Etsuro Ogata

Alfacalcidol Inhibits Bone Resorption and Stimulates Formation in an Ovariectomized Rat Model of Osteoporosis: Distinct Actions from Estrogen

Although alfacalcidol has been widely used for the treatment of osteoporosis in certain countries, its mechanism of action in bone, especially in the vitamin D–replete state, remains unclear. Here we provide histomorphometric as well as biochemical evidence that alfacalcidol suppresses osteoclastic bone resorption in an ovariectomized rat model of osteoporosis. Furthermore, when compared with 17β‐estradiol, a representative antiresorptive drug, it is evident that alfacalcidol causes a dose‐dependent suppression of bone resorption, and yet maintains or even stimulates bone formation, as reflected in increases in serum osteocalcin levels and bone formation rate at both trabecular and cortical sites. 17β‐Estradiol, which suppresses bone resorption to the same extent as alfacalcidol, causes a parallel reduction in the biochemical and histomorphometric markers of bone formation. As a final outcome, treatment with alfacalcidol increases bone mineral density and improves mechanical strength more effectively than 17β‐estradiol, with a more pronounced difference in cortical bone. We conclude that estrogens depress bone turnover primarily by suppressing bone resorption and, as a consequence, bone formation as well, whereas alfacalcidol “supercouples” these processes, in that it suppresses bone resorption while maintaining or stimulating bone formation.

ED-71, a vitamin D analog, is a more potent inhibitor of bone resorption than alfacalcidol in an estrogen-deficient rat model of osteoporosis

Although active vitamin D is used in certain countries for the treatment of osteoporosis, the risk of causing hypercalcemia/hypercalciuria means that there is only a narrow therapeutic window, and this has precluded worldwide approval. The results of our previous animal studies have suggested that the therapeutic effect of active vitamin D on bone loss after estrogen deficiency can be dissociated at least partly from its effect of enhancing intestinal calcium absorption and suppressing parathyroid hormone (PTH) secretion. To test this, we compared the effects of ED-71, a hydroxypropoxy derivative of 1alpha,25-dihydroxyvitamin D3, with orally administered alfacalcidol, on bone mineral density (BMD) and the bone remodeling process as a function of their effects on calcium metabolism and PTH, in a rat ovariectomy (ovx) model of osteoporosis. ED-71 increased bone mass at the lumbar vertebra to a greater extent than alfacalcidol, while enhancing calcium absorption (indicated by urinary calcium excretion) and decreasing serum PTH levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers. These results suggest that active vitamin D exerts an antiosteoporotic effect by inhibiting osteoclastic bone resorption while maintaining osteoblastic function, and that these anticatabolic/anabolic effects of active vitamin D take place independently of its effects on calcium absorption and PTH. The demonstration that ED-71 is more potent in these properties than alfacalcidol makes it an attractive candidate as an antiosteoporotic drug.

The Advantage of Alfacalcidol Over Vitamin D in the Treatment of Osteoporosis

Abstract. Although alfacalcidol is widely used in the treatment of osteoporosis, its mechanism of action in bone is not fully understood. Alfacalcidol stimulates intestinal calcium (Ca) absorption, increases urinary Ca excretion and serum Ca levels, and suppresses parathyroid hormone (PTH) secretion. It remains to be clarified, especially under vitamin D-replete conditions, whether alfacalcidol exerts skeletal effects solely via these Ca-related effects, whether the resultant suppression of PTH is a prerequisite for the skeletal actions of alfacalcidol, and, by inference, whether alfacalcidol has an advantage over vitamin D in the treatment of osteoporosis. To address these issues, we (1) compared the effects of alfacalcidol p.o. (0.025–0.1 μg/kg BW) vis-à-vis vitamin D3 (50–400 μg/kg BW) on bone loss in 8-month-old, ovariectomized (OVX) rats as a function of their Ca-related effects, and (2) examined whether the skeletal effects of alfacalcidol occur independently of suppression of PTH, using parathyroidectomized (PTX) rats continuously infused with hPTH(1–34). The results indicate that (1) in OVX rats, alfacalcidol increases BMD and bone strength more effectively than vitamin D3 at given urinary and serum Ca levels: larger doses of vitamin D3 are required to produce a similar BMD-increasing effect, in the face of hypercalcemia and compromised bone quality; (2) at doses that maintain serum Ca below 10 mg/dl, alfacalcidol suppresses urinary deoxypyridinoline excretion more effectively than vitamin D3; and (3) alfacalcidol is capable of increasing bone mass in PTX rats with continuous infusion of PTH, and therefore acts independently of PTH levels. It is suggested that alfacalcidol exerts bone-protective effects independently of its Ca-related effects, and is in this respect superior to vitamin D3, and that the skeletal actions of alfacalcidol take place, at least in part, independently of suppression of PTH. Together, these results provide a rationale for the clinical utility of alfacalcidol and its advantage over vitamin D3 in the treatment of osteoporosis.

Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor κB (NF‐κB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] is a bone‐resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1α,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony‐stimulating factor (M‐CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol‐ or estrogen‐treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle‐treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D‐treated mice as well as estrogen‐treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone‐selective analogs may be useful for the management of osteoporosis.

CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) Regulates Osteoblast Differentiation

ABSTRACT Differentiation of committed osteoblasts is controlled by complex activities involving signal transduction and gene expression, and Runx2 and Osterix function as master regulators for this process. Recently, CCAAT/enhancer-binding proteins (C/EBPs) have been reported to regulate osteogenesis in addition to adipogenesis. However, the roles of C/EBP transcription factors in the control of osteoblast differentiation have yet to be fully elucidated. Here we show that C/EBP homologous protein (CHOP; also known as C/EBPζ) is expressed in bone as well as in mesenchymal progenitors and primary osteoblasts. Overexpression of CHOP reduces alkaline phosphatase activity in primary osteoblasts and suppresses the formation of calcified bone nodules. CHOP-deficient osteoblasts differentiate more strongly than their wild-type counterparts, suggesting that endogenous CHOP plays an important role in the inhibition of osteoblast differentiation. Furthermore, endogenous CHOP induces differentiation of calvarial osteoblasts upon bone morphogenetic protein (BMP) treatment. CHOP forms heterodimers with C/EBPβ and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBPβ, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBPβ and prevents osteoblast differentiation but promotes BMP signaling in a cell-type-dependent manner. Thus, endogenous CHOP may have dual roles in regulating osteoblast differentiation and bone formation.

1,25-dihydroxyvitamin D3 as well as its analogue OCT lower blood calcium through inhibition of bone resorption in hypercalcemic rats with continuous parathyroid hormone-related peptide infusion.

The effects of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] and its analogue 22‐oxa‐1,25(OH)2D3 (22‐oxacalcitriol) (OCT) on calcium and bone metabolism were examined in an animal model of hypercalcemia with continuous infusion of parathyroid hormone–related peptide (PTHrP), to determine whether active vitamin D could counteract the skeletal action of PTHrP in addition to its reported effect in suppressing the production of PTHrP in cancer cells. Parathyroid glands were removed from 8‐week‐old Sprague–Dawley rats to eliminate the confounding effects of endogenous PTH. Animals were then continuously infused with human PTHrP(1–34) at a constant rate via osmotic minipumps for 2 weeks, and at the same time treated orally or intravenously with OCT or 1,25(OH)2D3 four to nine times during the 2‐week period. Under these conditions, OCT and, surprisingly, 1,25(OH)2D3 alleviated hypercalcemia in a dose‐dependent manner. 1,25(OH)2D3 and OCT suppressed the urinary excretion of deoxypyridinoline, although they did not affect renal calcium handling, suggesting that the antihypercalcemic effect is attributable to the inhibition of bone resorption. These active vitamin D compounds also counteracted the effects of PTHrP at the proximal renal tubules, as reflected by a decrease in phosphate excretion. Histomorphometric analysis of bone revealed a dose‐related decrease in parameters of bone resorption. These results suggest that 1,25(OH)2D3 as well as OCT has the potential to alleviate hypercalcemia, at least in part, through the inhibition of bone resorption in hypercalcemic rats with constant PTHrP levels. We propose that the main function of active vitamin D in high bone‐turnover states is to inhibit bone resorption, and this may have important implications for the understanding of the role of active vitamin D in the treatment of metabolic bone diseases, such as osteoporosis.

Role of Interleukin-6 in Uncoupling of Bone In Vivo in a Human Squamous Carcinoma Coproducing Parathyroid Hormone-Related Peptide and Interleukin-6

OCC tumor has been established from a human squamous carcinoma associated with humoral hypercalcemia of malignancy (HHM) and shown to overproduce parathyroid hormone‐related peptide (PTHrP) and cause aggressive hypercalcemia when implanted into nude rats. In the present study, we have demonstrated by reverse transcription‐polymerase chain reaction and Northern blot analysis that OCC tumor also overexpressed interleukin 6 (IL‐6) mRNA and that tumor‐bearing animals exhibited a marked increase in plasma IL‐6 as well as PTHrP concentrations. When a monoclonal antibody against human IL‐6 was injected to block the activities of tumor‐derived IL‐6, bone loss in tumor‐bearing animals was significantly prevented. Quantitative bone histomorphometric analysis revealed that treatment with anti–IL‐6 antibody caused a substantial decrease in both osteoclast number and eroded surface (as parameters of bone resorption) and also a significant increase in the mineral apposition rate, but little effect on the osteoblastic surface. These results provide in vivo evidence suggesting that in tumors coproducing IL‐6 and PTHrP, IL‐6 is involved not only in the acceleration of osteoclastic bone resorption but also, at least in part, in the suppression of osteoblastic functions in HHM syndrome.

Parathyroid hormone-related protein as a potential target of therapy for cancer-associated morbidity

Proinflammatory cytokines are involved in the genesis of cancer‐associated cachexia. Parathyroid hormone‐related protein (PTHrP) is the causative agent in humoral hypercalcemia of malignancy (HHM) and is frequently secreted from various kinds of solid tumors as well as from adult T‐cell leukemia/lymphoma. PTHrP, like PTH, acts on PTH receptor type 1 (PTH1R). Activation of PTH1R may lead to stimulation of secretion of proinflammatory cytokines. It is expected, therefore, that PTHrP constitutes a key factor in the activation of the proinflammatory and cachectogenic cytokine network and consequently in the development of cachexia in patients with cancer.

p53-independent apoptosis is induced by the p19ARF tumor suppressor.

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.

Effect of Combination Treatment with a Vitamin D Analog (OCT) and a Bisphosphonate (AHPrBP) in a Nude Mouse Model of Cancer-Associated Hypercalcemia

Hypercalcemia represents one of the important paraneoplastic syndromes affecting morbidity and mortality of cancer patients. We and others have demonstrated that vitamin D analogs with little calcemic activities suppress the transcription of the parathyroid hormone‐related peptide (PTHrP) gene, a major humor responsible for cancer hypercalcemia, and thereby prevent the development of hypercalcemic syndrome. The present study was undertaken: to compare the therapeutic efficacy of a vitamin D analog, 22‐oxa‐1,25‐dihydroxyvitamin D3 (OCT), and a bisphosphonate (disodium 3‐amino‐1‐hydroxypropylidene‐1,1‐bisphosphonate pentahydrate [AHPrBP]), an inhibitor of osteoclastic bone resorption, on cancer‐induced hypercalcemia; and to see if the effect could be enhanced by combination treatment, using a nude mouse model implanted with a human pancreas carcinoma (FA‐6). After a single intravenous administration, OCT (5 μg/kg of body weight [BW]) was as effective as AHPrBP (10 mg/kg of BW) in lowering blood ionized calcium levels in tumor‐bearing nude mice, and their combination further enhanced the therapeutic effect. Although AHPrBP lost its efficacy after repeated injections, OCT was still effective after the third administration. The therapeutic effect of OCT in cancer hypercalcemia was observed in four other human tumors, including another pancreas carcinoma (PAN‐7), two squamous cell carcinomas of the lung (KCC‐C1 and LC‐6), and a squamous carcinoma of the pharynx (PHA‐1), all of which elaborated PTHrP into the circulation. Treatment with OCT resulted in a decrease in circulating PTHrP levels by approximately 50% in two representative models. However, the mechanism underlying the antihypercalcemic effect of OCT seemed complex, involving inhibition of PTHrP production, suppression of excessive bone resorption, and an antitumor activity. OCT also markedly inhibited the body weight loss with tumor growth, while AHPrBP, which exhibited a similar antihypercalcemic effect, was less effective than OCT in preventing cachexia. The anticachectic activity of their combination did not exceed that of OCT alone, suggesting a hypercalcemia‐dependent as well as an independent mechanism of cancer cachexia. It is concluded that OCT may be useful, either as a single agent or in combination with bisphosphonates, for the treatment of cancer‐associated hypercalcemia and cachexia.