyrate and bile acids on apoptosis of human colon adenoma cells: differential activation of PKC and MAP kinases[END_REF] and that there is a link between PKC loss and a reduction in responsiveness of human adenocarcinoma tissues to apoptosis induced by butyrate [START_REF] Craven | Loss of protein kinase C delta isozyme immunoreactivity in human adenocarcinomas[END_REF].

The in vitro anti-tumorigenic effects of butyrate on colon cancer cells is well known but, in vivo, colon cancers grow despite the high butyrate concentration in the colonic lumen.

Cancer cells must develop mechanisms to escape butyrate effects, including possible alterations in its uptake. Comparison of uptake in butyrate-resistant and sensitive cells only reveals minor differences regarding the effect of inhibitors, energy dependence, and signaling through PKCδ. However, resistant cells show higher MCT1 and lower MCT4 expression, lack of response to butyrate treatment in high-affinity transport and MCT1 protein expression.

These changes could also be related to a modification in the metabolism of the cells.

Employing cDNA microarrays we have previously shown that the expression of some key glycolytic enzymes is modified in BCS-TC2.BR2 cells compared with parental BCS-TC2 cells [START_REF] López De Silanes | Acquisition of resistance to butyrate enhances survival after stress and induces malignancy of human colon carcinoma cells[END_REF].

In summary, we have described, for the first time, the presence of two components for specific butyrate uptake in colon adenocarcinoma cells that cooperate to enable its uptake at both high and low concentrations to maintain cell functionality. Low-affinity uptake is mediated by a butyrate-bicarbonate antiporter along with a possible contribution of AE2 and MCT4. The high-affinity/low-capacity activity, described and characterized here for the first time, is an energy-and PKCδ-dependent proton-monocarboxylate cotransport mainly mediated by MCT1. It functions under low butyrate concentrations and may be relevant for the survival of colon carcinoma cells in tumor regions with low glucose and butyrate availability. The physiological relevance of the high-affinity transport is confirmed by its presence in human normal colonic cells. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 1 Primers used for RT-PCR analysis

S t a g e 2

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(

  200 mM). No modifications in the specific transport were detected after raising the osmolarity (40 mM mannitol) or in the presence of the thiol-reagent NaHS or the inhibitor of S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society MCT-transporters, α-cyanocinnamate derivative 4-CHC (Figure 1B). In contrast 1 or 2 mM DIDS increased transport ~2.3-fold. As DIDS blocks several anion exchangers in the membrane [8]

  the first time. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

S t a g e 2

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 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1

 1 Figure 1 Kinetics of [ 14 C]butyrate uptake and effect of different agents in BCS-TC2 cells. Measurement of butyrate transport at 10-150 mM butyrate. (A) Butyrate transport at 37°C (black circles) and 4°C (white circles). Discontinuous lines represent the nonlinear regression of data at 37°C showing a hyperbolic and a linear component. (B) Effect of structuralanalogues, inhibitors and ions on butyrate uptake. Cells were preincubated at the indicated agent concentration or in sodium-free buffer (-Na + ) for 15 min before measuring uptake in the presence of 20 mM butyrate. Data represent specific uptake expressed as a percentage of the control uptake; data are the mean ± SD of four independent experiments with triplicate samples (**p<0.01).

Figure 2

 2 Figure 2 Kinetics of [ 14 C]butyrate uptake and effect of pH and different agents in BCS-TC2 cells at low butyrate concentrations. Experiments were essentially performed as indicated in Figure 1. (A) Butyrate transport at 37°C (black circles) is shown at concentrations below 200 μM or 5 mM (inset). Discontinuous lines represent the nonlinear regression of data showing the hyperbolic and linear components. (B) Effect of pH on butyrate uptake at 40 μM butyrate. (C) Effect of structural analogues, inhibitors and ions on butyrate uptake. Data represent specific uptake and are expressed as percentage of the control; values in the presence of inhibitors were significantly lower than the control (p<0.01) with the exception of mannitol and DIDS; data are the mean ± SD of four independent experiments with triplicate samples.

Figure 3

 3 Figure 3 Kinetics of [ 14 C]butyrate uptake in human colon adenocarcinoma cells and human normal colon cells at low butyrate concentrations. Experiments were essentially performed as indicated in Figure 1. Butyrate transport at 37°C is shown at concentrations below 5 mM (A) or 200 μM (B) in human colon adenocarcinoma cells. (C) Butyrate transport in normal colonic cells (FHC) at concentrations below 200 μM or 5 mM (inset). Discontinuous lines represent the nonlinear regression of data showing the hyperbolic and linear components. In all the cases, kinetic parameters determined from the S t a g e 2 ( a ) P O S T -P R I N T

Figure 4

 4 Figure 4 Effect of different agents on [ 14 C]butyrate uptake in butyrate-resistant BCS-TC2.BR2 cells. Uptake assays were carried out at 20 mM (A) and 40 μM (B) butyrate after preincubation with the indicated agent or in sodium-free buffer (-Na + ) for 15 min. Data represent specific uptake and are expressed as percentage of the control; data are the mean ± SD of four independent experiments with triplicate samples (*p<0.05, **p<0.01).

Figure 5

 5 Figure 5 Energy requirement and effect of PKC inhibitors and activators in [ 14 C]butyrate uptake in butyrate-sensitive and resistant cells. Specific uptake at 40 μM butyrate was analyzed after 15 min preincubation in the presence of the indicated concentrations of antimycin A, rottlerin, BSM, PMA and ingenol-3-angelate (I3A). Data are expressed as percentage of control uptake in the absence of these agents and are the mean ± SD of three independent experiments with triplicate samples (*p<0.05, **p<0.01).

Figure 6

 6 Figure 6 Effect of butyrate treatment in its uptake and MCT1 expression in butyratesensitive and resistant cells. (A) Specific uptake was analyzed in BCS-TC2 (black circles) and BCS-TC2.BR2 cells (white circles) after preincubation (left panel) up to 4 days in the presence of 4 mM butyrate or (right panel) after 4-day treatment with up to 10 mM butyrate. Data are expressed as percentage of specific uptake after preincubation in the absence of butyrate, and correspond to mean values ± SD of three independent experiments with triplicate samples. (B) Representative Western blots corresponding to MCT1 expression under the experimental conditions described in (A). Vinculin expression is also shown. (C) Densitometric analysis of MCT1 levels using vinculin expression for normalization. Data correspond to mean values ± SD of four independent experiments.

Figure 7

 7 Figure 7 Expression of different transporters in human colon adenocarcinoma cells.RT-PCR analysis of transporters potentially involved in butyrate uptake in colon adenocarcinoma cells; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as control. The number of PCR cycles is indicated.S t a g e 2 ( a ) P O S T -P R I N T

Figure 8

 8 Figure 8 MCT1 silencing using specific siRNAs and influence on [ 14 C]butyrate uptake in BCS-TC2 cells. (A) BCS-TC2 cells were transfected with 500 nM scrambled siRNA (S) or with 250 nM or 500 nM siRNA #1 (si1) or #2 (si2), respectively. 72 h after transfection, cells were homogenized and analyzed by Western blot using MCT1 or vinculin specific antibodies. Representative blots are shown. (B) Densitometric analysis of MCT1 levels using vinculin expression for normalization. Data correspond to mean values ± SD of four independent experiments. (C) Specific transport at 40 μM butyrate was analyzed in parallel untransfected and transfected cultures. Data correspond to mean values ± SD of three independent experiments using triplicate samples. S t a g e 2 ( a ) P O S T -P R I N T

5 S

 5 Figure 1

Figure 8 S

 8 Figure 4

  14

Competition studies and treatment with inhibitors and activators

	Biochemical Journal Immediate Publication. Published on 31 Aug 2007 as manuscript BJ20070374
	regression analysis after subtraction of butyrate uptake at 4°C, or fitted to an equation
	considering a hyperbolic transport with an added linear component (using only uptake data at
	37°C): v 0 =(V max •[B]/(K m +[B]))+(K'•[B]), where [B] is butyrate concentration and K' is the
	All values were corrected by subtracting time zero radioactivity. Protein concentration was determined in each well after butyrate extraction by solubilization of the monolayers with 0.1 M NaOH in PBS and using the D C Protein Assay (Bio-Rad, Madrid, Spain). Butyrate uptake kinetics were determined by adding unlabeled butyrate to the standard uptake buffer containing constant tracer amounts of [1-14 C]butyrate. Kinetic data were either analyzed for simple Michaelis-Menten kinetics using hyperbolic nonlinear slope of the linear component. For competition experiments, acetate, L-lactate and unlabeled butyrate were used. For studies employing transport inhibitors, a 15-min pre-treatment was carried out in buffer B at 37ºC with the different agents (4-CHC, NaHS, and DIDS; Sigma). The effects of bicarbonate, sodium, antimycin A, mannitol, amiloride, acetazolamide, phorbol 12-myristate 13-acetate S t a g e 2 ( a ) P O S T -P R I N T (PMA), ingenol-3-angelate (all from Sigma), rottlerin, and bisindolylmaleimide I (BSM)	THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070374
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	© 2007 The Authors Journal compilation © 2007 Biochemical Society

C]butyrate (American Radiolabeled Chemicals, St. Louis, MO; 56 mCi/mmol, 1 mCi/ml) and cells were incubated for 30 min at 37 or 4ºC. Cells were then washed twice with ice-cold buffer B, [1-14 C]butyrate was extracted with 1 ml ethanol for 30 min, and radioactivity was measured.

Table 2 Kinetic parameters of the high-affinity/low-capacity [ 14 C]butyrate transport and phosphatase alkaline activity in human colon adenocarcinoma cells

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	Cell line	V max x 10 4 (nmol•μg -1 •min -1 )	K m (μM)	ALP activity( a ) (mU/mg)
	BCS-TC2	0.71 ± 0.04	17.9 ± 4.0	27.3 ± 1.3
	BCS-TC2.BR2	0.99 ± 0.06	14.0 ± 3.6	65.8 ± 2.6
	HT-29	2.93 ± 0.11	13.9 ± 2.5	6.8 ± 0.8
	Caco-2	4.70 ± 0.18	23.0 ± 3.6	162.0 ± 2.7
	FHC	3.42 ± 0.22	14.5 ± 4.9	466.0 ± 18.1
	( a ) Alkaline phosphatase (ALP) activity was determined in the same conditions as butyrate
	uptake.

© 2007 The Authors Journal compilation © 2007 Biochemical Society

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© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by grants BMC2002-01407 and BFU2005-02671 from the Ministerio de Educación y Ciencia (Spain).

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