Mitsuhide Noshiro

Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase

A cDNA encoding 25‐hydroxyvitamin D3 24‐hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA wax 3.2 kbp long and contained a 1542‐bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical or mitochondrial enzymes in the N‐terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family or P450, COS‐7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH‐dependent 24‐hydroxylation of 25‐hydroxy‐vitamin D3 in the presence or adrenodoxin and NADPH‐adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24‐hydroxylation activity caused by administration or vitamin D3 into rats was accompanied by an increase of the mRNA.

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen

A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

Differential Effects of Various Growth Factors and Cytokines on the Syntheses of DNA, Type I Collagen, Laminin, Fibronectin, Osteonectin/Secreted Protein, Acidic and Rich in Cysteine (SPARC), and Alkaline Phosphatase by Human Pulp Cells in Culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor‐β (TGF‐β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3‐ to 10‐fold. Western and Northern blots showed that TGF‐β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet‐derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [3H]thymidine incorporation into DNA. TGF‐β, EGF, and TNF‐α had less effect on DNA synthesis, whereas IL‐1β inhibited DNA synthesis. These findings demonstrated that TGF‐β, bFGF, EGF, PDGF, TNF‐α, and IL‐1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998.

Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria

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.

Basic Helix-loop-helix Protein DEC1 Promotes Chondrocyte Differentiation at the Early and Terminal Stages

The mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-β or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-β and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation.

DEC1 Modulates the Circadian Phase of Clock Gene Expression

ABSTRACT DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E′ boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E′-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E′ box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1−/− mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.

RGD-CAP (βig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin α1β1

Abstract In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human βig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-β. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or β-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg 2+ or Mn 2+ ), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin α 1 or β 1 subunit, but not to the α 2 , α 3 , α 5 or β 2 subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the α 5 subunit, but not the mAb to the α 1 subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin α 1 β 1 that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.

Molecular cloning of cDNA for cholesterol 7α-hydroxylase from rat liver microsomes: Nucleotide sequence and expression

A complete cDNA clone encoding cholesterol 7 alpha-hydroxylase was isolated from a rat liver cDNA library by the use of specific antibodies to the enzyme. The isolated cDNA clone was 3.6 kbp long and contained a 1509-bp open reading frame encoding 503 amino acid residues (Mr = 56,880). The identity of the cDNA was confirmed by expression of cholesterol 7 alpha-hydroxylase activity and the immunoreactive protein in COS cells transfected with pSVL expression vector carrying the cDNA insert. The primary structure of cholesterol 7 alpha-hydroxylase deduced from the nucleotide sequence of the cDNA indicated that the enzyme constitutes a novel P-450 family.Cholesterol 7α‐hydroxylase; Cytochrome P‐450; cDNA cloning; (COS cell)

Multiple Mechanisms Regulate Circadian Expression of the Gene for Cholesterol 7α-Hydroxylase (Cyp7a), a Key Enzyme in Hepatic Bile Acid Biosynthesis:

Cholesterol 7α-hydroxylase (CYP7A) and sterol 12α-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, Hnf4α, Pparα, Lxrα, Rev-erbα, and Rev-erbβ—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-erbα, and Rev-erb β—potent regulators for Cyp7a expression—were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a—Lxrα, Hnf4α, Pparα, and E4bp4—had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBα/β, DBP, LXRα, and HNF4α increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARα were moderate negative regulators. Furthermore, knockdown of REV-ERBα/β with siRNA suppressed Cyp7a transcript levels, and in the electrophoretic mobility shift assay, REV-ERBα/β 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-ERBα/β—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-ERBα/β, LXRα, HNF4α DEC2, E4BP4, and PPARα.

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.