Cocultures Of Bone Marrow Cells Research Articles

Harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo

Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive bone resorption is involved in the pathogenesis of bone-related disorders such as osteoporosis, arthritis and periodontitis. To obtain new antiresorptive agents, we searched for natural compounds that can inhibit osteoclast differentiation and function. We found that harmine, a β-carboline alkaloid, inhibited multinucleated osteoclast formation induced by receptor activator of nuclear factor-κB ligand (RANKL) in RAW264.7 cells. Similar results were obtained in cultures of bone marrow macrophages supplemented with macrophage colony-stimulating factor and RANKL, as well as in cocultures of bone marrow cells and osteoblastic UAMS-32 cells in the presence of vitamin D 3 and prostaglandin E 2. Furthermore, harmine prevented RANKL-induced bone resorption in both cell and bone tissue cultures. Treatment with harmine (10 mg/kg/day) also prevented bone loss in ovariectomized osteoporosis model mice. Structure–activity relationship studies showed that the C3–C4 double bond and 7-methoxy group of harmine are important for its inhibitory activity on osteoclast differentiation. In mechanistic studies, we found that harmine inhibited the RANKL-induced expression of c-Fos and subsequent expression of nuclear factor of activated T cells (NFAT) c1, which is a master regulator of osteoclastogenesis. However, harmine did not affect early signaling molecules such as ERK, p38 MAPK and IκBα. These results indicate that harmine inhibits osteoclast formation via downregulation of c-Fos and NFATc1 induced by RANKL and represses bone resorption. These novel findings may be useful for the treatment of bone-destructive diseases.

OA/Gpnmb Mutation Impairs Osteoblast and Osteoclast Differentiation and Function

Osteoactivin/Gpnmb (OA/Gpnmb) is a glycoprotein identified in bone, and later found to be strongly localized to bone‐forming osteoblasts. There exists in mice a naturally occurring mutation for OA/Gpnmb, which is attributable to premature stop codon resulting in a truncated 150 amino acid OA/Gpnmb protein. Micro‐CT analysis of aged mice show decreased bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb. Th.) in OA/Gpnmb mutant mice when compared to wild‐type, implying that OA/Gpnmb mutant mice are more osteoporotic. Immunoflourescent analysis has shown OA/Gpnmb protein in mutant osteoblasts (OB) is retained in the peri‐nuclear/cis‐Golgi area. Electron microscopy analysis of osteoblasts showed no morphological differences in mutant osteoblasts versus control, suggesting OA/Gpnmb retention is not cytotoxic. qPCR mRNA and western blot analyses showed increased RANKL expression in OB from OA/Gpnmb mutant. Co‐culture of bone marrow cells showed marked increase in osteoclast (OC) numbers and size in mutant cultures when compared to WT. Treatment of WT and Mutant osteoblasts with recombinant OA/Gpnmb protein resulted in enhanced osteoclast number and size in vitro in WT and but not in mutant OB, suggesting an inherent defect to in mutant compared to WT OC. Collectively, these data suggest OA/Gpnmb acts as a regulator of osteoblast‐osteoclast differentiation and function.Grant Funding Source: NIAMS

Trichostatin A inhibits osteoclastogenesis and bone resorption by suppressing the induction of c-Fos by RANKL

Histone deacetylases are enzymes involved in the remodeling of chromatin structure, in the regulation of transcriptional activity, and in epigenetic integrity. Histone deacetylase inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) have emerged as potent anticancer drugs that have proved useful in preclinical and early clinical trials. The role of histone deacetylase inhibitors in regulating osteoclast differentiation, however, is not well established. In this study, we analyzed the effects of TSA on osteoclast differentiation induced by the differentiation factor RANKL (receptor activator of NF-κB ligand). TSA strongly inhibited osteoclast formation in coculture of bone marrow cells and osteoblasts without reducing RANKL expression in osteoblasts. Furthermore, TSA suppressed RANKL-induced osteoclast formation from primary bone marrow-derived macrophages. TSA was only effective when present during the early stage of osteoclast differentiation. This effect was accompanied by a significant decrease in the RANKL-stimulated induction of c-Fos and NFATc1, which are key transcription factors during early osteoclastogenesis. The ectopic introduction of c-Fos and a constitutively active form of NFATc1 reversed the TSA-induced antiosteoclastogenic effect. Consistent with the in vitro results, TSA inhibited lipopolysaccharide- and interleukin-1-induced bone resorption and osteoclast formation in an in vivo model. Taken together, our findings suggest a novel action of TSA: inhibiting RANKL-induced osteoclast formation by suppressing the induction of the osteoclastogenic transcription factor c-Fos. Also, the inhibitory effect of TSA on bone destruction in vivo suggests that histone deacetylase inhibitors may be novel therapeutics for treating typical bone diseases.

Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK‐mediated signaling

Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast-like cells were induced in the co-cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti-osteoprotegerin (OPG) antibody was added to the co-cultures. mRNA levels of receptor activator of nuclear factor-kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D(3). Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force-induced OPG up-regulation. Inhibition of extracellular signal-regulated kinase (ERK) signaling by a pharmacological inhibitor (10 microM PD98059) or by siERK transfection suppressed the force-induced OPG up-regulation along with the augmentation of osteoclast-like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation.

Risedronate Directly Inhibits Osteoclast Differentiation and Inflammatory Bone Loss

Risedronate, a nitrogen-containing bisphosphonate, is widely used in the clinical field for the treatment of osteoporosis. Risedronate is known to exert its effects through binding to hydroxyapatite in bone tissue, inhibiting osteoclastic activity, and inducing apoptosis of osteoclasts. The purpose of this study was to determine the effects of risedronate on osteoclast differentiation in vitro and on an inflammatory bone loss model in vivo. Risedronate inhibited osteoclast differentiation in co-culture of bone marrow cells (BMCs) and osteoblasts, and suppressed receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from bone marrow-derived macrophages (BMMs) in a dose-dependent manner without toxicity. Risedronate significantly inhibited expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 induced by RANKL. To examine the effect of risedronate on bone loss in vivo, we used a mouse model of lipopolysaccharide (LPS)-mediated bone loss. Micro-CT analysis of the femurs showed that LPS treatment caused bone loss. However, bone loss was significantly attenuated in mice administered with risedronate. Taken together, we conclude that risedronate exerts beneficial effects on osteoporosis by inhibiting osteoclast differentiation both directly and indirectly. In infectious conditions, the inhibitory effect of risedronate on bone erosion was excellent. Thus risedronate could be a treatment option for osteoporosis caused by inflammatory and infectious conditions.

Pyridone 6, A Pan-Janus-Activated Kinase Inhibitor, Suppresses Osteoclast Formation and Bone Resorption through Down-Regulation of Receptor Activator of Nuclear Factor-.KAPPA.B (NF-.KAPPA.B) Ligand (RANKL)-Induced c-Fos and Nuclear Factor of Activated T Cells (NFAT) c1 Expression

It has been reported that Janus tyrosine kinase (JAK)-dependent signaling pathways play a critical role in the pathogenesis of numerous malignancies and immune reactions, and inhibition of JAK has been implicated in cell growth inhibition. The role which JAK has on osteoclast differentiation and anti-bone resorptive activity is not well understood. In this study, we investigated the effects of a pan-JAK inhibitor, pyridone 6, on osteoclast differentiation and bone-resorption in vitro and ex vivo. Pyridone 6 inhibited osteoclast differentiation in mouse bone marrow macrophage (BMM) cultures stimulated by the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and co-cultures of bone marrow cells and osteoblasts. Pyridone 6 suppressed the expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 in BMMs. It also inhibited the bone resorptive activity of mature osteoclasts that was accompanied by disruption of actin rings. Pyridone 6 also suppressed I-kappaB degradation and extracellular signal-regulated kinase (ERK) in mature osteoclasts, suggesting that these are the key molecules that pyridone 6 targets in the inhibition of osteoclast function. These results demonstrate inhibition of JAK may be useful for the treatment of bone-resorptive diseases, such as osteoporosis.

Naringin Suppresses Osteoclast Formation and Enhances Bone Mass in Mice

Naringin is a flavonoid commonly found in citrus fruits. Previous studies have supported a positive association between fruit consumption and health including bone quality. To clarify the role of naringin in bone turnover, we examined the effect of naringin on bone mass in mice, and on osteoclast formation induced by bone-resorbing cytokine. When mice were orally administered naringin for 2 weeks, femoral bone mineral density (BMD) was clearly elevated compared with control mice. BMD in distal and middle portion was significantly enhanced, but proximal BMD was not changed by naringin administration. In soft-X-ray analysis, a marked accumulation of cancellous bone could be detected in distal femoral metasphysis in mice treated with naringin. In cocultures of bone marrow cells and osteoblasts, naringin dose-dependently suppressed the number of osteoclasts formed by treatment with interleukin-1 (IL-1). The size of osteoclasts formed in the presence of naringin was smaller than that induced by IL-1. Naringin enhances bone mass possibly by suppression of osteoclast formation in mice, suggesting the role of the citrus flavonoid on bone health and to prevent bone diseases such as osteoporosis.

Tanshinone IIA suppresses inflammatory bone loss by inhibiting the synthesis of prostaglandin E 2 in osteoblasts

Tanshinone IIA isolated from Danshen is widely used in Oriental medicine. However, the action of tanshinone IIA in inflammatory bone-resorptive diseases remains unknown. Here we examined the effect of tanshinone IIA in inflammation-mediated osteoclastic bone resorption. Tanshinone IIA inhibited osteoclast differentiation in cocultures of bone marrow cells and calvarial osteoblasts. Tanshinone IIA regulated the expression of receptor activator of NF-κB ligand and osteoprotegerin in osteoblasts treated with lipopolysaccharide (LPS). Also, tanshinone IIA inhibited prostaglandin E 2 (PGE 2) synthesis by inhibiting Cyclooxygenase-2 (COX-2) expression induced by LPS. Furthermore, tanshinone IIA greatly suppressed bone loss in the mouse models of bone loss. Our findings suggest that tanshinone IIA inhibits osteoclast formation by inhibiting COX-2/PGE 2 signaling and by suppressing bone erosion in vivo. These results suggest that tanshinone IIA may be of therapeutic value as an anti-bone-resorptive drug in the treatment of bone-related disease.

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Skeletal remodeling is a finely orchestrated process coupling bone formation to bone resorption. The dynamics of coupling is regulated by the microenvironment at the bone remodeling site, which in turn is influenced by the intercellular communication between cells like osteoclasts and osteoblasts. Understanding the dynamics of coupling is important in devising new therapeutic approaches to the treatment of skeletal diseases characterized by disturbances in the bone remodeling process. In this study, we report the localization of bone morphogenetic proteins (BMPs) in osteoclasts generated from primary cocultures of bone marrow cells from mouse femur and tibia with mouse calvarial osteoblasts, using immunocytochemistry and in situ hybridization. Positive staining was seen in osteoclasts for BMP-2, -4, -6, and -7. Real-time PCR was used to quantitatively confirm the expression of transcripts for BMP-2, BMP-4, and BMP-6 mRNA in murine osteoclasts. Finally, the presence of BMP-2, -4, -6, and-7 proteins was confirmed in osteoclast lysates by Western blotting. Overall, our data suggest a possible direct role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites.

Functions of Notch signaling-dependent Thy1-expressing dendritic cells in immune response (B164)

Abstract Dendritic cells (DCs) are one of key regulators in immune response through antigen presentation and producing several cytokines. Although recent studies have shown that several DC subsets have different phenotypes and functions, it is still unclear how differentiation of various DC subsets is regulated and determined. We here investigated whether Notch signaling contributes to DCs development and their functions. Thy1-expressing DCs (Thy1-DCs) dominantly differentiated by co-culture of bone marrow cells and OP9 cells overexpressing Delta1 in the presence of GM-CSF. Thy1-DCs had different phenotypes from those of reported DCs and developed in Notch signaling dependent manner. The Thy1 expressing DCs were also found in vivo and the proportion of Thy1-DCs was markedly increased in both spleen and liver of mice infected with Trypanosoma cruzi (T.cruzi). The Thy1-DCs from mice infected with T. cruzi were capable of producing high amount of IFN-gamma. Considering that IFN-gamma is a crucial cytokine for excluding T.cruzi, Notch signaling might have an important role in controlling T.cruzi infection by expanding Thy1-DCs.

Prostaglandin E receptor EP4 antagonist suppresses osteolysis due to bone metastasis of mouse malignant melanoma cells

We examined the effects of prostaglandin E (PGE) receptor subtype EP4 antagonist on bone metastasis of cancer to clarify PGE’s role in bone metastasis. Metastatic regions were detected in femurs accompanying severe bone loss in mice injected with B16 malignant melanoma cells. Administration of EP4 antagonist restored the bone loss induced by B16 melanoma. Adding B16 cells induced osteoclast formation in the coculture of bone marrow cells and osteoblasts without any exogenous bone-resorbing factor, and EP4 antagonist completely suppressed the osteoclast formation induced by B16 cells. Therefore, EP4 antagonist is a possible candidate for the therapy of bone metastasis of cancer.

Expression of Mouse Osteoclast K-Cl Co-Transporter-1 and Its Role During Bone Resorption

To assess the role of Cl- transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl- co-transporters (KCCs). KCC1 and chloride channel-7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)-butylindazone (DIOA), KCC1 antisense oligo-nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl- extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl- extrusion mechanism accompanying the H+ extrusion during bone resorption. Mice with deficient chloride (Cl-) channels, ClC7, show severe osteopetrosis, resulting from impairment of Cl- extrusion during osteoclastic bone resorption. However, the expression and functional role of Cl- transporters other than ClC7 in mammalian osteoclasts is unknown. The aim of this study was to determine expression of K+/Cl- co-transporters (KCCs) and their functional role for bone resorption in mouse osteoclasts. Mouse osteoclasts were derived from cultured bone marrow cells with macrophage-colony stimulating factor (M-CSF) and RANKL or from co-culture of bone marrow cells and primary osteoblasts. We examined the expression of Cl- transporters using RT-PCR, immunochemical, and Western blot methods. The effects of Cl- transport inhibitors on H+ and Cl- extrusion were assessed by measuring intracellular H+ ([H+]i) and Cl- ([Cl-]i). The effects of inhibitors, antisense oligo-nucleotides, and siRNA for Cl- transporters on bone resorption activities were evaluated using a pit formation assay. Mouse osteoclasts express not only ClC7 but also K+/Cl- co-transporter mRNA. The existence of KCC1 in the cell membrane of mouse osteoclasts was confirmed by immunochemical staining and Western blot analysis. KCC inhibitors and Cl- channels blockers increased [Cl-]i and [H+]i in resorbing osteoclasts, suggesting that the suppression of Cl- extrusion through KCC and Cl- channels leads to reduced H+ extrusion activity. The combination of both inhibitors greatly suppressed these extrusion activities. KCC inhibitors and Cl- channel blockers also decreased osteoclastic bone resorption in our pit area essay. Furthermore, KCC1 antisense oligo-nucleotides and siRNA suppressed osteoclastic pit formation as well as treatment of ClC7 inhibitors. These results indicate that K+/Cl- co-transporter-1 expressed in mouse osteoclasts acts as a Cl- extruder and plays an important role for H+ extrusion during bone resorption.

Transgenic Mice Overexpressing Macrophage Migration Inhibitory Factor (MIF) Exhibit High-Turnover Osteoporosis

The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP-3, -9, and -13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo. The role of macrophage migration inhibitory factor (MIF) in in vivo bone remodeling remains unelucidated. We describe disordered bone metabolism in transgenic mice overexpressing MIF. For in vivo study, muCT, bone histomorphometry, blood and urine biochemical data, and gene expression of MIF transgenic (MIF Tg) mice and littermate wildtype (WT) mice were examined. For in vitro study, osteoclastogenesis in the co-culture of bone marrow cells and osteoblasts from MIF Tg and WT were assessed. muCT analyses revealed a significant reduction in the trabecular bone of distal femur in MIF Tg at 8-12 weeks of age. Histomorphometric analysis revealed increase in several measures of bone formation. Osteoclastogenesis was not influenced by the origin of bone marrow cells or osteoblasts. Urine level of deoxypyridinoline/creatinine and the mRNA levels of matrix metalloproteinase (MMP) -3, -9, and -13 in femurs were elevated in MIF Tg. Overexpression of MIF causes high-turnover osteoporosis in mice. The increased expression of MMPs in bone was suggested, at least in part, as one cause of this phenotype, because MMPs plays important roles for bone resorption without affecting the formation of osteoclasts. This model provides evidence of the role played by MIF in bone remodeling and balance.

Effect of Monoterpenes on the Formation and Activation of Osteoclasts In Vitro

Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.

Oral squamous cell carcinoma cells induce osteoclast differentiation by suppression of osteoprotegerin expression in osteoblasts

The invasion of oral squamous cell carcinoma (SCC) cells into the mandibular bone is a common clinical problem. It has been reported that BHY cells, a human oral SCC cell line, are capable of invading mandibular bone of nude mice. These results led us to examine possible mechanisms of osteoclastogenesis induced by BHY cells using in vitro culture systems. When BHY cells were cocultured with mouse bone marrow cells (BMCs), only few osteoclasts were formed, even though BHY cells express the receptor activator of NF-kappaB ligand (RANKL). However, adding BHY cells to a coculture of mouse primary osteoblasts (POBs) and BMCs markedly induced osteoclastogenesis in the absence of osteotropic factors. Furthermore, another oral SCC cell line, HSC-2, which does not express RANKL, also induced osteoclastogenesis in our cocultures. These effects were significantly, but not completely, inhibited by adding osteoprotegerin (OPG). In addition, we also found that TNFalpha released from these cells partially contributes to osteoclastogenesis via a RANKL-independent mechanism. Adding BHY or HSC-2 cells suppressed mouse OPG mRNA expression and protein production by POBs in cocultures of POBs and human oral SCC cells. This finding is consistent with the result that BHY cells and HSC-2 cells did not enhance osteoclastogenesis in cocultures of BMCs and POBs from OPG-deficient mice. Immunohistochemical analysis showed a reduction of OPG expression in osteolytic lesions as compared to normal lesions from oral SCC patients. Therefore, oral SCC-induced suppression of OPG expression in POBs appears critical for osteoclastogenesis, rather than expression of RANKL in SCC cells.

Osteopontin-deficiency suppresses growth of B16 melanoma cells implanted in bone and osteoclastogenesis in co-cultures.

Tumor metastasis and invasion to bone is one of major medical issues in our modern societies. Osteopontin deficiency decreased tumor invasion in bone based on knockout mouse study. In bone, osteopontin is a positive factor to increase tumor invasion. Osteopontin is an arginine-glycine-aspartate (RGD)-containing protein and is recognized by integrin family members. Osteopontin promotes cell attachment to bone, where it is abundantly present. Because osteopontin levels were reported to be elevated in patients bearing highly metastatic tumors, this molecule has been implicated in the metastasis of tumors. However, the effect of osteopontin on the invasion of tumor cells in bone microenvironment has not been clear. The purpose of this paper is to elucidate the effect of host osteopontin on the behavior of tumor cells in bone. Bone marrow ablation was conducted in the femora of mice, and B16 melanoma cells were injected directly into the ablated bone marrow space of the osteopontin-deficient and wildtype mice. Invasion foci of B16 melanoma cells in the cortical bone was observed 7 weeks after tumor cell implantation. The number of the foci was 5-fold less in osteopontin-deficient mice compared with that in wildtype mice. In wildtype mice, trabecular bone formation was not observed in the ablated marrow space where tumor cells were injected. In contrast, significant levels of trabecular bone were observed in the marrow space of osteopontin-deficient mice even after tumor cells were injected. To examine cellular mechanisms underlying these observations, co-cultures of bone marrow cells and B16 cells were conducted. While the presence of B16 cells promoted TRACP+ cell development in wildtype bone marrow cells, such enhancement in TRACP+ cell formation by the co-cultures with B16 cells was reduced in the case of bone marrow cells from osteopontin-deficient mice. Osteopontin deficiency reduced the bone loss caused by tumor cell implantation into the bone marrow space.

Effects of kindled seizures upon hematopoiesis in rats

Objective: Studies conducted in epilepsy patients and experimental animals have suggested a linkage between seizure activity and alterations in immune functions. However, little is known about the underlying mechanisms. The present study sought to determine whether chronic seizures result in changes in hematopoietic functions that contribute to alterations in immune function. Materials and methods: Sprague–Dawley rats were implanted with electrodes in the basal amygdala or frontal cortex for induction of focal seizures by kindling. After inducing stage 5 seizures for 30 days, rats were sacrificed and assays for colony-forming units granulocyte/macrophage (CFU-GM) were performed to study progenitor cell functions. Long-term culture-initiating culture (LTC-IC) assays were employed to determine the effects of kindling upon bone marrow stroma. A Western blot for caspase-3 and CFU-GM assays from peripheral blood were used to determine the cause of reduced cellularity of bone marrow. Results: Kindled seizures of the basal amygdala resulted in decreases in bone marrow cellularity and hyperproliferation of colony-forming cells in peripheral blood and bone marrow. Modified LTC-IC assays, where co-cultures of bone marrow cells and stroma from experimental animals were employed, revealed that hyperproliferation of progenitor cells was not associated with alterations in stromal functions. The changes observed in this study were associated with seizure foci in the basal amygdaloid complex but not the frontal cortex. Conclusion: Kindled seizures of the basal amygdala induce hyperproliferation of bone marrow progenitor cells, suggesting that alterations in immunological functions observed following seizure activity may be due to changes in hematopoietic functions. Such changes appear to be site specific within the brain.

Estradiol enhances osteolytic lesions in mice inoculated with human estrogen receptor-negative MDA-231 breast cancer cells in vivo.

The effect of 17-beta-estradiol (E2) on the induction of osteolytic lesions by estrogen receptor (ER)-negative breast cancer cells was investigated in 4-week-old female nude mice. Exposure to exogenous E2 was found to increase osteolytic areas on radiographs up to 5.3 times in mice inoculated intracardially with MDA-231 human breast cancer cells. The MDA-231 cells were ER-negative, both before inoculation, and after isolation from osteolytic lesions, and the corresponding cell cultures were insensitive to E2. The induction of skeletal lesions by E2 in this mouse model was mainly effectuated at the early development of bone metastases, since exposure to E2 for 8 days around MDA-231 inoculation increased osteolysis to the same level, as did E2 given throughout the entire 31-day experimental period, and because E2-exposure for just the final 14 days had no effect. Independently of exposure to E2, histology revealed cancer cells in hind limp long bones of approximately 80% of the mice, and tumors were absent in non-skeletal organs. In vitro studies showed that the number and activity of osteoclasts generated from mouse bone marrow cells were increased 5-6 times when co-cultured with MDA-231 cells. Addition of 0.1-10 nM E2 further dose-dependently increased the osteoclastogenesis and associated bone resorption in these co-cultures. In conclusion, E2 was found to increase the morbidity in mice inoculated with ER-negative MDA-231 cells, and to stimulate osteoclast formation and bone resorption in co-cultures of bone marrow cells and MDA-231, suggesting that the progression of osteolytic metastases by ER-negative breast cancer cells can be induced by E2 due to stimulation of osteoclastogenesis.

The role of LFA-1 in osteoclast development induced by co-cultures of mouse bone marrow cells and MC3T3-G2/PA6 cells.

Interactions between leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) influence the development of osteoclasts. However, little is known about how these adhesion molecules are involved in the process of osteoclast development. This study evaluated the role of LFA-1 and its ligands in osteoclast development and bone resorption. Co-cultures of bone marrow cells from LFA-1-deficient mice and MC3T3-G2/PA6 (PA6) cells were cultured in the presence of 1alpha,25(OH)2D3 and dexamethasone for 7 days. The number of TRAP-positive cells that were generated by bone marrow cells from LFA-1-deficient mice was smaller than that generated by bone marrow cells from wild-type mice. In addition, the bone-resorbing activity of osteoclast-like cells that were generated from LFA-1-deficient mice was lower than that generated by osteoclast-like cells from wild-type mice. Immunofluorescence flow cytometry showed that osteoclast stromal PA6 cells expressed the cell adhesion molecules, ICAM-1 and VCAM-1. When monoclonal antibodies to mice VCAM-1, CD11b or CD18 were added separately to the co-culture system, the number of TRAP-positive cells that were generated from LFA-1-deficient mice was 20-30% smaller than that generated from wild-type mice. The formation of TRAP-positive cells from both LFA-1 deficient and wild-type mice was especially inhibited by anti-CD18 antibody, in comparison to the addition of normal IgG serum. These results suggest that LFA-1 adhesion molecules play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. CD18 appears to be a key adhesion molecule in cell-to-cell contacts during the early stage of osteoclast development.

A Novel Interaction between Thyroid Hormones and 1,25(OH) 2D 3 in Osteoclast Formation

Thyroid hormones enhance osteoclast formation and their excess is an important cause of secondary osteoporosis. 3,5,3′-Triiodo- l-thyronine (T3) induced the mRNA expression of receptor activator of nuclear factor-κB ligand (RANKL), which is a key molecule in osteoclast formation, in primary osteoblastic cells (POB). This effect was amplified in the copresence of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). Although T3 alone did not induce octeoclasts in coculture of bone marrow cells with POB, T3 enhanced 1,25(OH) 2D 3-induced osteoclast formation. Thyroxine (T4) also enhanced 1,25(OH) 2D 3-induced osteoclast formation. These data suggested that T4 was locally metabolized to T3 for its action, since T4 is a prohormone with little hormonal activity. The mRNA expression of type-2 iodothyronine deiodinase (D2), which is responsible for maintaining local T3 concentration, was induced by 1,25(OH) 2D 3 dose- and time-dependently. Our data would facilitate our understanding of the mechanism of osteoclast formation by thyroid hormones and suggest a novel interaction between thyroid hormones and 1,25(OH) 2D 3.