Biomedical subjects

T Akatsu

Publications and source records attributed to T Akatsu.

At least 55 records · Page 3Linked to original sources

PubMed1993

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.

Animals↗

PubMed1993

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)

Acid Phosphatase↗

PubMed1993

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.

Animals↗

PubMed1992

Lack of bone resorption in osteosclerotic (oc/oc) mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

In a co-culture system of mouse spleen cells and osteoblastic cells, we have demonstrated that a suitable microenvironment must be provided by osteoblastic cells in order for osteoclast-like multinucleated cell (MNC) formation. Using this co-culture system, we examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. Numerous tartrate-resistant acid phosphatase (TRAP, an osteoclast marker enzyme)-positive MNCs were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] both in co-cultures of oc/oc spleen cells and normal osteoblastic cells and in those of normal spleen cells and oc/oc osteoblastic cells. TRAP-positive MNCs derived from normal spleen cells tended to spread out on culture dishes, whereas those from oc/oc spleen cells remained as small, compact MNCs. When TRAP-positive MNCs enriched from co-cultures of normal spleen cells and oc/oc osteoblastic cells were cultured on dentine slices, they formed numerous resorption pits with ruffled borders and clear zones. In contrast, none of the TRAP-positive MNCs derived from oc/oc spleen cells formed either ruffled borders or resorption pits. These results indicate that the lack of bone resorption in oc/oc mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

Acid Phosphatase↗

PubMed1992

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.

Animals↗

PubMed1992

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.

Acid Phosphatase↗

PubMed1991

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.

Acid Phosphatase↗

PubMed1991

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)

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

A stable analogue of thromboxane A2, 9,11-epithio-11,12-methanothromboxane A2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture.

Thromboxane A2 (TXA2) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, thromboxane B2 (TXB2). TXA2 has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA2 have not yet been proved because of its highly unstable nature. Recently, a chemically stable analogue of TXA2, 9,11-epithio-11,12-methanothromboxane A2 (STA2), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E2 (PGE2), a well-known bone resorbing agent, STA2 time- and dose-dependently stimulated the release of 45Ca from prelabelled mouse calvariae. Both STA2 and PGE2 induced the accumulation of cAMP in mouse calvariae. The TXA2 antagonist, ONO-3708, inhibited STA2-induced release of 45Ca. TXB2 induced neither bone resorption nor cAMP accumulation. When mouse marrow cells were cultured with STA2 for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA2 added. Again TXB2 showed no effect on osteoclast-like cell formation. These results indicate a role for TXA2 in some form of bone resorption.

Animals↗

PubMed1991

Power spectral analysis of blood pressure variability in traumatic quadriplegic humans.

A study was performed on seven traumatic neurologically complete quadriplegic (QP) males and seven age-matched healthy males (control) while they were at rest in the supine position in a climatic chamber (temperature 30 degrees C, relative humidity 60%). Arterial blood pressure waveforms were measured by a continuous noninvasive blood pressure-monitoring system based on arterial tonometry. Furthermore, the spontaneous beat-to-beat systolic blood pressure (SBP) variabilities of subjects were investigated by means of autoregressive power spectral analysis. As shown by earlier studies with an invasive (intra-arterial) blood pressure-monitoring system, in the control group there were two major spectral components: a high-frequency (HF) component [center frequency 0.27 +/- 0.02 (SE) Hz eq, power 0.9 +/- 0.2 mmHg2] and a low-frequency (LF) component (0.10 +/- 0.01 Hz eq, 5.2 +/- 1.4 mmHg2). On the contrary, in the QP group only the HF component was observed (0.28 +/- 0.03 Hz eq, 3.2 +/- 1.4 mmHg2). The results suggest that 1) in the QP subject the disappearance of the LF component in the SBP variability (i.e., the Mayer waves in humans) is presumably caused by the interruption of the spinal pathways linking supraspinal cardiovascular centers with the peripheral sympathetic outflow and 2) the cervical spinal sympathetic pathways may be instrumental in the genesis of the Mayer waves in humans.

Adult↗

PubMed1991

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.

Acid Phosphatase↗

PubMed1990

IL-6 is produced by osteoblasts and induces bone resorption.

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.

Alkaline Phosphatase↗

PubMed1990

Cloning of an osteoblastic cell line involved in the formation of osteoclast-like cells.

Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast-like cells from spleen cells in mice. Osteoclasts were defined as tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP-positive MNCs) in which specific calcitonin receptors were identified by autoradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP-positive MNCs in response to 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] in co-cultures with spleen cells. A cell line, KS-4, was identified with the greatest potency in inducing osteoclast-like cell formation in co-culture with spleen cells. The capacity of KS-4 cells to produce this effect was much greater than that of two bone marrow-derived stromal cell lines (MC3T3-G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1 alpha, 25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805-1813, 1989). Parathyroid hormone (PTH) increased cAMP production in KS-4 cells, and PTH and interleukin-1 alpha also induced TRACP-positive MNCs in co-cultures with spleen cells. Contact between living KS-4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS-4 cells were killed by fixation. Separate cultures of either spleen cells or KS-4 cells formed no TRACP-positive MNCs. KS-4 cells synthesized predominantly type I collagen, formed bone nodules without added of beta-glycerophosphate in a long-term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS-4 cells have properties consistent with progression toward the osteoblast phenotype and represent a single cell line with the ability to promote osteoclast formation by a contact-requiring process.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Inhibition of renal membrane adenylate cyclase by extract of pancreatic cancer associated with humoral hypercalcemia of malignancy.

The parathyroid hormone (PTH)-like activity, defined by the stimulation of cAMP production in MC3T3E1 cells, in the extract of a pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM) was eluted in two peaks (I and II) by reverse phase HPLC. Both peaks dose-dependently inhibited the binding of human (h) PTH(1-34) to canine renal membrane in an essentially similar fashion to hPTH(1-34) or PTH-related protein (rP). In the renal membrane, neither of these peaks stimulated adenylate cyclase (AC) but rather dose-dependently inhibited AC activity stimulated by hPTH(1-34), PTH-rP(1-34) or forskolin. In rat renal cortical slices, however, both peaks could exhibit their own stimulatory effect and did not inhibit PTH or forskolin-stimulated cAMP production. It has been concluded that a factor which inhibits AC activity, probably reflecting the direct action at catalytic site, can occasionally be produced with PTH-like factor. Although PTH-like and AC-inhibiting activities were very close on reverse phase HPLC, currently the interrelation between these two activities is not clear. It may be important to be aware of the presence of such a factor in the evaluation of the bioassay data employing a broken cell preparation, which is often used to assess the PTH-like activity of tumor products.

Adenylyl Cyclase Inhibitors↗

PubMed1990

Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells.

We previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. In this study, we developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells (10(3)-10(5) cells per well) obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive mononuclear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. When hemopoietic cells suspended in a collagen-gel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. All of the colonies consisted of nonspecific esterase-positive cells. The monocyte-depleted population prepared from peripheral blood failed to form colonies, whereas the monocyte-enriched population produced a large number of TRAPase-positive colonies. In addition, alveolar macrophages formed TRAPase-positive colonies most efficiently on the ST2 cell layers in the presence of the two hormones. Salmon 125I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. When direct contact between the peripheral blood cells and the ST2 cells was inhibited by a collagen-gel sheet, no TRAPase-positive cells were formed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a suitable microenvironment is provided by bone marrow-derived stromal cells.

Acid Phosphatase↗

PubMed1990

[Cytokine].

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Animals↗

PubMed1989

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.

Animals↗