Ken-ichi Miyamoto

Expression Cloning and Characterization of a Transporter for Large Neutral Amino Acids Activated by the Heavy Chain of 4F2 Antigen (CD98)

A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopusoocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.

A Vitamin D Receptor Gene Polymorphism in the Translation Initiation Codon: Effect on Protein Activity and Relation to Bone Mineral Density in Japanese Women

The effect of a T‐C transition polymorphism at the translation initiation codon of the human vitamin D receptor (VDR) gene on the biological function of the encoded protein was investigated. Of 239 Japanese women volunteers subjected to genotype analysis for this polymorphism, 32 (13%) were genotype MM (the M allele is ATG at the putative translation start site), 75 (31%) were genotype mm (the m allele is ACG at the putative translation start site), and 132 (55%) were genotype Mm. The bone mineral density (BMD) in the lumbar spine (L2–L4) was determined for 110 healthy premenopausal women from the volunteers and was shown to be 12.0% greater (p < 0.05) for mm homozygotes than for MM homozygotes. Synthesis of the proteins by the M and m alleles from the cloned cDNAs in vitro and in transfected COS‐7 cells revealed them to have a size of 50 and 49.5 kD, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is consistent with initiation of translation of the M allele‐encoded protein from an ATG codon located at nucleotides +10 to +12 in the conventional open reading frame. The extent of vitamin D–dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells was ∼1.7‐fold greater for the m type VDR than for the M type protein. These results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.

Human L-type amino acid transporter 1 (LAT1) : characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

THE POLYMORPHISM IN THE CAUDAL-RELATED HOMEODOMAIN PROTEIN Cdx-2 BINDING ELEMENT IN THE HUMAN VITAMIN D RECEPTOR GENE

The major physiological activity of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] is the regulation of calcium absorption in the small intestine, and the level of vitamin D receptor (VDR) is an important factor in this regulation. In a previous study, we indicated that the caudal‐related homeodomain Cdx‐2 played an important role in the intestine‐specific transcription of the human VDR gene. In this study, the polymorphism was identified in the core sequence 5′‐ATAAAAACTTAT‐3′ in the Cdx‐2 binding site in the VDR gene promoter. In 261 Japanese women with genotyped VDR polymorphisms, 48 were genotype Cdx‐A (adenine at −3731 nucleotides [nt] relative to the transcription start site of human VDR gene 5‐ATAAAAACTTAT), 82 were genotype Cdx‐G (guanine at −3731 nt, 5′‐GTAAAAACTTAT‐3′), and 131 were genotype Cdx‐A/G (heterozygote). In postmenopausal Japanese women, the bone mineral density (BMD) in the lumbar spine (L2‐L4) with the Cdx‐G homozygote was 12% lower than that with the Cdx‐A homozygote (p < 0.05). In electrophoretic gel mobility shift assay (EMSA), the oligonucleotide with Cdx‐G allele markedly decreased the binding to Cdx‐2 compared with that in the Cdx‐A allele. The transcriptional activity of the VDR promoter with Cdx‐G allele was decreased to 70% of the Cdx‐A allele. In addition, in the herpes simplex virus thymidine kinase promoter, the Cdx‐2 binding element with the G allele showed significantly lower transcriptional activity than that of the A allele. Thus, the polymorphism in the Cdx‐2 binding site of the VDR gene (Cdx‐polymorphism) would affect the expression of VDR in the small intestine. In addition, this polymorphism may modulate BMD in postmenopausal Japanese women.

Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1

BACKGROUND & AIMS Dietary regulation is one of the most important factors of intestinal peptide transport. However, the cellular and molecular mechanisms of dietary regulation of the intestinal peptide transport system remain unknown. This study investigated the molecular mechanism of transcriptional activation of intestinal peptide transporter (PepT1) gene by the dietary protein. The promoter region of the rat PepT1 gene was isolated and characterized. METHODS PepT1 messenger RNA levels were determined by Northern blot analysis. In transient transfection experiments, effects of amino acid and dipeptide on luciferase activity were investigated. RESULTS The proximal promoter region of the rat PepT1 gene has a TATA-like box and a GC box sequence. The luciferase activities of the clone -351 RPT-LUC responded to particular amino acids (phenylalanine, arginine, and lysine) and dipeptides (Gly-Sar, Gly-Phe, Lys-Phe, and Asp-Lys). An AP-1 binding site and an amino acid-responsible element were present at -295 and -277 nucleotides relative to the transcription start site in this region. CONCLUSIONS These results suggest that the up-regulation of dipeptide transport activity by dietary protein is caused by transcriptional activation of the PepT1 gene by selective amino acids and dipeptides in the diet.

Identification of an Amino Acid Transporter Associated with the Cystinuria-related Type II Membrane Glycoprotein

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b0,+ amino acid transporter). The transporter designated BAT1 (b0,+-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y+L, and system x−C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na+-independent transport of cystine as well as basic and neutral amino acids with the properties of system b0,+. The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

The caudal-related homeodomain protein Cdx-2 regulates vitamin D receptor gene expression in the small intestine

The actions of 1,25‐dihydroxyvitamin D3 (1,25(OH)2 D3) are mediated through the nuclear vitamin D receptor (VDR). The regulation of VDR abundance plays an important role in determining the magnitude of the target cell response to 1,25(OH)2D3. The major physiological activity of 1,25(OH)2D3 is the regulation of calcium absorption in the small intestine, and the level of VDR is an important factor in this regulation. However, the characterization of VDR gene expression in the small intestine remains unknown. In the present study, we investigated the regulation of the human VDR (hVDR) gene expression in the small intestine. The 4.0 kb of the 5′‐flanking region of the hVDR gene promoter was cloned and characterized by the measurement of luciferase activity and an electrophoretic mobility‐shift assay (EMSA). With the EMSA, we found that Cdx‐2 (a homeodomain protein‐related caudal) binds to the sequence 5′‐ATAAAAACTTAT‐3′ at −3731 to −3720 bp (hVD‐SIF1) relative to the transcription start site of the hVDR promoter. This sequence is very similar to the human sucrase‐isomaltase footprint 1 (SIF1) element. With a competition analysis and specific antibodies for Cdx‐2, we demonstrated that Cdx‐2 is able to activate VDR gene transcription by binding to this element. The mutation of the hVD‐SIF1 sequence in the hVDR gene promoter markedly suppressed the transactivation of the reporter gene in Caco‐2 cells. In addition, the DNA fragment (−3996 to −3286) containing the hVD‐SIF1 binding site increased transcription when placed upstream of the herpes simplex virus thymidine kinase promoter. These findings suggest that Cdx‐2 plays an important role in the intestine‐specific transcription of the hVDR gene.

CCR5 Plays a Critical Role in Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Regulating Both Macrophage Recruitment and M1/M2 Status

C-C motif chemokine receptor (CCR)2 and its ligand, monocyte chemoattractant protein (MCP)-1, are pivotal for adipose tissue macrophage (ATM) recruitment and the development of insulin resistance. However, other chemokine systems also may play a role in these processes. In this study, we investigated the role of CCR5 in obesity-induced adipose tissue inflammation and insulin resistance. We analyzed expression levels of CCR5 and its ligands in white adipose tissue (WAT) of genetically (ob/ob) and high-fat (HF) diet–induced obese (DIO) mice. Furthermore, we examined the metabolic phenotype of Ccr5−/− mice. CCR5 and its ligands were markedly upregulated in WAT of DIO and ob/ob mice. Fluorescence-activated cell sorter analysis also revealed that DIO mice had a robust increase in CCR5+ cells within ATMs compared with chow-fed mice. Furthermore, Ccr5−/− mice were protected from insulin resistance, glucose intolerance, and hepatic steatosis induced by HF feeding. The effects of loss of CCR5 were related to both reduction of total ATM content and an M2-dominant shift in ATM polarization. It is noteworthy that transplantation of Ccr5−/− bone marrow was sufficient to protect against impaired glucose tolerance. CCR5 plays a critical role in ATM recruitment and polarization and subsequent development of insulin resistance.

Role of the vitamin D receptor in FGF23 action on phosphate metabolism

FGF23 (fibroblast growth factor 23) is a novel phosphaturic factor that influences vitamin D metabolism and renal re-absorption of Pi. The goal of the present study was to characterize the role of the VDR (vitamin D receptor) in FGF23 action using VDR(-/-) (VDR null) mice. Injection of FGF23M (naked DNA encoding the R179Q mutant of human FGF23) into VDR(-/-) and wildtype VDR(+/+) mice resulted in an elevation in serum FGF23 levels, but had no effect on serum calcium or parathyroid hormone levels. In contrast, injection of FGF23M resulted in significant decreases in serum Pi levels, renal Na/Pi co-transport activity and type II transporter protein levels in both groups when compared with controls injected with mock vector or with FGFWT (naked DNA encoding wild-type human FGF23). Injection of FGF23M resulted in a decrease in 25-hydroxyvitamin D 1a-hydroxylase mRNA levels in VDR(-/-) and VDR(+/+) mice, while 25-hydroxyvitamin D 24-hydroxylase mRNA levels were significantly increased in FGF23M-treated animals compared with mock vector control- or FGF23WT-treated animals. The degree of 24-hydroxylase induction by FGF23M was dependent on the VDR, since FGF23M significantly reduced the levels of serum 1,25(OH)2D3 [1,25-hydroxyvitamin D3] in VDR(+/+) mice, but not in VDR(-/-) mice. We conclude that FGF23 reduces renal Pi transport and 25-hydroxyvitamin D 1a-hydroxylase levels by a mechanism that is independent of the VDR. In contrast, the induction of 25-hydroxyvitamin D 24-hydroxylase and the reduction of serum 1,25(OH)2D3 levels induced by FGF23 are dependent on the VDR.