Biomedical subjects

Yukio Kato

Publications and source records attributed to Yukio Kato.

At least 55 records · Page 3Linked to original sources

PubMed2004

Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells.

UNLABELLED: We isolated and expanded BMSCs from human alveolar/jaw bone at a high success rate (70%). These cells had potent osteogenic potential in vitro and in vivo, although their chondrogenic and adipogenic potential was less than that of iliac cells. INTRODUCTION: Human bone marrow stromal cells (BMSCs) have osteogenic, chondrogenic, and adipogenic potential, but marrow aspiration from iliac crest is an invasive procedure. Alveolar BMSCs may be more useful for regenerative medicine, because the marrow can be aspirated from alveolar bone with minimal pain. MATERIALS AND METHODS: In this study, alveolar bone marrow samples were obtained from 41 patients, 6-66 years of age, during the course of oral surgery. BMSCs were seeded and maintained in culture with 10% FBS and basic fibroblast growth factor. In addition, BMSCs were induced to differentiate into osteoblasts, chondrocytes, or adipocytes in appropriate medium. RESULTS AND CONCLUSION: From a small volume (0.1-3 ml) of aspirates, alveolar BMSCs expanded at a success ratio of 29/41 (70%). The success rate decreased with increasing donor age, perhaps because of age-dependent decreases in the number and proliferative capacity of BMSCs. The expanded BMSCs differentiated into osteoblasts under osteogenic conditions in 21-28 days: the mRNA levels of osteocalcin, osteopontin, and bone sialoprotein, along with the calcium level, in alveolar BMSC cultures were similar to those in iliac cultures. However, unlike iliac BMSC, alveolar BMSC showed poor chondrogenic or adipogenic potential, and similar differences were observed between canine alveolar and iliac BMSCs. Subsequently, human alveolar BMSCs attached to beta-tricalcium phosphate were transplanted into immunodeficient mice. In transplants, new bone formed with osteoblasts and osteocytes that expressed human vimentin, human osteocalcin, and human GAPDH. These findings suggest that BMSCs have distinctive features depending on their in vivo location and that alveolar BMSCs will be useful in cell therapy for bone diseases.

Adipocytes↗

PubMed2004

Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit. This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene. The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.

Alkaline Phosphatase↗

PubMed2004

PDZK1 directly regulates the function of organic cation/carnitine transporter OCTN2.

Urinary excretion of cationic xenobiotics is believed to be mediated by organic cation transporter (OCT and OCTN) families expressed on both basolateral and brush-border membranes of renal tubules, although the molecular mechanisms for targeting of these transporters to each membrane are poorly understood. Here, to examine the regulatory mechanisms for cell-surface expression and function of these transporters, we evaluated the interaction of these transporters with several PDZ proteins. A pull-down study using recombinant C-terminal proteins of OCTs and OCTNs identified a specific interaction of apical transporters OCTN1 and OCTN2, but not basolateral transporters OCT1 and OCT2, with PDZK1, intestinal and kidney-enriched PDZ protein, and Na+/H+ exchanger regulatory factor 2 (also called E3KARP, SIP-1, or TKA-1). Both yeast two-hybrid and pull-down studies suggested a requirement of the last four amino acids in OCTN1 and OCTN2 for the interaction. The interaction of PDZK1 with the C terminus of OCTN2 was also confirmed in a pull-down study using kidney brush-border membrane vesicles. Immunohistochemical analysis revealed that both PDZK1 and OCTN2 are colocalized in brush-border membranes of the kidney. Finally, double transfection of OCTN2 with PDZK1 stimulated the uptake by OCTN2 of its endogenous substrate carnitine, and this increase could be accounted for by the 6-fold increase in transport capacity. Such an increase was not observed for OCTN2 with deletion of the last four amino acids. Biotinylation study of surface proteins revealed minimal effect of PDZK1 on cell-surface expression of OCTN2. The present findings are the first to identify PDZK1 as a functional regulator of OCTN2 through direct interaction with the C terminus.

Amino Acid Sequence↗

PubMed2004

Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system.

Dec2, a member of the basic helix-loop-helix superfamily, is a recently confirmed regulatory protein for the clockwork system. Transcripts of Dec2, as well as those of its related gene Dec1, exhibit a striking circadian oscillation in the suprachiasmatic nucleus, and Dec2 inhibits transcription from the Per1 promoter induced by Clock/Bmal1 [Honma, Kawamoto, Takagi, Fujimoto, Sato, Noshiro, Kato and Honma (2002) Nature (London) 419, 841-844]. It is known that mammalian circadian rhythms are controlled by molecular clockwork systems based on negative-feedback loop(s), but the molecular mechanisms for the circadian regulation of Dec2 gene expression have not been clarified. We show here that transcription of the Dec2 gene is regulated by several clock molecules and a negative-feedback loop. Luciferase and gel retardation assays showed that expression of Dec2 was negatively regulated by binding of Dec2 or Dec1 to two CACGTG E-boxes in the Dec2 promoter. Forced expression of Clock/Bmal1 and Clock/Bmal2 markedly increased Dec2 mRNA levels, and up-regulated the transcription of the Dec2 gene through the CACGTG E-boxes. Like Dec, Cry and Per also suppressed Clock/Bmal-induced transcription from the Dec2 promoter. Moreover, the circadian expression of Dec2 transcripts was abolished in the kidney of Clock/Clock mutant mice. These findings suggest that the Clock/Bmal heterodimer enhances Dec2 transcription via the CACGTG E-boxes, whereas the induced transcription is suppressed by Dec2, which therefore must contribute to its own rhythmic expression. In addition, Cry and Per may also modulate Dec2 transcription.

5' Flanking Region↗

PubMed2004

Differential involvement of multidrug resistance-associated protein 1 and P-glycoprotein in tissue distribution and excretion of grepafloxacin in mice.

The involvement of multidrug resistance-associated protein 1 (Mrp1) and P-glycoprotein (mdr1) in the tissue distribution and excretion of grepafloxacin (GPFX), a fluoroquinolone antibiotic, was investigated using gene-deficient mice [mdr1a(-/-), mdr1a/1b(-/-), and mrp1(-/-)]. The plasma concentration-time profile of GPFX in mrp1(-/-) was nearly identical to that in mrp1(+/+), whereas that in mdr1a/1b(-/-) was higher than that in mdr1a/1b(+/+). The urinary clearance of GPFX in mdr1a/1b(-/-) was lower than that in mdr1a/1b(+/+), suggesting that the urinary excretion of GPFX is at least partially mediated by mdr1. The tissue-to-plasma concentration ratios during the beta-phase (K(p beta),) was significantly higher in the heart, trachea, kidney, spleen, and brown fat of mrp1(-/-) than those in mrp1(+/+). In MRP1-transfected LLC-PK1 cells, the efflux of GPFX after preloading into the cells was higher than that observed in the parent cell lines. These results suggest that GPFX is a substrate of MRP1 and that its distribution to these tissues might be limited by Mrp1. On the other hand, a higher K(p beta), and of GPFX in mdr1a(-/-) mdr1a/1b(-/-) compared with mdr1a/1b(+/+) was observed only in the brain. GPFX was efficiently distributed to the lung parenchyma cells and pulmonary airspaces, including the epithelial lining fluid and macrophages that are the pharmacological target of GPFX, although the contribution of Mdr1 and Mrp1 to such distribution seems to be minor. Thus, the present findings reveal that the disposition of GPFX is at least in part governed by these two ABC transporters and that both Mrp1 and Mdr1 are involved in the limited distribution of GPFX to the distinct tissues, including pharmacological and/or toxicological targets by an active efflux mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Lectins induce resistance to proteases and/or mechanical stimulus in all examined cells--including bone marrow mesenchymal stem cells--on various scaffolds.

Transplantation of bone marrow mesenchymal stem cells (MSC), chondrocytes, osteoblasts, or muscle cells promotes regeneration. However, these cells adhere poorly to some scaffolds--depending upon the scaffold material--and are often damaged by proteases or mechanical stimuli at site of transplantation. We found, however, that MSC, chondrocytes, and osteoblasts--along with some other cells--that were exposed to phaseolus vulgaris erythroagglutinin (PHA-E) or concanavalin A (ConA) increased their adhesion capacity on plastic tissue culture dishes and on plates of hydroxyapatite, titanium and poly-DL-lactic-co-glycolic acid (PLGA), and that these cells, moreover, built up resistance to proteases and/or mechanical stimuli. Thus, lectins may have great potential in tissue engineering and cell therapy.

Animals↗

PubMed2004

In vitro increase in intracellular calcium concentrations induced by low or high extracellular glucose levels in ependymocytes and serotonergic neurons of the rat lower brainstem.

Pancreatic glucokinase (GK)-like immunoreactivities are located in ependymocytes and serotonergic neurons of the rat brain. The present study investigated in vitro changes in intracellular calcium concentrations ([Ca(2+)](i)) in response to low (2 mm) or high (20 mm) extracellular glucose concentrations in isolated cells from the wall of the central canal (CC), raphe obscurus nucleus (ROb), ventromedial hypothalamus (VMH), and lateral hypothalamic area (LHA) in male rats. An increase in [Ca(2+)](i) was found in cells from the CC (21.1% or 9.8% of ependymocytes), ROb (10.9% or 14.5% of serotonergic neurons), VMH (7.8% and 25.2% of neurons), and LHA (20% or 15.7% of neurons), when extracellular glucose levels were changed from 10 to either 2 or 20 mm, respectively. Most of the ependymocytes and serotonergic neurons responding to the glucose changes were immunoreactive to the anti-GK in the CC (96.8% for low glucose and 100% for high glucose) and ROb (100% for low and high glucose). The [Ca(2+)](i) increase was blocked with calcium-free medium or L-type calcium channel blocker. Cells with an increase in [Ca(2+)](i) in response to low glucose did not respond to high glucose and vice versa. Inhibition of GK activity with acute alloxan treatment blocked low or high glucose-induced [Ca(2+)](i) increases in most GK-immunoreactive cells from the CC or ROb. The glucose-sensitive [Ca(2+)](i) increase in neurons of the VMH and LHA was also alloxan-sensitive, but no cells taken from the VMH and LHA were immunoreactive to the antibody used. The present study further indicates that ependymocytes of the CC and serotonergic neurons in the ROb are also sensitive to the changes in extracellular glucose in a GK-dependent manner, but that the subtype of GK in these cells could be different from that in the VMH and LHA.

Alloxan↗

PubMed2004

A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix.

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.

Adipocytes↗

PubMed2004

A novel autofeedback loop of Dec1 transcription involved in circadian rhythm regulation.

An autofeedback loop associated with transcription of clock gene(s), Per(s), is generally accepted as the molecular machinery of circadian rhythm generation, in which CLOCK/BMAL act as positive regulators and PER/CRY as negative ones. We show here an autofeedback loop of Dec1 encoding a basic helix-loop-helix transcription factor: CLOCK/BMAL increased the promoter activity of Dec1, and DEC1 and DEC2 as well as PERs and CRYs suppressed the induced expression. Three CACGTG E-boxes are responsible for both the activation and the suppression of Dec1 transcription. Forced expression of Clock/Bmal increased endogenous Dec1 mRNA level, and overexpression of Dec1 resulted in suppression of Dec2, Per2, and Dbp expression. The level of Dec1 expression in the heart of Clock/Clock mutant mice was continuously low throughout the day. These findings suggest that Dec1 is positively regulated by CLOCK/BMAL and is involved in circadian rhythm regulation by suppressing CLOCK/BMAL-induced gene expression. The autofeedback loop of Dec1 may be interlocked with the core feedback loop of Per in some situations.

Animals↗

PubMed2004

Transporter-mediated renal handling of nafamostat mesilate.

Nafamostat mesilate (NM) is a serine-protease inhibitor that is rapidly eliminated from the circulation and accumulated in the kidney. This study was conducted to characterize the mechanism of NM transport in the kidney because a serious side effect of NM-induced hyperkalemia may be related to accumulation of NM in the kidney. Measurements of uptake of NM in vivo by the kidney uptake index (KUI) method and of transport in an in vitro-cultured LLC-PK1 cell system suggested the involvement of an organic cation transporter (OCT). To clarify the involvement of OCTs located in the basolateral membrane of proximal tubules, we evaluated NM transport by OCTs expressed in Xenopus laevis oocytes. The IC(50) values of NM on [(14)C]TEA ([(14)C]tetraethylammonium) uptake by rOCT1, rOCT2, and hOCT2 were 50, 0.5, and 20 microM, respectively, and NM was concluded to be a substrate of OCTs. To investigate the transport of NM across the brush-border membrane, we examined the uptake of NM into brush-border membrane vesicles (BBMVs) isolated from rat renal cortex. NM was taken up into the BBMVs, and the uptake was decreased by unlabeled NM and temperature, implying that a transporter(s) is also involved in NM transport across the apical membrane. NM was not a substrate of hOCTN1, hOCTN2, or P-gp, implying the involvement of some unknown transporter(s). Thus, renal accumulation of NM can be explained by the involvement of the basolateral OCTs, though the influence of the apical membrane transporter remains to be clarified.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Characterization of renal excretion mechanism for a novel diuretic, M17055, in rats.

M17055 was developed as a novel diuretic that inhibits both Na(+), K(+), and 2Cl(-) cotransport at the thick ascending Henle's loop and Na(+) reuptake at the distal tubule. It is secreted at the renal proximal tubules. The purpose of the present study was to characterize the renal excretion mechanism of M17055. We used the renal cortical slices and brush border membrane vesicles (BBMVs) to investigate the transport mechanisms across the basolateral and brush border membranes, respectively. M17055 uptake by rat renal slices increased with time and was saturable. Several organic anions including probenecid, para-aminohippurate (PAH), and estrone-3-sulfate, decreased M17055 uptake. The uptake of M17055 was also observed into HEK293 cells expressing rat OAT1, and was inhibited by PAH. M17055 uptake by BBMVs was time-dependent, saturable, osmolarity-sensitive, and inhibited by several organic anions, but not by PAH. These results suggest that plural organic anion transport systems are involved in M17055 transport via both basolateral and brush border membranes of proximal tubule epithelial cells, a part of the renal uptake being mediated by OAT1.

Animals↗

PubMed2004

Functional characterization of human organic cation transporter OCTN1 single nucleotide polymorphisms in the Japanese population.

The organic cation transporter OCTN1 (SLC22A4) is expressed ubiquitously, with strong expression in kidney, trachea, bone marrow, and fetal liver, and it mediates transport of organic cations in a pH-dependent manner. Recent studies have identified single nucleotide polymorphisms (SNPs) of OCTN1 in the Japanese population. Two SNPs present in the exon regions, c1063t and g1531a, cause amino acid mutation, Thr306Ile (T306I) and Gly462Glu (G462E), respectively. We examined the influence of these SNPs on the intracellular localization, protein expression, and transport activity of OCTN1. Immunocytochemical analysis showed similar localizations of OCTN1 in cellular membranes of HEK293 cells transiently transfected with an expression plasmid DNA for OCTN1 or its SNP allelic variants. The Km and Vmax values for tetraethylammonium (TEA) uptake by T306I were similar to those of the wild-type even when the Vmax value was normalized for the expression level of OCTN1 protein. In contrast, G462E had almost negligible transport activity, although the protein expression level of G462E was equivalent to that of the wild-type. We conclude that the SNP that causes the single amino acid mutation T306I does not affect TEA transport activity, whereas the mutation G462E abrogates the TEA transport activity, presumably affecting the physiological function of OCTN1 and/or the pharmacological characteristics of its substrates.

Asian People↗

PubMed2004

Molecular cloning, nucleotide sequencing and characterization of the flagellin gene from isolates of urease-positive thermophilic Campylobacter.

A primer pair which was expected to generate an amplicon of the estimated size (approximately 1700 base pair (bp)) of the flaA gene for Campylobacter jejuni amplified products of approximately 1450 bp for 33 of the 44 isolates of urease-positive thermophilic Campylobacter (UPTC). The primer pair, however, failed to amplify fragments for 11 isolates of UPTC, for all of the 12 isolates of urease-negative C. lari and for one isolate of C. coli. Nevertheless, it successfully amplified fragments of approximately 1700 bp for five isolates of C. jejuni and for nine isolates of C. coli. Thus, the fragments of the flaA gene of UPTC were shorter than those of C. jejuni and C. coli. After PCR amplification and nucleotide sequencing of the flaA genes from five UPTC NCTC isolates, the putative open reading frames (ORFs) were found to range from 1461 to 1479 bp. The amino acid and nucleotide sequence alignments demonstrated that the PCR clones contained the flaA gene; however, our data indicated that this locus was markedly shorter in the UPTC organisms examined, as they were approximately 85 amino acid residues shorter, mainly corresponding to approximate residue numbers 390-470 of the large variable region of C. jejuni 81116. Heterogeneity was indicated in the molecular mass of the flagellin purified from the isolates examined. Flagellin of UPTC was demonstrated to be genotypically and phenotypically smaller than those of C. jejuni.

Amino Acid Sequence↗

PubMed2004

Estrogen regulation of nitric oxide synthesis in the porcine oocyte.

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.

Animals↗

PubMed2004

Screening of the interaction between xenobiotic transporters and PDZ proteins.

PURPOSE: Xenobiotic transporters have been proposed to be involved in membrane penetration of various therapeutic agents. As little information is available on molecular mechanism of their functional regulation, we have attempted to clarify the protein-protein interactions of such transporters as a first step to identify their regulators. METHODS: Yeast two-hybrid screening was performed to examine the interaction between carboxylic terminus of various xenobiotic transporters and PDZ (PSD95, D1g and ZO1) domain-containing proteins. The interaction and functional regulation were also evaluated in pull-down, immunoprecipitation and transport studies. RESULTS: Specific interaction with PDZ proteins was identified for several xenobiotic transporters including PEPT1, PEPT2, OCT3, OCTN1, OCTN2, OAT4, OATP-A, OATP-D, and OATP-F. The potent interaction was observed between PEPT2 and PDZK1, and deletion of the last four amino acids of the PEPT2 C-terminus almost completely abrogated such interaction. Recombinant PEPT2 C-terminus fusion protein can bind to purified His6-tagged PDZK1, confirming the involvement of two of four PDZ domains within PDZK1 in the interaction. Alanine-scanning mutation in PEPT2 revealed the presence of a consensus sequence (-T-X-L) that is responsible for the PDZK1 interaction. Transfection of PDZK1 increased the uptake of glycylsarcosine by PEPT2, whereas such stimulation was not observed for PEPT2 with the last four amino acids deleted. CONCLUSIONS: These results first identified the interaction between PDZ proteins and the cytosolic tail of various xenobiotic transporters. PDZK1 directly interacts with PEPT2, exerting functional regulation of its transporting activity. The current findings imply the localization of PEPT2 within a protein network constructed from PDZK1 and other transporter proteins.

Carrier Proteins↗

PubMed2004

Usefulness of bone marrow aspiration for definite diagnosis of Asian variant of intravascular lymphoma: four autopsied cases.

The Asian variant of intravascular lymphoma (AIVL) is characterized by hemophagocytic syndrome, pancytopenia and hepatosplenomegaly but usually lacks any neurological abnormality and skin lesions, which are typical features of classical intravascular lymphoma (IVL). An ante-mortem diagnosis of AIVL is difficult due to the absence of visible lymphoma lesions and unspecific clinical manifestations. A definite diagnosis relies on the presence of neoplastic B cells in the lumina of small vessels. Paraffin block samples of aspirated bone marrow clots were obtained from 4 patients with clinically suspected IVL and subjected to immunohistopathological analysis. All samples exhibited CD 20+ or CD 79a+ lymphoma cells proliferating intravascularly as well as erythrocytic hemophagocytosis. The distribution of neoplastic cells in the structure of the bone marrow allowed IVL to be distinguished from bone marrow invasions due to other types of lymphoma. We demonstrated the successful establishment of a definite ante-mortem diagnosis of AIVL in 3 of 4 patients by the rapid and simple method of using aspirated bone marrow samples.

Aged↗

PubMed2004

Rhythmic expression of DEC1 and DEC2 in peripheral tissues: DEC2 is a potent suppressor for hepatic cytochrome P450s opposing DBP.

The mammalian master molecular clock consisting of several clock gene products in the suprachiasmatic nucleus (SCN) drives circadian rhythms in behaviour and physiology. Molecular clocks consisting of the same components also exist in various peripheral organs. DEC1 and DEC2, basic helix-loop-helix transcription factors, were recently reported to be involved in the central clock in the SCN. We examined the expression profile of DEC1 and DEC2 in the periphery and their roles in the regulation of oscillating target genes in the liver. Levels of DEC1 and DEC2 mRNA exhibited a day-night variation in various peripheral tissues of rats. In the liver, their expression was high during the subjective night. Transfection assays showed that DEC2, but not DEC1, suppressed the transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A), overwhelming the potent enhancement by D-site binding protein (DBP). Electrophoretic mobility shift assays indicated that DEC2 binds to the E-box (CACATG) at the -219/-214 region of CYP7A. The transcriptional activities of the other sterol metabolizing cytochrome P450s (Cyps), CYP8B and CYP51, were also suppressed by DEC2 but not DEC1. DEC2, but not DEC1, works as a direct output mediator that transmits the circadian signals to the hepatic functions, including the CYP7A, CYP8B, and CYP51 expression.

Animals↗

PubMed2004

Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation. Interaction with BMAL1.

The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature 419, 841-844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loop-helix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein-protein interaction with BMAL1 and DNA binding to the E-box.

ARNTL Transcription Factors↗