Functioning adrenocortical oncocytoma: the first documented case producing interleukin-6 and review of the literature.

Adrenocortical oncocytoma is an extremely rare and predominantly non-functioning tumor. We herein report the first case of an adrenocortical oncocytoma that produces interleukin (IL)-6. A 38-yr-old woman was referred for treatment of a 4-cm adrenal mass. Laboratory test results showed elevated inflammatory parameters. Intriguingly, IL-6 serum level was also high at 30 pg/ml (normal 0-4 pg/ml). The patient underwent laparoscopic right adrenalectomy. Microscopic examination showed that the tumor was an adrenocortical oncocytoma with a unique peripheral lymphoid cuff with germinal centers. Electron microscopy demonstrated that the cytoplasm of the neoplastic cells was packed with numerous abnormal mitochondria. Three observations lead us to consider that this tumor was the primary source of serum IL-6. First, the IL-6 level in blood collected from the right adrenal vein was highest (527 pg/ml) among intra-operative blood samples. Second, neoplastic cells stained positively for IL-6. Third, the serum IL-6 returned to normal levels immediately after surgery.

Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.

Prolonged decrease of serum calcium concentration by murine gamma-interferon in hypercalcemic, human tumor (EC-GI)-bearing nude mice.

Murine gamma-interferon (MuIFN-gamma) is a potent inhibitor of bone resorption induced by interleukin 1 and parathyroid hormone-related protein in vitro. To investigate whether MuIFN-gamma is also effective in vivo, the cytokine was injected s.c. into hypercalcemic, tumor (EC-GI)-bearing nude mice, in which parathyroid hormone-related protein and interleukin 1 alpha are synergistically responsible for causing humoral hypercalcemia. When MuIFN-gamma was injected s.c. at a dose of 1 to 20 x 10(4) units for 5 days consecutively, serum calcium concentrations in the tumor-bearing mice decreased in a dose-dependent manner. The minimal effective dose was 5 x 10(4) units/mouse. Unlike calcitonin, which decreased the serum calcium concentration for only 1 to 2 days despite continuous daily injections, MuIFN-gamma decreased it for more than 7 days even after the injections had been stopped. Human gamma-interferon was completely ineffective. The decrease in serum calcium concentration was accompanied by a decrease in urinary calcium excretion. Histological examination of the femur revealed a decreased number of osteoclasts in the MuIFN-gamma-treated mice. Furthermore, MuIFN-gamma, when injected into nude mice or normal mice at a dose of 15 x 10(4) units for 3 days, almost completely abolished the formation of multinucleated osteoclast-like cells in vitro. These findings suggest that MuIFN-gamma suppresses the formation and maturation of osteoclasts and inhibits osteoclastic bone resorption, resulting in the prolonged decrease of serum calcium concentration seen in hypercalcemic, tumor-bearing nude mice. Therefore, bone resorption inhibitors like MuIFN-gamma, which ameliorate humoral hypercalcemia without an escape phenomenon, are potentially useful for the treatment of malignancy-associated hypercalcemia.

Role of nonenzymatic glycosylation of type I collagen in diabetic osteopenia.

Formation of advanced glycation end products (AGEs) in extracellular matrix (ECM) is implicated in the development of chronic diabetic complications. However, the involvement of AGEs in diabetic bone disease has not been well established. We have examined whether AGEs are increased in the bone collagen of streptozotocin-induced diabetic rats in vivo and whether glycation of type I collagen affects the functions of osteoblastic cells in vitro. During 12 weeks of observation, AGEs in collagen extracted from the tibiae of diabetic rats increased in a time-dependent manner and were significantly higher than controls at every time point. In vitro, the incubation of collagen with glucose-6-phosphate resulted in a time-dependent increase of AGEs. When osteoblastic cells isolated from fetal rat calvaria were cultured on AGE-modified type I collagen, it dose-dependently inhibited phenotypic expressions of osteoblasts. Among osteoblastic parameters, nodule formation was the most sensitive, being inhibited by approximately 70% by the glycation of collagen for only 1 week. Alkaline phosphatase activity and osteocalcin secretion were inhibited by 20-30% and 15-70%, respectively, by the glycation of collagen for 1-5 weeks. These results indicate that AGE-modified collagen affects osteoblastic cell differentiation and function in vitro and suggest that similar changes occurring in vivo may contribute to diabetic osteopenia.

Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures.

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.

A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation.

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures. In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor beta (TGF-beta). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.

New resorption assay with mouse osteoclast-like multinucleated cells formed in vitro.

We previously reported a procedure to obtain a preparation containing a large number of mouse osteoclast (OCL)-like multinucleated cells (MNCs) formed in cocultures of mouse osteoblastic and bone marrow cells in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. The MNCs satisfied major criteria of OCLs, such as tartrate-resistant acid phosphatase (TRAP) activity, acid production, calcitonin (CT) receptors, and the ability to form resorption pits on bone slices. In this report, we describe a simple resorption assay system using MNC preparations. After culturing MNC preparations or disaggregated rat OCL preparations on dentin slices, they were stained with Mayer's hematoxylin. The stained area corresponded exactly with the resorption pits visualized by scanning electron microscopy and were measured using an image analysis system attached to a light microscope. Pit formation by MNCs was gradually enhanced by reducing the medium pH (pH 7.5 < 7.2 < 6.9). The plan area resorbed by MNCs increased linearly for up to 72 h. These results are very similar to those obtained with OCL preparations. In multiple standard assays with MNC preparations, more than 250 MNCs could be placed on a dentin slice, and the total area resorbed to a level of up to 9% of the whole surface within 48 h. In contrast, in multiple assays with OCL preparations, it was not easy to place more than 50 OCLs on a slice and the resorbed area was only 0.7% of the surface.(ABSTRACT TRUNCATED AT 250 WORDS)

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)-methylene-bisphosphonate 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.

Role of colony-stimulating factors in osteoclast development.

Effects of various colony-stimulating factors (CSFs) [interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), and granulocyte CSF (G-CSF)] on osteoclast-like cell formation were examined in two different culture systems: the one-step mouse marrow culture system and the two-step coculture system of mouse primary osteoblastic cells with the bone marrow cells collected from the colonies that formed in the methylcellulose in the presence of the CSFs. In the one-step mouse marrow cultures, none of the CSFs stimulated the formation of tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts)-positive multinucleated cells (MNCs). Furthermore, the CSFs other than G-CSF inhibited in a dose-dependent manner the TRAP-positive MNC formation induced by 1 alpha-25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. In contrast, when marrow cells were first cultured in semisolid methylcellulose in the presence of a CSF and the recovered marrow cells from the semisolid cultures were subsequently cocultured with primary osteoblastic cells in the presence of 1 alpha,25-(OH)2D3, numerous TRAP-positive MNCs were formed. [125I]salmon calcitonin specifically bound to TRAP-positive cells formed in this two-step culture system. Over 90% of the TRAP-positive mononuclear cells and MNCs accumulated [125I]calcitonin. M-CSF was the most potent in inducing TRAP-positive MNCs, followed by GM-CSF, IL-3, and G-CSF in that order. No TRAP-positive cells were formed in the absence of either osteoblastic cells or 1 alpha,25-(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of prostaglandins in interleukin-1-induced bone resorption in mice in vitro.

The mechanism of bone resorption induced by interleukin 1 (IL-1) was examined in mice using three different in vitro assay systems: a fetal long bone organ culture system, a bone marrow culture system, and a coculture system of primary osteoblastic cell populations and spleen cells. In the organ culture system, recombinant human IL-1 alpha (rhIL-1 alpha) increased both bone resorption and osteoclast number. Both were partially suppressed in the presence of indomethacin. In the marrow culture, both rhIL-1 alpha and rhIL-1 beta stimulated osteoclastlike cell formation, which was completely inhibited by adding indomethacin concurrently. Furthermore, there was a good correlation between the number of osteoclastlike cells formed and the amount of prostaglandin E2 (PGE2) released into the culture media. This indicates that PGE2 is involved in the mechanism of IL-1-mediated osteoclastlike cell formation. In the coculture of primary osteoblastic cell populations and spleen cells, rhIL-1 again stimulated osteoclastlike cell formation, which was inhibited by adding indomethacin. In the cocultures in which direct interaction between osteoblastic cells and spleen cells was inhibited, PGE2 synthesis was similarly increased but no osteoclastlike cells were formed. These results indicate that IL-1 induces osteoclast formation by a mechanism involving PG (most likely PGE2). Furthermore, direct interaction between osteoclast progenitors and osteoblastic cells is required in the osteoclast recruitment induced by IL-1.

Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages.

The effects of parathyroid hormone (1-34) (PTH (1-34) on osteoblast differentiation were investigated using primary osteoblast-like cells isolated from newborn mouse calvaria. The osteoblast-like cells cultured at low cell densities, in which the cells remained in a subconfluent state at the end of culture, were exposed for 7 days to PTH. This stimulated alkaline phosphatase (ALP) activity in a dose-dependent manner. In contrast, PTH dose-dependently inhibited both ALP activity and osteocalcin production in cells inoculated at high cell densities, in which they had reached a confluent state before the end of culture. The changes of ALP activity by PTH were accompanied with the expression of ALP messenger RNA. PTH induced no changes of the hydroxyproline content in the cell layer when the cells were exposed to the hormone at a subconfluent state, but reduced the content at a postconfluent state. The stimulation of ALP activity by PTH at a preconfluent state was retained even after the removal of PTH from the culture media. The opposite effect of PTH, observed between the preconfluent and the postconfluent state, was reproduced by adding dibutyryl cyclic adenosine monophosphate (cAMP) or forskolin, but not by adding phorbol myristate acetate. In a colony-forming unit fibroblastic (CFU-F) assay, using bone marrow cells isolated from tibiae of 10-week-old mice, PTH induced no changes in the total number of CFU-Fs, but increased the proportion of ALP-positive colonies. These results indicate that PTH exerts opposite effects on the phenotypic expression of osteoblasts, depending on their differentiation stages of osteoblasts. PTH may preferentially stimulate osteoblast differentiation in immature osteoblasts but inhibit it in more mature cells.

Glucocorticoid regulation of calcitonin receptor in mouse osteoclast-like multinucleated cells.

Abundant multinucleated cells (MNCs) are formed in cocultures of mouse osteoblastic cells and marrow cells in the presence of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3], and these cells have the properties of osteoclasts (OCs). In this study using the mammalian OCs, we tried to clarify the role of glucocorticoids (GCs) in calcitonin receptors (CTR) and CT-responsive cAMP production in OCs. Dexamethasone (DEX) dose and time dependently enhanced the specific binding of [125I]salmon calcitonin (sCT). When the MNCs were preincubated with DEX for 24 h, the effect was evident at 10(-9) M and the maximum effect was obtained at 10(-7) M. The effect developed over 12-48 h at doses of 10(-9) and 10(-6) M DEX. The numbers of CTR-positive mononuclear cells and MNCs were not altered by the DEX treatment. Prednisolone and triamcinolone reproduced the DEX effect, but 17 beta-estradiol, progesterone, testosterone, aldosterone, and 1 alpha, 25(OH)2D3 did not. RU486, a GC receptor antagonist, attenuated the effect of DEX to enhance the specific binding of [125I]sCT. From a Scatchard plot analysis, DEX enhanced CTR number (212 +/- 64%) with a minimal change in the affinity to sCT. Autoradiographic studies using [125I]sCT showed that DEX enhanced the density of the grains on the tartrate-resistant acid phosphatase (TRAP)-positive MNCs and mononuclear cells, but not on other types of cells. DEX preincubation also enhanced sCT-stimulated but not prostaglandin E2- or forskolin-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

The mouse mammary tumor cell line, MMT060562, produces prostaglandin E2 and leukemia inhibitory factor and supports osteoclast formation in vitro via a stromal cell-dependent pathway.

Osteoclastic bone resorption increases at the site of bone metastasis, but little is known about how tumor cells induce osteoclast (OC) recruitment in the bone marrow microenvironment. To clarify this point, we examined the effects of various mouse tumor cells on OC recruitment using cocultures of tumor cells and mouse marrow cells. The mouse mammary tumor cell lines, MMT060562 (MMT), BALB/c-MC, Jyg-MC(A), or other nonmammary tumor cell lines, LLC and B16, were cocultured with mouse marrow cells, and OC recruitment from marrow cells was determined by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNCs) formed. Of the tumor cells examined, MMT and BALB/c-MC stimulated OC formation, but other tumor cells did not. OC formation with MMT was dependent on the number of MMTs inoculated, and only ten cells per well were sufficient to induce OC development. OCs appeared on day 4, and the number reached a maximum on days 5-8 and decreased thereafter. TRAP(+) MNCs induced by MMT satisfied the major criteria of OCs, such as the presence of calcitonin receptors and the ability to resorb calcified tissues. The majority of OCs were formed adjacent to the stromal cells, which were positive for alkaline phosphatase. When spleen cells were cocultured with MMT, no OCs were formed. In contrast, when osteoblastic cells were added to cocultures of spleen cells and MMT, many OCs were formed. The cultured media (CM) of MMT induced OC formation in mouse marrow cultures. Neither parathyroid hormone-like nor interleukin 1-like activity was present in the CM. MMT constitutively produced prostaglandin E2 (PGE2) and OC formation in cocultures was completely inhibited by indomethacin. Fractionation of the CM of MMT by ultrafiltration indicated that the OC-inducing activities were present not only in the fraction with molecular weight below 3 kDa but also in the fraction with molecular weight above 3 kDa. OC-inducing activity with high molecular weight was eluted around 50 kDa by Bio-Gel P-60 column chromatography. The active fractions also possessed leukemia inhibitory factor (LIF) activity, and OC-inducing activity of the peak fraction was inhibited in the presence of anti-LIF neutralizing antibody. The results of this study indicated that MMTs release PGE2 and LIF, which in turn stimulate OC formation via a stromal cell-dependent pathway. These culture systems will help to clarify the mechanisms by which tumor cells induce OC formation in a bone marrow microenvironment.

Structure-activity relationships in calcitonin gene-related peptide: cyclic AMP response in a preosteoblast cell line (KS-4).

Synthetic analogs of chicken and human CGRP were used to define structure-function relationships in a murine osteoblast precursor cell line, KS-4, that exhibits a substantial cyclic AMP response to CGRP but no response to calcitonin. Human CGRP had 40% of the activity of chicken CGRP, but [Asp-14]-human CGRP was equipotent with chicken CGRP. Similar observations were made previously for calcium- and phosphate-lowering effects. Des-1-Ala,deamino CGRP compounds of both human and chicken origin were three-fold more potent than the respective native CGRPs. The [4-F-Phe)-37]-chicken CGRP analog had a four-fold enhanced activity. In all of the 13 analogs either truncated amino-terminally or missing the (2-7)-disulfide bridge, biologic activity was greatly reduced (0.1% of chicken CGRP), as it was in chicken CGRP-(1-36)-OH, which lacks the C-terminal amino acid. The same analog has been shown to retain a hypocalcemic effect, which is most likely mediated through calcitonin receptors. In confirmation of their antagonistic effect reported in liver, analogs that lack the N-terminal ring structure exhibited significant antagonistic activity. The data illustrate the specificity of CGRP receptors in promoting cyclic AMP formation in osteoblast-like cells. Such CGRP analogs will be useful in investigating structure-function relationships of CGRP.

Chinese hamster ovary cells expressing alpha4beta1 integrin stimulate osteoclast formation in vitro.

It is reported that Chinese hamster ovary cells transfected with human alpha4 cDNA (alpha4CHOs) and expressing functional alpha4beta1 integrin developed bone metasasis in nude mice. To clarify the role of alpha4beta1 integrin in bone metastasis, in terms of tumor-mediated bone destruction, we examined whether alpha4CHOs 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 alpha4CHOs 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 alpha4CHOs. TRAP(+) MNCs induced by alpha4CHOs possessed calcitonin receptors and resorbed calcified tissues. In cocultures, alpha4CHOs 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 alpha4beta1 integrin. TRAP(+) MNC formation was not stimulated in cocultures where direct contact between alpha4CHOs and bone marrow cells was inhibited by membrane filters. Alpha4CHOs 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 alpha4CHOs, bone marrow cells, and cocultures of alpha4CHOs 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 alpha4CHOs. Anti-human alpha4 and anti-mouse VCAM-1 antibodies inhibited TRAP(+) MNC formation induced by alpha4CHOs. These results indicate that alpha4CHOs stimulated TRAP(+) MNC formation through direct cell-to-cell interaction between alpha4beta1 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.

Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors.

Malignancy-associated hypercalcemia is mainly caused by excessive production of parathyroid hormone-related protein (PTHrP) by the tumor. Using anti-PTHrP-(1-34) monoclonal murine antibody (anti-PTHrP MoAb), we studied whether repeated injection of the homologous antibody would continuously decrease the serum calcium concentration in hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors, leading to prolongation of their survival time. Daily SC injections of anti-PTHrP MoAb decreased the serum calcium concentration almost to within the normal range in nude mice bearing transplanted human PTHrP-producing tumors (T3M-1, EC-GI, PC-3, and FA-6) but not in a nude mouse bearing a transplanted parathyroid carcinoma. The antibody did not affect FA-6 tumor growth either in vitro or in vivo. Pancreatic carcinoma cells (FA-6), which caused the most severe hypercalcemia, were inoculated into 6-week-old nude mice. When severe hypercalcemia (approximately 19 mg/dl) had developed, daily SC injection of anti-PTHrP MoAb was started. Within 18 days of this time point, all untreated tumor-bearing mice (n = 10) died of hypercalcemia and cachexia, whereas all the treated mice (n = 10) showed an increase in body weight and survived for at least 25 days. Histologic examination of the treated mice revealed a marked decrease in osteoclastic bone resorption, without toxicologic findings in the kidney and liver. These results suggest that passive immunization against PTHrP can continuously ameliorate the hypercalcemia and markedly prolong the survival time of severely hypercalcemic, tumor-bearing mice. If a human monoclonal antibody against PTHrP-(1-34) could be developed, then passive immunization would be potentially one of the most effective therapies for patients with malignancy-associated hypercalcemia due to excessive production of PTHrP.

Preparation and characterization of a mouse osteoclast-like multinucleated cell population.

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.

Regulation by calcitonin and glucocorticoids of calcitonin receptor gene expression in mouse osteoclasts.

We previously studied regulation of the calcitonin (CT) receptor (CTR) by glucocorticoid (GC) and CT in cultures of mature mouse osteoclast-like cells (OCLs). The present studies were designed to examine the interaction of CT and GC in regulation of the CTR in osteoclasts and the molecular mechanisms involved. Treatment of OCLs with 10(-7) M dexamethasone (Dex) increased the CTR number in a time-dependent manner, whereas treatment with 10(-9) M salmon CT (sCT) reduced CTR number; neither treatment changed receptor affinity. Dex pretreatment somewhat antagonized the CT-induced reduction in [125I]sCT specific binding. Dex increased, and sCT pretreatment decreased, the sCT-responsive adenylate cyclase activity in parallel with the change in receptor binding. Dex treatment resulted in an increase in CTR messenger RNA (mRNA) levels, as assessed by reverse transcription-PCR, indicating that the increased CTR number was mediated by de novo CTR synthesis. This effect was specific to GCs and was not reproduced by mineralocorticoids or sex steroids. Treatment with sCT resulted in a rapid and profound reduction in CTR mRNA expression, and this reductions was somewhat delayed by Dex pretreatment. OCLs were treated with 5,6-dichloro-1 beta-D-ribofuranosyl benzimidazole to enable estimation of the mRNA decay rates in the absence of ongoing transcription. The stability of CTR mRNA was similar to the control value in Dex-treated OCLs, suggesting that the effect of Dex may be due to changes in transcriptional activity. Interestingly, transcriptional inhibition by 5,6-dichloro-1 beta-D-ribofuranosyl benzimidazole abolished the ability of CT to reduce CTR mRNA levels, suggesting that CT may act by increasing the rate of CTR mRNA decay, and that this effect requires ongoing transcription. The 3'-untranslated region of the mouse CTR mRNA contains four copies of the AUUUA motif, as well as other A/U-rich sequences, which have been shown to determine the stability of other mRNA transcripts. The stability results were consistent with the results of the nuclear transcript run-on assay, which indicated that treatment with Dex enhanced the rate of transcription, whereas CT had no effect. These results show that GC and CT influence CTR expression by distinct mechanisms and provide the basis for identification of the cellular factors involved.

Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells.

We have reported that osteoblastic cells are required for differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts. In the present study we examined the pathogenesis of the osteoclast deficiency in osteopetrotic (op/op) mice using a coculture system of spleen cells and osteoblastic cells. When spleen cells obtained from op/op or normal (+/?) littermates of op/+ parent mice were cocultured with osteoblastic cells obtained from calvaria of normal ddy strain mice, numerous tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) were formed in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Most of the TRAP-positive MNCs bound [125I]salmon calcitonin. This suggests that there is no abnormality in the osteoclast progenitors present in the splenic tissues of op/op mice. When osteoblastic cells from +/? littermates were cocultured with normal spleen cells from ddy mice, TRAP-positive MNCs were similarly formed in response to 1 alpha,25(OH)2D3. In contrast, in cocultures of op/op osteoblastic cells with normal spleen cells, no TRAP-positive cells appeared, even in the presence of 1 alpha,25(OH)2D3. The op/op mutation was recently reported to exist in the coding region of the macrophage colony-stimulating factor (M-CSF) gene. Adding M-CSF and 1 alpha,25(OH)2D3 to the coculture with op/op osteoblastic cells induced the appearance of TRAP-positive MNCs with calcitonin receptors. These results clearly indicate that osteoclast deficiency in op/op mice is due to a defect in the local microenvironment in bone, in which M-CSF produced by osteoblastic cells plays a critical role in osteoclast development.

Parathyroid hormone (PTH)-related protein is a potent stimulator of osteoclast-like multinucleated cell formation to the same extent as PTH in mouse marrow cultures.

Induction of osteoclast-like multinucleated cells (MNCs) by various fragments of PTH-related protein (PTHrP) was examined in mouse marrow cultures. Osteoclast-like MNCs were defined as tartrate-resistant acid phosphatase (TRACP)-positive MNCs with calcitonin receptors. In all experimental protocols examined, PTHrP-(1-34) induced TRACP-positive MNCs at almost the same rate as PTH-(1-34). PTHrP-(1-29) was less potent than PTHrP-(1-34). PTHrP-(1-25) and PTHrP-(1-14) had no effect. PTHrP-(1-34) was more potent than PTH-(1-34) in increasing the accumulation of cAMP, but the former appeared to lose its activity more rapidly than the latter. Isobutylmethylxanthine increased the effect of PTHrP-(1-34) and PTH-(1-34) in inducing TRACP-positive MNCs. Furthermore, the calcium ionophore A23187 significantly increased the formation of TRACP-positive MNCs. The effect of PTH-(1-34) and PTHrP-(1-34) in inducing TRACP-positive MNCs was potentiated by adding A23187 but suppressed by adding verapamil simultaneously. The inhibition by verapamil was overcome by adding A23187. [Nle8,18,Tyr34]PTH-(3-34)amide inhibited the effect of not only PTH-(1-34) but also PTHrP-(1-34) in inducing both the accumulation of cAMP and the TRACP-positive MNC formation. These results show that PTHrP is a potent stimulator of osteoclast-like MNC formation to almost the same extent as PTH. It increases the number of osteoclast-like MNCs by a mechanism involving cAMP and calcium ions, and is most likely mediated through the same receptor. The controversial results of the bone-resorbing activity of PTH and PTHrP reported so far may be explained by the differences in the relative potencies of the respective hormones in increasing the intracellular cAMP and calcium ions and by the shorter half-life of PTHrP in culture medium.