Mitogen-activated protein kinases mediate interleukin-1beta-induced receptor activator of nuclear factor-kappaB ligand expression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Interleukin-1beta-stimulated receptor activator of nuclear factor-kappaB ligand (RANKL) expression in human periodontal ligament cells is partially mediated by endogenous prostaglandin E2, whereas mitogen-activated protein kinases (MAPKs) are implicated in regulating various interleukin-1-responsive genes. We investigated herein the involvement of MAPKs in interleukin-1beta-stimulated RANKL expression in human periodontal ligament cells. MATERIAL AND METHODS: Human periodontal ligament cells were pretreated separately with specific inhibitors of MAPKs, including extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and subsequently treated with interleukin-1beta. Following each treatment, the phosphorylation of each MAPK, the expression of RANKL, and the production of prostaglandin E2 were determined. RANKL activity was evaluated using an assay to determine the survival of prefusion osteoclasts. RESULTS: Interleukin-1beta induced RANKL expression at the mRNA and protein levels, as well as RANKL activity in human periodontal ligament cells. Interleukin-1beta also activated extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Pretreatment with each MAPK inhibitor partially, but significantly, suppressed interleukin-1beta-induced RANKL expression and its activity, as well as prostaglandin E2 production. CONCLUSION: In human periodontal ligament cells, three types of MAPK inhibitor may abrogate RANKL expression and activity induced by interleukin-1beta, directly or indirectly through partial suppression of prostaglandin E2 synthesis. In addition, extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase signals may co-operatively mediate interleukin-1beta-stimulated RANKL expression and its activity in those cells.

[The mechanism which calcitonin activates vitamin D].

Calcitropic hormones, such as calcitonin and vitamin D, are one of the most common drugs used in the treatment of osteoporosis. Recently, three enzymes involved in vitamin D metabolism (25-hydroxylase, 24-hydroxylase, 1alpha-hydroxylase) were cloned and enabled us to analyze the vitamin D metabolism at a molecular level. We summarize the mechanism which CT activates vitamin D.

[The regulation of vitamin D metabolism and PTH secretion by phosphorus].

Vitamin D and PTH are major hormones that regulate calcium metabolism. Also, the serum level of phosphorus is known to change according to the level of calcium and to regulate the vitamin D and PTH. Recently, cloning of 1alpha- hydroxylase cDNA which is the key enzyme in vitamin D metabolism, enabled us to examine the effects of phosphorus in vitamin D metabolism at a molecular level. Furthermore, the direct effect of phosphorus in PTH synthesis is being elucidated in recent reports. In this paper, we summarized the regulation of vitamin D and PTH by phosphorus.

Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP receptors.

SaOS-4/3, a subclone of the human osteosarcoma cell line SaOS-2, established by transfecting the human parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS-4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony-stimulating factor (M-CSF). Expression of messenger RNAs (mRNAs) for receptor activator of NF-kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane-associated and secreted forms of M-CSF by SaOS-4/3 cells was up-regulated in response to PTH. SaOS-4/3 cells constitutively expressed OPG mRNA, expression of which was down-regulated by PTH. To elucidate the mechanism of PTH-induced osteoclastogenesis, SaOS-4/3 cells were spot-cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M-CSF, osteoclasts were induced exclusively inside the colony of SaOS-4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS-4/3 cells in coculture treated with a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M-CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed only inside the colony of SaOS-4/3 cells. Adding M-CSF alone failed to support osteoclast formation in the spot coculture. PTH-induced osteoclast formation occurring inside the colony of SaOS-4/3 cells was not affected by the concentration of M-CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS-4/3 cells. These findings suggest that the up-regulation of RANKL/ODF expression is an essential step for PTH-induced osteoclastogenesis, and membrane- or matrix-associated forms of both M-CSF and RANKL/ ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.

Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function.

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.

Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D3-1alpha-hydroxylase gene in normocalcemic rats.

Regulation of vitamin D metabolism has long been examined by using vitamin D-deficient hypocalcemic animals. We previously reported that, in a rat model of chronic hyperparathyroidism, expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (CYP27B1) mRNA was markedly increased in renal proximal convoluted tubules. It is believed that the major regulator for the expression of renal CYP27B1 is parathyroid hormone (PTH). However, in the normocalcemic state, the mechanism to regulate the renal CYP27B1 gene could be different, since plasma levels of PTH are very low. In the present study, the effect of PTH and calcitonin (CT) on the expression of renal CYP27B1 mRNA was investigated in normocalcemic sham-operated rats and normocalcemic thyroparathyroidectomized (TPTX) rats generated by either PTH or CaCl2 infusion. A single injection of CT dose-dependently decreased the expression of vitamin D receptor mRNA in the kidney of normocalcemic sham-TPTX rats. Concomitantly, CT greatly increased the expression of CYP27B1 mRNA in the kidney of normocalcemic sham-TPTX rats. CT also increased the expression of CYP27B1 mRNA in the kidney of normocalcemic TPTX rats. Conversion of serum [3H]1alpha,25(OH)2D3 from 25-hydroxy[3H]vitamin D3 in vivo was also greatly increased by the injection of CT into sham-TPTX rats and normocalcemic TPTX rats, but not into hypocalcemic TPTX rats. In contrast, administration of PTH did not induce the expression of CYP27B1 mRNA in the kidney of vitamin D-replete sham-TPTX rats and hypocalcemic TPTX rats. PTH increased the expression of renal CYP27B1 mRNA only in vitamin D-deficient hypocalcemic TPTX rats. These results suggest that CT plays an important role in the maintenance of serum 1alpha,25(OH)2D3 under normocalcemic physiological conditions, at least in rats.

Three-dimensional structure-function relationship of vitamin D: side chain location and various activities.

The various biological activities of side-chain mobility restricted analogs, four diastereomers at C(20) and C(22) of 22-methyl-1alpha,25-dihydroxyvitamin D3, were evaluated. The relationship between structure and the various activities of the analogs was discussed in terms of the active space region concept that we previously suggested.

Potential role of cbfa1, an essential transcriptional factor for osteoblast differentiation, in osteoclastogenesis: regulation of mRNA expression of osteoclast differentiation factor (ODF).

The role of Cbfa1 (core binding factor alpha1), an essential transcriptional factor for osteoblast differentiation, in osteoclastogenesis was investigated in vitro and in vivo using Cbfa1-deficient calvarial cells and mice. Co-cultures of calvarial cells isolated from embryos with three different Cbfa1 genotypes (Cbfa1+/+, Cbfa1+/- and Cbfa1-/-) and normal spleen cells generated TRAP-positive multinucleated osteoclast-like cells (OCLs) in response to 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and dexamethasone, but the number and bone-resorbing activity of OCLs formed in co-culture with Cbfa1-/- calvarial cells were significantly decreased in comparison with those formed in co-cultures with Cbfa1+/+ or Cbfa1+/- calvarial cells. The expression of osteoclast differentiation factor/osteoprotegerin ligand (ODF/OPGL) mRNA was increased by the treatment with 1alpha, 25(OH)2D3 and dexamethasone in calvarial cells from Cbfa1+/+ and Cbfa1+/- mouse embryos, but not from Cbfa1-/- embryos. In contrast, the expression of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) mRNA was inhibited by 1alpha,25(OH)2D3 and dexamethasone similarly in all three types of calvarial cells. ODF/OPGL and OPG/OCIF mRNAs were highly expressed in the tibia and femur of Cbfa1+/+ and Cbfa1+/- embryos. In the tibia and femur of Cbfa1-/- embryos, however, ODF/OPGL mRNA was undetectable and the expression of OPG/OCIF mRNA was also decreased compared with those in Cbfa1+/+ and Cbfa1+/- embryos. These results suggested that Cbfa1 is somehow involved in osteoclastogenesis through regulation of ODF/OPGL.

Two novel 1alpha-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type I1.

BACKGROUND: Vitamin D dependency rickets type I (VDDR-I) is an autosomal recessive disorder in which 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha-hydroxylase) activity in renal proximal tubules is deficient. VDDR-I is recognized throughout the world, but occurs more frequently in a subset of the French-Canadian population. We and others have recently cloned the human 1alpha-hydroxylase cDNA and gene, making it possible to screen for mutations. The first VDDR-I mutations were reported in one American and four Japanese patients. In this study, we screened for 1alpha-hydroxylase mutations in French-Canadian patients with VDDR-I. METHODS: The nine exons of the 1alpha-hydroxylase gene were amplified by polymerase chain reaction (PCR) from genomic DNA of four unrelated French-Canadian patients with VDDR-I and their parents, and sequenced. RESULTS: Three of the patients were homozygous for a single base-pair deletion (G) at position 262 in the cDNA that lies in exon 2, and causes a premature termination codon upstream from the putative ferredoxin- and heme-binding domains. The fourth patient was homozygous for a 7-bp insertion (CCCCCCA) at position 1323 of the cDNA that lies in exon 8, and causes a premature termination upstream from the putative heme-binding domain. In each family, obligate carriers have one copy of the mutant allele. These mutations, which could be detected by PCR-restriction fragment length polymorphism and polyacrylamide gel electrophoresis of the PCR products, were not found in 25 normal French-Canadians. CONCLUSION: We describe two novel 1alpha-hydroxylase mutations that are consistent with loss of function in four French-Canadian patients with VDDR-I and suggest that the 1alpha-hydroxylase mutations arise from more than one founder in this population.

Mg2+ binding and catalytic function of sphingomyelinase from Bacillus cereus.

The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/Km for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of kcat for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

[Cloning and expression of 25-hydroxyvitamin D3-1 alpha-hydroxylase gene].

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1 alpha-hyroxylase, was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When 1 alpha-hydroxylase cDNA was transfected into COS-7 cells, they expressed 25-hydroxyvitamin D3-1 alpha-hydroxylase activity. The sequence analysis showed that 1 alpha-hydroxylase cDNA consisted of 2469 bp in length and contained an open reading frame encoding 501 amino acids. The expression of 1 alpha-hydroxylase mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3 (rats fed a low Ca diet), expression of 1 alpha-hydroxylase mRNA was greatly enhanced in the renal proximal convoluted tubules. These results clearly demonstrate that the expression of 1 alpha-hydroxylase is regulated at a transcriptional level. The DNA flanking the 5'-sequence of the mouse 1 alpha-hydroxylase gene has been cloned and sequenced. The promoter has 3 potential CRE sites, 2 perfect and 1 imperfect AP-1 sites, while no DR-3 was detected. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, while forskolin stimulates it by 3-fold. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1 alpha-hydroxylase by acting on the promoter of the 1 alpha-hydroxylase gene.

Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alpha-hydroxylase gene promoter.

The DNA flanking the 5' sequence of the mouse 1alpha-hydroxylase gene has been cloned and sequenced. A TATA box has been located at -30 bp and aCCAAT box has been located at -79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1alpha-hydroxylase by acting on the promoter of the 1alpha-hydroxylase gene.

Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.

Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 alpha-hydroxylase.

The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase. The cDNA clone was obtained from a human kidney cDNA library by cross-hybridization with a previously cloned rat cDNA probe. The cDNA consists of 2469 bp and encodes a protein of 508 amino acids that shows 82.5% sequence identity with the rat enzyme. A computer-aided homology search revealed that 1 alpha-hydroxylase shares a relatively high homology with vitamin D3 25-hydroxylase (about 40% amino acid identity). Northern blot analysis showed that the 2.5-kb mRNA is most abundant in kidney. The gene for human 1 alpha-hydroxylase spans approximately 6 kb, is composed of nine exons, and is present as a single copy. This molecular cloning makes it possible to investigate the genetic mechanism of diseases related to calcium metabolism, including vitamin D-dependency rickets type I.

1alpha,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) hydroxylates the carbon at the end of the side chain (C-26) of the C-24-fluorinated analog of 1alpha,25-dihydroxyvitamin D3.

The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.

Functional assessment of two vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase gene.

Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5'-upstream region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene at -151/-137 and -259/-245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter -291/+9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat -169/-155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element -169/-155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous beta-globin promoter. The fragment -291/-102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element -169/-155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.

Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1 alpha-hydroxylase.

Conversion of 25-hydroxyvitamin D3 (25(OH)D3) to the active vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) is catalyzed by 25(OH)D3, 1 alpha-hydroxylase(1 alpha-hydroxylase). It has been suggested that this enzyme is cytochrome P450 (P450). We purified 1 alpha-hydroxylase 430-fold from cholate-solubilized kidney mitochondria of vitamin D-deficient chickens by utilizing hydrophobic and ion-exchange column chromatographies. Enzymatic activity was assessed by measuring on HPLC the formation of 1 alpha,25(OH)2D3 from 25(OH)D3 in the assay mixture containing NADPH, adrenodoxin reductase, adrenodoxin as a reducing system. The purified enzyme showed a CO-difference spectrum characteristic of P450. The molecular activity of this preparation was calculated to be 8.7 pmol/min/pmol P450. This value was higher by more than 87-fold than those reported so far. The present preparation was found to contain several proteins on SDS-PAGE. Among them, only the 54-kD protein became undetectable when kidney mitochondria from normal and vitamin D-replete chickens, where 1 alpha-hydroxylase activities were 15 and 0% of that found in vitamin D-deficient chicken, respectively, were used as the starting enzyme sources. Furthermore, the band intensity of the 54-kD protein accounted for the spectrophotometrically determined amount of P450 in the preparation. These results suggest that the 54-kD protein is 1 alpha-hydroxylase.

Regulation of vitamin D-responsive gene expression by fluorinated analogs of calcitriol in rat osteoblastic ROB-C26 cells.

We compared the activation of vitamin D-responsive genes by 24,24-difluorocalcitriol [F2-1 alpha,25(OH)2D3] and 26,26,26,27,27,27-hexafluorocalcitriol [F6-1 alpha,25(OH)2D3] with that by calcitriol [1 alpha,25(OH)2D3] in rat osteoblastic ROB-C26 cells. F2-1 alpha,25(OH)2D3 and F6-1 alpha, 25(OH)2D3 were ten times more potent than 1 alpha,25(OH)2D3 in inducing the expression of 1 alpha, 25(OH)2D3-24-hydroxylase (24-OHase) mRNA 6 h after adding vitamin D compounds. The lower affinity of these two fluorinated analogs compared with that of 1 alpha,25(OH)2D3 for vitamin D binding protein in serum (serum DBP) seemed to be partly involved in their increased ability to activate the 24-OHase gene. A time course study revealed that the expression of the 24-OHase and osteopontin mRNAs in the cells incubated with 1 alpha, 25(OH)2D3 and F2-1 alpha,25(OH)2D3 attained maximal levels at 6 h for 24-OHase mRNA and 18 h for osteopontin mRNA, the both decreased thereafter. On the contrary, F6-1 alpha,25(OH)2D3 increased the expression of 24-OHase and osteopontin exponentially until 72 h. While F2-1 alpha,25(OH)2[1 beta-3H]D3 was catabolized quickly by ROB-C26 cells, F6-1 alpha,25(OH)2[1 beta-3H]D3 was slowly and quantitatively converted into putative 26,26,26,27,27,27-hexafluoro-23S-hydroxy[1 beta-3H]calcitriol (F6-1 alpha,23S,25(OH)3[1 beta-3H]D3). This may explain why the time-course profiles of the accumulation of mRNAs for 24-OHase and osteopontin differed in the cells exposed to the fluorinated analogs. In addition to the longer retention, unknown up-regulating mechanisms appeared to be involved in the exponential activation of the 24-OHase and osteopontin genes induced by F6-1 alpha,25(OH)2D3.

A possible role of vitamin D receptors in regulating vitamin D activation in the kidney.

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases. Previously, we reported that renal proximal convoluted tubules, the major site of 1 alpha, 25-dihydroxyvitamin D3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1 alpha, 25-dihydroxyvitamin D3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities. In rat models of enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3, expression of vitamin D receptors and 25-hydroxyvitamin D3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts. In vitamin D-deficient rats with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity, expression of vitamin D receptor mRNA in renal proximal convoluted tubules was also down-regulated, indicating that the down-regulation of vitamin D receptor mRNA is not the result of the enhanced production of 1 alpha, 25-dihydroxyvitamin D3. In Japanese quail models with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by sex steroids, expression of vitamin D receptor mRNA was also down-regulated in the kidney but not in the duodenum. These results suggest that the down-regulation of vitamin D receptors plays a critical role in production of 1 alpha, 25-dihydroxyvitamin D3 in renal proximal convoluted tubules.

Microgravity generated by space flight has little effect on the growth and development of chick embryonic bone.

Seven days' space flight of fertilized chicken eggs pre- incubated for 7 and 10 days on earth caused no differences in the morphology of osteoblasts, osteoclasts, and osteocytes of humerus and tibia from those of control embryos. Bone-resorbing and -forming activities of the femur were not different between control and flight groups. As a consequence, calcium and phosphorus contents of the femora between control and flight groups were not changed. Alkaline phosphatase activity of 3 different regions (resting cartilage, growth cartilage, and cortical bone) of tibia showed no significant difference between control and flight groups. No significant difference of gene expressions of hepatocyte growth factor and receptors of fibroblast growth factor was observed in perichondrium, trabecula, and skeletal muscles and tendons of hind limbs between control and flight groups. Unlike the results of previous space flight experiments in which young growing mammals were used, these morphological and biochemical results indicate that microgravity has little effect on bone metabolism of the chick embryo.