[How is bone formed and resorbed?-- molecular mechanisms of bone formation and resorption].

Bone has developed as a storage of calcium as well as a supporting tissue in vertebrates. Bone is a complex tissue in which resorption and formation take place throughout life. This process is called bone remodeling. Osteotrophic hormones such as 1 alpha,25-dihydroxyvitamin D3[1 alpha,25 (OH) 2D3], parathyroid hormone (PTH) and calcitonin maintain serum calcium homeostasis within a narrow range of 9 to 10 mg/dl by regulating intestinal absorption of calcium and bone remodeling. Bone tissue contains various types of cells, of which bone-forming osteoblasts and bone-resorbing osteoclasts are mainly responsible for bone remodeling. Osteoblasts arise from common progenitors with chondrocytes, myotubes and adipocytes. Recently, four research groups independently identified core-binding protein alpha-1(Cbfa-1) as a key transcription factor for osteoblast differentiation and bone formation, since Cbaf-1 knockout mice completely lacked bone formation due to maturation arrest of osteoblasts. In contrast, multinucleated osteoclasts are primarily responsible for bone resorption. The recent discovery of new members of tumor necroses factor (TNF) receptor-ligand family has indicated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast formation. Osteoblasts/stromal cells express a new member of the TNF ligand family "osteoclast differentiation factor (ODF)" as a membrane-associated factor. Osteoclast progenitors which express ODF receptor(RANK) recognize ODF through cell-to-cell interaction with osteoblasts/stromal cells, then differentiate into osteoclats. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble decoy receptor for ODF. Thus, ODF, RANK and OPG/OCIF are the three key molecules for osteoclast formation. The discovery of Cbfa-1 and ODF may establish a new way to treat several metabolic bone diseases caused by abnormal bone formation and resorption.

Divergent regulation of 1,25-dihydroxyvitamin D3 on human bone marrow osteoclastogenesis and myelopoiesis.

The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has influence over osteoclastogenesis and myelopoiesis, but the regulational mechanism is not well-defined. In this report, formation of osteoclast-like (OCL) cells from primitive myeloid colony-forming cells (PM-CFC) as mediated by 1,25(OH)2D3 was examined. Our results present in this report clearly show that 1,25(OH)2D3 dose-dependently stimulated OCL cell formation when added to suspension cultures of individually replated PM-CFC colonies. Marrow cells were plated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or the human bladder carcinoma cell line 5637 conditioned medium (5637 CM) as the source of colony-stimulating activity. The 1,25(OH)2D3 effect of osteoclast differentiation was associated with a concomitant decrease in clonogenic growth of myelopoietic progenitors in response to colony-stimulating activity. Secondly, the effect of adding the known stimulator of hematopoiesis, interleukin-1beta (IL-1beta) and/or 1,25(OH)2D3 on human myeloid colony growth was assessed. IL-1beta enhanced the formation of primitive myeloid colonies in response to GM-CSF by 160%. On the other hand, 1,25(OH)2D3 dose-dependently inhibited both GM-CSF- and 5637 CM-driven myeloid colony formation by as much as 90% at 100 nM. Addition of IL-1beta to GM-CSF-stimulated cultures dampened the inhibitory effect of 1,25(OH)2D3. The inhibition of myeloid clonogenic growth by 1,25(OH)2D3 was almost abolished (89%) by simultaneously adding anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha MoAb) to the culture medium. These results collectively suggest divergent roles for 1,25(OH)2D3 in osteoclastogenesis and myelopoiesis, promoting the differentiation of OCL cells from primitive myeloid cells but inhibiting the proliferation of later myeloid progenitor cells. This inhibition of myeloid progenitors may be mediated by TNF-alpha.

Tacalcitol.

Tacalcitol is a vitamin D3 analogue which is available in Japan as a 2 micrograms/g ointment for twice daily application and in Western markets as a 4 micrograms/g ointment for once daily application. Tacalcitol inhibits proliferation, and induces the differentiation, of keratinocytes. In addition, it appears to modulate inflammatory and immunological mediators in the skin which may be involved in the aetiology of psoriasis. No significant systemic drug absorption occurs after application of tacalcitol to the skin. Results of clinical trials indicate that topical tacalcitol is effective in the management of stable plaque psoriasis (and possibly pustular forms of the disease), and has a similar efficacy to topical betamethasone valerate in this setting. Application of tacalcitol ointment 4 micrograms/g once daily for up to 8 weeks did not cause hypercalcaemia or hypercalciuria. Mild local skin irritation has been reported in a variable proportion of patients (< or = 12%).

Use of ketoconazole to probe the pathogenetic importance of 1,25-dihydroxyvitamin D in absorptive hypercalciuria.

Ketoconazole was used to probe the pathogenetic importance of the serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentration in 19 patients with well characterized absorptive hypercalciuria (AH). Patients were studied while receiving a constant metabolic diet before and after 2 weeks of ketoconazole administration (600 mg daily). Twelve of the patients were classified as ketoconazole responders, because in conjunction with a reduction of serum 1,25-(OH)2D from 113 +/- 36 to 70 +/- 26 pmol/L, intestinal 47Ca absorption decreased from 76.3 +/- 8.1% to 61.9 +/- 7.7%, and 24-h urinary Ca excretion declined from 7.6 +/- 1.4 to 5.7 +/- 1.1 mmol (P < 0.001 each). In these patients, intestinal 47Ca absorption was directly correlated with serum 1,25-(OH)2D levels and 24-h Ca excretion. In another group of 7 patients, termed ketoconazole nonresponders, despite reduction of 1,25-(OH)2D from 122 +/- 36 to 84 +/- 17 pmol/L (P = 0.015), there was no significant change in intestinal Ca absorption (76.0 +/- 8.2% to 72.1 +/- 10.6%) or 24-h urinary Ca excretion (7.3 +/- 1.3 to 7.2 +/- 1.0 mmol). In these patients, neither intestinal Ca absorption nor urinary Ca excretion was correlated with serum 1,25-(OH)2D levels. It, thus, appears that AH is a heterogeneous disorder comprised of both vitamin D-dependent and vitamin D-independent subsets. Although useful to probe the pathogenesis of AH, chronic treatment with ketoconazole is not recommended because of its generalized effects in inhibiting steroid synthesis.

Osteocalcin and its message: relationship to bone histology in magnesium-deprived rats.

These studies examine effects of brief Mg deprivation on bone histomorphometry and on secretion and synthesis of the specific osteoblast product, osteocalcin. Osteocalcin mRNA was determined in calvaria-derived cells and was correlated to circulating concentrations of the protein after 8 days of Mg deprivation. Circulating osteocalcin was decreased in Mg-deprived rats when compared with pair-fed normal rats after 8 days of Mg deprivation, with no significant changes in calcium or 1,25-dihydroxyvitamin D [1,25(OH)2D]. Serial sampling demonstrated a difference in osteocalcin levels by 2 days of Mg deprivation, before any changes in circulating calcium or parathyroid hormone were present. Although circulating osteocalcin is decreased in Mg-deprived animals, levels can be stimulated with 1,25(OH)2D3. Osteocalcin mRNA is reduced after Mg deprivation, suggesting that low circulating levels are due, at least in part, to reduced osteocalcin synthesis. Mg-deprived rats had diminished bone volume and abnormal histological features consistent with disorganized and chaotic bone remodeling. These findings indicate that low-Mg intake during growth can alter the quality and quantity of bone and suggest that Mg deprivation may contribute to the development of osteoporosis.

Hydroxycholecalciferols modulate parathyroid hormone and calcitonin sensitive adenylyl cyclase in bone and kidney of rats. A possible physiological role for 24,25-dihydroxy vitamin D3.

In particulate fractions from rat bone cells, but not from kidney, 24,25-(OH)2 D3 inhibits in a dose dependent manner (1 nM and above) the parathyroid hormone (PTH)-activated adenylyl cyclase. In contrast, 24,25-(OH)2D3 enhances the calcitonin (CT) stimulated cyclase in bone, but attenuates the CT-induced cyclase response in kidney. In supranormal concentrations 1,25-(OH)2D3 is also able to reduce the PTH-stimulated adenylyl cyclase in bone. In comparison, neither vitamin D3 metabolite interferes with stimulation of adenylyl cyclase from pituitary cell membranes by thyroliberin (TRH) or vasoactive intestinal polypeptide (VIP). These findings may have important therapeutical consequences in preventing excessive PTH action and bone demineralization.

[Endocrine regulation of phosphorus and calcium metabolism during pregnancy in domestic ruminants]. / Régulation endocrinienne du métabolisme phosphocalcique au cours de la gestation chez les ruminants domestiques.

In pregnant domestic ruminants (cows, ewes, goats) foetal plasma calcium and inorganic phosphorus concentrations are higher than those measured in the dam. The foetus regulates its own calcaemia and phosphataemia. Changes in maternal plasma calcium levels have no significant effect on foetal calcaemia. Calcium and phosphorus are transported from the dam to the foetus according to a one-way process, the transport from the foetus to the dam being negligible. An important part of the calcium transferred to the foetus comes from the maternal skeleton. The true molecular mechanisms involved in placental transport of calcium are still unknown. This is an active transport, stimulated by vitamin D metabolites (of maternal, foetal or placental origin) and maternal prolactin. Maternal calcitonin protects the skeleton of the pregnant (and lactating) female ruminant against excessive demineralization, partly by modulating placental transport of calcium during periods of intense mineralization of foetal skeleton.

Identification of 1,24(R)-dihydroxyvitamin D3-like bone-resorbing lipid in a patient with cancer-associated hypercalcemia.

A lipid indistinguishable from 1,24(R)-dihydroxyvitamin D3 [1,24(R)-(OH)2D3] was found in serum and tumor extracts from a hypercalcemic patient with a small cell carcinoma of the lung. The lipid comigrated with authentic 1,24(R)-(OH)2D3 on high performance liquid chromatography using both straight and reverse phase columns and competed with tritiated 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3)] for binding to intestinal 1,25-(OH)2D3 receptor. Increasing doses of the lipid factor from tumor and authentic 1,24(R)-(OH)2D3 gave parallel responses in a bone resorption assay, as assessed by 45Ca release from prelabeled mouse calvaria. The lipid factor from the patient's serum and authentic 1,24(R)-(OH)2D3 had identical biological activities in the receptor binding and bone resorption assays. In addition, the mechanisms of action of this lipid factor and 1,24(R)-(OH)2D3 were indistinguishable. Bone resorption by both was inhibited by calcitonin, and neither the lipid factor nor authentic 1,24(R)-(OH)2D3 affected cAMP content in osteoblast-like bone cells derived from mouse calvaria. The estimated concentrations of the 1,24(R)-(OH)2D3-like lipid, expressed as 1,24(R)-(OH)2D3 were 11 ng/g tumor wet wt by the receptor binding assay and 9.2 ng/g tumor wet wt by the bone resorption assay. The mean serum concentration was 1.4 +/- 0.3 (+/- SD) ng/ml (n = 3) by the receptor binding assay. No activity was detected in either bioassay when extracts of nontumor tissues from this patient or tumor extracts and sera from one hypercalcemic and four normocalcemic cancer patients were tested. The mean serum 1,25-(OH)2D level was low (6.4 +/- 0.5 pg/ml; n = 2), and serum 1,24(R),25-(OH)3D in this patient was high (103 pg/ml) compared to normocalcemic cancer patients, in whom the mean serum 1,25-(OH)2D level was 27 +/- 12 pg/ml (n = 4) and the 1,24(R),25(OH)3D level was 28 +/- 1.3 pg/ml (n = 4). Thus, the 1,24(R)-(OH)2D3-like lipid may be a substrate for metabolic conversion to 1,24(R),25-(OH)3D in vivo. These results provide evidence for the presence of a novel metabolite of vitamin D3, 1,24(R)-(OH)2D3. Detection of this bone-resorbing lipid in both tumor and serum suggests, but does not prove, that the tumor secreted this bioactive lipid into the circulation and that the high level of circulating bone-resorbing lipid was related to the hypercalcemia in this patient.

Demonstration that the vitamin D metabolite 1,25(OH)2-vitamin D3 and not 24R,25(OH)2-vitamin D3 is essential for normal insulin secretion in the perfused rat pancreas.

It has previously been shown that vitamin D deficiency impairs arginine-induced insulin secretion from the isolated, perfused rat pancreas (Science 1980; 209:823-25). Since vitamin D is known to be metabolized to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) and 24R,25-dihydroxyvitamin D3 (24,25[OH]2D3), it is essential to clarify which vitamin D metabolite has the important role of enhancing insulin secretion. In this report, a comparison is made of the relative efficacy of 3-wk repletion with vitamin D3 (980 pmol/day), 1,25(OH)2D3 (39 pmol/day or 195 pmol/day), and 24,25(OH)2D3 (650 pmol/day) on arginine-induced insulin secretion from the isolated, perfused rat pancreas; in this experiment, the daily caloric intake of the animals receiving vitamin D or its metabolites was controlled by pair feeding to the caloric intake of the vitamin D-deficient rats. 1,25(OH)2D3 repletion was found to completely restore insulin secretion to the levels seen in vitamin D3-replete, pair-fed controls in both the first and second phases, while 24R,25(OH)2D3 only partially improved insulin secretion, and then only in the first phase. Changes of both serum calcium levels and dietary caloric intake after vitamin D metabolite administration are concluded to play a lesser role on the enhancement of insulin secretion, since, in a separate experiment, vitamin D-deficient rats with normal serum calcium levels did not show recovery of insulin secretion equivalent to the vitamin D-replete animals under conditions of dietary pair feeding. These results suggest that 1,25(OH)2D3 but not 24,25(OH)2D3 plays an essential role in the normal insulin secretion irrespective of the dietary caloric intake and prevailing serum calcium levels.

Parathyroid hormone, calcitonin, and vitamin D metabolites during normal fracture healing in humans. A preliminary report.

The metabolism of calcium is regulated by hormones: parathyroid hormone, (PTH) calcitonin (CT), and vitamin D metabolites. To study the physiologic role of these hormones during the process of fracture healing in humans, the blood levels of PTH, CT, 25-(OH)-D3, 24,25-(OH)2-D3, and calcium were determined in 13 young patients with fractures of long bones. The parameters were measured first on admission and again after six to eight weeks. A group of healthy volunteers of similar age and sex served as control subjects. Plasma calcium level on the day of fracture was significantly reduced, 8.50 +/- 0.23 mg% (p less than 0.001). Serum CT level on the day of fracture was significantly increased, 0.18 +/- 0.02 ng/ml (p less than 0.05), and it continued to increase during the healing period, up to 0.23 +/- 0.02 ng/ml (p less than 0.001) after six to eight weeks. A significant rise was noted in plasma level of 24,25-(OH)2-D3, from 2.02 +/- 0.42 ng/ml on the day of fracture to 2.84 +/- 0.41 ng/ml six weeks later (p less than 0.05). No significant changes were found in serum PTH and plasma 25-(OH)-D3 levels on the day of fracture or during the healing process. The results suggest a possible physiologic active role for CT and 24,25-(OH)2-D3 in fracture healing in humans.

Vitamin D: metabolism and biological actions.

In the last few years, it has become clear that the vitamin D endocrine system is comprised of many more target cells and tissues than were imagined a decade ago; in addition to the intestine, kidney, and bone, the vitamin D endocrine system now includes the beta cells of the pancreas, breast tissue, placenta, the pituitary gland, cells of the reticuloendothelial system, and several other cells and tissues. The complexity of the metabolic pathway by which the active metabolite(s) of vitamin D are produced and further metabolized has emerged, as has the complexity of its regulation.

Bone mineralization in infants.

The major physiologic control of bone mineralization in infancy involves calcium and phosphorus. Ca and P metabolism in turn is affected by endogenous stores at birth and the ability to deliver and absorb exogenous sources of these minerals. Calciotropic hormones (parathyroid hormone, calcitonin and 1,25 dihydroxyvitamin D) modulate the response of major end organs - intestine, kidney and bone - to balance the need to maintain a relatively stable extracellular biochemical environment with the need for adequate mineralization of the bone. Many other factors such as glucocorticoids under pathological circumstances may disturb bone mineralization; however, the mechanisms by which they control bone mineralization in infancy under physiologic circumstances is ill understood. Clinical bone demineralization occurs primarily in infants born with extreme prematurity. In the presence of conventional vitamin D supplementation, deficiency of calcium and phosphorus appears to play a major role in its causation.

[Comparative evaluation of biological activity of 1 alpha,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol in rats]. / Sravnitel'noe izuchenie biologicheskoi aktivnosti 1 alpha,25-dioksikholekal'tsiferola i 24,25-dioksikholekal'tsiferola u krys.

Deficiency of vitamin D in rats led to impairment of Ca2+ absorption in intestine, hypocalcemia, decrease in specific weight of femur diaphyses, decrease in content of Ca2+ and in the ratio Ca2+/hydroxyproline in diaphyses and epiphyses. These patterns were normalized after daily administration of cholecalciferol (vitamin D3) at a dose of 500 ng (20 IU) into rats within 5 days. I alpha, 25-dihydroxycalciferol at a daily dose of 30 ng stimulated absorption of Ca2+ in intestine, increased the specific weight of diaphyses and content of Ca2+ in diaphysis, but content of Ca2+ in blood and epiphyses was not normalized. Increase in 1,25 (OH)2D3 dose up to 300 ng caused a decrease in the rate of mineralization of diaphyses and epiphyses with simultaneous elevation of the Ca2+ content in blood. 24, 25-dihydroxycalciferol at a dose of 250 ng stimulated effectively both diaphyses and epiphyses mineralization, at the same time the effect was also maintained after 10-fold increase of the dose.