Neonatal Mouse Calvarial Bones Research Articles

Neonatal Mouse Bone Supports Growth Of Primary Myeloma Cells Ex Vivo and Assessment Of Responses To Antimyeloma Drugs

A barrier to optimizing treatment for multiple myeloma (MM) is the lack of a convenient assay to determine drug-responsiveness in individual patients. Although primary MM cells are routinely obtained from patient bone marrow aspirates, CD138+ cells die too rapidly in culture to be analyzed. We hypothesized that primary MM cell survival requires the bone microenvironment to provide the necessary soil for the growth of the cancer cell seeds. Immunodeficient animal models contain a complete multicellular bone marrow microenvironment, including extracellular matrix, and will support engraftment of patient MM cells. However, intact animal models are too slow, complex and expensive to provide timely and clinically useful predictions of individual tumor behavior and responses to drugs.We hypothesized that a well-established model for studying bone in culture could be adapted to support the growth of primary MM cells and to study myeloma bone disease. We developed an ex vivo organ coculture assay (EVOCA), with neonatal mouse calvarial bones providing the soil to support tumor cell growth. Human MM cell lines and primary MM cells were successfully cocultured with immune-naïve neonatal hemi-calvariae for up to 2 weeks. EVOCA was optimized to support bone and MM cell survival and analyzed by histology and by PCR with species-specific primers for changes in expression of tumor (human) and host (mouse) genes. Four human MM cell lines (8226, H929, U266 and JJN3) and primary CD138+ cells from 5 patients (1 freshly isolated and 4 from previously frozen cells) survived for > one week in calvarial cultures, using as few as 104 cells. Both primary MM cells and CD138+ cells were detected by immunohistochemistry for human CD138 after 2 weeks and by species-specific RT-PCR for the human housekeeping gene RPL32. Appropriate responses of mouse bones to MM were observed at 2-4 days, including increased osteolysis by histology, RANK ligand mRNA by PCR and osteoclast numbers by TRAP staining.To determine responsivness of MM to anti-myeloma drugs, cells were cultured with calvariae for 48 hours to establish engraftment, then the EVOCA was treated with 10 uM lenalidomide, 10 nM bortezomib, or 10nM carfilzomib. Anti-myeloma treatments attenuated the bone responses including reduction of RANKL. Primary MM cells from different patients varied in responsiveness to different drugs. Among 5 samples tested, primary cells obtained at the time of diagnosis were more responsive to anti-myeloma drugs in blunting induction of RANK ligand compared to MM cells obtained at the time of relapse.EVOCA, based on neonatal mouse calvariae supporting human MM cells, provides a useful platform for studies of myeloma bone disease. The assay can be run and analzyed in two weeks with 104CD138+ cells (freshly isolated or from frozen stock) per well, making it practical to determine individual patient sensitivity to a panel of currently available anti-myeloma drugs at initial diagnosis or at relapse. This could permit rational selection of personalized MM therapy. EVOCA could also be used to develop novel myeloma drugs that target tumor or bone by efficiently testing them in a physiologically relevant setting. Disclosures:No relevant conflicts of interest to declare.

Inhibition of the insulin-like growth factor-1 receptor enhances effects of simvastatin on prostate cancer cells in co-culture with bone.

Prostate cancer (PC) bone metastases show weak responses to conventional therapies. Bone matrix is rich in growth factors, with insulin-like growth factor-1 (IGF-1) being one of the most abundant. IGF-1 acts as a survival factor for tumor cells and we speculate that bone-derived IGF-1 counteracts effects of therapies aimed to target bone metastases and, consequently, that therapeutic effects could be enhanced if given in combination with IGF-1 receptor (IGF-1R) inhibitors. Simvastatin inhibits the mevalonate pathway and has been found to induce apoptosis of PC cells. The aims of this study were to confirm stimulating effects of bone-derived IGF-1 on PC cells and to test if IGF-1R inhibition enhances growth inhibitory effects of simvastatin on PC cells in a bone microenvironment. The PC-3 and 22Rv1 tumor cell lines showed significantly induced cell growth when co-cultured with neonatal mouse calvarial bones. The tumor cell IGF-1R was activated by calvariae-conditioned media and neutralization of bone-derived IGF-1 abolished the calvarium-induced PC-3 cell growth. Treatment of PC-3 and 22Rv1 cells with simvastatin, or the IGF-1R inhibitor NVP-AEW541, reduced tumor cell numbers and viability, and induced apoptosis. Combined simvastatin and NVP-AEW541 treatment resulted in enhanced growth inhibitory effects compared to either drug given alone. Effects of simvastatin involved down-regulation of IGF-1R in PC-3 and of constitutively active androgen receptor variants in 22Rv1 cells. In conclusion, we suggest that IGF-1 inhibition may be a way to strengthen effects of apoptosis-inducing therapies on PC bone metastases; a possibility that needs to be further tested in pre-clinical models.Electronic supplementary materialThe online version of this article (doi:10.1007/s12307-013-0129-z) contains supplementary material, which is available to authorized users.

Osteoclast progenitor cells present in significant amounts in mouse calvarial osteoblast isolations and osteoclastogenesis increased by BMP-2

Enzymatically released cells from neonatal mouse calvarial bones are frequently used as primary mouse osteoblasts for in vitro studies. We, here, report that although these cells lack mRNA expression of the osteoclastic genes Calcr, Acp5 and Mmp-9 at early time points after their isolation, these transcripts are gradually upregulated when the calvarial osteoblast cultures are differentiated to more mature osteoblasts in long term cultures. Similarly, Calcr, Acp5, Mmp-9, as well as Rank and Nfatc1 mRNA expressions are robustly increased when the osteoblast cultures were induced to differentiate by treatment with bone morphogenetic protein (BMP-2). The increased Calcr mRNA resulted in functionally active calcitonin receptors. Enhanced osteoblastic differentiation was associated with increased Rankl mRNA expression and decreased Opg and Cfs1 mRNA expression. Treatment of the osteoblastic cells with BMP-2 or RANKL, either on plastic dishes or bone slices, resulted in the formation of multinucleated tartrate-resistant acid phosphatase positive cells, which were able to excavate resorption pits and release CTX from the bones. In contrast, increased osteoblastic differentiation induced by BMP-2 in the mouse calvarial osteoblastic cell line MC3T3-E1 was not associated with increased mRNA expression of Calcr, Acp5, Rank, c-Fms or Oscar. Interestingly, Ctsk mRNA was increased during osteoblastic differentiation in both mouse calvarial osteoblast cultures and in MC3T3-E1 cultures. Also osteoblasts isolated from mouse long bones by outgrowth from explant cultures were contaminated with osteoclast progenitors able to differentiate into bone resorbing osteoclasts. These data indicate that mouse calvarial osteoblast cultures contain osteoclast progenitor cells, which will be differentiated along the osteoclastic lineage during osteoblastic differentiation. Moreover, the data show that BMP-2 not only stimulates osteoblastic differentiation but can also induce osteoclastogenesis through increased RANKL.

Stimulation of bone resorption in calvarial bones by toll-like2 receptor through enhanced rankl

BackgroundBone loss in inflammatory diseases like periodontitis, rheumatoid arthritis, septic arthritis and loosened joint prosthesis or tooth implants is being considered a consequence of cytokine induced RANKL and subsequent enhanced...

Retinoids Stimulate Periosteal Bone Resorption by Enhancing the Protein RANKL, a Response Inhibited by Monomeric Glucocorticoid Receptor

Increased vitamin A (retinol) intake has been suggested to increase bone fragility. In the present study, we investigated effects of retinoids on bone resorption in cultured neonatal mouse calvarial bones and their interaction with glucocorticoids (GC). All-trans-retinoic acid (ATRA), retinol, retinalaldehyde, and 9-cis-retinoic acid stimulated release of (45)Ca from calvarial bones. The resorptive effect of ATRA was characterized by mRNA expression of genes associated with osteoclast differentiation, enhanced osteoclast number, and bone matrix degradation. In addition, the RANKL/OPG ratio was increased by ATRA, release of (45)Ca stimulated by ATRA was blocked by exogenous OPG, and mRNA expression of genes associated with bone formation was decreased by ATRA. All retinoid acid receptors (RARα/β/γ) were expressed in calvarial bones. Agonists with affinity to all receptor subtypes or specifically to RARα enhanced the release of (45)Ca and mRNA expression of Rankl, whereas agonists with affinity to RARβ/γ or RARγ had no effects. Stimulation of Rankl mRNA by ATRA was competitively inhibited by the RARα antagonist GR110. Exposure of calvarial bones to GC inhibited the stimulatory effects of ATRA on (45)Ca release and Rankl mRNA and protein expression. This inhibitory effect was reversed by the glucocorticoid receptor (GR) antagonist RU 486. Increased Rankl mRNA stimulated by ATRA was also blocked by GC in calvarial bones from mice with a GR mutation that blocks dimerization (GR(dim) mice). The data suggest that ATRA enhances periosteal bone resorption by increasing the RANKL/OPG ratio via RARα receptors, a response that can be inhibited by monomeric GR.

Glucocorticoid Regulation of Osteoclast Differentiation and Expression of Receptor Activator of Nuclear Factor-κB (NF-κB) Ligand, Osteoprotegerin, and Receptor Activator of NF-κB in Mouse Calvarial Bones

In the present study, dexamethasone treatment of neonatal mouse calvarial bones increased mRNA expression of tartrate-resistant acid phosphatase, calcitonin receptor (CTR), cathepsin K, carbonic anhydrase II, osteoprotegerin (OPG), and receptor activator of nuclear factor-kappaB (RANK) as well as mRNA and protein expression of RANK ligand (RANKL). The increase in OPG mRNA noted with dexamethasone was in contrast to 1,25(OH)(2)-vitamin D3 (D3) treatment, which decreased OPG expression. Stimulation of (45)Ca release by dexamethasone and hydrocortisone in calvariae was blocked by OPG. Stimulation of RANKL, RANK, OPG, and CTR mRNA expression by dexamethasone in calvariae was blocked by the glucocorticoid receptor antagonist RU 38,486. Greater than additive potentiations of CTR mRNA and RANKL mRNA and protein were observed when D3 and dexamethasone were combined. Vitamin D receptor mRNA was increased by dexamethasone and D3, whereas glucocorticoid receptor (GR) mRNA was decreased by dexamethasone and unaffected by D3. No synergistic interaction between dexamethasone and D3 on either vitamin D receptor or GR mRNA expression was noted. The data demonstrate that dexamethasone-induced bone resorption in calvarial bones is associated with increased differentiation of osteoclasts and regulation of the RANKL-RANK-OPG system. The increase in OPG expression and the decrease of GR expression noted with dexamethasone offer an explanation for why bone breakdown in mouse calvariae treated with glucocorticoids is less than that caused by resorptive agents like D3. The synergistic stimulation of RANKL by dexamethasone and D3 offers an explanation of how glucocorticoids and D3 interact to potentiate bone resorption.

Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities.

Electric stimulation has been used successfully to treat a wide range of bone disorders. However, the mechanism by which the electric fields can influence the bone cells behavior remains poorly understood. The purpose of this research was to assess the possible mechanism of the stimulatory effect of pulsed electromagnetic field (PEMF) on bone cells. A PEMF with a frequency of 15 Hz (1 G [0.1 mT]; electric field strength 2 mV/cm) were applied to neonatal mouse calvarial bone cell cultures for 14 days. The temporal effects of PEMF on the osteoblasts were evaluated by the status of proliferation, differentiation, mineralization, and gene expression on the 3rd, 5th, 7th, and 14th days of culture. Our results demonstrated that PEMF stimulation significantly increased the osteoblasts' proliferation by 34.0, 11.5, and 13.3% over the control group after 3, 5, and 7 days' culture. Although the alkaline phosphatase (ALP) staining and the mineralization nodules formation did not change, the ALP activity of the bone cells decreased significantly after PEMF stimulation. Under the PEMF stimulation, there was no effect on the extracellular matrix synthesis, while the osteoprotegerin (OPG) mRNA expression was up regulated and the receptor activator of NF-kappaB ligand (RANKL) mRNA expression were down regulated, compared to the control. In conclusion, the treatment by PEMF of osteoblasts may accelerate cellular proliferation, but did not affect the cellular differentiation. The effect of PEMF stimulation on the bone tissue formation was most likely associated with the increase in the number of cells, but not with the enhancement of the osteoblasts' differentiation.

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1α,25-dihydroxyvitamin D 3 and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 μM) completely inhibited the calcium release induced by IL-1β, TNF-α, and LPS. The resorptive effect of 1α,25-dihydroxyvitamin D 3 was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E 2. Celecoxib (0.1 μM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE 2 in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1β, TNF-α, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.

Bone resorbing activity released from zymosan-activated mouse peritoneal macrophages - the role of prostanoids and interleukin-1

To study the effect of zymosan on the release of osteoclast stimulating activity from macrophages. Calvarial bones from neonatal mice and peritoneal macrophages were incubated in the absence and presence of zymosan for 72 h and supernatants harvested for subsequent analysis of bone resorbing activities and prostaglandin concentration. Bone resorption was assessed in vitro by analysing the release of 45Ca and 3H from neonatal mouse calvarial bones prelabelled in vivo by injections of [45Ca]CaCl2 or [3H]-proline. Prostaglandin E2 (PGE2) and I2 (PGI2) were analyzed using radioimmunoassays. Supernatants from macrophages treated with zymosan stimulated the release of 45Ca and 3H. The amount of bone resorbing activity present in the macrophage supernatants was dependent on the concentration of zymosan (0.1-100 microg/ml), as well as the number of macrophages present. The 45Ca release induced by zymosan treated macrophages was inhibited by three different inhibitors of osteoclastic bone resorption (calcitonin, acetazolamide, amino bisphosphonate). The bone resorbing activity released by the zymosan-activated macrophages was lost after ultrafiltration using a filter with a molecular weight cut off of 30,000 Daltons, but retained when using a filter with a cut off of 3000 Daltons. Time-course studies of the production of bone resorbing activity in macrophages showed that activity increased during the first hour of exposure to zymosan and then reached a plateau for 96 h. PGE2 and PGI2 release from macrophages was increased during the first three hours of exposure to zymosan. This prostanoid production, together with bone resorbing activity, was abolished by indomethacin. The bone resorbing activity present 3-72 h after zymosan exposure, however, was not inhibited by indomethacin. Bone resorption stimulated by conditioned media from zymosan treated macrophages after 3 h was inhibited by 60-75% in the presence of anti IL-1alpha, 0-20% by anti IL-1beta, and completely by antisera neutralizing both IL-1alpha and IL-1beta. In addition, an IL-1 receptor antagonist abolished the stimulatory effect of conditioned media from zymosan treated macrophages. These data indicate that treatment of mouse peritoneal macrophages with zymosan results in production of activities capable of stimulating bone resorption in vitro. The activity released initially appears to be due to a zymosan induced burst of prostanoid production, while the activity released during prolonged exposure to zymosan is due primarily to IL-1alpha.

Gutta-percha-stimulated mouse macrophages release factors that activate the bone resorptive system of mouse calvarial bone.

This study investigated the capacity of macrophages exposed to gutta-percha particles to produce factors affecting bone metabolism. Peritoneal mouse macrophages were isolated and incubated with and without gutta-percha particles, and the supernatants were assessed for bone resorbing activity by adding macrophage-conditioned media to cultures of neonatal mouse calvarial bones. Bone resorption was measured by mineral mobilization (45Ca release) and matrix degradation (3H from [3H]proline labelled bones). The results showed that supernatant from gutta-percha-stimulated macrophages enhanced bone resorption. This effect was related to the amount of gutta-percha, and the concentration and time of exposure to the conditioned media. Stimulated macrophages released enhanced amounts of prostaglandins E2 and I2; however, indomethacin, which inhibits the prostanoid response, had no effect on bone resorbing activity. The stimulatory effect on bone resorption was inhibited by calcitonin, interleukin-1 receptor antagonistic protein, and by antiserum neutralizing mouse interleukin-1alpha(IL-1alpha), but not by anti-IL-1beta. Filtration experiments revealed that the molecules involved in the resorption activity had an apparent molecular weight between 3000 and 30,000 Da. These experiments show that mouse peritoneal macrophages, when exposed to gutta-percha particles, release factors which have a bone resorbing activity that is primarily due to enhanced production of IL-1alpha.

The cellular actions of interleukin-11 on bone resorption in vitro.

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.

Bone resorption induced by A23187 is abolished by indomethacin: implications for second messenger utilised by parathyroid hormone

Parathyroid hormone acts on the osteoblast to induce osteoclastic bone resorption. Parathyroid hormone utilises cyclic AMP as a second messenger in osteoblasts, but may also cause an increase in cytoplasmatic free calcium ions ([Ca 2+] i) in the same cell. To investigate the role of osteoblastic [Ca 2+] i in the induction of bone resorption, we have compared the effects of parathyroid hormone and the Ca 2+-ionophore, A23187, as well as the adenylate cyclase stimulating agent, forskolin, and the phorbol ester, phorbole 12,13 dibutyrate (PDB), on bone resorption in neonatal mouse calvarial bones. Parathyroid hormone (0.1 and 1 nM) dose dependently stimulated the release of prelabelled 45Ca 2+ in 72 h culture. Parathyroid hormone-induced bone resorption was not affected by the addition of 1 μM indomethacin to the incubation media, and was therefore, not mediated by local prostaglandin formation. A23187 stimulated the release of 45Ca 2+ at 1–10 nM. Above 100 nM, A23187 inhibited bone resorption. The A23187 (3 and 10 nM)-induced bone resorption was abolished by the cyclooxygenase inhibitor, indomethacin (1 μM), indicating that the stimulatory effect was mediated via prostaglandin formation. The adenylate cyclase stimulating agent, forskolin, dose dependently stimulated bone resorption at and above 1 μM. There was no additive or synergistic effect of forskolin and A23187 on 45Ca 2+ release. Forskolin-induced bone resorption was, as with parathyroid hormone but in contrast to ionophore-induced bone resorption, not abolished by indomethacin (1 μM). The protein kinase C activator, PDB, at 10 and 1000 nM stimulated the release of prelabelled 45Ca 2+. The stimulatory effect of the protein kinase C stimulating phorbol ester, PDB, on bone resorption was abolished by the addition of indomethacin. In summary, bone resorption induced by a Ca 2+-ionophore is abolished by indomethacin. This indicates that bone resorbing agents known to increase [Ca 2+] i subsequently enhance local prostaglandin formation. Bone resorption induced by the protein kinase C activator, PDB, was also abolished by indomethacin, whereas, forskolin and parathyroid hormone-induced bone resorption was unaffected. These data indicate that cyclic AMP, but not [Ca 2+] i, is involved as a second messenger in parathyroid-induced bone resorption.

Prostaglandin-Independent Stimulation of Bone Resorption in Mouse Calvariae and in Isolated Rat Osteoclasts by Thyroid Hormones (T4, and T3)

The thyroid hormones, thyroxine (T4) and triiodothyronine (T3), were found to enhance both neonatal mouse calvarial bone resorption and pit formation on bovine slices by isolated rat osteoclasts. Dosage-dependent release of 45Ca from mouse calvarial bones was observed after 120 hr of culture with 10(-6)-10(-8) MT4 and 10(-6)-10(-10) M T3. Maximum treatment/control ratios of 45Ca release were recorded for 10(-7) M T4 and 10(-8) MT3. Inhibition of 45Ca release stimulated by 10(-8) M T3 was observed in the presence of 30 nM salmon calcitonin at 48 hr and 120 hr of culture with no indication of "escape" by T3-treated bones. In contrast, stimulation of 45Ca release from mouse calvarial bones by 10(-7) MT4 and 10(-8) MT3 was not inhibited by 10(-6) M indomethacin. Formation of PGE2 and PGI2 (evaluated by measuring 6-keto-PGF1alpha) by mouse calvariae was also not increased by 10(-8) MT3 after 120 hr of culture. Furthermore, no increases in cAMP formation were observed in calvarial bone cultures after either 10 min or 24 hr of exposure to 10(-8) MT3. However, significant inhibition of 45Ca release stimulated by 10(-8) M T3 was found at 120 hr in the presence of 10(-3) M hydroxyurea. When isolated rat osteoclasts were cultured in the presence of 10(-7) MT3, a 1.4-fold stimulation of pit number was observed. Pit formation was not affected by addition of 10(-6) M indomethacin to either the control or T3-treated cultures. These data suggest that the stimulation of bone resorption in neonatal mouse calvariae and activation of isolated rat osteoclasts by the thyroid hormones is not related to either prostaglandin or cAMP formation. In mouse calvariae, the effect on bone resorption of the thyroid hormones is dependent on increased cellular replication, perhaps of osteoclast precursors, or other bone cells involved in the resorptive process.

Insulin-like growth factor I does not stimulate bone resorption in cultured neonatal mouse calvarial bones.

Insulin-like growth factor I (IGF-I) has documented anabolic effects on osteoblasts, whereas its influence on osteoclasts and on bone resorption is unclear. We have investigated the effects of IGF-I on osteoclast recruitment and bone resorption in vitro. IGF-I (at and above 1 nM) stimulated the formation of multinucleated tartrate-resistant acid phosphatase positive cells in murine bone marrow cultures, incubated for 9 days. The number of multinucleated cells increased to 540 +/- 160% of control (mean +/- SEM) in cultures treated with 10 nM IGF-I. IGF-I (0.1-100 nM) had no effect by itself on 45Ca-release from prelabelled neonatal mouse calvarial bones. However, IGF-I (100 nM) had an inhibitory effect on bone resorption induced by prostaglandin E2 and 1,25(OH)2D3. These findings indicate that IGF-I enhances the formation of osteoclasts-like cells in long-term bone marrow cultures. In bone organ cultures, however, IGF-I has an inhibitory effect on stimulated bone resorption, suggesting that IGF-I inhibits existing osteoclasts and, alternatively, that IGF-I interferes with the osteoblast-derived factor(s) that stimulate existing osteoclasts.

Stimulation of neonatal mouse calvarial bone resorption by the glucocorticoids hydrocortisone and dexamethasone.

In vitro stimulation of bone resorption was observed with the glucocorticoids hydrocortisone and dexamethasone. Dosage-dependent release of 45Ca from neonatal mouse calvarial bones was found for both steroids, with half-maximal responses for hydrocortisone and dexamethasone of 0.3 and 0.08 microM, respectively. Significant release of stable calcium (Ca2+), inorganic phosphate (Pi), and the lysosomal enzyme beta-N-acetylglucosaminidase was noted following treatment of mouse calvariae with either 1 microM hydrocortisone or 1 microM dexamethasone. Additionally, both 1 microM hydrocortisone and 1 microM dexamethasone elicited release of 3H from calvarial bones prelabeled with [3H]proline. The stimulation of bone resorption by the glucocorticoids, as assessed by 45Ca release, was sustained over 120 h of culture. Inhibition of 45Ca release from calvariae treated with either 1 microM hydrocortisone or 0.1 microM dexamethasone was observed with 0.01-30 nM salmon calcitonin (sCT), 0.1 mM acetazolamide, and 0.1 mM of the bisphosphonate AHPrBP. Inhibition of glucocorticoid-induced bone resorption by sCT occurred without "escape from calcitonin-induced inhibition." The 45Ca release stimulated by 1 microM hydrocortisone and 0.1 microM dexamethasone was also inhibited by 10 microM progesterone in a competitive manner and by 1 microM of the antiglucocorticoid RU38486, both of which are modulators of glucocorticoid binding. Prostaglandin E2 (PGE2) formation by 10 nM parathyroid hormone (PTH) in neonatal mouse calvarial bones was inhibited by both 1 microM hydrocortisone and 1 microM dexamethasone, but neither compound altered basal PGE2 formation. Exposure of calvarial bones to the mitotic inhibitors hydroxyurea and mitomycin C inhibited 45Ca release stimulated by 1 microM hydrocortisone and 1 microM dexamethasone. In contrast, addition of 1 ng/ml of recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF) had no effect on 45Ca release elicited by the glucocorticoids. These results suggest that hydrocortisone and dexamethasone stimulate osteoclastic resorption in neonatal mouse calvariae by a receptor-mediated mechanism that is dependent on cellular replication.

Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro

In a previous study of the cellular mechanism underlaying Wolff's law we showed that mechanical stimulation by intermittent hydrostatic compression (IHC) increases bone formation in cultured fetal mouse calvariae compared to non-stimulated cultures. To test whether mechanical stimuli may modulate bone-specific gene expression, we studied the effect of IHC on alkaline phosphatase (AP) expression and enzyme activity as well as collagen and actin mRNA levels in neonatal mouse calvariae and calvarial bone cells. Two cell populations, one resembling osteoprogenitor (OPR) cells and another resembling osteoblasts (OB) were obtained from calvariae by sequential digestion. IHC was applied by intermittently (0.3 Hz) compressing the gas- phase of a closed culture chamber (peak stress 13 kPa, peak stress rate 32.5 kPas −1). In control cultures of calvariae as well as OB and OPR cells, AP activity and AP-, collagen-, and actin-mRNA levels all decreased after one or more days, with the exception of OPR cell collagen expression which increased during culture. IHC treatment upregulated AP, collagen and actin expression and AP activity in calvariae and OB cells, but decreased collagen expression in OPR cells. These results suggest that treatment with IHC promotes the osteoblastic phenotype in bone organ cultures and in osteoblasts. Osteoprogenitor cells seem to react somewhat differently to mechanical stress than osteoblasts. The loss of bone-specific gene expression under control culture conditions, in the absence of mechanical stimuli, suggests that the mechanical environment is important in maintaining the differentiated phenotype of bone cells, and that IHC treatment partially restores this environment in bone cell- and organ cultures.

Studies on two new vitamin D analogs, EB 1089 and KH 1060: Effects on bone resorption and osteoclast recruitment in vitro

We have investigated the effects on bone resorption of two new potent antiproliferative vitamin D3 analogs, EB 1089 and KH 1060, by studying recruitment of osteoclasts in murine bone marrow cultures and 45Ca release from prelabeled neonatal mouse calvarial bones. Binding studies to vitamin D receptor protein, from human osteosarcoma MG-63 cells, demonstrated k d values of 8.5 × 10 −11 for 1α,25(OH) 2D 3, 6.5 × 10 −11 for KH 1060, and 2.7 × 10 −10 for EB 1089. 1α,25(OH) 2D 3 and EB 1089 were equipotent stimulators of osteoclast recruitment in murine bone marrow cultures, with EC 50 at 10 −10 mol/L, whereas KH 1060 was about tenfold more potent with an EC 50 at 10 −11 mol/L. In serum-free media, 1α,25(OH) 2D 3 enhanced 45Ca release from neonatal mouse calvarial bones with EC 50 at 10 −11 mol/L, but in the presence of 10% fetal calf serum (FCS) the stimulatory effect was significantly diminished, with a threshold value at 10 −10 mol/L. EB 1089 stimulated bone resorption with an estimated EC 50 at 3 × 10 −11 mol/L, whereas KH 1060 was about tenfold more potent than 1α,25(OH) 2D 3, and stimulated bone resorption with an EC 50 at 10 −12 mol/L. The effects of EB 1089 and KH 1060 on 45Ca release were not significantly affected by the addition of 10% FCS. Addition of vitamin D binding protein to serum-free incubations of neonatal mouse calvarial bones significantly inhibited the bone resorbing effect of 1α,25(OH) 2D 3, but did not affect EB 1089 and KH 1060 induced 45Ca release. These data show that the k d values for the binding to Vitamin D receptors are similar to the EC 50 values for stimulation of bone resorption and osteoclast differentiation, in vitro, for 1α,25(OH) 2D 3, EB 1089, and KH 1060. The discrepancy in the effect of FCS on 45Ca release between 1α,25(OH) 2D 3, and the new analogs suggests that EB 1089 and KH 1060 do not bind to vitamin D binding protein, indicating that pharmacokinetic differences may partially explain their less calcemic effects in vivo.

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by 45Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.

Osteoblasts mediate insulin-like growth factor-I and -II stimulation of osteoclast formation and function.

Insulin-like growth factor-I (IGF-I) and IGF-II have powerful, well defined effects on osteoblastic cells, stimulating their proliferation and inducing collagen synthesis, but the role of IGF-I and -II in modulating osteoclast differentiation and activity remains unclear. We first examined the bone-resorptive effects of IGF-I and IGF-II by assessing 45Ca2+ release from neonatal mouse calvarial bones. Both IGFs dose dependently stimulated bone resorption, with an EC50 of 8 x 10(-9) M for IGF-I and 2 x 10(-8) M for IGF-II. We then tested the effects of the IGFs on bone resorption by rat isolated osteoclasts cultured on ivory slices. Neither IGF-I nor IGF-II stimulated isolated osteoclast activity. However, in the presence of either primary mouse osteoblasts or human osteosarcoma MG 63 cells, both IGFs enhanced osteoclast resorptive activity, with an EC50 of 5 x 10(-10) M for IGF-I and 10(-9) M for IGF-II. Stimulation was not mediated by BALB/c/3T3 cells, a nonosteoblastic cell line. The effects of the IGFs were blocked by alpha IR-3, an antibody to the type I IGF receptor, but not by beta-galactosidase, a lysosomal enzyme that competes with IGF-II for the type II IGF receptor. We then examined the effects of the IGFs on the formation of osteoclast-like multinucleate cells (MNCs) in mouse bone marrow cultures. IGF-I and -II dose dependently increased the number of tartrate-resistant acid phosphatase (TRAP)-positive MNCs, although their effects were less than that of 1,25-dihydroxyvitamin D3 (a hormone that induces osteoclast differentiation). No TRAP-positive MNCs appeared in the absence of these hormones. Like authentic osteoclasts, the TRAP-positive MNCs formed in response to IGF-I and -II bound [125I]salmon calcitonin. When mouse bone marrow cells were cultured on ivory slices in the presence of either IGF-I or IGF-II for 10 days, numerous resorption lacunae were formed. beta-Galactosidase had no effect on IGF-mediated osteoclast formation. These results are strong evidence that both IGF-I and IGF-II stimulate bone resorption in vitro by enhancing osteoclast formation and function. Our data also suggest that the IGFs act through the intermediary of osteoblastic cells to stimulate osteoclast activity and that the type I, but not the type II, IGF receptor is involved in their responses. We propose that the local production of IGF-I and IGF-II may modulate both osteoblast-osteoclast interactions and osteoclast formation and play an important role in bone remodeling.

Helodermin, helospectin, and PACAP stimulate cyclic AMP formation in intact bone, isolated osteoblasts, and osteoblastic cell lines

Helodermin and helospectin are peptides structurally similar to vasoactive intestinal polypeptide (VIP) which were recently isolated from the salivary gland venom of the lizard Heloderma suspectum. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been isolated from ovine hypothalamus and also shows sequence homology to VIP. A helodermin-like peptide has been detected by combined immunohistochemical and immunochemical techniques in the thyroid C-cells. In the present study, lizard helodermin was found to cause a time- and dose-dependent stimulation of cyclic AMP (cAMP) formation in neonatal mouse calvarial bones. Also, helospectin I, PACAP 27, and the C-terminally extended PACAP 38 stimulated cAMP accumulation in the mouse calvariae. The cAMP rise in response to helodermin was comparable to that induced by VIP, both in terms of potency and magnitude of the response. Helodermin, helospectin I, PACAP 27, and PACAP 38, at concentrations of 1 mumol/liter, stimulated cAMP accumulation in enzymatically isolated mouse calvarial bone cells. A significant response to all peptides was observed in both early and late released bone cells isolated from the calvariae, with low and high alkaline phosphatase activity, respectively. Helodermin and VIP stimulated cAMP accumulation in the cloned mouse calvarial osteoblastic cell line MC3T3-E1, in rat (UMR 106-01), and human (Saos-2) osteoblastic osteosarcoma cell lines, but not in the rat osteosarcoma cell line ROS 17/2.8. The effect of helodermin was synergistically and dose-dependently enhanced by forskolin (0.1 and 1 mumol/liter). These data show that bone cells, including osteoblasts, respond to several peptides of the VIP family, including helodermin, helospectin I, PACAP 27, and PACAP 38.(ABSTRACT TRUNCATED AT 250 WORDS)