Kyoko Morita

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.

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.

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.

Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil–induced injury

BACKGROUND & AIMS The oligopeptide transport system of the small intestine is resistant to mucosal injury. The mechanism of this resistance was investigated by examining the activity level and expression of the peptide transporter PepT1 in the intestine of rats treated with 5-fluorouracil. METHODS The expression and localization of PepT1 were examined by immunoblot analysis of brush border membrane vesicles and immunohistochemical analysis of intestinal sections with PepT1-specific rabbit polyclonal antibodies. Also, Northern blot analysis was used for the expression of PepT1 messenger RNA (mRNA). RESULTS Although the amounts of sucrase and an Na+-dependent glucose transporter protein in intestinal vesicles decreased markedly after 5-fluorouracil treatment, the amount of PepT1 protein remained largely unaffected. Immunohistochemical analysis also showed that the PepT1 immunoreactivity level was preserved in the brush border membrane of the remaining villi of 5-fluorouracil-treated rats. Levels of amino acid, glucose, and phosphate transporter mRNAs were profoundly depressed in 5-fluorouracil-treated animals, whereas the level of PepT1 mRNA conversely increased. CONCLUSIONS The resistance of intestinal peptide transport to tissue injury may be attributable to increased synthesis of PepT1 rather than to a change in the kinetic properties of the residual absorbing cells.

Serotonin transporter mRNA expression in peripheral leukocytes of patients with major depression before and after treatment with paroxetine

Serotonin transporter (5HTT) is thought to be involved in the pathophysiology of major depression and the target of antidepressants. We hypothesized that 5HTT mRNA levels in peripheral leukocytes may be associated with depressive states and the therapeutic response to antidepressant treatments. Fifteen patients with major depression and age-, sex-matched control subjects were studied. 5HTT mRNA levels were determined with quantitative real-time PCR method. 5HTT mRNA levels in leukocytes were significantly higher in depressive patients at baseline (before medication) than in control subjects. 5HTT mRNA levels were decreased significantly after 8 weeks of paroxetine medication compared with those at baseline. Our investigation suggested that the increased expression of 5HTT mRNA in peripheral leukocytes may be related with the pathophysiology of depression and its reduction after treatment may reflect the adaptive change induced by the antidepressant.

Identification of marker genes for differential diagnosis of chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a clinically defined condition characterized by long-lasting disabling fatigue. Because of the unknown mechanism underlying this syndrome, there still is no specific biomarker for objective assessment of the pathological fa-tigue.We have compared gene expression profiles in peripheral blood between 11 drug-free patients with CFS and age- and sex-matched healthy subjects using a custom microarray carrying complementary DNA probes for 1,467 stress-responsive genes. We identified 12 genes whose mRNA levels were changed significantly in CFS patients. Of these 12 genes, quantitative real-time PCR validated the changes in 9 genes encoding granzyme in activated T or natural killer cells (GZMA), energy regulators (ATP5J2, COX5B, and DBI), proteasome subunits (PSMA3 and PSMA4), putative protein kinase c inhibitor (HINT), GTPase (ARHC), and signal transducers and activators of transcription 5A (STAT5A). Next, we performed the same microarray analysis on 3 additional CFS patients and 20 other patients with the chief complaint of long-lasting fatigue related to other disorders (non-CFS patients) and found that the relative mRNA expression of 9 genes classified 79% (11/14) of CFS and 85% (17/20) of the non-CFS patients. Finally, real-time PCR measurements of the levels of the 9 involved mRNAs were done in another group of 18 CFS and 12 non-CFS patients. The expression pattern correctly classified 94% (17/18) of CFS and 92% (11/12) of non-CFS patients. Our results suggest that the defined gene cluster (9 genes) may be useful for detecting pathological responses in CFS patients and for differential diagnosis of this syndrome.

Expression analysis of psychological stress-associated genes in peripheral blood leukocytes

In this study, we have developed a microarray including 1467 cDNAs that were selected to specifically measure stress response in peripheral blood leukocytes. Venous blood was collected from 10 graduate students 2 h before and 2 or 24 h after an open presentation for their Ph.D. The mRNA levels in leukocytes were compared with those prepared 4 weeks before the presentation. Hierarchical cluster showed that distinct groups of genes uniformly changed their expression values in response to the stress. Bayesian t test identified significantly up-regulated 49 genes and down-regulated 21 genes. Most of them are categorized into cytokines, cytokine receptors, growth- or apoptosis-related molecules, and heat shock proteins, suggesting that stressful life events trigger acute responses in leukocytes. Our results suggest that gene expression profile in peripheral blood leukocytes may be a potentially useful method for the assessment of complex stress responses.

Gene expression signature in peripheral blood cells from medical students exposed to chronic psychological stress

To assess response to chronic psychological stress, gene expression profiles in peripheral blood from 18 medical students confronting license examination were analyzed using an original microarray. Total RNA was collected from each subject 9 months before the examination and mixed to be used as a universal control. At that time, most students had normal scores on the state-trait anxiety inventory (STAI). However, STAI scores were significantly elevated at 2 months and at 2 days before the examination. Pattern of the gene expression profile was more uniform 2 days before than 2 months before the examination. We identified 24 genes that significantly and uniformly changed from the universal control 2 days before the examination. Of the 24 genes, real-time PCR validated changes in mRNA levels of 10 (PLCB2, CSF3R, ARHGEF1, DPYD, CTNNB1, PPP3CA, POLM, IRF3, TP53, and CCNI). The identified genes may be useful to assess chronic psychological stress response.