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Mechanism of the Regulation of Organic Cation/Carnitine Transporter 1 (SLC22A4) by Rheumatoid Arthritis-Associated Transcriptional Factor RUNX1 and Inflammatory Cytokines

Department of Molecular Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Japan (T.M., M.H., D.K., I.T.); and Central Research Laboratories, Kissei Pharmaceutical Co. Ltd., Azumino, Japan (K.M.)

(Received July 19, 2006; accepted November 21, 2006)

	Abstract

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Recently, it was reported that the organic cation/carnitine transporter 1 (OCTN1, SLC22A4) is associated with chronic inflammatory diseases, such as rheumatoid arthritis (RA) and Crohn's disease. OCTN1 in humans is expressed in synovial tissues of individuals with rheumatoid arthritis. Furthermore octn1 in mice is expressed in inflamed joints with collagen-induced arthritis, a model of human arthritis, but not in the joints of normal mice. OCTN1 should be involved in the inflammatory disease and in the present study, the regulatory mechanism of OCTN1 expression was characterized using the human fibroblast-like synoviocyte cell line MH7A, derived from RA patients. A luciferase-reporter gene assay and gel shift assay demonstrated that RUNX1, which is an essential hematopoietic transcription factor associated with acute myeloid leukemia and is related to RA and Sp1, is involved in the regulation of OCTN1 promoter activity. Inflammatory cytokines such as interleukin-1ß and tumor necrosis factor- increased the expression of OCTN1 mRNA. Furthermore, overexpression of nuclear factor-B (NF-B) activated promoter activity of OCTN1. These results clearly demonstrate that expression of OCTN1 is regulated by various factors, including RUNX1, inflammatory cytokines, and NF-B, all of which are also related to the pathogenesis of RA. Further studies on the physiological substrate(s) of OCTN1 should be done to clarify the roles of OCTN1 in these diseases.

The chromosomal region 5q31 that includes the OCTN1 gene is of particular interest, because it contains many genes involved in immune and inflammatory systems and also because it is suggested to be a susceptible locus for several inflammatory or autoimmune diseases, such as rheumatoid arthritis (RA) and Crohn's disease that have been reported recently on the basis of the frequency of specific single-nucleotide polymorphisms (SNPs) in the intron 1 of the OCTN1 gene in Japanese, Greekm and English patients with RA and Crohn's disease (Tokuhiro et al., 2003; Gazouli et al., 2005; Russell et al., 2006), and the frequency of SNPs in the exons of the OCTN1 gene in patients with Crohn's disease (Peltekova et al., 2004; Yamazaki et al., 2004). SNPs in intron 1 of OCTN1 affect the expression of OCTN1 by lowering the affinity for the transcription factor RUNX1 (AML1) (Tokuhiro et al., 2003), which is expressed mainly in hematopoietic cells. In addition, the RUNX1 gene was independently associated with three autoimmune disorders: systemic lupus erythematosus, psoriasis, and RA (Prokunina et al., 2002; Helms et al., 2003; Tokuhiro et al., 2003). It was also reported that OCTN1 was expressed in the inflamed joints of mice with collagen-induced arthritis, a model of human arthritis, but not in the joints of normal mice (Tokuhiro et al., 2003). RA is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity (Dendorfer et al., 1994). In particular, the macrophage-like synoviocytes and fibroblast-like synoviocytes of the hyperplastic lining layer exhibit an activated phenotype in RA (Buchan et al., 1988; Panayi et al., 1992; Arend and Dayer, 1995). These cells are a major source of several inflammatory cytokines, such IL-1, tumor necrosis factor- (TNF), and IL-6 proteins, which play crucial roles in the pathophysiology of RA (Firestein et al., 1990; Dendorfer et al., 1994). Both IL-1ß and TNF stimulate fibroblast-like synoviocytes to secrete mediators, such as matrix metalloproteinases (Pillinger et al., 2003) and vascular endothelial growth factor (Jackson et al., 1997), which regulate inflammation and connective tissue degradation, respectively. Cytokines also stimulate the NF-B signaling pathway (Barchowsky et al., 2000), which in turn regulates the expression of a wide array of proinflammatory molecules. In addition, monoclonal anti-TNF antibody, IL-1ß receptor antagonist, and NF-B decoy oligonucleotides have been successfully used to block the activity of TNF, IL-1ß and NFB both in experimental models and human trials (Tomita et al., 2000). Therefore, elucidating the regulatory mechanism of OCTN1 expression is useful for future treatment of RA and Crohn's disease.

Accordingly, in the present study, the regulatory mechanism of OCTN1 expression was characterized, focusing especially on promoter analysis, using human fibroblast-like synoviocyte cell line MH7A derived from RA patients as a model (Miyazawa et al., 1998, Miyazawa et al., 1998). We used several methods to identify the basal transcription factor of OCTN1 and factors, including inflammatory cytokines such as IL-1ß and NF-B, which modulate the expression of OCTN1, because they are known to be closely related to RA (Hiramitsu et al., 2006).

	Materials and Methods

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Materials. [-³²P]ATP (3000 Ci/mmol) was purchased from GE Healthcare Bio-Sciences (Piscataway, NJ). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and Wako Pure Chemicals (Osaka, Japan). Antibodies for Sp-1, RUNX1, and NF-B (NF-B p50) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Culture and Cytokine Treatment. The cell line (MH7A) was purchased from RIKEN BioResource Center (Tsukuba, Japan) and cultured in RPMI 1640 medium (Sigma-Aldrich), containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA) in a humidified incubator at 37°C under 5% CO₂. At 24 or 48 h before harvesting of the cells, the culture medium was replaced with fresh RPMI 1640 and supplemented with phosphate-buffered saline (PBS), with or without IL-1ß or TNF as required.

Identification of the OCTN1 Transcription Start Site. Determination of the transcription start site of the OCTN1 gene was performed by the 5'-rapid amplification of cDNA ends method in accordance with the manufacturer's protocol (Clontech, Mountain View, CA). The first-round polymerase chain reaction (PCR) was performed using the Marathon cDNA adaptor-specific primer AP1 and OCTN1 gene-specific primer GSP1 (synthesized by Hokkaido System Science Co., Ltd., Sapporo, Japan) (Table 1). The second-round PCR was performed using the Marathon cDNA adaptor-specific primer AP2 and OCTN1 gene-specific primer GSP2 (synthesized by Hokkaido System Science Co., Ltd.) (Table 1). The reactions were performed using the following conditions: 95°C for 5 min and then 35 or 25 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 5 min. A PCR product of approximately 0.20 kilobases was obtained, subcloned into pGEM-T easy vector (Promega, Madison, WI), and sequenced.

Cloning of Human OCTN1 Promoter. The 5' region of the OCTN1 gene was PCR-amplified using human genomic DNA (Clontech) as a template, using upstream primer p-2544, downstream primer p+92 (both synthesized by Hokkaido System Science Co., Inc.) (Table 1), and Ex Taq DNA polymerase (Takara Shuzo Co. Ltd., Shiga, Japan), on the basis of the reported OCTN1 gene sequence (accession number AB007448 [GenBank] ). All amplified fragments were sequenced and confirmed to not have any mutation. The nuclear base sequence obtained finally for the promoter region of OCTN1 was submitted to GenBank (accession number AB252052 [GenBank] ). Because the upstream and downstream primers were designed to include an internal HindIII restriction site and an internal XhoI site, respectively, the resulting PCR products were digested with HindIII and XhoI and ligated into the luciferase reporter gene vector pGL3-Basic (Promega).

Nuclear Extract Preparation and Gel Mobility Shift Assay. Nuclear extracts were prepared from MH7A cells based on the method of Schreiber et al. (1989). Briefly, MH7A cells (1 x 10⁶ cell) were washed with 10 ml of PBS and pelleted by centrifugation at 1500g for 5 min. The pellet was resuspended in 1 ml of PBS, transferred into a 1.5-ml tube, and centrifuged again by 10,000g for 15 s. The resultant cell pellet was resuspended in 400 µlof ice-cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml leupeptine) by gentle pipetting. The cells were allowed to swell on ice for 15 min, after which 25 µl of a 10% solution of Nonidet P-40 was added, and the mixture was vigorously mixed by vortex mixer for 10 s. The resultant cell homogenate was centrifuged at 10,000g for 30 s. The nuclear pellet obtained was resupended in 50 µl of aliquot of ice-cold buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml leupeptine), and the tube was vigorously rocked at 4°C for 15 min on a shaking platform. The obtained cell lysate was centrifuged for 5 min at 10,000g at 4°C, and the resultant supernatant was used as nuclear extract.

Sense and antisense oligonucleotides containing putative Sp1, RUNX1 and NFB binding sites were synthesized (Hokkaido System Science Co., Ltd.). Their sequences are shown in Table 1 and mapped on the OCTN1 promoter sequence in Fig. 1. Gel mobility shift assays were carried out as described previously (Maeda et al., 2005). Briefly, double-stranded synthetic oligonucleotide was used as a probe. T4 polynucleotide kinase (Toyobo Co., Ltd., Osaka, Japan), and [-³²P]adenosine triphosphate (3000 Ci/mmol) were used to label the 5' end of the sense strand oligonucleotide. Binding reactions were initiated by incubating nuclear extracts from MH7A cells (10–15 µg of protein) with 750 ng of poly(dI·dC) (GE Healthcare, Bio-Sciences) and 100 µg of bovine serum albumin (Sigma-Aldrich Co.) for 20 min at room temperature. Double-stranded probe was then added, and incubation was continued for another 30 min at room temperature. The specific antibodies for Sp1, RUNX1 and NFB (NF-B p50) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

View larger version (68K): [in this window] [in a new window]   FIG. 1. Nucleotide sequence and structure of the 5'-flanking region of the OCTN1 gene. The nucleotide sequence of the 5'-flanking region of the human OCTN1 gene from –2544 to +92 nt is shown. Nucleotide numbers are relative to the transcription start site. Consensus binding sites for putative regulatory elements are underlined, and the respective transcription factors are given above the sequence. Flags denote the size of the deletional constructs used for functional promoter studies.

Transfections and Luciferase Assay. The deletion-mutant luciferase-reporter plasmids were constructed by PCR using specific primers. Because the upstream and downstream primers were designed to include an internal HindIII restriction site and an internal XhoI site, respectively, the resulting PCR products were digested with HindIII and XhoI and ligated into the luciferase reporter gene vector pGL3-Basic (Promega). Reporter gene constructs were transfected using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, cells were plated in 24-well plates at 0.5 x 10⁵ cells/well for 24 h before transfection. Before addition of DNA-liposome complexes, the cells were rinsed with serum-free medium. For each transfection, reporter constructs (0.8 µg) were cotransfected with 0.08 µg of pRL-TK vector (Promega) that included TK promoter and Renilla luciferase as an internal control in 0.5 ml of serum-free medium by incubating at 37°C for 6 h. After 6 h, the culture medium was changed to medium containing 10% fetal bovine serum, and the cells were incubated for 48 h at 37°C, rinsed twice with PBS, and harvested using passive lysis buffer (Promega). For luciferase assays, cell extracts were mixed with luciferase assay reagent (Promega) for detection in a luminometer (Berthold Technologies Bad Wildbad, Germany). Relative luciferase activities were shown as the ratio of firefly/Renilla luciferase activities, and the results were presented as the mean ± S.E.M. of four to seven independent transfection experiments. Sp1 expression vector was a kind gift from Dr. Alexandrea Stewart (University of Pittsburgh, Pittsburgh, PA), which was reported previously (Kadonaga et al., 1987). RUNX1 expression vector was prepared with LA Taq DNA polymerase, on the basis of the reported RUNX1 gene sequence (Lutterbach and Hiebert, 2000), and ligated into expression vector pcDNA3.1 (Invitrogen). NF-B expression vectors were kind gifts from Dr. Seiichi Tanuma (Tokyo University of Science).

Site-Directed Mutagenesis. Site-directed mutagenesis of the binding sites of Sp1 (GC-box), RUNX1, and NF-B was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the oligonucleotides shown in Table 1. Briefly, an OCTN1-derived –180/+92 construct or a –2544/+89 construct containing a mutation was generated by PCR using two complementary oligonucleotides mutated in the Sp1 binding site or RUNX1 binding site or NF-B binding site (sequence mut Sp1: 5'-GGTCCTTGGGGGCGGGCGGGCG-3'; mut RUNX1: 5'-CCGGGGATGGGGGTGTTTTCCCAAGTGT-3'; and mut NF-B: 5'-CAACTTTGAAAATAAATTCCCCCAGTGGGC-3' in Table 1) and Pfu DNA polymerase (Stratagene). The product was digested with DpnI to remove the parental DNA template and to identify the DNA containing the mutation. The mutated plasmids obtained were termed mut Sp1, mut RUNX1, and mut NF-B for mutants in binding sites of Sp1, RUNX1, and NF-B, respectively, and all mutants were sequenced.

Reverse Transcription-Polymerase Chain Reaction. Total RNA was prepared from MH7A cells using ISOGEN (Wako Pure Chemicals). The total RNA content was determined by measuring the absorbance at 260 nm. The mRNA level was analyzed using semiquantitative reverse transcription-PCR. Single-strand cDNAs were constructed using an oligo(dT) primer (Invitrogen) and Improm-II reverse transcriptase (Promega). These cDNAs provided templates for PCRs using specific primers (Table 1) at a denaturation temperature of 94°C for 30 s, an annealing temperature of 58 to 62°C for 30 to 60 s, and an elongation temperature of 72°C for 30 s in the presence of dNTPs and Ex Taq polymerase (Takara Shuzo Co. Ltd.). Annealing time and temperature were changed as required, depending on the genes. The PCR cycle numbers were titrated for each primer pair to confirm that amplification was performed within a linear range. PCR products were analyzed by 2% agarose gel (w/v) electrophoresis, and the gels were stained with ethidium bromide for visualization. mRNA levels were quantified by densitometry using light capture (Atto Co., Tokyo, Japan). PCR amplification data were normalized with respect to glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The sets of primers specific for the nucleotide sequences of the transporters are shown in Table 1. The quantitation of each gene was repeated at least three times using RNA sources isolated from independently cultured cells.

Statistical Analysis. The statistical significance of differences was determined by Student's t test or one-way analysis of variance with Dunnett's post hoc test, with p < 0.05 as the criterion. Data are shown as mean ± S.E.M.

	Results

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Localization of the Transcriptional Initiation Site and Promoter Region of the OCTN1 Gene. To localize the promoter region of the OCTN1 gene, the transcription start site was identified by the 5'-rapid amplification of cDNA ends methods. By using a downstream oligonucleotide (Table 1) that corresponded to nucleotides +142 to +160 and +271 to +292 of the OCTN1 cDNA sequence as published by Tamai et al. (1997) (accession number AB007448 [GenBank] ), a PCR product was amplified and sequenced (see Supplemental Data). The obtained sequences suggested that the transcription initiation site was 20 base pairs upstream from the start of the published OCTN1 cDNA sequences (accession number AB252052 [GenBank] ).

Analysis of the 5'-Flanking Region of the Human OCTN1 Gene. On the basis of the above finding, the upstream genomic region of the OCTN1 was identified using the Human Genome Resource of the National Center for Biotechnology Information and the National Institutes of Health. A 2544-base pair fragment of the 5'-flanking sequence of the OCTN1 gene was obtained by PCR amplification (Fig. 1). PCR was performed with primers p-2544 and p+92 (Table 1) and human genomic DNA as a template. Figure 1 shows the sequence of the 5'-flanking 2544 nt relative to the transcription start site up to the translation start site. Potential transcription factor recognition sites were identified by using the program TRANSFAC 4.0 (BIOBASE GmbH, Wolfenbüttel, Germany) and revealed that this region lacks canonical TATA and CAAT boxes, but contains several putative transcription factor binding sites such as the GC box (Sp1 binding site) at –59 to –50, RUNX1 at –65 to –60, –391 to –386, –1119 to –1114, –1383 to –1377, –1563 to –1558, –1958 to –1953, –1996 to –1991, and –2457 to –2452, GATA at –2167 to –2162, and NF-Bat –2243 to –2234, as shown in Fig. 1.

Constitutive Activity of the OCTN1 Enhancer-Promoter Region in MH7A Cells. To functionally characterize the OCTN1 promoter activity, a series of promoter deletion mutants were constructed in a reporter gene vector pGL3-Basic that expresses firefly luciferase. Promoter activities of all deleted constructs between p-2544 Luc and p-61 Luc were compared with that of pGL3-Basic (Fig. 2). Transfection of the p-2544 Luc plasmid into MH7A cells, which contained the promoter region from –2544 to +92 nts, stimulated luciferase activity approximately 35-fold compared with pGL3-Basic (no promoter insert) (Fig. 2). Other constructs, p-2142, p-1682, and p-1263 Luc, stimulated luciferase activity 40-fold compared with pGL3-Basic. The constructs p-699 Luc, p-189 Luc, and p-150 Luc stimulated luciferase activity 25-, 15-, and 20-fold compared with pGL3-Basic, respectively. A shorter construct that stimulated luciferase activity, p-150 Luc, which includes the RUNX1 binding site (Fig. 1), was sufficient to confer residual promoter activity in MH7A cells. The shortest construct, p-61 Luc, stimulated luciferase activity 3-fold. Accordingly, first of all, we focused on p-189 Luc, because it included a putative Sp1 binding site (GC box) and a RUNX1 binding site. Although p-189 Luc showed lower promoter activity than p-150 Luc, it did maintain promoter activity.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Deletion analysis of the 5'-flanking region of the human OCTN1 gene in MH7A cells. Deletional fragments in the 5'-upstream region of the human OCTN1 gene are illustrated at the left. Each deleted promoter fragment was ligated into the luciferase reporter plasmid pGL3-Basic. Numbers indicate distance in base pairs from the transcription start site. The MH7A cells were transiently transfected with eight chimeric promoter constructs ranging from nt –2544 to +92 relative the transcription start site. Transfection efficiency was normalized by cotransfection of pRL-TK, and the promoter activity was measured as relative light units of firefly luciferase per unit of Renilla luciferase. Promoter activity is shown as the factor of induction of luciferase relative to background activity measured in cells transfected with pGL3-Basic alone. Results are expressed as the means ± S.E.M. of four to seven independent transfection experiments. , , and show putative GC box, NF-B, and RUNX1 binding sites, respectively.

View larger version (62K): [in this window] [in a new window]   FIG. 3. Gel mobility shift competition assay and supershift analysis of Sp1, RUNX1, and NF-B-bound protein complexes. Gel mobility shift assays were carried out with nuclear extract from MH7A cells. Lanes 1, 4, 6, and 9, radiolabeled probe alone; lane 2, 5, 7, and 10, probe with 5 pmol of unlabeled oligonucleotide (self competition); and lanes 3, 8, and 11, probe with Sp1, RUNX1, or NFB specific antibodies. The two or three shifted protein complexes are labeled from largest to smallest as H and L or H, M, and L for NF-B and Sp1, respectively. The arrows and asterisk indicate the supershifted complexes and RUNX1 complex, respectively.

Effect of Mutation of Sp1 Binding Sites on OCTN1 Expression. To assess the importance of Sp1 binding to OCTN1 promoter regions for basal promoter function, mutations of the Sp1 binding sites were introduced into p-189 Luc by site-directed mutagenesis. The resulting reporter gene plasmids p-mut Sp1 Luc contained mutations of nucleotides at –162 and –161 (GG to TT) (Table 1). Introduction of these mutations abrogated Sp1 binding in mobility shift assays (Fig. 3, lanes 4 and 5). GC box mutation did not change the luciferase activity compared with wild type, whereas the activity of mutant p-mut Sp1 Luc was no longer stimulated by overexpression of Sp1 (Fig. 4). These results demonstrate that Sp1 is associated with OCTN1 transcriptional regulation but is not involved in basal transcriptional regulation of OCTN1.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Effect of Sp1 on OCTN1 expression in MH7A cells. A, schematic sequences of the –189 and –150 regions are shown. B, MH7A cells were cotransfected with the p-189 Luc, p-150 Luc, or p-mut Sp1 Luc reporter gene construct plus either Sp1 expression plasmid () or empty vector as a control (). Luciferase activity is shown as the ratio of firefly/Renilla luciferase activities, and data represent the means ± S.E.M. of four to seven independent transfection experiments. *, statistically significant difference by Student's t test (p < 0.05).

View larger version (18K): [in this window] [in a new window]   FIG. 5. Effect of RUNX1 on OCTN1 promoter activity in MH7A cells. A, schematic sequences of the –150 and –61 regions are shown. B, MH7A cells were cotransfected with the p-1682 Luc, p-1263 Luc, p-699 Luc, p-189 Luc, p-150 Luc, p-61 Luc, or p-mut RUNX1 Luc reporter gene constructs plus either RUNX1 expression plasmid () or empty vector as a control (). Luciferase activity is shown as the ratio of firefly/Renilla luciferase activities, and data represent the means ± S.E.M. of four to seven independent transfection experiments. *, statistically significant difference by Student's t test (p < 0.05).

Effects of IL-1ß and TNF on the Expression of OCTN1 and OCTN2. IL-1ß and TNF are inflammatory cytokines and are associated with RA. Therefore, we examined the effects of IL-1ß and TNF on transcriptional activation of OCTN1 and OCTN2. As shown in Fig. 6, expression of OCTN1 mRNA was activated 2- to 3-fold relative to the control by treatment with 1 ng/ml IL-1ß or 10 ng/ml TNF for 24 and 48 h, respectively (n = 3, normalized to G3PDH, p < 0.05). However, expression of OCTN2 mRNA was affected by neither IL-1ß nor TNF (Fig. 6). Accordingly, OCTN1 and OCTN2 are presumed to be regulated by distinct mechanisms. The mechanism of up-regulation of OCTN1 by IL-1ß was further analyzed using promoter constructs. Only p-2544 Luc that included the NF-B binding site construct was activated by IL-1ß (Fig. 7).

View larger version (20K): [in this window] [in a new window]   FIG. 6. Effects of IL-1ß and TNF on OCTN1 mRNA expression in MH7A cells. MH7A cells were cultured in either the absence () or presence of IL-1ß (1 ng/ml) () or TNF (10 ng/ml) () for 24 h (A) or 48 h (B) before harvest, respectively. mRNA levels of OCTN1 and OCTN2 were analyzed by semiquantitative reverse transcription-PCR. mRNA expression is shown as the ratio of OCTNs mRNA/G3PDH mRNA and data represent the means ± S.E.M. of three independent experiments. *, Statistically significant difference from the control by one-way analysis of variance with Dunnett's post hoc test (p < 0.05).

View larger version (20K): [in this window] [in a new window]   FIG. 7. Effect of IL-1ß on promoter activity of OCTN1 in MH7A cells. A, schematic sequences of the –2544 and –2142 regions are shown. B, MH7A cells were cultured in either the absence () or presence of IL-1ß (1 ng/ml) () for 24 h. Luciferase activity is shown as the ratio of firefly/Renilla luciferase activities, and data represent the means ± S.E.M. of four to nine independent transfection experiments. *, significant difference by Student's t test (p < 0.05).

View larger version (19K): [in this window] [in a new window]   FIG. 8. Effect of NFB on promoter activity of OCTN1 in MH7A cells. MH7A cells were cotransfected with p50 and/or p65 expression vectors together with the OCTN1 reporter gene construct (p-2544 Luc). Luciferase activity is shown as the ratio of firefly/Renilla luciferase activities, and data represent the means ± S.E.M. of five independent transfection experiments. *, statistically significant difference by one-way analysis of variance with Dunnett's post hoc test (p < 0.05).

View larger version (15K): [in this window] [in a new window]   FIG. 9. Effect of mutagenesis of the NF-B binding site on promoter activity of OCTN1 in MH7A cells. MH7A cells were transfected with OCTN1 reporter gene construct (p-2544 Luc or p-mut NF-B Luc). A, MH7A cells were cultured in either the absence () or presence of IL-1ß (1 ng/ml) () for 24 h. Luciferase activity is shown as the ratio of firefly/Renilla luciferase activities, and data represent the means ± S.E.M. of four to nine independent transfection experiments. B, MH7A cells were cotransfected with p50 and/or p65 expression vectors together with the OCTN1 reporter gene construct (p-mut NF-B Luc). Luciferase activity is shown as the ratio of firefly/Renilla luciferase, and data represent the mean ± S.E.M. of five independent transfection experiments. *, significant difference by one-way analysis of variance with Dunnett's post hoc test (p < 0.05).

	Discussion

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It is known that the expression of serum amyloid A is induced by IL-1ß after activation by Sp1 and a sequence binding factor (Ray et al., 1999). Moreover, the DNA binding affinity and amount of Sp1 were increased by IL-1ß (Ray et al., 1999). However, in the present study, luciferase activity of p-189 Luc that includes the Sp1 binding site was not induced by IL-1ß (Fig. 7). Therefore, it appears that the expression of OCTN1 was not induced via Sp1 after treatment with IL-1ß.

Previously, it was demonstrated that SNPs in intron 1 of OCTN1 affected the expression of OCTN1 by lowering the affinity for RUNX1 (Tokuhiro et al., 2003), which is an essential hematopoietic transcription factor (Roumier et al., 2003). So, we investigated the role of RUNX1 in the OCTN1 promoter region. The present results suggested that RUNX1 binds to the putative proximal RUNX1 binding site on the OCTN1 promoter and activates the promoter (Figs. 3 and 5). However, RUNX1 was not associated with basal promoter activity of OCTN1, because mutation of RUNX1 in the OCTN1 promoter region led to a partial loss of the promoter activity (Fig. 5). Previous findings suggested that the expression of OCTN1 is negatively regulated by RUNX1 via intron 1 (Tokuhiro et al., 2003). However, the present results indicate that RUNX1 activated the OCTN1 promoter via the proximal RUNX1 binding site (Fig. 5). RUNX1 activated luciferase activity of p-150 Luc but not of p-61 Luc (Fig. 5B). In addition, we confirmed that the region from the –62 to –67 nt is a functional site for RUNX1 binding, considering the results of the gel mobility shift assay and site-directed mutagenesis (Figs. 3 and 5). Therefore, OCTN1 expression is likely to be regulated by RUNX1 via the –62 to –67 nt region. At present, the mechanism by which RUNX1 is associated with OCTN1 expression via the RUNX1 binding sites at the promoter region and intron 1 of the OCTN1 gene is not clear. It is possible that different cofactors are associated with RUNX1 for the regulation of OCTN1 via those binding sites at the promoter and intron 1. Accordingly, further studies on the role of RUNX1 in regulating of OCTN1 expression are needed.

Human OCTN1 is strongly expressed in hematopoietic tissues, such as bone marrow and fetal liver as well as kidney but not in adult liver (Tamai et al., 1997). In addition, when the expression of OCTN1 was suppressed, the proliferation and differentiation of K562 cells, a human leukemia cell line, were inhibited (T. Nakamura, S. Sugiura, D. Kobayashi, K. Yoshida, H. Yabuuchi, A. Aizawa, T. Maeda, and I. Tamai, unpublished data). Moreover, RUNX1 knockout embryos develop normal blood islands and show progress through the yolk sac phase of hematopoiesis, but die between embryonic day 11 and 12.5 (Okuda et al., 1996). Before death, the liver rudiment contains primitive nucleated erythrocytes but lacks all definitive erythroid, myeloid, and megakaryocytic cells, indicating a complete block of the development of the definitive hematopoietic program in the absence of RUNX1. Therefore, it is possible that the expression of OCTN1 is regulated by RUNX1 in fetal liver as well as in rheumatoid tissue, and OCTN1 is associated with proliferation and differentiation of hematopoietic stem cells. Moreover, because p-150 Luc, which includes the proximal RUNX1 binding site, was not activated by IL-1ß (Fig. 7), the expression of OCTN1 regulated by RUNX1 may be independent of the cascade of inflammatory cytokines. Therefore, it is possible that the role of RUNX1 is the tissue-specific regulation of certain genes.

In the present study, we also examined the effect of inflammatory cytokines on the expression of OCTN1, because these cytokines are related to RA. There is a report that the level of OCTN1 mRNA was increased 2-fold by TNF, but no such response was observed with OCTN2 mRNA in fibroblast-like synoviocytes from individuals with RA (Tokuhiro et al., 2003). The selective increase of OCTN1 by TNF is consistent with our results using MH7A cells (Fig. 6). Up-regulation of OCTN1 promoter activity by IL-1ß was shown to involve positions of the –2244 to –2253 nt by gel mobility shift assay, deletion analysis, and overexpression of NF-B (Figs. 3, 7, and 8). IL-1ß/TNF stimulated the translocation of p65 and p50 from the cytosol to the nucleus of human RA synovial fibroblasts (Gomez et al., 2005). IL-1ß and TNF play important roles in sideration and progress of RA, such as proliferation of synoviocytes, production of protease, and induction of adhesion proteins. In fact, RA is a chronic inflammatory disease characterized by the proliferation of the synovial membrane into a highly vascularized tissue known as pannus and inflammation. The pannus consists of several distinct cell types, which include resident fibroblast-like synoviocytes and infiltrating mononuclear cells capable of producing inflammatory cytokines such as IL-1, TNF, and IL-6 (Feldmann et al., 1996). The present study showed that OCTN1 expression was regulated by inflammatory cytokines related to RA. Therefore, it is possible that OCTN1 is associated with proliferation of the synovial membrane and fibroblast-like synoviocyte cells. However, there is no direct evidence of a contribution of OCTN1 to sideration and progression of RA at present.

The present study demonstrated that OCTN1 expression is regulated by RUNX1 and inflammatory cytokines, such as IL-1ß and NF-B. Although RUNX1 and these cytokines are known to be associated with RA, the mechanisms through which they are involved remain unclear. Their physiological roles and the mechanism through which OCTN1 acts in RA and Crohn's disease might be explained by considering the roles of ergothioneine, that was recently found to be a substrate of OCTN1 (Grundemann et al., 2005). Colognato et al. (2006) reported that ergothioneine, which is a good substrate of OCTN1, should inhibit apoptosis by induced oxidant stress in PC12 cells. In addition, we investigated the expression of OCTN1 and transport of ergothioneine by OCTN1 in PC12 cells. As a result, the transport of ergothioneine was mainly accounted for by OCTN1 in PC12 cells (T. Nakamura, K. Yoshida, H. Yabuuchi, T. Maeda, and I. Tamai, unpublished data). Moreover, it was reported that reactive oxygen species play a significant role in the pathogenesis of RA (Ozturk et al., 1999). Therefore, it is possible that the expression of OCTN1 is induced in inflammation to suppress the oxygen species by transporting antioxidants, such as ergothioneine. Further analysis will also be required to specify the substrate(s) of OCTN1 to establish its physiological role in inflammation and in the pathogenesis of RA other than ergothioneine.

In conclusion, we showed that OCTN1 expression is regulated by several transcription factors, i.e., Sp1, RUNX1, and NFB, and the regulation by these factors might explain the involvement of OCTN1 in autoimmune diseases, such as RA and Crohn's disease.

	Acknowledgments

 
We thank Dr. Alexandrea Stewart and Dr. Seiichi Tamuma for their generous gifts of the expression vectors of Sp1 and NF-B, respectively.

	Footnotes

 
This study was supported in part by a Grant in Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science, and Technology, Japan, and the Foundation for the Advancement of Science and Technology.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.012112.

ABBREVIATIONS: OCTN, organic cation/carnitine transporter; RA, rheumatoid arthritis; SNP, single-nucleotide polymorphism; IL, interleukin; TNF, tumor necrosis factor-; NF-B, nuclear factor-B; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotide; Luc, luciferase. ET, ergothioneine

The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Ikumi Tamai, Department of Molecular Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamasaki, Noda, Chiba, Japan; E-mail: tamai{at}rs.noda.tus.ac.jp

	References

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