Murine Bone Marrow Cultures Research Articles

Osteoblast-specific overexpression of human interleukin-7 rescues the bone mass phenotype of interleukin-7–deficient female mice

Interleukin-7 is a critical cytokine for lymphoid development and a direct inhibitor of in vitro osteoclastogenesis in murine bone marrow cultures. To explore the role of IL-7 in bone, we generated transgenic mouse lines bearing the 2.3-kb rat collagen 1α1 promoter driving the expression of human IL-7 specifically in osteoblasts. In addition, we crossed these mice with IL-7-deficient mice to determine if the alterations in lymphopoiesis, bone mass, and osteoclast formation observed in the IL-7 knockout (KO) mice could be rescued by osteoblast-specific overexpression of IL-7. Here, we show that mice overexpressing human IL-7 in the osteoblast lineage showed increased trabecular bone volume in vivo by µCT and decreased osteoclast formation in vitro. Furthermore, targeted overexpression of IL-7 in osteoblasts rescued the osteopenic bone phenotype and B-cell development of IL-7 KO mice but did not have an effect on T lymphopoiesis, which occurs in the periphery. The bone phenotypes in IL-7 KO mice and targeted IL-7-overexpressing mouse models were observed only in females. These results likely reflect both direct inhibitory effects of IL-7 on osteoclastogenesis in vivo and sex-specific differences in responses to IL-7.

Control of Megakaryocyte Expansion and Bone Marrow Fibrosis by Lysyl Oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.

The skeletal effects of the tyrosine kinase inhibitor nilotinib

Nilotinib is a tyrosine kinase inhibitor (TKI) developed to manage imatinib-resistance in patients with chronic myeloid leukemia (CML). It inhibits similar molecular targets to imatinib, but is a significantly more potent inhibitor of Bcr-Abl. Nilotinib exhibits off-target effects in other tissues, and of relevance to bone metabolism, hypophosphataemia has been reported in up to 30% of patients receiving nilotinib. We have assessed the effects of nilotinib on bone cells in vitro and on bone metabolism in patients receiving nilotinib for treatment of CML. We firstly investigated the effects of nilotinib on proliferating and differentiating osteoblastic cells, and on osteoclastogenesis in murine bone marrow cultures and RAW264.7 cells. Nilotinib potently inhibited osteoblast proliferation (0.01-1uM), through inhibition of the platelet-derived growth factor (PDGFR). There was a biphasic effect on osteoblast differentiation such that it was reduced by lower concentrations of nilotinib (0.1-0.5uM), with no effect at higher concentrations (1uM). Nilotinib also potently inhibited osteoclastogenesis, predominantly by stromal-cell dependent mechanisms. Thus, nilotinib decreased osteoclast development in murine bone marrow cultures, but did not affect osteoclastogenesis in RAW264.7 cells. Nilotinib treatment of osteoblastic cells increased expression and secretion of OPG and decreased expression of RANKL. In 10 patients receiving nilotinib, levels of bone turnover markers were in the low-normal range, despite secondary hyperparathyroidism, findings that are similar to those in patients treated with imatinib. Bone density tended to be higher than age and gender-matched normal values. These data suggest that nilotinib may have important effects on bone metabolism. Prospective studies should be conducted to determine the long-term effects of nilotinib on bone density and calcium metabolism.

Transforming growth factor β signaling inhibitor, SB-431542, induces maturation of dendritic cells and enhances anti-tumor activity

The transforming growth factor β (TGFβ) stimulates tumor progression and metastasis. Secretion of TGFβ by tumor cells also suppresses an antitumor immune response in which dendritic cells (DCs) play an important role to activate cytotoxic T lymphocytes (CTLs). Herein we report that the small molecule TGFβ signaling inhibitor SB-431542, induces DC maturation in vitro and triggers antitumor activity in vivo. We added SB-431542 to cultures of murine bone-marrow derived DCs (BM-DCs) derived from BALB/c mice and human DCs generated from peripheral monocytes (human DCs) at different concentrations in triplicates and examined expression of co-stimulatory molecules by FACS and production of Interleukin-12 (IL-12) by ELISA. SB induced phenotypic maturation of BM-DCs and human DCs and improved their abilities to produce IL-12 in a dose-dependent manner. SB-431542 also augmented capacity of murine and human DCs to activate naive T cells in allogeneic mixed lymphocyte reaction. Interestingly, SB-431542 augmented the capacity of BM-DCs and human DCs to incorporate FITC-conjugated dextran. Intraperitoneal administration of SB-431542 initiated 3 and 7 days after the implantation of colon-26 cancer cells into the peritoneal cavity of BALB/c mice significantly induced CTL activity against colon-26. We incubated human DCs with SB-431542 and cell lysate of scirrhous gastric cancer cell line OCUM-8, and then examined CTL activities against OCUM-8. CD8 T cells activated by human DCs treated with SB-431542 showed modest augmentation CTL activity against cancer cells. Furthermore, pretreatment of human DCs with SB-431542 upregulated cytotoxic activity against K562 cells, suggesting SB should have potential to activate DCs to natural killer cells. In conclusion, TGFβ receptor I kinase inhibitor such as SB-431542 might induce anti-tumor immune response in immuno-tolerant patients associated with TGFβ activity.

Mechanisms involved in the inhibition of osteoclast generation by the benzothiophene SERM LY117018

Selective estrogen receptor modulators are compounds that act via estrogen receptors in different tissues to mediate either estrogenic or estrogen antagonistic effects in osteoporosis patients. The aim of this study was to assess the effect of the selective estrogen receptor modulator LY117018, a raloxifene analogue, on osteoclastogenesis in vitro. In primary murine bone marrow cultures osteoclasts, defined as TRAP-positive multinucleated cells, were induced by 10(-8) M 1,25-dihydroxyvitamin D(3). LY117018 at concentrations between 10(-12) M and 10(-9) M significantly reduced the number of osteoclasts. At higher concentrations no effect of LY117018 on osteoclast generation was observed. LY117018 enhanced alkaline phosphatase activity of mouse calvaria osteoblasts at a concentration of 10(-14) to 10(-7) M, but had no influence on the proliferation and transcription of RANKL and osteoprotegerin. In order to study the effect of the compound on the production of cytokines that can stimulate bone resorption, spleen cells were incubated with LY117018. Data from four-color flow cytometric analysis indicate a significantly decreased frequency of tumor necrosis factor-α positive CD8(+) cells after treatment with LY117018. These findings suggest that LY117018 can significantly inhibit the generation of osteoclasts and stimulate osteogenic differentiation in vitro. Suppression of tumor necrosis factor-α production by LY117018 may contribute to its anti-osteoclastogenic effect.

Abstract 3449: Tungsten induces DNA damage and alters growth of developing B lymphocytes

Abstract Tungsten is used widely in modern life in everything from household goods to technologically advanced material. However, little is known about the consequences of tungsten exposure. The lack of information concerning the toxicities associated with tungsten has been highlighted recently when high tungsten levels were discovered at the sites of several pediatric leukemia clusters. In these clusters, the majority of the children developed acute lymphoblastoid leukemia of the preB lymphocyte subtype. Thus, we investigated whether tungsten exposure alters the growth of preB cells and defined signaling pathways induced by tungsten. We exposed a co-culture system of the BU-11 preB cell line grown on BMS2 stromal cells to tungsten at doses found at the sites of the leukemia clusters. Growth, cell cycle, and apoptosis were determined in both cell types. While no significant changes in any of these parameters were observed in the BMS2 stromal cells, tungsten treatment for 48 hours decreased the BU-11 cell number, which correlated with an accumulation of cells in G0/G1 and increased apoptosis. Unlike polycyclic aromatic hydrocarbons, tungsten induced apoptosis in BU-11 cells grown in rIL-7 and therefore, is independent of the stromal cell layer. These results were confirmed in primary murine bone marrow cultures and in human peripheral blood mononuclear cells. Tungsten can polymerize forming polytungstates in water and soil. Interestingly, these polytungstates are significantly more effective at inhibiting growth in BU-11 and BMS2 cells. In order to begin analysis of signaling pathways induced by tungsten, cDNA microarrays were performed in a leukemic cell line following 24 hours of tungsten treatment and integrated pathway analyses were performed. Genes involved in DNA damage and ER stress were highly upregulated following tungsten exposure and validated by qPCR. An increase in DNA damage was confirmed by COMET assay. These results suggest that the developing B lymphocyte population is sensitive to tungsten-induced toxicities and that tungsten exposure may contribute to leukemogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3449.

Murine hematopoietic blast colony-forming cells and their progeny have distinctive membrane marker profiles

Two distinct bone marrow-derived blast colony-forming cells can generate colonies of lineage-restricted progenitor cells in agar cultures of murine bone marrow. Both cell types selectively had a Kit(+) ScaI(+) phenotype distinguishing them from most lineage-restricted progenitor cells. Multicentric blast colony-forming cells stimulated by stem cell factor plus interleukin-6 (IL-6) (BL-CFC-S) were separable from most dispersed blast colony-forming cells stimulated by Flt3 ligand and IL-6 (BL-CFC-F) using CD34 and Flt3R probes. Multicentric BL-CFC-S cofractionated with colony-forming units, spleen (CFU-S) supporting the possibility that the 2 cells may be identical. The colony populations generated by BL-CFC-S were similar in their phenotype and proliferative capacity to progenitor cells in whole bone marrow but the progeny of BL-CFC-F were skewed with an abnormally high proportion of Kit(-) Flt3R(+) cells whose clonogenic cells tended to generate only macrophage progeny. Both blast colony populations had a high percentage of GR1(+) and Mac1(+) cells but BL-CFC-F colonies also contained a significant population of B220(+) and IL-7R(+) cells relevant to the superior ability of BL-CFC-F colony cells to generate B lymphocytes and the known dependency of this process on Flt3 ligand and IL-7. The commitment events and phenotypic changes during the generation of differing progenitor cells in blast colonies can now be clonally analyzed in a convenient in vitro culture system.

Bone morphogenetic protein 2 enhances PGE 2-stimulated osteoclast formation in murine bone marrow cultures

Bone morphogenetic protein 2 (BMP-2) is used clinically to stimulate bone formation and accelerate fracture repair. Adding prostaglandin (PG) E 2 or PGE 2 receptor agonists to BMP-2 has been proposed to improve BMP-2 efficacy. However, this may enhance bone resorption, since PGE 2 can increase receptor activator of NF-κB ligand (RANKL) expression and decrease osteoprotegerin (OPG) expression in osteoblasts, and the RANKL:OPG ratio is critical for osteoclast formation. We used bone marrow (BM) cultures and BM macrophage (BMM) cultures from outbred CD1 mice to examine effects on osteoclast formation of BMP-2 and PGE 2. In BM cultures, which contain both osteoblastic and osteoclastic lineage cells, BMP-2 (100 ng/ml) alone did not increase osteoclast formation but enhanced the peak response to PGE 2 by 1.6–9.6-fold. In BMM cultures, which must be treated with RANKL because they do not contain osteoblastic cells, BMP-2 did not increase osteoclast formation, with or without PGE 2. Our results suggest that BMP-2 can increase osteoclast formation in response to PGE 2 by increasing the RANKL:OPG ratio in osteoblasts, which may have therapeutic implications for the use of BMP-2.

Effects of biomaterials on osteoblastogenesis and osteoclastogenesis in murine bone marrow cultures

Effects of biomaterials on osteoblastogenesis and osteoclastogenesis in murine bone marrow cultures

A comparative study on BMP-induced osteoclastogenesis and osteoblastogenesis in primary cultures of adult rat bone marrow cells

Bone remodeling is a process consisting of bone formation and resorption. The present study compared the relative osteoclastic and osteoblastic potency of bone morphogenetic proteins (BMP)-2, -4, -5, -6, and -7 in primary murine bone marrow cultures. All five BMPs stimulated, to varying degree, formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in a time- and protein concentration-dependent manner. The TRAP staining intensity correlated positively with the number of nuclei per TRAP-positive cell and the mRNA levels of colony-stimulating factor (CSF-1), receptor activator of nuclear factor κB ligand (RANKL), TRAP, and cathepsin K. Under osteogenic conditions, all five BMPs stimulated AP activity and mineralized bone nodule formation in a protein concentration-dependent manner in the same primary murine bone marrow cell culture system. These findings should be useful in designing treatment strategies for bone regeneration.

Cysteinyl leukotrienes mediate the enhancing effects of indomethacin and aspirin on eosinophil production in murine bone marrow cultures

Prostaglandin E(2) (PGE(2)) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone-marrow cultures. Because cysteinyl leukotrienes (cys-LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone-marrow were due to blockade of PGE(2) production alone, or involved further promotion of cys-LTs production/signalling. BALB/c liquid bone-marrow cultures were established with IL-5, alone or associated with indomethacin, aspirin, or cys-LTs. The effects of preventing cys-LT production or signalling were assessed. Indomethacin and aspirin counteracted the suppression of eosinophil production by exogenous PGE(2). LTD(4), LTC(4) and LTE(4) enhanced IL-5-dependent eosinophil production and further counteracted the effect of exogenous PGE(2). The 5-lipoxygenase activating protein (FLAP) inhibitor, MK886, a leukotriene synthesis inhibitor, zileuton, the CysLT(1) receptor antagonists, MK571 and montelukast, or inactivation of the LTC(4) synthase gene, abolished effects of indomethacin and aspirin. MK886 and zileuton were ineffective but MK571 and montelukast were effective, against LTD(4). Indomethacin, aspirin and LTD(4) failed to enhance eosinophil production in bone-marrow from CysLT1 receptor-deficient mice. Indomethacin, aspirin and LTD(4) no longer counteracted the effects of exogenous PGE(2) in the presence of MK571 and montelukast. MK886, MK571 and montelukast had no effect by themselves, or in association with PGE(2). Dependence on the FLAP/5-lipoxygenase/LTC(4) synthase pathway and receptor signalling shows that cyclo-oxygenase inhibitors act here through endogenous cys-LTs. While PGE(2) does not act by suppressing cys-LT production, cys-LTs override PGE(2) signalling. Eosinophil production is therefore coordinately regulated by both pathways.

Deletion of Aspartate 182 in OPG Causes Juvenile Paget' Disease by Impairing Both Protein Secretion and Binding to RANKL

Mutations in the OPG gene cause idiopathic hyperphosphatasia. We characterized the effects of one such mutation and found that the mutant OPG is poorly secreted and has reduced biological activity compared with the wildtype protein. Therefore, correct structure and cellular processing of OPG is essential for normal bone remodeling. Inactivating mutations in osteoprotegerin (OPG) cause juvenile Paget's disease (JPD). We recently reported a family with JPD in which affected members were homozygous for an in-frame mutation resulting in the deletion of aspartate 182 in OPG. Here we report the structural and functional characterization of the OPGdeltaD182 mutant protein. Inhibition of osteoclastogenesis by the recombinant OPG proteins was studied in a murine bone marrow culture. Binding of wildtype and mutant OPG to RANKL was measured in two experimental systems: glutathione-S-transferase (GST) pull-down assay and surface plasmon resonance. Site-directed mutagenesis was used to study the glycosylation of OPGdeltaD182 in two potential glycosylation sites adjacent to the deleted aspartate residue at position 182. ELISA and Western blots were used to determine OPG concentrations in cell lysates and conditioned media from transiently transfected cells. OPGdeltaD182 inhibited the generation of osteoclasts less effectively than the wildtype protein and had a reduced ability to bind to RANKL. The apparent higher molecular weight of OPGdeltaD182 compared with the wildtype is a result of hyperglycosylation of asparagine residues at positions 178 and 183. Glycosylation at N183 has the potential to disrupt OPG structure by interfering with disulphide bond formation and correct protein folding. Transient transfection experiments in SaOS2 cells suggest that OPGdeltaD182 is retained within the cell, a typical response to unstable or incorrect protein folding. Taken together, these data suggest that the deletion of aspartate 182 impairs both the secretion and activity of OPG, which in turn provides an explanation for the increased osteoclastogenesis and high bone turnover observed in JPD patients with this mutation.

Activated platelets retain their potential to induce osteoclast-like cell formation in murine bone marrow cultures

Supernatants immediately obtained after platelet activation can induce osteoclast-like cell formation in murine bone marrow cultures. Here we report that activated platelets retain their potential to induce osteoclast-like cell formation over a 3-day period with repeated washing, when co-cultured with murine bone marrow cells. Supernatants obtained from washed platelets 3 days following their activation with thrombin, caused the differentiation of haematopoietic progenitors into osteoclast-like cells. The platelet-derived soluble factor(s) responsible for the induction of osteoclastogenesis can be retained in an ultrafilter with a nominal molecular weight limit of 10 kDa, and loose their activity when incubated at 99°C. Indomethacin, which inhibits cyclooxygenase activity, and osteoprotegerin, a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL), suppressed the formation of osteoclast-like cells in this model. The in vitro findings presented here suggest that activated platelets can induce osteoclast-like cell formation via a prostaglandin and RANKL-dependent mechanism over a time period corresponding to the existence of a blood clot.

RANKL-stimulated osteoclast-like cell formation in vitro is partially dependent on endogenous interleukin-1 production

Receptor activator of NF-κB ligand (RANKL) and interleukin-1 (IL-1) individually plays a critical role in the differentiation and activation of osteoclasts in bone. In addition, both RANKL and IL-1 activate similar signal transduction pathways including p38 MAP kinase and c-Jun NH 2 terminal kinase (JNK). We examined if endogenously produced IL-1 influenced osteoclast-like cell (OCL) formation in murine bone marrow and bone marrow monocyte (BMM) cultures that were stimulated with M-CSF and RANKL. RANKL stimulated OCL formation in a dose-dependent manner in bone marrow cultures, and this response was significantly inhibited by IL-1 RA (100 ng/ml), a specific IL-1 antagonist. Interleukin-1 further increased OCL formation in BMM cultures that were treated with M-CSF (30 ng/ml) and RANKL (1, 3, 10 and 30 ng/ml). In addition, BMM cultures from IL-1 type I receptor-deficient mice, which do not respond to IL-1, demonstrated significantly less OCL formation compared to wild-type BMM cultures. We examined the time course and dose response of IL-1alpha protein expression by ELISA in BMM cultures that were treated with or without M-CSF and RANKL. RANKL dose dependently stimulated IL-1α protein significantly (up to 46%) in 6-day cultures. The interaction of RANKL and IL-1 on osteoclastogenesis did not appear significantly dependent on prostaglandin synthesis since PGE 2 expression in the conditioned medium of BMM cultures was nearly undetectable and the PGHS-2 specific inhibitor, NS-398, was without effect. We also investigated the effect of IL-1 on p38 MAP kinase and JNK in BMM cultures. The combination of RANKL and IL-1 had additive effects on JNK but not p38 MAP kinase compared to results in cultures treated with RANKL or IL-1 alone. In addition, SP600125, a specific JNK inhibitor, markedly reduced OCL formation in BMM cultures that were treated with RANKL or the combination of RANKL and IL-1. These findings demonstrate that endogenously produced IL-1 augments the response of bone marrow cells to RANKL, and this effect appears mediated by mechanisms that are associated with enhancement of JNK activity.

Imatinib and Leptomycin B Are Effective in Overcoming Imatinib-Resistance Due to Bcr-Abl Amplification or Clonal Evolution but Not Due to Bcr-Abl Kinase Domain Mutation.

Constitutive activity of the Bcr-Abl kinase has been shown to be the causative event in the molecular pathogenesis of CML and Ph+ ALL. Even though Imatinib, a small molecule inhibitor of the Abl kinase, is very effective in treating the disease, development of resistance to this drug is a common phenomenon in advanced stage disease. Using Leptomycin B (LMB), a nuclear export blocker, Vigneri et al. have shown that Bcr-Abl kinase activity trapped in the nucleus induces cell death, thus being a new potential treatment option for Bcr-Abl expressing diseases. To test whether this approach might be effective in the case of imatinib resistant leukaemia several Bcr-Abl positive, imatinib-resistant Ba/F3 clones were established and characterized: 1. Ba/F3 cells resistant due to a point mutation in the Abl kinase domain at position T315I. 2. Ba/F3 cells resistant due to amplification of the Bcr-Abl kinase. 3. Ba/F3 cell clones resistant to imatinib with clear features of clonal evolution. All four cell lines were tested for the efficacy of a combined treatment approach with imatinib and leptomycin B. This combination was very effective in inducing apoptosis in imatinib resistant Ba/F3 cells displaying Bcr-Abl amplification. One out of two cell lines with signs of clonal evolution also responded to this combination treatment. However, imatinib resistant cells expressing the Bcr-AblT315I mutant kinase were completely resistant to this approach. Thus, these results are in agreement with the proposed hypothesis of Vigneri and wang that the combined effect of leptomycin B and imatinib requires Bcr-Abl kinase inhibition (which is not achieved in cells expressing Bcr-Abl T315I) by imatinib and nuclear entrapment by LMB To check the feasibility of this combined treatment for purging purposes, in vitro colony forming assays were performed with mixed cultures of Bcr-Abl positive BaF3 cells and primary murine bone marrow. All colonies formed after combined treatment with imatinib and LMB were derived from BM cells, indicating selective toxicity towards Bcr-Abl positive cells while sparing primary murine bone marrow cells. We are currently evaluating the effectivity of the combined LMB/imatinib treatment as purging strategy in a murine bone marrow transplantation model.

Membranous osteogenesis system modeled with KUSA-A1 mature osteoblasts

Several stromal cells were established from murine bone marrow cultures. One of the KUSA subclones, KUSA-A1 cells, displays osteogenic characteristics in vitro and in vivo. The calcium deposition, osteocalcin release, and parathyroid hormone (PTH) responsiveness of KUSA-A1 cells indicate that they are mature osteoblasts or osteocytes. Bone had formed in subcutaneous tissue 1 week after subcutaneous injection of cells into immunodeficient mice. The osteogenesis by KUSA-A1 was not mediated by chondrogenesis and thus was considered to be membranous ossification. These unique characteristics of KUSA-A1 cells provide an opportunity to analyze the process of membranous ossification in detail.

Lactoferrin - A Novel Bone Growth Factor

Lactoferrin is an iron-binding glycoprotein that belongs to the transferrin family. It is present in breast milk, in epithelial secretions, and in the secondary granules of neutrophils. In healthy subjects lactoferrin circulates at concentrations of 2-7 x 10(-6) g/ml. Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Recently, we have shown that lactoferrin can also promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor inhibiting apoptosis induced by serum withdrawal. Lactoferrin also affects osteoclast formation and, in murine bone marrow culture, lactoferrin potently inhibits osteoclastogenesis. In vivo, local injection of lactoferrin above the hemicalvaria of adult mice results in substantial increases in the dynamic histomorphometric indices of bone formation and bone area. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through LRP1, a member of the family of low-density lipoprotein receptor-related proteins that are primarily known as endocytic receptors. Using confocal laser scanning microscopy, we demonstrated that fluorescently labeled lactoferrin is endocytosed and can be visualized in the cytoplasm of primary osteoblastic cells. Lactoferrin also induces activation of p42/44 MAPK signaling in primary osteoblasts, but the two pathways seem to operate independently as activation of MAPK signaling, but not endocytosis, is necessary for the mitogenic effect of lactoferrin. We conclude that lactoferrin may have a physiological role in bone growth and healing, and a potential therapeutic role as an anabolic factor in osteoporosis.

Parathyroid Hormone (1–34) Treatment Induces Hematopoietic Precursor Cell Expansion in Murine Long-Term Bone Marrow Culture.

Parathyroid hormone (PTH) induces activation and increases the number of osteoblasts. An increase the stem cells number (Lin-Sca1+cKit+) was observed in mice after 4 weeks of PTH treatment, that suggest osteoblasts participation in regulation of hematopoiesis (Calvi, et al., 2003, Nature, 425, 841). Long-term bone marrow culture (LTBMC) was used for the study of PTH influence on hematopoietic progenitors of different stages of maturation. Rat PTH (1–34) (final concentration, 10−7M) was supported during whole culture period. Cell number, colony-forming units in culture (CFU-GM, CFU-C-21 days) and cobblestone area forming cells (CAFC) were measured during 10 weeks. The number of mature cells in culture did not changed during PTH treatment. The number of studied progenitors did not changed significantly during 3 and 6 weeks of PTH treatment. The number of CFU-GM, CFU-C-21 days and CAFC 14 days, CAFC 21 days, CAFC 28 days increased 7 –10 fold in suspension fraction of LTBMC after 10 weeks of PTH treatment.Table 1. Number of different precursor cells in suspension fraction of LTBMC after PTH treatment.To evaluate the possibility of stem cells expansion by using of PTH treated adherent cell layers (ACL) of LTBMC, the PTH-treated for 4 and 8 weeks cultures, were irradiated with 40 Gy and seeded with 2 × 106 bone marrow cells depleted of adherent cells. Following 24 hours the number of survived CAFC 8 – 28 was analyzed. On PTH-treated ACL the number of CAFC 21 increased 2,7 ± 0,4 fold as compared with fresh bone marrow or culture with non-treated ACLs. The number of CAFC 28, which characterizes the number of marrow repopulating cells, increased 2 fold only after cultivation on ACL treated with PTH for 8 weeks.Table 2. CAFC number in bone marrow cultivated 24 hours on PTH treated ACL of LTBMCThe data suggest possibility to increase stem cell expansion ex vivo on pharmacologically manipulated microenvironment.Time of cultivation, weeksSpecificity of progenitorNumber of precursors, per 100000 cellsControlControlPTH treatment3CFU-GM50 ± 5.277 ± 5.66CFU-GM33 ± 431 ± 5.910CFU-GM20 ± 4.8147.5 ± 3.53CFU-C-2155 ± 5.976.6 ± 11.210CFU-C-2111.5 ± 1.7102.0 ± 2.03CAFC-721.620.36CAFC-725.926.910CAFC-70.93.03CAFC-211.010.696CAFC-210.430.4810CAFC-210.283.113CAFC-280.320.556CAFC-280.120.1410CAFC-280.10.82Age of CAFC (days)Number of CAFC per 2 x 10^6 bone marrow cellsnot treatedPTH (4 weeks)PTH (8 weeks)CAFC-7424.1804.5647.5CAFC-14262.5529.8295.2CAFC-2193.5272.7226.2CAFC-2838.330.179.2

Prostaglandin E2 and dexamethasone regulate eosinophil differentiation and survival through a nitric oxide- and CD95-dependent pathway

Apoptosis, involving both CD95/CD95L interactions and their modulation by nitric oxide (NO), is central to regulation of mature eosinophil numbers. However, its role in regulating eosinophil production from bone-marrow precursors is unknown. We examined the effects of prostaglandin E 2 (PGE 2) and dexamethasone on eosinophil differentiation and survival in murine bone-marrow cultures, and their relationship to: NO production as well as CD95/CD95L-dependent apoptosis. Bone-marrow cultures were established with IL-5, alone or in association with PGE 2, dexamethasone or both. PGE 2 (10 −7 M) inhibited eosinophil differentiation by selectively inducing apoptosis in developing eosinophils. Dexamethasone (10 −7 M) protected developing eosinophils from PGE 2-induced apoptosis. Since dexamethasone prevents induction of nitric oxide synthase (NOS), we evaluated the role of NO in the effects of both PGE 2 and dexamethasone. NO donors (SNAP and SNP) down-modulated eosinophil precursor responses to IL-5. SNAP induced apoptosis through a dexamethasone-resistant mechanism. The NOS inhibitors, Nω-nitro- l-arginine and aminoguanidine, blocked the effects of PGE 2 on developing eosinophils. PGE 2 was ineffective in bone-marrow from knockout mice lacking inducible NOS. PGE 2 up-regulated CD95 and CD95L expression in developing eosinophils. Neither PGE 2 nor SNAP were effective in cultures from CD95L-deficient gld mice. These data suggest that PGE 2 induces apoptosis in developing eosinophils through inducible NOS, leading to NO-dependent activation of the CD95L/CD95 pathway, while dexamethasone antagonizes the effects of PGE 2 on the same targets.

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.