Toshimasa Shinki

Vitamin D and bone

It is now well established that supraphysiological doses of 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF‐κB ligand (RANKL) in response to several bone‐resorbing factors including 1α,25(OH)2D3 to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF‐κB (RANK) recognize RANKL through cell‐to‐cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage‐colony stimulating factor (M‐CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1α,25(OH)2D3 for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH‐induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1α,25(OH)2D3 on the PTH‐induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1α,25(OH)2D3 increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone‐resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1α,25(OH)2D3 rather suppress the PTH‐induced bone resorption in vivo, supporting the concept that 1α,25(OH)2D3 or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism. J. Cell. Biochem. 88: 259–266, 2003.

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‐κB 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.

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 β-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.

Mouse primary osteoblasts express vitamin D3 25-hydroxylase mRNA and convert 1α-hydroxyvitamin D3 into 1α,25-dihydroxyvitamin D3

We examined whether 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) is metabolized into 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in bone. Northern blot analysis indicated that the expression of vitamin D3 25-hydroxylase mRNA was highest in the liver, followed by the duodenum, calvaria, lung, kidney, skin and long bone, and lowest in the spleen. Of the bone cell fractions isolated from fetal mouse calvaria by a sequential enzymatic digestion, fraction 3, which consisted of mostly osteoblastic cells, showed the highest expression of vitamin D3 25-hydroxylase mRNA. When either cultured bone cells of fraction 3 or mouse calvaria were incubated with [3H]-1 alpha (OH)D3, a radioactive peak which comigrated at the same position as authentic 1 alpha,25(OH)2D3 was found on an HPLC chromatogram. The radioactive fraction obtained from the conditioned media of fetal mouse calvaria was tentatively identified as 1 alpha,25(OH)2D3 by cochromatography with authentic 1 alpha,25(OH)2D3 on three different HPLC systems and a thermal isomerization analysis. These results indicate that 1 alpha(OH)D3 is hydroxylated at the 25-position in bones, resulting in the local synthesis of 1 alpha,25(OH)2D3 from 1 alpha(OH)D3 in the skeletal tissues.

1α,25-Dihydroxyvitamin D3 receptors and their action in embryonic chick chondrocytes

SummaryThe role of vitamin D in the maturation of epiphyseal chondrocytes was investigated in the developing chick embryo. Cartilage tissues were divided into two parts: resting cartilage and growth cartilage. A cytosol component to which 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) is specifically bound first appeared in the growth cartilage on day 15, rapidly increased, and attained a maximum on day 19. The calcium content of the growth cartilage also began to increase on day 15 and continued to increase in parallel with the 1α,25(OH)2D3 receptor levels. Glycosaminoglycan (GAG) synthesis by the growth cartilage cells increased from day 11–17 and rapidly declined thereafter reciprocally with the increase in calcium and receptor levels. In the resting cartilage, no cytosol receptor for 1α,25(OH)2D3 was detected up to hatching time. The calcium content and GAG synthesis in the resting cartilage were very low and did not change appreciably throughout development. No receptor-like macromolecule for 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) was recognized in either the resting or growth cartilage. 1α,25(OH)2D3 added to the culture of chondrocytes from the epiphyseal growth cartilage inhibited GAG synthesis and stimulated its release from the cell layer into the medium in a dose-dependent manner. Thesein vitro effects of 1α,25(OH)2D3 were not observed in chondrocytes obtained from 13-day-old growth cartilage and 19-day-old resting cartilage. 25-Hydroxyvitamin D3 and 24R,25(OH)2D3 had no effect on chondrocytes in any of the preparations. These results suggest that 1α,25(OH)2D3 is directly involved in the maturation of chondrocytes and possibly in the calcification of growth cartilage.

Extremely high circulating levels of 1α,25-dihydroxyvitamin D3 in the marmoset, a new world monkey

Compared to most mammals, the marmoset, a new world monkey, requires particularly large amounts of vitamin D to maintain normal growth. We compared serum concentrations of vitamin D metabolites in marmosets with rhesus monkeys and humans. The circulating levels of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in marmosets were 4 to 10 times higher than those in rhesus monkeys and humans. But none of the marmosets exhibited hypercalcemia. In two marmosets which had suffered bone fractures, the 1 alpha,25(OH)2D3 levels were particularly elevated. These results suggest that the marmoset has an end-organ resistance to 1 alpha,25(OH)2D3.

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene.

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.

Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases

vitamin D is 25-hydroxylated in the liver, before being activated by 1alpha-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min(-1)), vitamin D2 (0.16 min(-1)), and 1alpha-hydroxyvitamin D3 (2.2 min(-1)). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min(-1)) more efficiently than vitamin D2 (0.86 min(-1)). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2.

In vivo administration of 1,25‐dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose‐dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)2D3 was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 μg/kg of 1,25(OH)2D3 inhibited the PTH‐induced RANKL mRNA expression, but 0.5 μg/kg of the vitamin did not inhibit it. Regulator of G protein signaling‐2 (RGS‐2) gene expression was suppressed by 1,25(OH)2D3 dose‐dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)2D3 suppressed PTH‐induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)2D3 stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH‐induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor‐mediated signaling. J. Cell. Biochem. 90: 267–277, 2003.

Effects of Geranylgeranoic Acid in Bone: Induction of Osteoblast Differentiation and Inhibition of Osteoclast Formation

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3‐E1 cells, but also up‐regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1α,25(OH)2D3‐ or prostaglandin E2 (PGE2)‐induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony‐stimulating factor (M‐CSF) and soluble receptor activator of nuclear factor κB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence‐accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.