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Tissue-Specific mRNA Expression Profiles of Human Solute Carrier 35 Transporters
Drug Metab. Pharmacokinet. 24 (1): 91–99 (2009).
Regular Article Tissue-Specific mRNA Expression Profiles of Human Solute Carrier 35 Transporters Masuhiro NISHIMURA1, Satoshi SUZUKI2, Tetsuo SATOH2 and Shinsaku NAITO1,* 1Research 2Non-Profit
and Development Center, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan Organization Human & Animal Bridging Research Institute, Ichikawa General Hospital, Chiba, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: Pairs of forward and reverse primers and TaqMan probes specific to each of 23 human solute carrier 35 (SLC35) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The mRNA expression of SLC35As, SLC35Bs, SLC35Cs, SLC35D1, SLC35D2, SLC35Es, and SLC35F5 was found to be ubiquitous in both adult and fetal tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina. SLC35F1 mRNA was expressed at high levels in the adult and fetal brain. SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland. Both SLC35F3 and SLC35F4 mRNAs were expressed at the highest levels in the adult cerebellum. Further, individual differences in the mRNA expression levels of human SLC35 transporters in the liver were also evaluated. Our newly determined expression profiles were used to study the gene expression in 31 adult human tissues, 6 fetal human tissues, and 3 cell lines, and tissues with high transcriptional activity for human SLC35 transporters were identified. These results are expected to be valuable for research concerning the clinical diagnosis of disease. Keywords: SLC35 transporter; mRNA expression; tissue distribution; quantification; human
sulfated proteoglycans and glycoproteins.3) SLC35C1 encodes a GDP-Fuc transporter.1) SLC35D1 transports UDP-glucuronic acid/UDP-N-acetylgalactosamine.4) No function has been identified for any of the four members of SLC35 transporter subfamily E (E1-E4). Although no function has been identified for any of the five members of SLC35 transporter subfamily F (F1-F5), Matsuyama et al.5) have reported that SLC35F5 has significantly different expression profiles in 5-fluorouracil-nonresponding and -responding tumors. Glucuronidation, which is catalyzed by UDPglucuronosyltransferases (UGTs), is an important detoxification reaction. UGTs catalyze the transfer of glucuronic acid from UDP-glucuronic acid to many xeno- and endobiotic aglycone substrates. The substrates
Introduction Members of the human solute carrier 35 (SLC35) transporter family, which encode for nucleotide sugar transporters, have been divided into six subfamilies (A-F).1,2) With regard to their subcellular localization, members of the SLC35 transporter family are predominately expressed in the lumen of the endoplasmic reticulum (ER) and the Golgi apparatus.3) For each of the SLC35 transporter subfamilies A-D, the function of at least one member has been identified. SLC35A1 transports CMP-sialic acid,1) SLC35A2 transports UDP-galactose,1) and SLC35A3 transports UDP-N-acetylgalactosamine.1) SLC35B3 encodes a 3?-phosphoadenosine 5?phosphosulfate transporter involved in the formation of
Received; June 4, 2008, Accepted; August 8, 2008 *To whom correspondence should be addressed: Shinsaku NAITO, Ph.D., Preclinical Assessment Department, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan. Tel. ＋81-88-685-1151, Fax. ＋81-88-686-8176, E-mail: naitousn＠ otsukakj.co.jp
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required by all glycosyltransferases are sugars activated by the addition of a nucleoside mono- or diphosphate (UDP, GDP, or CMP) forming a nucleotide-sugar. The UGTs are integral membrane proteins of the ER,6) and therefore, the water-soluble UDP-glucuronic acid, which is synthesized in the cytosol, needs to be transported into the ER. For example, due to its cellular localization (in the ER) and substrate specificity (the transport of UDPglucuronic acid and UDP-N-acetylgalactosamine), a human nucleotide sugar transporter isoform called SLC35D1 has been suggested to function as a supplier of UDP-glucuronic acid for glucuronidation.4) Information concerning gene expression in various tissues and cell lines may be important in assessing the feasibility of measuring the mRNA expression levels of target genes in target tissues and cell lines. However, the tissue distribution of the mRNA expression of large numbers of human SLC35 transporters has not been evaluated under the same experimental conditions. The present study was therefore undertaken to investigate the mRNA expression levels of 23 human SLC35 transporters using high-sensitivity real-time reverse transcription PCR (RTPCR) in total RNA from single and pooled specimens of 31 adult human tissues, from pooled specimens of 6 fetal human tissues, and from 3 cell lines derived from adult
Table 1. Nomenclature of target genes Abbreviation
Trivial Name(s)/Synonym(s)
SLC35A1
CST, hCST, CMPST
SLC35A2
UGT, UGAT, UGT1, UGT2, UGTL, UGALT
SLC35A3
DKFZp781P1297
SLC35A4
MGC2541
SLC35A5
FLJ11130, FLJ20730, FLJ25973, DKFZp434E102
SLC35B1
UGTREL1
SLC35B2
SLL, PAPST1, UGTrel4
SLC35B3
CGI–19, PAPST2, C6orf196
SLC35B4
YEA, YEA4, FLJ14697
SLC35C1
FUCT1, FLJ11320, FLJ14841
SLC35C2
CGI–15, OVCOV1, C20orf5, FLJ37039, MGC20633, MGC32079, MGC39183, BA394O2.1
SLC35D1
UGTREL7, KIAA0260, MGC138236
SLC35D2
hfrc, HFRC1, SQV7L, UGTrel8, MGC117215, MGC142139
SLC35D3
FRCL1, MGC102873, bA55K22.3
SLC35E1
FLJ14251, MGC44954
SLC35E2
KIAA0447, MGC104754, MGC117254, MGC126715, MGC138494, DKFZp686M0869
SLC35E3
BLOV1
SLC35E4
MGC129826
SLC35F1
C6orf169, FLJ13018, dJ230I3.1
SLC35F2
HSNOV1, FLJ13018, DKFZp667H1615
SLC35F3
FLJ37712
SLC35F4
C14orf36, FLJ37712, c14_5373
SLC35F5
FLJ22004
human tissues. The nomenclature of the transporters evaluated in the present study is summarized in Table 1.
Materials and Methods Materials: Total RNA prepared from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) was purchased from CLONTECH Laboratories, Inc. (Hilden, Palo Alto, CA, USA). Further, total RNA prepared from the livers of individual subjects supplied by the National Disease Research Interchange (NDRI) in the USA were obtained from the Non-Profit Organization Human & Animal Bridging Research Institute (HAB) (Chiba, Japan). Informed consent to use these tissues for research purposes was obtained from all donors. The total RNA source information for these human tissues is shown in Table 2. The present study was approved by the Ethics Committee of Otsuka Pharmaceutical Factory, Inc. Yeast tRNA was purchased from Life Technologies, Inc. (Rockville, MD, USA), and the TaqMan One-Step RT-PCR Master Mix Reagents Kit was purchased from Applied Biosystems (Foster City, CA, USA). All other chemicals used in this study were of reagent grade. Oligonucleotides: The pairs of primers and the TaqMan probes for peptidylprolyl isomerase A (PPIA) used for RT-PCR analysis employed sequences that have been reported previously.7) The pairs of primers and the TaqMan probes for the target mRNAs were designed based on the human mRNA sequence (Table 3) using Primer Express software (Applied Biosystems). The GenBank accession number and the position from the initiation codon are also shown in Table 3. Each primer and/or probe was homology searched by an NCBI BLAST search to ensure that it was specific for the target mRNA transcript. The primers and TaqMan probes were synthesized by the Genosys Division of SIGMA-ALDRICH JAPAN K.K. (Ishikari, Hokkaido, Japan). The TaqMan probes contained 6-carboxyfluorescein (FAM) at the 5? end and 6-carboxytetramethylrhodamine (TAMRA) at the 3? end and were designed to hybridize to a sequence located between the PCR primers. TaqMan RT-PCR conditions: Total RNA samples obtained from adult human tissues were diluted to 10 mg/mL with RNase-free water containing 50 mg/mL yeast tRNA. Total RNA at 30 ng per 20 mL of reaction mixture
Fetus Brain Heart Kidney Liver Spleen Thymus 21 34 34 63 34 12
Total RNA source (purchased from CLONTECH Adult Adipose tissue 18 22–61 years Adrenal gland* 62 15–61 years * Bladder 20 17–60 years Bone marrow 8 18–56 years * Brain (whole) 2 47, 55 years Cerebellum 24 16–70 years Colon 1 23 years * Heart 10 21–51 years * Kidney 14 18–59 years Liver* 1 51 years Lung (whole) 3 32–61 years * Mammary gland 1 27 years Ovary* 15 20–60 years * Pancreas 1 35 years Peripheral leukocytes* 14 19–52 years * Placenta 4 21–39 years Prostate* 32 21–50 years * Retina 29 20–60 years Salivary gland 24 16–60 years * Skeletal muscle 7 20–68 years Small intestine* 5 20–61 years Smooth muscle* 12 20–68 years Spinal cord 49 15–66 years Spleen* 1 44 years Stomach* 1 50 years Testis* 39 14–64 years Thymus* 3 20–38 years Thyroid gland 64 15–61 years Tonsil 34 22–61 years Trachea* — 18–54 years Uterus 8 23–63 years
26–40 12–31 12–31 22–40 12–31 22–34 weeks weeks weeks weeks weeks weeks
Total RNA source (obtained from HAB) Liver 1 46 years Liver 1 34 years Liver 1 24 years Liver 1 71 years Liver 1 55 years Liver 1 16 years Liver 1 80 years Liver 1 56 years Liver 1 45 years Liver 1 71 years Liver 1 70 years Liver 1 41 years Liver 1 52 years Liver 1 65 years Liver 1 49 years Liver 1 46 years Liver 1 46 years Liver 1 68 years Liver 1 16 years Liver 1 51 years Liver* 1 33 years Liver* 1 47 years Liver* 1 49 years Liver* 1 32 years Liver* 1 51 years Liver* 1 33 years Liver* 1 38 years Liver* 1 41 years Liver* 1 38 years Liver* 1 56 years female, male female, male female, male female, male male female, male female female, male female, male male female, male female female male female, male female male female, male female, male female, male female, male male female, male female male male male female, male female, male female, male female Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
female, female, female, female, female, female, Caucasian — Caucasian Caucasian Caucasian Caucasian
male male male male male male
male male male male male female male male female male female male male male female male male male male male male female male female female female male female male male
Caucasian Caucasian Hispanic Caucasian Hispanic Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Hispanic Hispanic Caucasian Hispanic Caucasian Caucasian Caucasian Caucasian
Laboratories, Inc.)
5?-TCAGTTTTTATGCTGTGTGCTGGAGTTACGC-3? (439–469) 5?-TCTCTCATCTACACCTTGCAGAATAACCTCCAG-3? (355–387) 5?-AAGGAACTTTCAGCTGGTTCTCAATTTGTAGGACTC-3? (493–528) 5?-TACATGGACCCCAGCACCTACCAGG-3? (325–349) 5?-AATACGCACCTAGGCAAGAAAGGATCCGAGA-3? (1145–1175) 5?-CAAACAGGCTCCAACCACATGATGC-3? (592–616) 5?-CTGCTGGCTATGCCAGCTTTATGGTACC-3? (128–155) 5?-TTCAACCTGACGGGTGTGGTGCTT-3? (670–693) 5?-TCATGTGGTTCTACCTCCTCATGAACATCATCA-3? (713–745) 5?-CTCTGGTGGACGAGCAACATGATGG-3? (964–988) 5?-ACGGCGCTTGACGTGGGCTTGT-3? (280–301) 5?-CCATTGCGTATTTCACAGGAGATGCACA-3? (698–725) 5?-TCAACAAGGCGCTGCTGACCACCT-3? (131–154) 5?-TCTTCAAGCGCTGCCTGCCCCT-3? (317–338) 5?-CAATACAGCCGGCAGAGCTACCCAAA-3? (745–770) 5?-TATGACGATGCTGTTTGTGGGTCTGATGAG-3? (429–458) 5?-CATCCCTTTATCAAGTGTGGGTAGGAGCCA-3? (497–526) 5?-TCAGTCTCACCTTTGGCACGTCCATG-3? (338–363) 5?-TAATCTCTGTGGCCCTAGGCCAGGTGTT-3? (182–209) 5?-ACAACTCTAACCAGTGTCCAGCTTTTGGATTGC-3? (394–426) 5?-ATTCATGGGAGTGAGGATTGTGGCCG-3? (813–838) 5?-CCCAGTTCACACAGTGGCATCAGTAGACA-3? (259–287) 5?-CTGCCAAGTTTTCCGGCATTGCTC-3? (77–100)
Race
5?-TAGCTTGGGCTGGTTTCCACT-3? (496–476) 5?-AGCGCTGTGGTCAGGATCTT-3? (461–442) 5?-AACATGCTGTGAGAACTGCCA-3? (550–530) 5?-CAGCTGTGCTTCCAATCTTGA-3? (382–362) 5?-ACTGGAACGCTCCCAAAGA-3? (1206–1188) 5?-GCAATGTCGACCAAAGGTTGA-3? (643–623) 5?-ACCGGTCTCCAGGTAGTTCTTC-3? (207–186) 5?-TGCACATAGTGCCAGGGAAA-3? (714–695) 5?-AAACACACCCCGGATGCAC-3? (774–756) 5?-GAGTCTTCTTCATCTCCCAGCC-3? (1045–1024) 5?-TTGTGTACAGCGAGACGGTGA-3? (343–323) 5?-TCAGCCCAGCCTTCAAACT-3? (755–737) 5?-TGCCATCTGTCCAATTCCAA-3? (198–179) 5?-TCTTGAGAACCAGGACGCC-3? (373–355) 5?-CATCATAGCGGTTCAAACTGTACG-3? (796–773) 5?-AACCCAAAACCACAGTTGCA-3? (481–462) 5?-TTGAGTTCACTTGTAATTCATGCTG-3? (553–529) 5?-CAGGGTGAACAGAGGTGTGGT-3? (444–424) 5?-GCTAGTCAAGCCAATTCCACAA-3? (243–222) 5?-AGCCATCAACACAGGAATCCC-3? (450–430) 5?-CATCACAATGCCAGCGATG-3? (867–849) 5?-ATAGGAGCAGAGGAATCTTCGGT-3? (329–307) 5?-TACACACACCATTTGCAGTCTTGT-3? (150–127)
Table 2. Total RNA Source Information for Various Tissues
The positions of the primers and probes were counted from the position of the initiation codon.
5?-CCGGACACTCAGCAAATTACA-3? (411–431) 5?-TATGTGGACACGCTCAAGCTC-3? (325–345) 5?-GCCCTCAGATTCTCAGCTTGATT-3? (468–490) 5?-AACCTGGTGATCTATCTTCAGCG-3? (301–323) 5?-ATAATGCCAGCAAGCCTCAA-3? (1118–1137) 5?-ACCACATGCGGGCTCATTA-3? (572–590) 5?-CTATGGTTCTTCCCATTTGTGG-3? (100–121) 5?-GACAGCACAACTGCACCAAA-3? (649–668) 5?-AATTCCCGTCATCGGAGTGA-3? (684–703) 5?-CTCTACTACGAGGAGACCAAGAGCTT-3? (937–962) 5?-AAGAGTGGCTCCCACAGCTC-3? (255–274) 5?-TTCATGATTCTGCCCACCCT-3? (676–695) 5?-GCTCCTTCCTCATCGTGCTT-3? (110–129) 5?-CCTCAGCCTGCCCATGTA-3? (294–311) 5?-GGCCGCAACAACATCTTAACA-3? (715–735) 5?-TTTCCTACCCACCCAACTTCC-3? (407–427) 5?-GGTGTTTGCTGCTCTTGGTG-3? (468–487) 5?-TCGCTGCCGAGTCCTACTG-3? (318–336) 5?-CAAAGTGCTGAACAGGGAGATG-3? (159–180) 5?-GTGATCGTCAGAGCCTACCAGTA-3? (370–392) 5?-TCATGGATCGTTCTCAGGGAC-3? (790–810) 5?-TCACTAGATGTAAACCAGGAGCCA-3? (230–253) 5?-GAGTTCTTCACCTCCTTTTAGACTGA-3? (48–73)
SLC35A1 (NM_006416) SLC35A2 (NM_005660) SLC35A3 (NM_012243) SLC35A4 (NM_080670) SLC35A5 (NM_017945) SLC35B1 (NM_005827) SLC35B2 (NM_178148) SLC35B3 (NM_015948) SLC35B4 (NM_032826) SLC35C1 (NM_018389) SLC35C2 (NM_173179) SLC35D1 (NM_015139) SLC35D2 (NM_007001) SLC35D3 (NM_001008783) SLC35E1 (NM_024881) SLC35E2 (NM_182838) SLC35E3 (NM_018656) SLC35E4 (NM_001001479) SLC35F1 (NM_001029858) SLC35F2 (NM_017515) SLC35F3 (NM_173508) SLC35F4 (NM_001080455) SLC35F5 (NM_025181)
—, no data available. *, The total RNA was the same as that used for the measurement of mRNA expression levels in our previous studies. Probe (Position)
Sex
Reverse primer (Position)
Age
Forward primer (Position)
Pool size
mRNA (GenBank No.)
Tissue
Table 3. Primers and Probes Used for RT-PCR Analysis
mRNA Expression Profiles of Human SLC35 Transporters
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was used for measurement of the target mRNA in each tissue. For the RT-PCR reaction, the TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems) containing 300 nM forward primer, 900 nM reverse primer, and 200 nM TaqMan probe was used at 20 mL per tube. The RT-PCR assay was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with the following profile: 1 cycle at 489 C for 30 min, 1 cycle at 959 C for 10 min, and 40 cycles each at 959 C for 15 sec and 609C for 1 min. The threshold cycle (Ct) was calculated by the instrument's software (7500 Fast System ver. 1.3.1). Data analysis: The relative expression of each mRNA was calculated by the DCt method (where DCt is the value obtained by subtracting the Ct value of PPIA mRNA from the Ct value of the target mRNA), as employed in previous studies.7–9) Specifically, the amount of target mRNA relative to PPIA mRNA is expressed as 2－(DCt). Data are expressed as the ratio of the target mRNA to PPIA mRNA.
Results and Discussion In the present study, we investigated the mRNA expression levels of 23 human SLC35 transporters in 31 adult human tissues, 6 fetal human tissues and 3 human cell lines. Analysis was conducted by RT-PCR using the Applied Biosystems 7500 Fast Real-Time PCR System in the presence of the TaqMan probe. In order to prepare the transporter calibration curves, the total RNA obtained from the tissue exhibiting the highest mRNA expression levels was used for the individual transporter isoforms. When preparing the calibration curves, various amounts ranging from 0.768 to 60,000 pg of total RNA were used. The lower limit of quantification for each mRNA ranged between 0.768 and 480 pg of total RNA per 20 mL of reaction mixture, and the upper limit of quantification for each mRNA was higher than 60,000 pg of total RNA per 20 mL of reaction mixture. In the present study, we selected PPIA, which showed low variability and was used as an endogenous control in our previous studies.7,9–11) The process used to select the best housekeeping gene has been described in detail in our previous studies.7,12) Similar to the findings of our previous studies,9,13) the highest and second highest Ct values for PPIA in various adult and fetal tissues and cell lines were 22.51 in the mammary gland and 21.08 in skeletal muscle, respectively, and the variation in the Ct values for PPIA as the internal control among various tissues was the smallest of the various housekeeping genes (data not shown). The mRNA expression levels of 23 human SLC35 transporters in various human adult and fetal tissues and cell lines are shown in Tables 4–9. All SLC35A mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 4). Ishida et al.14) have also reported that
Table 4. Expression of Human SLC35A Transporter mRNAs in Various Tissues Tissue
SLC35A1 SLC35A2 SLC35A3 SLC35A4 SLC35A5
Adult Adipose tissue
0.0627
0.00675
0.0482
0.0499
0.0672
Adrenal gland
0.0665
0.0138
0.0169
0.0447
0.0437
Bladder
0.0672
0.00692
0.0245
0.0311
0.0518
Bone marrow
0.0971
0.00641
0.0310
0.0214
0.0567
Brain
0.0359
0.00619
0.0106
0.0166
0.0610
Cerebellum
0.0412
0.00376
0.0173
0.0179
0.0557
Colon
0.0577
0.0118
0.0636
0.0361
0.0318
Heart
0.0691
0.00763
0.0331
0.0597
0.0629
Kidney
0.0367
0.0146
0.0355
0.0499
0.0583
Liver
0.0263
0.00985
0.0178
0.0168
0.0268
Lung
0.0828
0.00849
0.0424
0.0308
0.0542
Mammary gland
0.0878
0.00806
0.0404
0.0424
0.0270
Ovary
0.0482
0.00483
0.0307
0.0289
0.0527
Pancreas
0.0289
0.00673
0.0249
0.0249
0.0317
Peripheral leukocytes
0.0531
0.00551
0.0204
0.0273
0.0369
Placenta
0.0506
0.0433
0.0508
0.0634
0.148
Prostate
0.122
0.0163
0.0718
0.0390
0.0537
Retina
0.0540
0.00709
0.0343
0.0307
0.103
Salivary gland
0.0754
0.0154
0.0466
0.0341
0.0336
Skeletal muscle
0.115
0.0147
0.0524
0.290
0.0851
Small intestine
0.0213
0.00655
0.0593
0.0251
0.0256
Smooth muscle
0.0769
0.00473
0.0453
0.0244
0.0489
Spinal cord
0.0265
0.00572
0.0145
0.0205
0.0915
Spleen
0.0495
0.00726
0.0284
0.0448
0.0701
Stomach
0.0730
0.0120
0.0906
0.0369
0.0522
Testis
0.100
0.0114
0.0459
0.0661
0.154
Thymus
0.0217
0.00503
0.0203
0.0169
0.0216
Thyroid gland
0.0480
0.0109
0.0531
0.0326
0.0925
Tonsil
0.0273
0.00626
0.0160
0.0244
0.0315
Trachea
0.0711
0.0370
0.0808
0.0402
0.0575
Uterus
0.0839
0.00876
0.0427
0.0321
0.0741
Fetus Brain
0.0610
0.00666
0.0183
0.0129
0.0418
Heart
0.0421
0.00820
0.0181
0.0394
0.0396
Kidney
0.0308
0.0123
0.0165
0.0324
0.0354
Liver
0.0203
0.00985
0.0145
0.0195
0.0234
Spleen
0.0474
0.00873
0.0128
0.0333
0.0397
Thymus
0.0623
0.00646
0.0579
0.0198
0.0327
Cell lines HeLa
0.0123
0.00588
0.0216
0.0153
0.0149
Hep G2
0.0170
0.00945
0.0191
0.000303
0.0249
MDA 435
0.0250
0.00795
0.00605
0.0387
0.0286
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
95
mRNA Expression Profiles of Human SLC35 Transporters
Table 5. Expression of Human SLC35B Transporter mRNAs in Various Tissues Tissue
Table 6. Expression of Human SLC35C Transporter mRNAs in Various Tissues
SLC35B1
SLC35B2
SLC35B3
SLC35B4
Adipose tissue
0.0621
0.00124
0.0226
0.00969
Adipose tissue
0.0301
0.0902
Adrenal gland
0.0670
0.00197
0.0118
0.0163
Adrenal gland
0.00942
0.144
Adult
Tissue
SLC35C1
SLC35C2
Adult
Bladder
0.0418
0.00133
0.0183
0.0130
Bladder
0.0116
0.0872
Bone marrow
0.0715
0.000850
0.0181
0.00699
Bone marrow
0.00685
0.217 0.0640
Brain
0.0458
0.00134
0.00530
0.0299
Brain
0.00453
Cerebellum
0.0396
0.00106
0.00530
0.0243
Cerebellum
0.00668
0.0682
Colon
0.0391
0.00134
0.0185
0.00184
Colon
0.0168
0.0925
Heart
0.195
0.00213
0.0179
0.00800
Heart
0.00809
0.0961
Kidney
0.109
0.00231
0.0163
0.0206
Kidney
0.00496
0.111
Liver
0.113
0.000845
0.0127
0.0126
Liver
0.0105
0.0851
Lung
0.0529
0.00153
0.0130
0.00501
Lung
0.00752
0.0938
Mammary gland
0.217
0.000219
0.0253
0.00685
Mammary gland
0.0131
0.192
Ovary
0.0358
0.00148
0.00817
0.0150
Ovary
0.00901
0.0947
Pancreas
0.0896
0.00232
0.0129
0.00268
Pancreas
0.0116
0.148
Peripheral leukocytes
0.0469
0.00243
0.00528
0.00317
Peripheral leukocytes
0.0132
0.111
Placenta
0.155
0.00232
0.0311
0.0256
Placenta
0.0308
0.374
Prostate
0.0583
0.00152
0.0208
0.00876
Prostate
0.0102
0.129
Retina
0.0706
0.00145
0.00353
0.0234
Retina
0.00969
0.134
Salivary gland
0.0944
0.00303
0.0169
0.00278
Salivary gland
0.0136
0.144
Skeletal muscle
0.557
0.00682
0.0180
0.0230
Skeletal muscle
0.0212
0.228
Small intestine
0.115
0.00112
0.0111
0.00407
Small intestine
0.0255
0.0780
Smooth muscle
0.0405
0.00148
0.0175
0.00989
Smooth muscle
0.00682
0.0825
Spinal cord
0.0546
0.00197
0.00945
0.0114
Spinal cord
0.00624
0.0746
Spleen
0.0524
0.00120
0.0154
0.0120
Spleen
0.0109
0.111
Stomach
0.0977
0.00180
0.0257
0.00737
Stomach
0.0287
0.103
Testis
0.125
0.00209
0.0276
0.0157
Testis
0.0153
0.231
Thymus
0.0297
0.000693
0.00393
0.00392
Thymus
0.00395
0.0502
Thyroid gland
0.0836
0.00181
0.0194
0.00643
Thyroid gland
0.00530
0.0981
Tonsil
0.0487
0.000963
0.00834
0.00481
Tonsil
0.0148
0.0748
Trachea
0.108
0.00385
0.0216
0.00582
Trachea
0.0301
0.150
Uterus
0.0524
0.00183
0.0166
0.0129
Uterus
0.00952
0.110
Fetus Brain
0.0451
0.00173
0.00576
0.0119
Brain
0.0139
0.0533
Heart
0.0881
0.00238
0.00982
0.00602
Heart
0.00849
0.0813
Kidney
0.0474
0.00248
0.00999
0.0110
Kidney
0.00731
0.0964
Liver
0.0825
0.00164
0.00916
0.00371
Liver
0.00605
0.0769
Spleen
0.00765
0.0875
Thymus
0.00867
0.0822
Fetus
Spleen
0.0520
0.00157
0.0101
0.00784
Thymus
0.0368
0.00197
0.0132
0.00664
HeLa
0.0291
0.00143
0.00891
0.00353
Hep G2
0.0778
0.00240
0.00628
0.0107
MDA 435
0.0291
0.00285
0.00400
0.0206
Cell lines
Cell lines
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
HeLa
0.00534
0.0340
Hep G2
0.00749
0.113
MDA 435
0.00904
0.0836
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
96
Masuhiro NISHIMURA, et al.
Table 7. Expression of Human SLC35D Transporter mRNAs in Various Tissues Tissue
Table 8. Expression of Human SLC35E Transporter mRNAs in Various Tissues
SLC35D1
SLC35D2
SLC35D3
Adipose tissue
0.0239
0.0515
0.000249
Adipose tissue
0.126
0.0461
0.0190
0.00592
Adrenal gland
0.0271
0.0298
0.00354
Adrenal gland
0.0881
0.0520
0.0218
0.00423
Adult
Tissue
SLC35E1
SLC35E2
SLC35E3
SLC35E4
Adult
Bladder
0.0404
0.0602
0.0000415
Bladder
0.0499
0.0461
0.0147
0.00795
Bone marrow
0.0278
0.0300
0.00328
Bone marrow
0.0612
0.0538
0.0136
0.00195
Brain
0.0151
0.0326
0.00113
Brain
0.0845
0.0632
0.0148
0.0158
Cerebellum
0.0133
0.0113
0.000148
Cerebellum
0.0842
0.0619
0.0110
0.0117
Colon
0.0825
0.0822
0.00141
Colon
0.0822
0.0506
0.0103
0.0108
Heart
0.0318
0.0736
0.0000978
Heart
0.122
0.0420
0.0215
0.00817
Kidney
0.0552
0.106
0.0000095
Kidney
0.110
0.0387
0.0187
0.00290
Liver
0.128
0.0708
0.0000112
Liver
0.0385
0.0134
0.0136
0.000487 0.00471
Lung
0.0208
0.0595
0.000168
Lung
0.0994
0.0397
0.0151
Mammary gland
0.00628
0.0242
0.00283
Mammary gland
0.473
0.000730
0.0352
0.00267
Ovary
0.0368
0.0616
0.0000878
Ovary
0.0915
0.0751
0.0152
0.00576
Pancreas
0.0179
0.129
0.00188
Pancreas
0.164
0.0335
0.0350
0.00609
Peripheral leukocytes
0.0158
0.0363
0.000329
Peripheral leukocytes
0.0893
0.0720
0.0186
0.00476
Placenta
0.0356
0.0811
0.0000207
Placenta
0.459
0.0887
0.0173
0.00214
Prostate
0.0341
0.0627
0.000342
Prostate
0.204
0.0484
0.0213
0.00466
Retina
0.0504
0.0951
0.0260
Retina
0.122
0.0487
0.0246
0.0139
Salivary gland
0.0167
0.0422
0.0000351
Salivary gland
0.170
0.0511
0.0201
0.00419
Skeletal muscle
0.102
0.0335
0.000133
Skeletal muscle
0.603
0.0816
0.0711
0.134
Small intestine
0.0337
0.110
0.00117
Small intestine
0.0625
0.0355
0.00846
0.00331
Smooth muscle
0.0353
0.0557
0.0000403
Smooth muscle
0.111
0.0433
0.0134
0.00781
Spinal cord
0.0155
0.0623
0.000886
Spinal cord
0.0569
0.0295
0.0219
0.00817
Spleen
0.0269
0.0363
0.000181
Spleen
0.0825
0.0477
0.0111
0.00406
Stomach
0.0542
0.0842
0.000683
Stomach
0.119
0.0480
0.0123
0.00611
Testis
0.0711
0.0499
0.000577
Testis
0.110
0.0424
0.0161
0.00543
Thymus
0.0245
0.0142
0.0000089
Thymus
0.0581
0.0535
0.0169
0.00159
Thyroid gland
0.0249
0.107
0.0000233
Thyroid gland
0.103
0.0527
0.0126
0.00293
Tonsil
0.0183
0.0259
0.0000145
Tonsil
0.0756
0.0360
0.0101
0.00346
Trachea
0.0294
0.0799
0.000889
Trachea
0.192
0.0527
0.0241
0.00979
Uterus
0.0436
0.0730
0.0000329
Uterus
0.103
0.0715
0.0145
0.0111
Brain
0.0230
0.00660
0.00159
Brain
0.115
0.153
0.0166
0.0135
Heart
0.0340
0.0797
0.00139
Heart
0.102
0.0495
0.0110
0.00657
Kidney
0.0288
0.0751
0.0000935
Kidney
0.0878
0.0636
0.00985
0.00447
Liver
0.0589
0.117
0.00126
Liver
0.0442
0.0172
0.00812
0.000551
Spleen
0.0240
0.0354
0.000813
Spleen
0.0778
0.0552
0.00942
0.00781
Thymus
0.0544
0.0179
0.000164
Thymus
0.0848
0.0991
0.0192
0.00287
HeLa
0.00702
0.00626
0.0000074
HeLa
0.0233
0.0102
0.00491
0.000790
Hep G2
0.0292
0.190
BLQ
Hep G2
0.0794
0.0176
0.0167
0.0000052
MDA 435
0.00558
0.0430
0.0000173
MDA 435
0.0928
0.0124
0.00904
0.00315
Fetus
Fetus
Cell lines
Cell lines
BLQ, below the limit of quantification. Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
97
mRNA Expression Profiles of Human SLC35 Transporters
Table 9. Expression of Human SLC35F Transporter mRNAs in Various Tissues Tissue
SLC35F1
SLC35F2
SLC35F3
SLC35F4
SLC35F5
Adult Adipose tissue
0.00605
0.00826
0.000114
0.000210
0.176
Adrenal gland
0.00564
0.00233
0.000738
BLQ
0.431
Bladder
0.00632
0.00757
0.0000399
0.000138
0.154
Bone marrow
0.000234
0.00652
0.000104
0.0000535
0.112
Brain
0.0783
0.00351
0.0124
0.00282
0.0654
Cerebellum
0.0186
0.00132
0.0179
0.0128
0.0691
Colon
0.00617
0.0354
0.000171
0.0000550
0.139
Heart
0.00704
0.00284
0.0000187
0.0000497
0.258
Kidney
0.00182
0.0203
0.000356
0.000280
0.295
Liver
0.000180
0.000397
BLQ
BLQ
0.0845
Lung
0.000467
0.00949
0.000119
0.0000504
0.135
Mammary gland
0.00330
0.0203
0.00254
BLQ
0.218
Ovary
0.00114
0.0107
0.000146
0.000262
0.179
Pancreas
0.00214
0.0223
0.00562
0.000365
0.153
Peripheral leukocytes
BLQ
0.00628
0.000287
0.0000497
0.0492
Placenta
0.000196
0.0504
0.0000321
0.000208
0.292
Prostate
0.00864
0.0470
0.00121
0.00108
0.235
Retina
0.0336
0.0243
0.000295
0.00315
0.142
Salivary gland
0.00258
0.117
0.00181
0.0000461
0.206
Skeletal muscle
0.00224
0.00558
0.000899
0.000153
0.563
Small intestine
0.00586
0.0236
0.000360
0.0000487
0.103
Smooth muscle
0.00582
0.00503
BLQ
0.000295
0.132
Spinal cord
0.0296
0.00972
0.00443
0.00172
0.0677
Spleen
0.00174
0.00879
0.000157
BLQ
0.134
Stomach
0.00438
0.0216
0.000305
0.000121
0.174
Testis
0.00619
0.0201
0.000337
0.00534
0.269
Thymus
0.000203
0.00564
0.00118
BLQ
0.0411
Thyroid gland
0.00221
0.0147
0.000336
0.00174
0.231
Tonsil
0.000838
0.0146
0.000918
0.0000144
0.0944
Trachea
0.00273
0.0202
0.000896
0.0000834
0.230
Uterus
0.0131
0.0136
0.000549
0.000874
0.180
Fetus Brain
0.198
0.0220
0.00119
0.00179
0.0775
Heart
0.00888
0.00515
BLQ
0.0000860
0.184
Kidney
0.0103
0.0118
0.000196
0.000821
0.218
Liver
0.000323
0.000785
0.0000617
BLQ
0.0857
Spleen
0.00560
0.0150
0.0000552
0.0000339
0.111
Thymus
0.00121
0.00846
0.00206
0.0000115
0.114
HeLa
BLQ
0.0148
0.000401
0.0000048
0.0356
Hep G2
0.0000308
0.00564
BLQ
0.0000515
0.205
MDA 435
BLQ
0.0251
BLQ
0.0000092
0.0822
Cell lines
BLQ, below the limit of quantification. Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
SLC35A3 mRNA is expressed ubiquitously in human tissues. SLC35B1, SLC35B3, and SLC35B4 mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 5). SLC35B2 mRNA was also expressed ubiqui-
tously in both adult and fetal tissues, but was expressed at lower levels in adult bone marrow, liver, mammary gland, thymus, and tonsil (Table 5). SLC35C1 and SLC35C2 mRNAs were expressed ubiquitously in both
98
Masuhiro NISHIMURA, et al.
adult and fetal tissues (Table 6). Leach et al.15) have also reported that SLC35C2 mRNA is detected in all human tissues. SLC35D1 and SLC35D2 mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 7). On the other hand, Ishida et al.16) have reported that SLC35D2 mRNA is not detected in the brain, colon, or thymus. The differences between our results and their results may be due to the differences between the sensitivity of real-time reverse transcription PCR and Northern blot analysis. Muraoka et al.4) have also reported that SLC35D1 mRNA is expressed ubiquitously in human tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina (Table 7). SLC35E1 and SLC35E3 mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 8). SLC35E2 and SLC35E4 mRNAs were also expressed ubiquitously in both adult and fetal tissues, but SLC35E2 mRNA was expressed at lower levels in the adult mammary gland and SLC35E4 mRNA was expressed at lower levels in both the adult and fetal liver (Table 8). SLC35F1 mRNA was expressed at higher levels in the adult and fetal brain (Table 9). SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland (Table 9). Both SLC35F3 and SLC35F4 mRNA were expressed at the highest levels in the adult cerebellum (Table 9). SLC35F5 mRNA was expressed ubiquitously in both adult and fetal tissues (Table 9). SLC35 transporters were consistently expressed in HeLa cells, except for SLC35D3, SLC35E4, SLC35F1,
SLC35F3, and SLC35F4 (Tables 4–9). SLC35 transporters were consistently expressed in Hep G2 cells, except for SLC35A4, SLC35D3, SLC35E4, SLC35F1, SLC35F3, and SLC35F4 (Tables 4–9). SLC35 transporters were consistently expressed in MDA 435 cells, except for SLC35D3, SLC35F1, SLC35F3, and SLC35F4 (Tables 4–9). Individual differences in the mRNA expression of human SLC35 transporters in the liver are shown in Figure 1. The dispersion of the mRNA levels of each of the SLC35 transporters in the total RNA obtained from HAB sometimes differed by factors of several times, dozens of times, or more. The mRNA levels in the liver in the total RNA purchased from CLONTECH Laboratories, Inc., were the same as, slightly higher than, or slightly lower than those in the total RNA obtained from HAB. Therefore, with a few exceptions, it is suggested that the data shown in Table 4 provides valid information concerning the relative abundance of mRNAs in the liver and other tissues, although this is based on the results for one sample. In some reports, the expression levels of SLC35s (nucleotide sugar transporters) have been implicated in various stages of the development of human diseases. For example, transcripts of SLC35A2 specifically (but not of SLC35A1 or SLC35A3) were found to be more abundant in colon cancer cells than in non-malignant tissues obtained from the same patients.17) Furthermore, human
Fig. 1. Individual differences in the mRNA expression of human SLC35 transporters in the liver Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. Closed circles () are the same as the data in Table 4–9. Closed () and open () circles are the individual data for the total RNA purchased from CLONTECH Laboratories and the total RNA obtained from HAB, respectively. Small closed circles () and bars indicate the mean and standard deviation of the individual data (n＝30) for the total RNA obtained from HAB.
mRNA Expression Profiles of Human SLC35 Transporters
diseases that are closely related to the nucleotide sugar transporters have been reported by several groups.18–20) However, the roles of nucleotide sugar transporters in human diseases are still poorly understood. The results of the present study may be helpful to researchers in this field. In conclusion, the results of the present study provide valuable information concerning the tissue-specific profiles of the mRNA expression of 23 human SLC35 transporters. These findings should be useful for studies on the regulation of transport in research related to the clinical diagnosis of disease. In particular, the information obtained in this study is expected to be important in assessing the feasibility of measuring the mRNA expression levels of target genes in various tissues.
9)
10)
11)
12)
Acknowledgments: The authors would like to thank David G. Spear, M.D., for reviewing this manuscript.
13)
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Regular Article Tissue-Specific mRNA Expression Profiles of Human Solute Carrier 35 Transporters Masuhiro NISHIMURA1, Satoshi SUZUKI2, Tetsuo SATOH2 and Shinsaku NAITO1,* 1Research 2Non-Profit
and Development Center, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan Organization Human & Animal Bridging Research Institute, Ichikawa General Hospital, Chiba, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: Pairs of forward and reverse primers and TaqMan probes specific to each of 23 human solute carrier 35 (SLC35) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The mRNA expression of SLC35As, SLC35Bs, SLC35Cs, SLC35D1, SLC35D2, SLC35Es, and SLC35F5 was found to be ubiquitous in both adult and fetal tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina. SLC35F1 mRNA was expressed at high levels in the adult and fetal brain. SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland. Both SLC35F3 and SLC35F4 mRNAs were expressed at the highest levels in the adult cerebellum. Further, individual differences in the mRNA expression levels of human SLC35 transporters in the liver were also evaluated. Our newly determined expression profiles were used to study the gene expression in 31 adult human tissues, 6 fetal human tissues, and 3 cell lines, and tissues with high transcriptional activity for human SLC35 transporters were identified. These results are expected to be valuable for research concerning the clinical diagnosis of disease. Keywords: SLC35 transporter; mRNA expression; tissue distribution; quantification; human
sulfated proteoglycans and glycoproteins.3) SLC35C1 encodes a GDP-Fuc transporter.1) SLC35D1 transports UDP-glucuronic acid/UDP-N-acetylgalactosamine.4) No function has been identified for any of the four members of SLC35 transporter subfamily E (E1-E4). Although no function has been identified for any of the five members of SLC35 transporter subfamily F (F1-F5), Matsuyama et al.5) have reported that SLC35F5 has significantly different expression profiles in 5-fluorouracil-nonresponding and -responding tumors. Glucuronidation, which is catalyzed by UDPglucuronosyltransferases (UGTs), is an important detoxification reaction. UGTs catalyze the transfer of glucuronic acid from UDP-glucuronic acid to many xeno- and endobiotic aglycone substrates. The substrates
Introduction Members of the human solute carrier 35 (SLC35) transporter family, which encode for nucleotide sugar transporters, have been divided into six subfamilies (A-F).1,2) With regard to their subcellular localization, members of the SLC35 transporter family are predominately expressed in the lumen of the endoplasmic reticulum (ER) and the Golgi apparatus.3) For each of the SLC35 transporter subfamilies A-D, the function of at least one member has been identified. SLC35A1 transports CMP-sialic acid,1) SLC35A2 transports UDP-galactose,1) and SLC35A3 transports UDP-N-acetylgalactosamine.1) SLC35B3 encodes a 3?-phosphoadenosine 5?phosphosulfate transporter involved in the formation of
Received; June 4, 2008, Accepted; August 8, 2008 *To whom correspondence should be addressed: Shinsaku NAITO, Ph.D., Preclinical Assessment Department, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan. Tel. ＋81-88-685-1151, Fax. ＋81-88-686-8176, E-mail: naitousn＠ otsukakj.co.jp
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required by all glycosyltransferases are sugars activated by the addition of a nucleoside mono- or diphosphate (UDP, GDP, or CMP) forming a nucleotide-sugar. The UGTs are integral membrane proteins of the ER,6) and therefore, the water-soluble UDP-glucuronic acid, which is synthesized in the cytosol, needs to be transported into the ER. For example, due to its cellular localization (in the ER) and substrate specificity (the transport of UDPglucuronic acid and UDP-N-acetylgalactosamine), a human nucleotide sugar transporter isoform called SLC35D1 has been suggested to function as a supplier of UDP-glucuronic acid for glucuronidation.4) Information concerning gene expression in various tissues and cell lines may be important in assessing the feasibility of measuring the mRNA expression levels of target genes in target tissues and cell lines. However, the tissue distribution of the mRNA expression of large numbers of human SLC35 transporters has not been evaluated under the same experimental conditions. The present study was therefore undertaken to investigate the mRNA expression levels of 23 human SLC35 transporters using high-sensitivity real-time reverse transcription PCR (RTPCR) in total RNA from single and pooled specimens of 31 adult human tissues, from pooled specimens of 6 fetal human tissues, and from 3 cell lines derived from adult
Table 1. Nomenclature of target genes Abbreviation
Trivial Name(s)/Synonym(s)
SLC35A1
CST, hCST, CMPST
SLC35A2
UGT, UGAT, UGT1, UGT2, UGTL, UGALT
SLC35A3
DKFZp781P1297
SLC35A4
MGC2541
SLC35A5
FLJ11130, FLJ20730, FLJ25973, DKFZp434E102
SLC35B1
UGTREL1
SLC35B2
SLL, PAPST1, UGTrel4
SLC35B3
CGI–19, PAPST2, C6orf196
SLC35B4
YEA, YEA4, FLJ14697
SLC35C1
FUCT1, FLJ11320, FLJ14841
SLC35C2
CGI–15, OVCOV1, C20orf5, FLJ37039, MGC20633, MGC32079, MGC39183, BA394O2.1
SLC35D1
UGTREL7, KIAA0260, MGC138236
SLC35D2
hfrc, HFRC1, SQV7L, UGTrel8, MGC117215, MGC142139
SLC35D3
FRCL1, MGC102873, bA55K22.3
SLC35E1
FLJ14251, MGC44954
SLC35E2
KIAA0447, MGC104754, MGC117254, MGC126715, MGC138494, DKFZp686M0869
SLC35E3
BLOV1
SLC35E4
MGC129826
SLC35F1
C6orf169, FLJ13018, dJ230I3.1
SLC35F2
HSNOV1, FLJ13018, DKFZp667H1615
SLC35F3
FLJ37712
SLC35F4
C14orf36, FLJ37712, c14_5373
SLC35F5
FLJ22004
human tissues. The nomenclature of the transporters evaluated in the present study is summarized in Table 1.
Materials and Methods Materials: Total RNA prepared from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) was purchased from CLONTECH Laboratories, Inc. (Hilden, Palo Alto, CA, USA). Further, total RNA prepared from the livers of individual subjects supplied by the National Disease Research Interchange (NDRI) in the USA were obtained from the Non-Profit Organization Human & Animal Bridging Research Institute (HAB) (Chiba, Japan). Informed consent to use these tissues for research purposes was obtained from all donors. The total RNA source information for these human tissues is shown in Table 2. The present study was approved by the Ethics Committee of Otsuka Pharmaceutical Factory, Inc. Yeast tRNA was purchased from Life Technologies, Inc. (Rockville, MD, USA), and the TaqMan One-Step RT-PCR Master Mix Reagents Kit was purchased from Applied Biosystems (Foster City, CA, USA). All other chemicals used in this study were of reagent grade. Oligonucleotides: The pairs of primers and the TaqMan probes for peptidylprolyl isomerase A (PPIA) used for RT-PCR analysis employed sequences that have been reported previously.7) The pairs of primers and the TaqMan probes for the target mRNAs were designed based on the human mRNA sequence (Table 3) using Primer Express software (Applied Biosystems). The GenBank accession number and the position from the initiation codon are also shown in Table 3. Each primer and/or probe was homology searched by an NCBI BLAST search to ensure that it was specific for the target mRNA transcript. The primers and TaqMan probes were synthesized by the Genosys Division of SIGMA-ALDRICH JAPAN K.K. (Ishikari, Hokkaido, Japan). The TaqMan probes contained 6-carboxyfluorescein (FAM) at the 5? end and 6-carboxytetramethylrhodamine (TAMRA) at the 3? end and were designed to hybridize to a sequence located between the PCR primers. TaqMan RT-PCR conditions: Total RNA samples obtained from adult human tissues were diluted to 10 mg/mL with RNase-free water containing 50 mg/mL yeast tRNA. Total RNA at 30 ng per 20 mL of reaction mixture
Fetus Brain Heart Kidney Liver Spleen Thymus 21 34 34 63 34 12
Total RNA source (purchased from CLONTECH Adult Adipose tissue 18 22–61 years Adrenal gland* 62 15–61 years * Bladder 20 17–60 years Bone marrow 8 18–56 years * Brain (whole) 2 47, 55 years Cerebellum 24 16–70 years Colon 1 23 years * Heart 10 21–51 years * Kidney 14 18–59 years Liver* 1 51 years Lung (whole) 3 32–61 years * Mammary gland 1 27 years Ovary* 15 20–60 years * Pancreas 1 35 years Peripheral leukocytes* 14 19–52 years * Placenta 4 21–39 years Prostate* 32 21–50 years * Retina 29 20–60 years Salivary gland 24 16–60 years * Skeletal muscle 7 20–68 years Small intestine* 5 20–61 years Smooth muscle* 12 20–68 years Spinal cord 49 15–66 years Spleen* 1 44 years Stomach* 1 50 years Testis* 39 14–64 years Thymus* 3 20–38 years Thyroid gland 64 15–61 years Tonsil 34 22–61 years Trachea* — 18–54 years Uterus 8 23–63 years
26–40 12–31 12–31 22–40 12–31 22–34 weeks weeks weeks weeks weeks weeks
Total RNA source (obtained from HAB) Liver 1 46 years Liver 1 34 years Liver 1 24 years Liver 1 71 years Liver 1 55 years Liver 1 16 years Liver 1 80 years Liver 1 56 years Liver 1 45 years Liver 1 71 years Liver 1 70 years Liver 1 41 years Liver 1 52 years Liver 1 65 years Liver 1 49 years Liver 1 46 years Liver 1 46 years Liver 1 68 years Liver 1 16 years Liver 1 51 years Liver* 1 33 years Liver* 1 47 years Liver* 1 49 years Liver* 1 32 years Liver* 1 51 years Liver* 1 33 years Liver* 1 38 years Liver* 1 41 years Liver* 1 38 years Liver* 1 56 years female, male female, male female, male female, male male female, male female female, male female, male male female, male female female male female, male female male female, male female, male female, male female, male male female, male female male male male female, male female, male female, male female Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
female, female, female, female, female, female, Caucasian — Caucasian Caucasian Caucasian Caucasian
male male male male male male
male male male male male female male male female male female male male male female male male male male male male female male female female female male female male male
Caucasian Caucasian Hispanic Caucasian Hispanic Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Hispanic Hispanic Caucasian Hispanic Caucasian Caucasian Caucasian Caucasian
Laboratories, Inc.)
5?-TCAGTTTTTATGCTGTGTGCTGGAGTTACGC-3? (439–469) 5?-TCTCTCATCTACACCTTGCAGAATAACCTCCAG-3? (355–387) 5?-AAGGAACTTTCAGCTGGTTCTCAATTTGTAGGACTC-3? (493–528) 5?-TACATGGACCCCAGCACCTACCAGG-3? (325–349) 5?-AATACGCACCTAGGCAAGAAAGGATCCGAGA-3? (1145–1175) 5?-CAAACAGGCTCCAACCACATGATGC-3? (592–616) 5?-CTGCTGGCTATGCCAGCTTTATGGTACC-3? (128–155) 5?-TTCAACCTGACGGGTGTGGTGCTT-3? (670–693) 5?-TCATGTGGTTCTACCTCCTCATGAACATCATCA-3? (713–745) 5?-CTCTGGTGGACGAGCAACATGATGG-3? (964–988) 5?-ACGGCGCTTGACGTGGGCTTGT-3? (280–301) 5?-CCATTGCGTATTTCACAGGAGATGCACA-3? (698–725) 5?-TCAACAAGGCGCTGCTGACCACCT-3? (131–154) 5?-TCTTCAAGCGCTGCCTGCCCCT-3? (317–338) 5?-CAATACAGCCGGCAGAGCTACCCAAA-3? (745–770) 5?-TATGACGATGCTGTTTGTGGGTCTGATGAG-3? (429–458) 5?-CATCCCTTTATCAAGTGTGGGTAGGAGCCA-3? (497–526) 5?-TCAGTCTCACCTTTGGCACGTCCATG-3? (338–363) 5?-TAATCTCTGTGGCCCTAGGCCAGGTGTT-3? (182–209) 5?-ACAACTCTAACCAGTGTCCAGCTTTTGGATTGC-3? (394–426) 5?-ATTCATGGGAGTGAGGATTGTGGCCG-3? (813–838) 5?-CCCAGTTCACACAGTGGCATCAGTAGACA-3? (259–287) 5?-CTGCCAAGTTTTCCGGCATTGCTC-3? (77–100)
Race
5?-TAGCTTGGGCTGGTTTCCACT-3? (496–476) 5?-AGCGCTGTGGTCAGGATCTT-3? (461–442) 5?-AACATGCTGTGAGAACTGCCA-3? (550–530) 5?-CAGCTGTGCTTCCAATCTTGA-3? (382–362) 5?-ACTGGAACGCTCCCAAAGA-3? (1206–1188) 5?-GCAATGTCGACCAAAGGTTGA-3? (643–623) 5?-ACCGGTCTCCAGGTAGTTCTTC-3? (207–186) 5?-TGCACATAGTGCCAGGGAAA-3? (714–695) 5?-AAACACACCCCGGATGCAC-3? (774–756) 5?-GAGTCTTCTTCATCTCCCAGCC-3? (1045–1024) 5?-TTGTGTACAGCGAGACGGTGA-3? (343–323) 5?-TCAGCCCAGCCTTCAAACT-3? (755–737) 5?-TGCCATCTGTCCAATTCCAA-3? (198–179) 5?-TCTTGAGAACCAGGACGCC-3? (373–355) 5?-CATCATAGCGGTTCAAACTGTACG-3? (796–773) 5?-AACCCAAAACCACAGTTGCA-3? (481–462) 5?-TTGAGTTCACTTGTAATTCATGCTG-3? (553–529) 5?-CAGGGTGAACAGAGGTGTGGT-3? (444–424) 5?-GCTAGTCAAGCCAATTCCACAA-3? (243–222) 5?-AGCCATCAACACAGGAATCCC-3? (450–430) 5?-CATCACAATGCCAGCGATG-3? (867–849) 5?-ATAGGAGCAGAGGAATCTTCGGT-3? (329–307) 5?-TACACACACCATTTGCAGTCTTGT-3? (150–127)
Table 2. Total RNA Source Information for Various Tissues
The positions of the primers and probes were counted from the position of the initiation codon.
5?-CCGGACACTCAGCAAATTACA-3? (411–431) 5?-TATGTGGACACGCTCAAGCTC-3? (325–345) 5?-GCCCTCAGATTCTCAGCTTGATT-3? (468–490) 5?-AACCTGGTGATCTATCTTCAGCG-3? (301–323) 5?-ATAATGCCAGCAAGCCTCAA-3? (1118–1137) 5?-ACCACATGCGGGCTCATTA-3? (572–590) 5?-CTATGGTTCTTCCCATTTGTGG-3? (100–121) 5?-GACAGCACAACTGCACCAAA-3? (649–668) 5?-AATTCCCGTCATCGGAGTGA-3? (684–703) 5?-CTCTACTACGAGGAGACCAAGAGCTT-3? (937–962) 5?-AAGAGTGGCTCCCACAGCTC-3? (255–274) 5?-TTCATGATTCTGCCCACCCT-3? (676–695) 5?-GCTCCTTCCTCATCGTGCTT-3? (110–129) 5?-CCTCAGCCTGCCCATGTA-3? (294–311) 5?-GGCCGCAACAACATCTTAACA-3? (715–735) 5?-TTTCCTACCCACCCAACTTCC-3? (407–427) 5?-GGTGTTTGCTGCTCTTGGTG-3? (468–487) 5?-TCGCTGCCGAGTCCTACTG-3? (318–336) 5?-CAAAGTGCTGAACAGGGAGATG-3? (159–180) 5?-GTGATCGTCAGAGCCTACCAGTA-3? (370–392) 5?-TCATGGATCGTTCTCAGGGAC-3? (790–810) 5?-TCACTAGATGTAAACCAGGAGCCA-3? (230–253) 5?-GAGTTCTTCACCTCCTTTTAGACTGA-3? (48–73)
SLC35A1 (NM_006416) SLC35A2 (NM_005660) SLC35A3 (NM_012243) SLC35A4 (NM_080670) SLC35A5 (NM_017945) SLC35B1 (NM_005827) SLC35B2 (NM_178148) SLC35B3 (NM_015948) SLC35B4 (NM_032826) SLC35C1 (NM_018389) SLC35C2 (NM_173179) SLC35D1 (NM_015139) SLC35D2 (NM_007001) SLC35D3 (NM_001008783) SLC35E1 (NM_024881) SLC35E2 (NM_182838) SLC35E3 (NM_018656) SLC35E4 (NM_001001479) SLC35F1 (NM_001029858) SLC35F2 (NM_017515) SLC35F3 (NM_173508) SLC35F4 (NM_001080455) SLC35F5 (NM_025181)
—, no data available. *, The total RNA was the same as that used for the measurement of mRNA expression levels in our previous studies. Probe (Position)
Sex
Reverse primer (Position)
Age
Forward primer (Position)
Pool size
mRNA (GenBank No.)
Tissue
Table 3. Primers and Probes Used for RT-PCR Analysis
mRNA Expression Profiles of Human SLC35 Transporters
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was used for measurement of the target mRNA in each tissue. For the RT-PCR reaction, the TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems) containing 300 nM forward primer, 900 nM reverse primer, and 200 nM TaqMan probe was used at 20 mL per tube. The RT-PCR assay was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with the following profile: 1 cycle at 489 C for 30 min, 1 cycle at 959 C for 10 min, and 40 cycles each at 959 C for 15 sec and 609C for 1 min. The threshold cycle (Ct) was calculated by the instrument's software (7500 Fast System ver. 1.3.1). Data analysis: The relative expression of each mRNA was calculated by the DCt method (where DCt is the value obtained by subtracting the Ct value of PPIA mRNA from the Ct value of the target mRNA), as employed in previous studies.7–9) Specifically, the amount of target mRNA relative to PPIA mRNA is expressed as 2－(DCt). Data are expressed as the ratio of the target mRNA to PPIA mRNA.
Results and Discussion In the present study, we investigated the mRNA expression levels of 23 human SLC35 transporters in 31 adult human tissues, 6 fetal human tissues and 3 human cell lines. Analysis was conducted by RT-PCR using the Applied Biosystems 7500 Fast Real-Time PCR System in the presence of the TaqMan probe. In order to prepare the transporter calibration curves, the total RNA obtained from the tissue exhibiting the highest mRNA expression levels was used for the individual transporter isoforms. When preparing the calibration curves, various amounts ranging from 0.768 to 60,000 pg of total RNA were used. The lower limit of quantification for each mRNA ranged between 0.768 and 480 pg of total RNA per 20 mL of reaction mixture, and the upper limit of quantification for each mRNA was higher than 60,000 pg of total RNA per 20 mL of reaction mixture. In the present study, we selected PPIA, which showed low variability and was used as an endogenous control in our previous studies.7,9–11) The process used to select the best housekeeping gene has been described in detail in our previous studies.7,12) Similar to the findings of our previous studies,9,13) the highest and second highest Ct values for PPIA in various adult and fetal tissues and cell lines were 22.51 in the mammary gland and 21.08 in skeletal muscle, respectively, and the variation in the Ct values for PPIA as the internal control among various tissues was the smallest of the various housekeeping genes (data not shown). The mRNA expression levels of 23 human SLC35 transporters in various human adult and fetal tissues and cell lines are shown in Tables 4–9. All SLC35A mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 4). Ishida et al.14) have also reported that
Table 4. Expression of Human SLC35A Transporter mRNAs in Various Tissues Tissue
SLC35A1 SLC35A2 SLC35A3 SLC35A4 SLC35A5
Adult Adipose tissue
0.0627
0.00675
0.0482
0.0499
0.0672
Adrenal gland
0.0665
0.0138
0.0169
0.0447
0.0437
Bladder
0.0672
0.00692
0.0245
0.0311
0.0518
Bone marrow
0.0971
0.00641
0.0310
0.0214
0.0567
Brain
0.0359
0.00619
0.0106
0.0166
0.0610
Cerebellum
0.0412
0.00376
0.0173
0.0179
0.0557
Colon
0.0577
0.0118
0.0636
0.0361
0.0318
Heart
0.0691
0.00763
0.0331
0.0597
0.0629
Kidney
0.0367
0.0146
0.0355
0.0499
0.0583
Liver
0.0263
0.00985
0.0178
0.0168
0.0268
Lung
0.0828
0.00849
0.0424
0.0308
0.0542
Mammary gland
0.0878
0.00806
0.0404
0.0424
0.0270
Ovary
0.0482
0.00483
0.0307
0.0289
0.0527
Pancreas
0.0289
0.00673
0.0249
0.0249
0.0317
Peripheral leukocytes
0.0531
0.00551
0.0204
0.0273
0.0369
Placenta
0.0506
0.0433
0.0508
0.0634
0.148
Prostate
0.122
0.0163
0.0718
0.0390
0.0537
Retina
0.0540
0.00709
0.0343
0.0307
0.103
Salivary gland
0.0754
0.0154
0.0466
0.0341
0.0336
Skeletal muscle
0.115
0.0147
0.0524
0.290
0.0851
Small intestine
0.0213
0.00655
0.0593
0.0251
0.0256
Smooth muscle
0.0769
0.00473
0.0453
0.0244
0.0489
Spinal cord
0.0265
0.00572
0.0145
0.0205
0.0915
Spleen
0.0495
0.00726
0.0284
0.0448
0.0701
Stomach
0.0730
0.0120
0.0906
0.0369
0.0522
Testis
0.100
0.0114
0.0459
0.0661
0.154
Thymus
0.0217
0.00503
0.0203
0.0169
0.0216
Thyroid gland
0.0480
0.0109
0.0531
0.0326
0.0925
Tonsil
0.0273
0.00626
0.0160
0.0244
0.0315
Trachea
0.0711
0.0370
0.0808
0.0402
0.0575
Uterus
0.0839
0.00876
0.0427
0.0321
0.0741
Fetus Brain
0.0610
0.00666
0.0183
0.0129
0.0418
Heart
0.0421
0.00820
0.0181
0.0394
0.0396
Kidney
0.0308
0.0123
0.0165
0.0324
0.0354
Liver
0.0203
0.00985
0.0145
0.0195
0.0234
Spleen
0.0474
0.00873
0.0128
0.0333
0.0397
Thymus
0.0623
0.00646
0.0579
0.0198
0.0327
Cell lines HeLa
0.0123
0.00588
0.0216
0.0153
0.0149
Hep G2
0.0170
0.00945
0.0191
0.000303
0.0249
MDA 435
0.0250
0.00795
0.00605
0.0387
0.0286
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
95
mRNA Expression Profiles of Human SLC35 Transporters
Table 5. Expression of Human SLC35B Transporter mRNAs in Various Tissues Tissue
Table 6. Expression of Human SLC35C Transporter mRNAs in Various Tissues
SLC35B1
SLC35B2
SLC35B3
SLC35B4
Adipose tissue
0.0621
0.00124
0.0226
0.00969
Adipose tissue
0.0301
0.0902
Adrenal gland
0.0670
0.00197
0.0118
0.0163
Adrenal gland
0.00942
0.144
Adult
Tissue
SLC35C1
SLC35C2
Adult
Bladder
0.0418
0.00133
0.0183
0.0130
Bladder
0.0116
0.0872
Bone marrow
0.0715
0.000850
0.0181
0.00699
Bone marrow
0.00685
0.217 0.0640
Brain
0.0458
0.00134
0.00530
0.0299
Brain
0.00453
Cerebellum
0.0396
0.00106
0.00530
0.0243
Cerebellum
0.00668
0.0682
Colon
0.0391
0.00134
0.0185
0.00184
Colon
0.0168
0.0925
Heart
0.195
0.00213
0.0179
0.00800
Heart
0.00809
0.0961
Kidney
0.109
0.00231
0.0163
0.0206
Kidney
0.00496
0.111
Liver
0.113
0.000845
0.0127
0.0126
Liver
0.0105
0.0851
Lung
0.0529
0.00153
0.0130
0.00501
Lung
0.00752
0.0938
Mammary gland
0.217
0.000219
0.0253
0.00685
Mammary gland
0.0131
0.192
Ovary
0.0358
0.00148
0.00817
0.0150
Ovary
0.00901
0.0947
Pancreas
0.0896
0.00232
0.0129
0.00268
Pancreas
0.0116
0.148
Peripheral leukocytes
0.0469
0.00243
0.00528
0.00317
Peripheral leukocytes
0.0132
0.111
Placenta
0.155
0.00232
0.0311
0.0256
Placenta
0.0308
0.374
Prostate
0.0583
0.00152
0.0208
0.00876
Prostate
0.0102
0.129
Retina
0.0706
0.00145
0.00353
0.0234
Retina
0.00969
0.134
Salivary gland
0.0944
0.00303
0.0169
0.00278
Salivary gland
0.0136
0.144
Skeletal muscle
0.557
0.00682
0.0180
0.0230
Skeletal muscle
0.0212
0.228
Small intestine
0.115
0.00112
0.0111
0.00407
Small intestine
0.0255
0.0780
Smooth muscle
0.0405
0.00148
0.0175
0.00989
Smooth muscle
0.00682
0.0825
Spinal cord
0.0546
0.00197
0.00945
0.0114
Spinal cord
0.00624
0.0746
Spleen
0.0524
0.00120
0.0154
0.0120
Spleen
0.0109
0.111
Stomach
0.0977
0.00180
0.0257
0.00737
Stomach
0.0287
0.103
Testis
0.125
0.00209
0.0276
0.0157
Testis
0.0153
0.231
Thymus
0.0297
0.000693
0.00393
0.00392
Thymus
0.00395
0.0502
Thyroid gland
0.0836
0.00181
0.0194
0.00643
Thyroid gland
0.00530
0.0981
Tonsil
0.0487
0.000963
0.00834
0.00481
Tonsil
0.0148
0.0748
Trachea
0.108
0.00385
0.0216
0.00582
Trachea
0.0301
0.150
Uterus
0.0524
0.00183
0.0166
0.0129
Uterus
0.00952
0.110
Fetus Brain
0.0451
0.00173
0.00576
0.0119
Brain
0.0139
0.0533
Heart
0.0881
0.00238
0.00982
0.00602
Heart
0.00849
0.0813
Kidney
0.0474
0.00248
0.00999
0.0110
Kidney
0.00731
0.0964
Liver
0.0825
0.00164
0.00916
0.00371
Liver
0.00605
0.0769
Spleen
0.00765
0.0875
Thymus
0.00867
0.0822
Fetus
Spleen
0.0520
0.00157
0.0101
0.00784
Thymus
0.0368
0.00197
0.0132
0.00664
HeLa
0.0291
0.00143
0.00891
0.00353
Hep G2
0.0778
0.00240
0.00628
0.0107
MDA 435
0.0291
0.00285
0.00400
0.0206
Cell lines
Cell lines
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
HeLa
0.00534
0.0340
Hep G2
0.00749
0.113
MDA 435
0.00904
0.0836
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
96
Masuhiro NISHIMURA, et al.
Table 7. Expression of Human SLC35D Transporter mRNAs in Various Tissues Tissue
Table 8. Expression of Human SLC35E Transporter mRNAs in Various Tissues
SLC35D1
SLC35D2
SLC35D3
Adipose tissue
0.0239
0.0515
0.000249
Adipose tissue
0.126
0.0461
0.0190
0.00592
Adrenal gland
0.0271
0.0298
0.00354
Adrenal gland
0.0881
0.0520
0.0218
0.00423
Adult
Tissue
SLC35E1
SLC35E2
SLC35E3
SLC35E4
Adult
Bladder
0.0404
0.0602
0.0000415
Bladder
0.0499
0.0461
0.0147
0.00795
Bone marrow
0.0278
0.0300
0.00328
Bone marrow
0.0612
0.0538
0.0136
0.00195
Brain
0.0151
0.0326
0.00113
Brain
0.0845
0.0632
0.0148
0.0158
Cerebellum
0.0133
0.0113
0.000148
Cerebellum
0.0842
0.0619
0.0110
0.0117
Colon
0.0825
0.0822
0.00141
Colon
0.0822
0.0506
0.0103
0.0108
Heart
0.0318
0.0736
0.0000978
Heart
0.122
0.0420
0.0215
0.00817
Kidney
0.0552
0.106
0.0000095
Kidney
0.110
0.0387
0.0187
0.00290
Liver
0.128
0.0708
0.0000112
Liver
0.0385
0.0134
0.0136
0.000487 0.00471
Lung
0.0208
0.0595
0.000168
Lung
0.0994
0.0397
0.0151
Mammary gland
0.00628
0.0242
0.00283
Mammary gland
0.473
0.000730
0.0352
0.00267
Ovary
0.0368
0.0616
0.0000878
Ovary
0.0915
0.0751
0.0152
0.00576
Pancreas
0.0179
0.129
0.00188
Pancreas
0.164
0.0335
0.0350
0.00609
Peripheral leukocytes
0.0158
0.0363
0.000329
Peripheral leukocytes
0.0893
0.0720
0.0186
0.00476
Placenta
0.0356
0.0811
0.0000207
Placenta
0.459
0.0887
0.0173
0.00214
Prostate
0.0341
0.0627
0.000342
Prostate
0.204
0.0484
0.0213
0.00466
Retina
0.0504
0.0951
0.0260
Retina
0.122
0.0487
0.0246
0.0139
Salivary gland
0.0167
0.0422
0.0000351
Salivary gland
0.170
0.0511
0.0201
0.00419
Skeletal muscle
0.102
0.0335
0.000133
Skeletal muscle
0.603
0.0816
0.0711
0.134
Small intestine
0.0337
0.110
0.00117
Small intestine
0.0625
0.0355
0.00846
0.00331
Smooth muscle
0.0353
0.0557
0.0000403
Smooth muscle
0.111
0.0433
0.0134
0.00781
Spinal cord
0.0155
0.0623
0.000886
Spinal cord
0.0569
0.0295
0.0219
0.00817
Spleen
0.0269
0.0363
0.000181
Spleen
0.0825
0.0477
0.0111
0.00406
Stomach
0.0542
0.0842
0.000683
Stomach
0.119
0.0480
0.0123
0.00611
Testis
0.0711
0.0499
0.000577
Testis
0.110
0.0424
0.0161
0.00543
Thymus
0.0245
0.0142
0.0000089
Thymus
0.0581
0.0535
0.0169
0.00159
Thyroid gland
0.0249
0.107
0.0000233
Thyroid gland
0.103
0.0527
0.0126
0.00293
Tonsil
0.0183
0.0259
0.0000145
Tonsil
0.0756
0.0360
0.0101
0.00346
Trachea
0.0294
0.0799
0.000889
Trachea
0.192
0.0527
0.0241
0.00979
Uterus
0.0436
0.0730
0.0000329
Uterus
0.103
0.0715
0.0145
0.0111
Brain
0.0230
0.00660
0.00159
Brain
0.115
0.153
0.0166
0.0135
Heart
0.0340
0.0797
0.00139
Heart
0.102
0.0495
0.0110
0.00657
Kidney
0.0288
0.0751
0.0000935
Kidney
0.0878
0.0636
0.00985
0.00447
Liver
0.0589
0.117
0.00126
Liver
0.0442
0.0172
0.00812
0.000551
Spleen
0.0240
0.0354
0.000813
Spleen
0.0778
0.0552
0.00942
0.00781
Thymus
0.0544
0.0179
0.000164
Thymus
0.0848
0.0991
0.0192
0.00287
HeLa
0.00702
0.00626
0.0000074
HeLa
0.0233
0.0102
0.00491
0.000790
Hep G2
0.0292
0.190
BLQ
Hep G2
0.0794
0.0176
0.0167
0.0000052
MDA 435
0.00558
0.0430
0.0000173
MDA 435
0.0928
0.0124
0.00904
0.00315
Fetus
Fetus
Cell lines
Cell lines
BLQ, below the limit of quantification. Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
97
mRNA Expression Profiles of Human SLC35 Transporters
Table 9. Expression of Human SLC35F Transporter mRNAs in Various Tissues Tissue
SLC35F1
SLC35F2
SLC35F3
SLC35F4
SLC35F5
Adult Adipose tissue
0.00605
0.00826
0.000114
0.000210
0.176
Adrenal gland
0.00564
0.00233
0.000738
BLQ
0.431
Bladder
0.00632
0.00757
0.0000399
0.000138
0.154
Bone marrow
0.000234
0.00652
0.000104
0.0000535
0.112
Brain
0.0783
0.00351
0.0124
0.00282
0.0654
Cerebellum
0.0186
0.00132
0.0179
0.0128
0.0691
Colon
0.00617
0.0354
0.000171
0.0000550
0.139
Heart
0.00704
0.00284
0.0000187
0.0000497
0.258
Kidney
0.00182
0.0203
0.000356
0.000280
0.295
Liver
0.000180
0.000397
BLQ
BLQ
0.0845
Lung
0.000467
0.00949
0.000119
0.0000504
0.135
Mammary gland
0.00330
0.0203
0.00254
BLQ
0.218
Ovary
0.00114
0.0107
0.000146
0.000262
0.179
Pancreas
0.00214
0.0223
0.00562
0.000365
0.153
Peripheral leukocytes
BLQ
0.00628
0.000287
0.0000497
0.0492
Placenta
0.000196
0.0504
0.0000321
0.000208
0.292
Prostate
0.00864
0.0470
0.00121
0.00108
0.235
Retina
0.0336
0.0243
0.000295
0.00315
0.142
Salivary gland
0.00258
0.117
0.00181
0.0000461
0.206
Skeletal muscle
0.00224
0.00558
0.000899
0.000153
0.563
Small intestine
0.00586
0.0236
0.000360
0.0000487
0.103
Smooth muscle
0.00582
0.00503
BLQ
0.000295
0.132
Spinal cord
0.0296
0.00972
0.00443
0.00172
0.0677
Spleen
0.00174
0.00879
0.000157
BLQ
0.134
Stomach
0.00438
0.0216
0.000305
0.000121
0.174
Testis
0.00619
0.0201
0.000337
0.00534
0.269
Thymus
0.000203
0.00564
0.00118
BLQ
0.0411
Thyroid gland
0.00221
0.0147
0.000336
0.00174
0.231
Tonsil
0.000838
0.0146
0.000918
0.0000144
0.0944
Trachea
0.00273
0.0202
0.000896
0.0000834
0.230
Uterus
0.0131
0.0136
0.000549
0.000874
0.180
Fetus Brain
0.198
0.0220
0.00119
0.00179
0.0775
Heart
0.00888
0.00515
BLQ
0.0000860
0.184
Kidney
0.0103
0.0118
0.000196
0.000821
0.218
Liver
0.000323
0.000785
0.0000617
BLQ
0.0857
Spleen
0.00560
0.0150
0.0000552
0.0000339
0.111
Thymus
0.00121
0.00846
0.00206
0.0000115
0.114
HeLa
BLQ
0.0148
0.000401
0.0000048
0.0356
Hep G2
0.0000308
0.00564
BLQ
0.0000515
0.205
MDA 435
BLQ
0.0251
BLQ
0.0000092
0.0822
Cell lines
BLQ, below the limit of quantification. Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. The highest values among the various tissues are shown in boxes.
SLC35A3 mRNA is expressed ubiquitously in human tissues. SLC35B1, SLC35B3, and SLC35B4 mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 5). SLC35B2 mRNA was also expressed ubiqui-
tously in both adult and fetal tissues, but was expressed at lower levels in adult bone marrow, liver, mammary gland, thymus, and tonsil (Table 5). SLC35C1 and SLC35C2 mRNAs were expressed ubiquitously in both
98
Masuhiro NISHIMURA, et al.
adult and fetal tissues (Table 6). Leach et al.15) have also reported that SLC35C2 mRNA is detected in all human tissues. SLC35D1 and SLC35D2 mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 7). On the other hand, Ishida et al.16) have reported that SLC35D2 mRNA is not detected in the brain, colon, or thymus. The differences between our results and their results may be due to the differences between the sensitivity of real-time reverse transcription PCR and Northern blot analysis. Muraoka et al.4) have also reported that SLC35D1 mRNA is expressed ubiquitously in human tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina (Table 7). SLC35E1 and SLC35E3 mRNAs were expressed ubiquitously in both adult and fetal tissues (Table 8). SLC35E2 and SLC35E4 mRNAs were also expressed ubiquitously in both adult and fetal tissues, but SLC35E2 mRNA was expressed at lower levels in the adult mammary gland and SLC35E4 mRNA was expressed at lower levels in both the adult and fetal liver (Table 8). SLC35F1 mRNA was expressed at higher levels in the adult and fetal brain (Table 9). SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland (Table 9). Both SLC35F3 and SLC35F4 mRNA were expressed at the highest levels in the adult cerebellum (Table 9). SLC35F5 mRNA was expressed ubiquitously in both adult and fetal tissues (Table 9). SLC35 transporters were consistently expressed in HeLa cells, except for SLC35D3, SLC35E4, SLC35F1,
SLC35F3, and SLC35F4 (Tables 4–9). SLC35 transporters were consistently expressed in Hep G2 cells, except for SLC35A4, SLC35D3, SLC35E4, SLC35F1, SLC35F3, and SLC35F4 (Tables 4–9). SLC35 transporters were consistently expressed in MDA 435 cells, except for SLC35D3, SLC35F1, SLC35F3, and SLC35F4 (Tables 4–9). Individual differences in the mRNA expression of human SLC35 transporters in the liver are shown in Figure 1. The dispersion of the mRNA levels of each of the SLC35 transporters in the total RNA obtained from HAB sometimes differed by factors of several times, dozens of times, or more. The mRNA levels in the liver in the total RNA purchased from CLONTECH Laboratories, Inc., were the same as, slightly higher than, or slightly lower than those in the total RNA obtained from HAB. Therefore, with a few exceptions, it is suggested that the data shown in Table 4 provides valid information concerning the relative abundance of mRNAs in the liver and other tissues, although this is based on the results for one sample. In some reports, the expression levels of SLC35s (nucleotide sugar transporters) have been implicated in various stages of the development of human diseases. For example, transcripts of SLC35A2 specifically (but not of SLC35A1 or SLC35A3) were found to be more abundant in colon cancer cells than in non-malignant tissues obtained from the same patients.17) Furthermore, human
Fig. 1. Individual differences in the mRNA expression of human SLC35 transporters in the liver Data are expressed as the ratio of the target mRNA to PPIA mRNA. Experiments were performed in duplicate. Closed circles () are the same as the data in Table 4–9. Closed () and open () circles are the individual data for the total RNA purchased from CLONTECH Laboratories and the total RNA obtained from HAB, respectively. Small closed circles () and bars indicate the mean and standard deviation of the individual data (n＝30) for the total RNA obtained from HAB.
mRNA Expression Profiles of Human SLC35 Transporters
diseases that are closely related to the nucleotide sugar transporters have been reported by several groups.18–20) However, the roles of nucleotide sugar transporters in human diseases are still poorly understood. The results of the present study may be helpful to researchers in this field. In conclusion, the results of the present study provide valuable information concerning the tissue-specific profiles of the mRNA expression of 23 human SLC35 transporters. These findings should be useful for studies on the regulation of transport in research related to the clinical diagnosis of disease. In particular, the information obtained in this study is expected to be important in assessing the feasibility of measuring the mRNA expression levels of target genes in various tissues.
9)
10)
11)
12)
Acknowledgments: The authors would like to thank David G. Spear, M.D., for reviewing this manuscript.
13)
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