It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (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 1alpha,25(OH)(2)D(3) 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 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) 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 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.

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.

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 D 3 -1␣-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 thy- 1-3). Furthermore, renal CYP27B1 activity was strongly inhibited by the administration of 1␣,25(OH) 2 D 3 . Thus, it is suggested that the renal CYP27B1 activity is regulated mainly by a balance of the serum levels of 1␣,25(OH) 2 D 3 and PTH.Another calcium-regulating hormone, calcitonin (CT), was also shown to enhance renal conversion of 25(OH)D 3 into 1␣,25(OH) 2 D 3 in vitamin D-deficient rats (5-7). Lorenc et al. (6), however, showed that CT did not exhibit any effect on vitamin D metabolism, when animals were thyroparathyroidectomized (TPTX). From these results, they concluded that the stimulatory effect of CT was mediated by the endogenous secretion of PTH. The effect of CT on the 1␣,25(OH) 2 D 3 synthesis and its interaction with PTH are of considerable interest, since the stimulatory effect of PTH on 1␣,25(OH) 2 D 3 synthesis appears to be mediated by cAMP (4), and CT also stimulates cAMP production in the kidney, irrespective of the counteracting effect of CT on PTH in maintaining plasma calcium homeostasis (8).It is believed that 1␣,25(OH) 2 D 3 is responsible for inducing most of the biological functions of vitamin D 3 (1-3). Theoretically, derangements in the tissue and plasma levels of 1␣,25(OH) 2 D 3 could result from alterations in the rate of its synthesis or degradation or both. Recently, four laboratories independently succeeded in the molecular cloning of the cDNA for CYP27B1 (9-13). The cloning of the CYP27B1 cDNA has enabled us to examine the expression of mRNA of this enzyme. Using this probe, we found that the expression of CYP27B1 mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced production of 1␣,25(OH) 2 D 3 , expression of CYP27B1 mRNA was also greatly stimulated in renal proximal convoluted tubules (9). Brenza et al. (14) and Murayama et al. (15) also reported that a PTH-dependent positive regulatory element for the CYP27B1 gene expression is located in the promoter region of the gene.Regulation of vitamin D metabolism has long been examined by using hypocalcemic or hypophosphatemic vitamin D-deficient animals. It was difficult to examine the regulation of CYP27B1 activity in normocalcemic physiological conditions. 1␣,25(OH) 2 D 3 has been characterized as a negative regulator for the activity of renal...

SaOS