Takehiko Murakami

Osteoclastogenesis inhibitory factor exhibits hypocalcemic effects in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy.

Osteoclastogenesis inhibitory factor (OCIF) is a novel secreted protein that inhibits osteoclastogenesis both in vitro and in vivo. In this study, we examined the effects of OCIF on serum calcium (Ca) concentrations in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy. In normal mice, a single intraperitoneal injection of OCIF reduced serum Ca levels in a dose-dependent manner. Significant decrease in serum Ca (by 1.6 +/- 0.3 mg/dL, n = 5) was observed 2 h after the injection of OCIF at 20 mg/kg and the hypocalcemic effect continued for up to 12 h. Serum phosphate (Pi) concentrations also decreased in response to OCIF. Urinary excretion of Ca, Pi, and creatinine did not change significantly after injection of OCIF or vehicle. In hypercalcemic, tumor-bearing nude mice, a single intraperitoneal injection of OCIF at 20 mg/kg resulted in a dramatic decrease in serum Ca (maximal decrease 2.8 +/- 0.37 mg/dL, n = 11), which continued for up to 24 h. The results suggest that OCIF decreased serum Ca through its inhibitory effect on bone resorption. Furthermore, it is suggested that OCIF has therapeutic potential for the treatment of hypercalcemic conditions such as malignancy-associated hypercalcemia.

HYPOCALCEMIC EFFECT OF OSTEOCLASTOGENESIS INHIBITORY FACTOR/OSTEOPROTEGERIN IN THE THYROPARATHYROIDECTOMIZED RAT

Osteoclastogenesis inhibitory factor (OCIF), also termed as osteoprotegerin (OPG), is a soluble member of the tumor necrosis factor receptor family. Although OCIF/OPG is shown to inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo, its effect on serum calcium level remains to be determined. In this study we examined the acute effect of OCIF on thyroparathyroidectomized rats whose serum calcium concentrations were raised either by exogenous PTH or 1,25-(OH)2D3. When OCIF was administered at the start of PTH infusion, it attenuated the initial rise in serum calcium. When OCIF was administered into rats with established hypercalcemia, it decreased serum calcium rapidly (within 2 hr) and dramatically. OCIF did not increase urinary calcium excretion. These findings, especially the rapid onset of its hypocalcemic effect, suggest that OCIF not only inhibits the formation of osteoclasts but also affects the function and/or survival of mature osteoclasts at doses used in this study.

Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines.

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c‐MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo‐oxygenase‐2 (COX‐2), an inducible rate‐limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX‐2 inhibitor, JTE‐522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose‐dependent manner. Northern blotting showed a very high level of COX‐2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c‐MC, but it increased when BALB/c‐MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX‐2 protein in respective cultures were compatible with the results of COX‐2 mRNA. In vivo, BALB/c‐MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX‐2 protein. These findings suggest that COX‐2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.

Effect of acarbose on postprandial lipid metabolism in type 2 diabetes mellitus

The effect of acarbose, an alpha-glucosidase inhibitor, on postprandial glucose and lipid metabolism was investigated in patients with type 2 diabetes mellitus. Twenty patients (10 men and 10 women) with type 2 diabetes mellitus were studied. A test meal was taken with or without 100 mg of acarbose. The levels of plasma glucose, and serum immunoreactive insulin, lipids, apolipoproteins, and remnant-like particle cholesterol were investigated. Acarbose inhibited the postprandial increase of both plasma glucose and serum immunoreactive insulin. Acarbose also significantly suppressed the increase of serum triglycerides at 60, 90, and 120 min (P < 0.05 to P < 0.01), and the increase of serum remnant-like particle cholesterol at 60 and 120 min (P < 0.05). Acarbose inhibited the postprandial decline of apolipoprotein C-II, and decreased the postprandial serum apolipoprotein C-III level. These results suggest that acarbose may improve postprandial hyperlipidemia as well as postprandial hyperglycemia in patients with type 2 diabetes mellitus.

A Third‐Generation Bisphosphonate, YM175, Inhibits Osteoclast Formation in Murine Cocultures by Inhibiting Proliferation of Precursor Cells via Supporting Cell‐Dependent Mechanisms

The theory that bisphosphonates inhibit osteoclast formation through their effects on osteoblastic cells remains controversial. To confirm the inhibitory effect of bisphosphonates on osteoclast formation and gain some insights into the underlying mechanisms, we examined the effect of disodium dihydrogen (cycloheptylamino)‐methylenebisphosphonate monohydrate (YM175) on osteoclast‐like multinucleated cell (OCL) formation in various mouse coculture systems. When different origins of osteoclast precursors (bone marrow, spleen, or nonspecific esterase‐positive cells) were cocultured with the same supporting cells (calvarial osteoblasts), YM175 inhibited OCL formation similarly in all cultures. When the same osteoclast precursors (spleen cells) were cocultured with supporting cells of different origin, the results were variable. YM175 inhibited OCL formation almost completely in cocultures with calvarial osteoblasts or osteoblastic cell line KS4, while it did not, or only slightly, inhibit OCL formation in cocultures with stromal cell lines, ST2 or MC3T3‐G2/PA6. Temporal addition of YM175 in cocultures of spleen cells with osteoblastic cells revealed that YM175 was effective when it was present at an early phase of the culture period. Consistent with this observation, YM175 in the presence of osteoblastic cells inhibited proliferation of preosteoclastic cells, but did not inhibit the fusion of mononuclear prefusion osteoclasts. In conclusion, the inhibitory effect of YM175 on OCL formation was confirmed in various murine coculture systems, but the effect was dependent on the types of bone‐derived cells supporting osteoclastogenesis. The findings suggest that YM175 inhibits osteoclastogenesis by inhibiting the proliferation of osteoclast precursors through its action on supporting cells of osteoblast lineage rather than acting directly on osteoclast precursors.

Mouse mammary carcinoma cell line (BALB/c-MC) stimulates osteoclast formation from mouse bone marrow cells through cell-to-cell contact

We recently reported that numerous osteoclasts (OC) were formed in cocultures of some mouse cancer cell lines and bone marrow cells. In this study, we examined mechanisms by which one of the cell lines, BALB/c-MC, induces OC. BALB/c-MC dose dependently stimulated OC formation in cocultures. In cocultures where direct cell-to-cell contact between BALB/c-MC and bone marrow cells was inhibited by membrane filters, OC formation was not stimulated. The stimulation of OC formation in the coculture was completely abolished by adding 10(-7)-10(-6) mol/L indomethacin. The concentration of prostaglandin E2 (PGE2) in the culture media of cocultures with cell-to-cell contact was higher than that of cocultures without cell-to-cell contact or marrow cultures alone, and it reached levels sufficient to induce OC (11.9 +/- 5.3 ng/mL [about 3.4 x 10(-8) mol/L]). When BALB/c-MC or bone marrow cells were fixed with formalin and then cocultured with bone marrow cells or BALB/c-MC, respectively, the concentration of PGE2 in the culture media of cocultures of fixed BALB/c-MC and bone marrow cells increased, whereas that of cocultures of BALB/c-MC and fixed bone marrow cells did not increase. These results indicate that BALB/c-MC stimulate OC formation through direct cell-to-cell contact with bone marrow cells, and PGE2 released from bone marrow cells through direct cell-to-cell contact are involved in OC formation by the cell line.

Chinese Hamster Ovary Cells Expressing α4β1 Integrin Stimulate Osteoclast Formation In Vitro

It is reported that Chinese hamster ovary cells transfected with human α4 cDNA (α4CHOs) and expressing functional α4β1 integrin developed bone metasasis in nude mice. To clarify the role of α4β1 integrin in bone metastasis, in terms of tumor‐mediated bone destruction, we examined whether α4CHOs stimulate osteoclast formation in cocultures with mouse bone marrow cells. The number of osteoclast‐like cells identified as tartrate‐resistant acid phosphatase positive multinucleated cells (TRAP(+) MNCs) formed from bone marrow cells increased with the increasing number of α4CHOs cocultured. The effects of 1,25‐dihydroxyvitamin D3(1,25(OH)2D3) and prostaglandin E2 (PGE2) on TRAP(+) MNC formation were enhanced in cocultures with α4CHOs. TRAP(+) MNCs induced by α4CHOs possessed calcitonin receptors and resorbed calcified tissues. In cocultures, α4CHOs and bone marrow stromal cells were in contact with each other and bone marrow stromal cells expressed vascular cell adhesion molecule‐1 (VCAM‐1), which is one of the ligands for α4β1 integrin. TRAP(+) MNC formation was not stimulated in cocultures where direct contact between α4CHOs and bone marrow cells was inhibited by membrane filters. α4CHOs do not support TRAP(+) MNC formation in cocultures with spleen cells but do support TRAP(+) mononuclear cell and MNC formation from spleen cells in the presence of osteoblastic cells. Cultured media from α4CHOs, bone marrow cells, and cocultures of α4CHOs and bone marrow cells did not stimulate TRAP(+) MNC formation or enhance the effects of 1,25(OH)2D3 and PGE2 in bone marrow cultures. The concentrations of PGE2 and interleukin‐6 (IL‐6) in cultured media were not different between the cultures of bone marrow cells and the cocultures of bone marrow cells and α4CHOs. Anti‐human α4 and anti‐mouse VCAM‐1 antibodies inhibited TRAP(+) MNC formation induced by α4CHOs. These results indicate that α4CHOs stimulated TRAP(+) MNC formation through direct cell‐to‐cell interaction between α4β1 and VCAM‐1. It is suggested that in addition to various soluble factors regulating osteoclast formation, cell‐to‐cell interaction between tumor cells and bone marrow cells is important for inducing osteoclasts at the site of bone metastasis and leading to bone destruction.