[Show abstract][Hide abstract] ABSTRACT: DEC1 (BHLHB2/Stra13/Sharp2)-a basic helix-loop-helix transcription factor-is known to be involved in various biological phenomena including clock systems and metabolism. In the clock systems, Dec1 expression is dominantly up-regulated by CLOCK : BMAL1 heterodimer, and it exhibits circadian rhythm in the suprachiasmatic nucleus (SCN)-the central circadian pacemaker-and other peripheral tissues. Recent studies have shown that the strong circadian rhythmicity of Dec1 in the SCN was abolished by Clock mutation, whereas that in the liver was affected, but not abolished, by Clock mutation. Moreover, feeding conditions affected hepatic Dec1 expression, which indicates that Dec1 expression is closely linked with the metabolic functions of the liver. Among ligand-activated nuclear receptors examined, LXRalpha and LXRbeta with T0901317-agonist for LXR-were found to be potent enhancers for Dec1 promoter activity, and a higher expression level of LXRalpha protein was detected in the liver than in the kidney and heart. T0901317 increased the levels of endogenous Dec1 transcript in hepatoma cells. Chromatin immunoprecipitation assay indicated that LXRalpha bound to the Dec1 promoter, and an LXRalpha-binding site was identified. These observations indicate that hepatic DEC1 mediates the ligand-dependent LXR signal to regulate the expression of genes involved in the hepatic clock system and metabolism.
[Show abstract][Hide abstract] ABSTRACT: Cholesterol 7alpha-hydroxylase (CYP7A) and sterol 12alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a--including Dbp, Dec2, E4bp4, Hnf4alpha, Pparalpha, Lxralpha, Rev-erbalpha, and Rev-erbbeta--in the liver of wild-type and homozygous Clock mutant mice (Clock/Clock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wild-type mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hmgcr mRNA also lost its strong circadian rhythm in Clock/Clock , showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erbalpha, and Rev-erb beta--potent regulators for Cyp7a expression--were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxralpha, Hnf4alpha, Pparalpha, and E4bp4--had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBalpha/beta, DBP, LXRalpha, and HNF4alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARalpha were moderate negative regulators. Furthermore, knockdown of REV-ERBalpha/beta with siRNA suppressed Cyp7a transcript levels, and in the electrophoretic mobility shift assay, REV-ERBalpha/beta bound to the promoter of Cyp7a . These observations suggest that (1) active CLOCK is essential for the robust circadian expression of hepatic metabolic enzymes (Cyp7a, Cyp8b, and Hmgcr); (2) clock-controlled genes--DBP, DEC2, and REV-ERBalpha/beta--are direct regulators required for the robust circadian rhythm of Cyp7a; and (3) the circadian rhythm of Cyp7a is regulated by multiple transcription factors, including DBP, REV-ERBalpha/beta, LXRalpha, HNF4alpha DEC2, E4BP4, and PPARalpha.
No preview · Article · Sep 2007 · Journal of Biological Rhythms
[Show abstract][Hide abstract] ABSTRACT: To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.
No preview · Article · Oct 2006 · Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: A concanavalin-A-binding protein of 76 kDa was purified from the plasma membrane fraction of rabbit chondrocyte cultures. Amino acid sequencing of the N-terminal region and of tryptic peptides of the protein, in addition to sequencing of its cDNA revealed that this protein is highly similar to the tumour-associated antigen p97. Hence, it was concluded that this protein is the rabbit form of p97. Western blotting, Northern blotting and reverse-transcription PCR analyses indicated that rabbit p97 is expressed at high levels in cartilage and chondrocytes, but is barely detectable in the bone, liver, kidney, small intestine, eye, pancreas, heart, testis, skeletal muscle, spleen and fibroblasts. Immunocytochemical and immunohistochemical analyses demonstrated that p97 is expressed in the plasma membrane of chondrocytes. p97 transcript was detected in all zones of the cartilage but the level was relatively low in the hypertrophic zone. These findings suggest that p97 is involved in maintaining the cell surface characteristics of chondrocytes.
Preview · Article · Oct 1998 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Vitamin D3 is hydroxylated to 25-hydroxyvitamin D3 by a vitamin D3 25-hydroxylase (25-hydroxylase) located in liver mitochondria or endoplasmic reticulum. 25-hydroxyvitamin D3 is then transported to the kidney through the bloodstream. In kidney mitochondria, it is hydroxylated at the 1α-position by a 25-hydroxyvitamin D3 1α-hydroxylase to 1α,25-dihydroxyvitamin D3 in calcium-depleted animals or hydroxylated at the 24R-position to 24R,25-dihydroxyvitamin D3 by a 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) in animals that are calcium or 1,25-dihydroxyvitamin D3 repleted. Both 25- and 24-hydroxylases are purified and their cDNA clones are isolated. These hydroxylases constitute novel families in the P450 super family, that is, CYP27 for 25-hydroxylase and CYP24 for 24-hydroxylase. This chapter discusses the molecular cloning of these hydroxylases. Screening of cDNA libraries is performed by nucleic acid hybridization or by an immunological detection method. Construction of cDNA libraries, isolation of cDNA clones, and nucleotide sequencing are performed according to established methods.
No preview · Article · Dec 1997 · Methods in enzymology
[Show abstract][Hide abstract] ABSTRACT: Vitamin D3 is hydroxylated at position 25 by liver microsomal and mitochondrial vitamin D3 25-hydroxylases (25-hydroxylase). The microsomal enzyme purified from male rats is the same as CYP2C11, a male-specific P450. The mitochondrial enzyme is identical with 5β-cholestane-3α,7α,12α-triol (an intermediate in the conversion of cholesterol to cholic acid) 27-hydroxylase, and is categorized as CYP27. The product, 25-hydroxyvitamin D3 (25-OH-D3), is transported to the kidney through the bloodstream, where it is hydroxylated at the 1α-position to give 1α,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in calcium or 1,25-(OH)2D3 depleted animals. Both 25-and 24-hydroxylases are purified and their cDNA clones are isolated. These hydroxylases are members of the P450 superfamily that are b-type hemoproteins functioning as terminal monooxygenase in the oxygenation of various organic compounds. This chapter describes the assay and purification of both 25- and 24-hydroxylases.
No preview · Article · Feb 1997 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: 3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) [EC 126.96.36.199]2 plays important multifunctional roles in metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins and also in transforming the steroid nucleus for the biosynthesis of bile acids from cholesterol in liver. To gain insight into the details and physiological functions of 3 alpha-HSD in the bile acid biosynthetic pathway, cDNA clones of 3 alpha-HSD were isolated from rat liver lambda phage cDNA libraries by using specific antibodies to 3 alpha-HSD purified from rat liver. Transfection of the 3 alpha-HSD cDNA in Simian COS7 cells resulted in the expression of an immunoreactive protein to the antibodies against the purified enzyme, and the transfected cells exhibited activities for not only 7 alpha-hydroxy-5 beta-cholestan-3-one, the intermediate of bile acid biosynthesis, but also steroid hormones and 9,10-phenanthrenequinone. Northern blot analysis on poly(A)+ RNA by selective use of different cDNA fragments of the 5'-untranslated region, the coding region, and the 3'-untranslated region as probes revealed three hybridizable bands, 3.6, 2.7, and 2.5 kb, in liver and four bands, 3.6, 2.7, 2.5, and 1.8 kb, in ovary. Of these, the 2.7- and 1.8-kb bands were predominant in liver and ovary, respectively. Northern hybridization analysis also revealed that the coding region of the various sizes of mRNA seemed to be common. Southern blot analysis of genomic DNA by the selective use of the cDNA fragments as probes indicated that the various mRNA species were derived from a single gene, probably due to an alternative splicing mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
No preview · Article · Mar 1994 · Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.
[Show abstract][Hide abstract] ABSTRACT: To solve the problem of whether a common enzyme catalyzes both 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1 alpha-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol competitively inhibited 25-hydroxylation of 1 alpha-hydroxy-vitamin D3 and vice versa. From these results it was concluded that 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 1 alpha-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.
No preview · Article · Apr 1991 · Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The cDNA for vitamin D 25-hydroxylase in rat liver mitochondria was transfected in COS cells in order to confirm our previous postulation that both 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol 27-hydroxylation and vitamin D 25-hydroxylation are catalyzed by a common enzyme. As a result it was found that both enzyme activities could be reconstituted from the solubilized extract of mitochondria of these cells, NADPH, NADPH-adrenodoxin reductase and adrenodoxin, giving unequivocal evidence that the two enzyme activities are catalyzed by a common enzyme.
[Show abstract][Hide abstract] ABSTRACT: A cDNA clone encoding mitochondrial vitamin D3 25-hydroxylase was isolated from a rat liver cDNA library by the use of specific antibodies to the enzyme. The isolated cDNA clone was 1.9 kbp long and contained a 1599 bp open reading frame encoding 533 amino acid residues. The deduced primary structure contained a presequence typical for mitochondrial enzymes in the N-terminal region. The N-terminal sequence of the mature enzyme was determined to be Ala-Ile-Pro-Ala-Ala, which agrees perfectly with a portion of the deduced sequence, establishing the cleavage point of the precursor.
[Show abstract][Hide abstract] ABSTRACT: A reductase catalyzing the reduction of the 3-ketone group of 7α,12α-dihydroxy-5β-cholestan-3-one and 7α-hydroxy-5β-cholestan-3-one, which are the intermediates in the conversion of cholesterol to cholic acid and chenodeoxycholic acid, respectively, into the 3α-hydroxyl group, was purified about 250-fold as judged by the activity from the 100 000 × g supernatant of rat liver homogenate. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoretography was 32000. The absorption spectrum of the purified enzyme showed only a peak at 280 nm due to aromatic amino acids, precluding the presence of a chromophoric prosthetic group in the molecule. The enzyme showed activity toward a variety of substrates, including 3-oxo-5β-cholanoic acid, androsterone, 9,10-phenanthrenquinone, p-nitrobenzaldehyde, but not toward glucuronic acid, dl-glyceraldehyde, and glycolaldehyde. The optimal pH for the reduction of 7α-hydroxy-5β-cholestan-3-one was 7.4, and the cofactor required was either NADPH or NADH, though the former gave the higher activity. Judging from the chromatography behavior as well as substrate specificity, the enzyme was identified as 3α-hydroxysteroid dehydrogenase (3α-hydroxysteroid: NAD(P)+ oxidoreductase, EC 188.8.131.52).
No preview · Article · Jun 1986 · Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism