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.

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.

A cDNA clone encoding mitochondrial vitamin Ds 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 prescquence 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.Vitamin Ds 25-hydroxylase; Cytochrome P-450; cDNA cloning; 5B-Cholestane-3a, 7a, 12~triol27-hydroxylase

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, LXRα and LXRβ with T0901317—agonist for LXR—were found to be potent enhancers for Dec1 promoter activity, and a higher expression level of LXRα 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 LXRα bound to the Dec1 promoter, and an LXRα‐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.

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.Keywords : chondrocyte ; concanavalin A ; melanotransferrin; membrane-bound transferrin-like protein; p97.Concanavalin A, unlike other plant lectins, stimulates the synthesis of cartilage-matrix proteoglycan (aggrecan) by resting, articular and growth-plate chondrocytes in culture [1]. It also induces the syntheses of alkaline phosphatase and type-X collagen in resting chondrocyte cultures [2]. Concanavalin A induces the synthesis of cartilage in amphibian early gastrula ectoderm [3]. These findings suggest that some concanavalin-A-binding glycoproteins on the cell surface are involved in the control of chondrocyte differentiation.To characterize concanavalin-A-binding membrane proteins in chondrocytes, we labelled surface proteins of chondrocytes with 125 I and tested their concanavalin-A-binding ability. Among them, a 76-kDa protein was a major 125 I-labelled protein and bound to concanavalin-AϪSepharose. Previous studies have shown that a protein of approx. 80 kDa is expressed at very high levels in the outer surface of chick embryo chondrocytes, although it is undetectable in limb mesenchymal cells and dedifferentiated chondrocytes [4]. In the present study, we attempted to purify the 76-kDa concanavalin-A-binding protein and isolateCorrespondence to Y. Kato,