Identification and Characterization of a Diamine Exporter in Colon Epithelial Cells

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines. SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines. Polyamines are essential for normal cellular functions (1Cohen S.S. A Guide to the Polyamines. Oxford University Press, Oxford1998Google Scholar, 2Wang X. Ikeguchi Y. McCloskey D.E. Nelson P. Pegg A.E. J. Biol. Chem. 2004; 279: 51370-51375Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). They bind to intracellular polyanions such as nucleic acids and ATP and modulate their functions (3Igarashi K. Kashiwagi K. Biochem. Biophys. Res. Commun. 2000; 271: 559-564Crossref PubMed Scopus (754) Google Scholar). Intracellular polyamine content is increased in response to growth stimuli (4Kakinuma Y. Hoshino K. Igarashi K. Eur. J. Biochem. 1988; 176: 409-414Crossref PubMed Scopus (92) Google Scholar) and regulated by biosynthesis and degradation (5Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar). Uptake and export also play important roles in the regulation of cellular polyamine levels (5Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar). In recent years, polyamine transporters have been identified in bacteria, yeast, and protozoa, and their properties have been studied. In Escherichia coli, polyamine uptake is mediated by three systems, the spermidine-preferential uptake system PotABCD (6Furuchi T. Kashiwagi K. Kobayashi H. Igarashi K. J. Biol. Chem. 1991; 266: 20928-20933Abstract Full Text PDF PubMed Google Scholar, 7Kashiwagi K. Hosokawa N. Furuchi T. Kobayashi H. Sasakawa C. Yoshikawa M. Igarashi K. J. Biol. Chem. 1990; 265: 20893-20897Abstract Full Text PDF PubMed Google Scholar), the putrescine-specific uptake system Pot-FGHI (8Pistocchi R. Kashiwagi K. Miyamoto S. Nukui E. Sadakata Y. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 146-152Abstract Full Text PDF PubMed Google Scholar), and PuuP (9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Export of polyamines is mediated by PotE (10Kashiwagi K. Miyamoto S. Suzuki F. Kobayashi H. Igarashi K. Proc. Natl. Acad. Sci. U. S. 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Chem. 2005; 280: 9646-9652Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and TPO5 on the post-Golgi secretory vesicles (21Tachihara K. Uemura T. Kashiwagi K. Igarashi K. J. Biol. Chem. 2005; 280: 12637-12642Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) are polyamine exporters in yeast. A plasma membrane polyamine transporter, LmPot1, in protozoan parasite Leishmania major (22Hasne M.P. Ullman B. J. Biol. Chem. 2005; 280: 15188-15194Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) has been described. In these unicellular organisms, polyamine transport involves protein channels. In animal cells, polyamine uptake is mediated, at least in part, by a caveolar-dependent endocytic mechanism (23Belting M. Mani K. Jonsson M. Cheng F. Sandgren S. Jonsson S. Ding K. Delcros J.G. Fransson L.A. J. Biol. Chem. 2003; 278: 47181-47189Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and is positively regulated by K-RAS through phosphorylation of caveolin-1 protein (24Roy U.K. Rial N.S. Kachel K.L. Gerner E.W. Mol. Carcinog. 2008; 47: 538-553Crossref PubMed Scopus (70) Google Scholar). Export of the diamines putrescine and cadaverine have been characterized in several cells (25Hawel III, L. Tjandrawinata R.R. Fukumoto G.H. Byus C.V. J. Biol. Chem. 1994; 269: 7412-7418Abstract Full Text PDF PubMed Google Scholar, 26Hawel III, L. Tjandrawinata R.R. Byus C.V. Biochim. Biophys. Acta. 1994; 1222: 15-26Crossref PubMed Scopus (20) Google Scholar, 27Tjandrawinata R.R. Hawel III, L. Byus C.V. Biochem. Pharmacol. 1994; 48: 2237-2249Crossref PubMed Scopus (0) Google Scholar, 28Tjandrawinata R.R. Hawel III, L. Byus C.V. J. Immunol. 1994; 152: 3039-3052PubMed Google Scholar). However, export of polyamines from animal cells has not been characterized at the molecular level. We have described the biochemical properties of a diamine exporter (DAX) 2The abbreviations used are: DAXdiamine exporterCHOChinese hamster ovaryshRNAshort hairpin RNASATspermidine/spermine acetyltransferaseCHO-Tputrescine-tolerant CHOCHO-Sputrescine-sensitive CHOFBSfetal bovine serumMES4-morpholineethanesulfonic acidMSmass spectrometryHPLChigh performance liquid chromatographyGAPDHglyceraldehyde-3-phosphate dehydrogenaseODCornithine decarboxylaseNOnitric oxide. in Chinese hamster ovary (CHO) cells (29Xie X. Gillies R.J. Gerner E.W. J. Biol. Chem. 1997; 272: 20484-20489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and isolated putrescine-tolerant CHO (CHO-T) cells that appear to export putrescine at a higher rate than sensitive cells (30Pastorian K.E. Byus C.V. Exp. Cell Res. 1997; 231: 284-295Crossref PubMed Scopus (9) Google Scholar). To address the molecular mechanism of polyamine export, we compared membrane proteins of CHO-T with normal, putrescine-sensitive CHO (CHO-S) cells and found SLC3A2, a member of the solute carrier family (31Sala R. Rotoli B.M. Colla E. Visigalli R. Parolari A. Bussolati O. Gazzola G.C. Dall'Asta V. Am. J. Physiol. 2002; 282: C134-C143Crossref PubMed Google Scholar), as one of the proteins highly expressed in CHO-T cells. In this study, we evaluated the role of SLC3A2 in polyamine transport in a human colon cancer cell line. diamine exporter Chinese hamster ovary short hairpin RNA spermidine/spermine acetyltransferase putrescine-tolerant CHO putrescine-sensitive CHO fetal bovine serum 4-morpholineethanesulfonic acid mass spectrometry high performance liquid chromatography glyceraldehyde-3-phosphate dehydrogenase ornithine decarboxylase nitric oxide. Cell Culture—The human colorectal carcinoma cell line HCT116, which has an activating K-RAS mutation (G13V) in one of the K-RAS alleles (32Mariadason J.M. Arango D. Shi Q. Wilson A.J. Corner G.A. Nicholas C. Aranes M.J. Lesser M. Schwartz E.L. Augenlicht L.H. Cancer Res. 2003; 63: 8791-8812PubMed Google Scholar), and CHO cells were purchased from the American Type Culture Collection. CHO-T cells were isolated previously (30Pastorian K.E. Byus C.V. Exp. Cell Res. 1997; 231: 284-295Crossref PubMed Scopus (9) Google Scholar). The Hkh2 cell line, an isogenic clone of HCT116 that lacks the activated K-RAS allele, was kindly provided by Dr. Shirasawa, Research Institute, International Medical Center of Japan (33Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Science. 1993; 260: 85-88Crossref PubMed Scopus (609) Google Scholar). Dulbecco's modified essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin was used for culturing HCT116 and Hkh2 cells. G418 (0.6 mg/ml) was supplemented for Hkh2 cells. Modified essential medium supplemented with 10% FBS and 1% penicillin/streptomycin was used for growth of CHO cells. CHO-T cells were grown in medium supplemented with 15 mm putrescine. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. Preparation of Membrane Vesicles—Inside-out membrane vesicles were prepared by the method of Schaub et al. (34Schaub T. Ishikawa T. Keppler D. FEBS Lett. 1991; 279: 83-86Crossref PubMed Scopus (61) Google Scholar) and Saxena and Henderson (35Saxena M. Henderson G.B. J. Biol. Chem. 1995; 270: 5312-5319Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). One to 2 g of cells were suspended in hypotonic buffer (0.5 mm sodium phosphate, pH 7.0, 0.1 mm EGTA, and 0.1 mm phenylmethylsulfonyl fluoride) and stirred slowly at 4 °C for 16 h. Unbroken cells were pelleted by centrifugation at 3500 × g for 5 min at 4 °C, and the supernatant was subjected to centrifugation at 100,000 × g for 45 min at 4 °C. White fluffy material was collected and homogenized in hypotonic buffer with a 15-ml Potter-Elvehjem homogenizer. The homogenate was layered onto 14 ml of a 38% sucrose solution and centrifuged at 100,000 × g for 30 min at 4 °C. The turbid layer at the interface was collected into 25 ml of TS buffer (10 mm Tris-HCl, pH 7.4, 250 mm and mm and pelleted by centrifugation at 100,000 × g for 30 min at 4 °C. in 5 ml of TS vesicles were by the through a with a vesicles were by to a of with TS vesicles were in TS buffer and at °C. Putrescine Uptake by Membrane were as described by et al. (29Xie X. Gillies R.J. Gerner E.W. J. Biol. Chem. 1997; 272: 20484-20489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The containing of protein of membrane mm mm mm mm phosphate, 250 mm mm Tris-HCl, pH 7.4, mm and was prepared on The was at 37 °C, and putrescine uptake was by the on and with ml of buffer mm mm and mm pH The vesicles were collected by onto and four with on the was in a liquid to and membrane proteins were on an pH pH on a and using solution and highly expressed in CHO-T membrane were and with for 16 A. M. O. M. Chem. PubMed Scopus Google Scholar). The from the were by a mass with a high performance liquid chromatography and a were from a × with of material using a of and a at a rate of The was at and the was at °C. data was by S. A. A.J. III, 2002; PubMed Scopus Google Scholar) with a of an of mass an of an of 30 and a of on the mass data were as mass of mass mass of mass of of and of 5 one with mass by three of the of were with a that the of data with from protein III, J. Am. 1994; PubMed Scopus Google Scholar). mass was at mass and mass was at mass the The that were used for a are the as described previously B. D. J. Am. 2003; PubMed Scopus Google Scholar), with a an an and an A and a of were also used as for B. D. J. Am. 2003; PubMed Scopus Google Scholar). were the of the protein data from the Center for for human SLC3A2 was to Hkh2 cells using to the cells were selected by the resistance to 2 Knockdown of SLC3A2 was by RNA was isolated using the to the One of RNA was with and into using the The of SLC3A2, and glyceraldehyde-3-phosphate dehydrogenase were using of and for SLC3A2, and for and and for were with and in mm Tris-HCl, pH containing sodium and phenylmethylsulfonyl of protein were on a 10% were to a membrane were in 5% in containing for 30 min at SLC3A2, and were by the system using anti-SLC3A2 anti-SAT1 and as in were as described by et al. H. H. K. H. E. Y. PubMed Scopus Google Scholar) with One cells were in a and for 2 the medium was cells were with buffer containing 5 mm pH 7.4, mm mm mm mm and 5 mm and for 5 min at 37 °C in the Uptake was by the of 2 mm 2 mm 2 mm for cells were with buffer containing mm Cells were in and was using a cellular protein content was by the acid protein solution of in cells were homogenized in was by and polyamines were as described N. B. J. PubMed Scopus Google Scholar). was used for of protein by the protein Export in cells were in a and for Cells were three and in ml of Dulbecco's modified essential medium FBS at 37 °C. the medium was and diamines exported into the medium were by Cells were with and was using the method of et al. D. R. M. E. J. Shi A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with Hkh2 cells were from and with Cells were in buffer containing mm Tris-HCl, pH mm and One of cell were with decarboxylase at 4 °C for 16 h. were with protein for 16 at 4 °C and with were suspended with of buffer and for 5 were on a 10% to a and as described cells were on with and in 5% for 30 min at Cells were with for min at °C and for 30 at °C. Cells were with 1% bovine serum in and with antibody of anti-SLC3A2 and for 16 at 37 °C. Cells were with the buffer and with antibody of and from for 16 at 37 °C. Cells were and in was using a of in CHO-T and the polyamine we isolated CHO-T cells (30Pastorian K.E. Byus C.V. Exp. Cell Res. 1997; 231: 284-295Crossref PubMed Scopus (9) Google Scholar). These cells resistance to the high exogenous concentrations of the growth of non-selected cells was inhibited by than mm putrescine Putrescine uptake by membrane vesicles prepared from CHO-T cells was higher than the vesicles prepared from cells that the resistance of CHO-T cells to the high concentrations of putrescine was with putrescine We for proteins highly expressed in CHO-T cells, compared with cells, by proteomics Membrane proteins of CHO-T and cells were by and highly expressed in the plasma membrane of CHO-T were subjected to by liquid chromatography by mass the of several proteins that were highly expressed in the plasma membrane of CHO-T cells were transport SLC3A2, a of a acid transporter, and a acid polyamines and diamines are positively in we the transporter was in the export of and putrescine transport of the transporter in colon cancer cells polyamine transport an important role in colon E.W. Biochem. 2007; PubMed Scopus Google Scholar). We used the Hkh2 cell line, which is an isogenic clone of HCT116 colon carcinoma cells activated K-RAS allele. of a for SLC3A2 reduced and protein levels of SLC3A2 to and ornithine uptake by SLC3A2 knockdown cells and control cells. Putrescine uptake was higher in SLC3A2 knockdown cells than control cells, arginine uptake was uptake was not of SLC3A2 on Cell growth rate of SLC3A2 knockdown cells was SLC3A2 knockdown cells grew faster than control cells The of these cells to exogenous putrescine was SLC3A2 knockdown cells were sensitive to high concentrations of putrescine than control cells The polyamine of these cells in the of mm putrescine were Putrescine and were in SLC3A2 knockdown cells grown in medium supplemented putrescine. was not Intracellular levels of putrescine were increased these cells were in the of mm and levels were that SLC3A2 was in export of the of SLC3A2 knockdown on polyamine export from cells was SLC3A2 knockdown and control cells were in medium FBS for and polyamines exported into the medium were in and acetylated were exported at levels into the medium of SLC3A2 knockdown cells compared with control cells. of SLC3A2 by of polyamines is mediated by in colon cells, and K-RAS positively polyamine uptake through phosphorylation of caveolin-1 protein (24Roy U.K. Rial N.S. Kachel K.L. Gerner E.W. Mol. Carcinog. 2008; 47: 538-553Crossref PubMed Scopus (70) Google Scholar). K-RAS also to the regulation of intracellular polyamine content by H. D.E. Gerner E.W. Mol. Carcinog. 2004; PubMed Scopus Google Scholar). we SLC3A2 was regulated by K-RAS. The polyamine content in HCT116, a colon cancer cell line a K-RAS, and isogenic clone, which lacks the activated K-RAS allele, was and were in Hkh2 cells. and cadaverine levels in the cell line HCT116 cells grew faster than Hkh2 cells not The protein and levels of SLC3A2 were the protein and were also higher in Hkh2 compared with the isogenic HCT116 cells. These indicate that the expression of SLC3A2 was negatively regulated by K-RAS. the that and is also regulated by K-RAS. The protein levels of SAT1 were higher in Hkh2 cells, the levels were not that SAT1 expression is regulated by K-RAS at the in these expression of SLC3A2 was negatively regulated by K-RAS. The protein and levels of SLC3A2 and SAT1 in HCT116 and Hkh2 cells were by and as described and levels are as SLC3A2 with has been that SLC3A2 with and A. E. M. J. Cell Biol. 2007; PubMed Scopus Google Scholar) and that SAT1 with a of C. Pegg A.E. D. J. Cell Biol. 2004; PubMed Scopus Google Scholar). These to that SLC3A2 and SAT1 may a at the plasma membrane. To this the interaction of SLC3A2 and SAT1 was by and SLC3A2 and SAT1 were colocalized on the plasma membrane SAT1 was co-immunoprecipitated by anti-SLC3A2 antibody as was SLC3A2 with anti-SAT1 antibody. A of SAT1 was co-immunoprecipitated by antibody These indicate that SLC3A2 and SAT1 a on the plasma membrane and a for this In this study, we identified and characterized SLC3A2 as a of SLC3A2 was one of the proteins that were highly expressed in CHO-T cells compared with cells. Knockdown of SLC3A2 using increased putrescine uptake and arginine uptake in colon cancer cells. in SLC3A2 knockdown cells were and cell growth was concentrations of exogenous putrescine inhibited cell growth of SLC3A2 knockdown cells than in control cells. of medium with putrescine caused an increase in putrescine and in the cells, for export by (29Xie X. Gillies R.J. Gerner E.W. J. Biol. Chem. 1997; 272: 20484-20489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Knockdown of SLC3A2 reduced polyamine export from cells. These indicate that SLC3A2 is in the export of the diamine putrescine and and that SLC3A2 and SAT1 a protein on the plasma membrane. The between SLC3A2 and SAT1 may facilitate the export of acetylated the of SAT1 by the exporter containing SLC3A2 cellular polyamine levels high also suggests that may with SAT1 has been to to the plasma membrane M. J. S. E. J. 1999; PubMed Scopus Google Scholar). The interaction of SAT1 and the of to the plasma membrane and may a mechanism for of biosynthesis and of polyamines. in Hkh2 cells that the exporter that the SLC3A2 putrescine export and arginine a is to a of putrescine also is a for in the of is for polyamine uptake (23Belting M. Mani K. Jonsson M. Cheng F. Sandgren S. Jonsson S. Ding K. Delcros J.G. Fransson L.A. J. Biol. Chem. 2003; 278: 47181-47189Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). containing SLC3A2 may play an important role in regulation of cellular polyamine not by export of polyamines also by cellular arginine and the to polyamine is Putrescine accumulated in cells were in the of mm putrescine. and were in these cells that biosynthesis of and was by high levels of intracellular putrescine. of and of a from Putrescine to the for biosynthesis of and Gerner E.W. J. Physiol. 1994; PubMed Scopus Google Scholar), and this the mechanism for reduced and in these Knockdown of SLC3A2 the export of putrescine and as as and and are not for (29Xie X. Gillies R.J. Gerner E.W. J. Biol. Chem. 1997; 272: 20484-20489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), SLC3A2 may exporters with and The of high levels of in 4 to the growth of in We used medium FBS for the export FBS U. N. Y. Acad. Sci. Scopus Google Scholar). The putrescine export rate of was with the in putrescine in SLC3A2 knockdown and control cells of with mm putrescine FBS not appear to the of We found that K-RAS negatively regulated SLC3A2 and SAT1 K-RAS to intracellular polyamine by polyamine uptake (24Roy U.K. Rial N.S. Kachel K.L. Gerner E.W. Mol. Carcinog. 2008; 47: 538-553Crossref PubMed Scopus (70) Google Scholar) and biosynthesis H. D.E. Gerner E.W. Mol. Carcinog. 2004; PubMed Scopus Google Scholar, M. M. A. V. F. J. 2004; PubMed Scopus Google Scholar) and degradation N. D. Gerner E.W. Mol. Carcinog. 2004; PubMed Scopus Google Scholar). that K-RAS also polyamine export to increase cellular polyamine uptake and export have roles in colon as and polyamines this J. H. J. G.A. J. PubMed Scopus Google Scholar, C. A. D. 1999; PubMed Scopus Google Scholar). The of the is reduced by polyamines U.K. D.E. J.M. Gerner E.W. PubMed Scopus Google Scholar). polyamine transport may important for the of for cancer and provide the first of molecular characterization of polyamine export in colon cells. A is in is a of SLC3A2 and a The transporter export of putrescine and arginine activity. SAT1 may with SLC3A2 to and export of the and K-RAS negatively SLC3A2 and SAT1 We Dr. Shirasawa, Research Institute, International Medical Center of for kindly Hkh2 cells. data were by the by of the of the Cancer and of the University of with