Retroviral-mediated gene transfer of CSF-1 into op/op stromal cells to correct defective in vitro osteoclastogenesis

Colony-stimulating factor-1 (CSF-1) released by stromal cells in the bone microenvironment is essential for the proliferation of osteoclast progenitors. In op/op mutant mice, a thymidine insertion in the coding sequence of the CSF-1 gene results in CSF-1 deficiency that in turn leads to decreased osteoclast production and osteopetrosis. Because the osteopetrotic defect is due to the failure of stromal cells to produce CSF-1, we determined if retroviral-mediated gene transfer of the wild-type CSF-1 cDNA into op/op stromal cells would restore their ability to support osteoclast formation in vitro. A retroviral vector, L-CSF-1-SN, was constructed by inserting 1,867 bp of the wild-type CSF-1 cDNA into pLXSN. After transduction with L-CSF-1-SN or LXSN constructs, a stable PA317 packaging cell line that produced a high viral titre was isolated. Viral supernatant from this line was used to infect op/op bone marrow stromal cells. Stable L-CSF-1-SN op/op stromal clones overexpressed CSF-1 mRNA and released CSF-1 into conditioned medium, compared with no CSF-1 released by LXSN op/op stroma. The amount of CSF-1 produced by two clones was similar to the physiologic level released by normal littermate stroma. Southern blot analysis confirmed the presence of intact proviral sequences in transduced cells. In coculture assays, L-CSF-1-SN, but not LXSN, op/op stromal cells supported the formation of TRAP-positive multinucleated cells in the absence of exogenous CSF-1. These findings indicate that genetically engineered stromal cells may be used to improve defective osteoclastogenesis and suggest that targeting stromal cells to bone is a potentially useful therapeutic modality for treating bone disorders. J. Cell. Physiol. 176:323–331, 1998. © 1998 Wiley-Liss, Inc.     

Macrophage differentiation and granulomatous inflammation in osteopetrotic mice (op/op) defective in the production of CSF-1

Since the osteopetrotic (op/op) mouse was demonstrated to have a mutation within the coding region of the CSF-1 gene itself, it serves as a model for investigating the differentiation mechanism of macrophage populations in the absence of functional CSF-1. The op/op mice were severely monocytopenic and showed marked reduction and abnormal differentiation of tissue macrophages. Osteoclasts as well as marginal metallophilic macrophages and marginal zone macrophages in the spleen were absent. Most of the tissue macrophages were reduced in number and ultrastructurally immature. However, the degree of reduction in numbers of macrophages in the mutant mice was variable among tissues, suggesting that the heterogeneity of macrophages was generated by their different dependency on CSF-1. After daily CSF-1 injection, the numbers of monocytes, tissue macrophages, and osteoclasts were remarkably increased, and the macrophages showed morphological maturation. However, the numbers of macrophages in the ovary, uterus, and synovial membrane were not increased. In the bone marrow, macrophage precursors detected by monoclonal antibody ER-MP58 proliferated and differentiated into preosteoclasts and osteoclasts. In the spleen, marginal metallophilic macrophages and marginal zone macrophages developed slowly. In this manner, CSF-1 plays an important role in the development, proliferation, and differentiation of certain tissue macrophage populations and osteoclasts. In the op/op mice, Kupffer cells proliferated, transformed into epithelioid cells and multinucleated giant cells, and participated in glucan-induced granuloma formation. In CSF-1-treated op/op mice, the process of granuloma formation was similar to that in normal littermates due to increased monocytopoiesis and monocyte influx into the granulomas. These results indicate that CSF-1 is a potent inducer of the development and differentiation of CSF-1-dependent monocyte/macrophages, and that CSF-1-independent macrophages also play an important role in granuloma formation. Mol Reprod Dev 46:85–91, 1997. © 1997 Wiley Liss, Inc.     

Both cell-surface and secreted CSF-1 expressed by tumor cells metastatic to bone can contribute to osteoclast activation

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts, and is frequently produced by tumor cells. Here we hypothesize that the CSF-1 made by tumor cells contributes to bone destruction in osteolytic bone metastases. We show that high level CSF-1 protected osteoclasts from suppressive effects of transforming growth factor β (TGF-β). r3T cells, a mouse mammary tumor cell line that forms osteolytic bone metastases, express abundant CSF-1 in vitro as both a secreted and a membrane-spanning cell-surface glycoprotein, and we show that both the secreted and the cell-surface form of CSF-1 made by r3T cells can support osteoclast formation in co-culture experiments in the presence of RankL. Mice with r3T bone metastases have elevated levels of both circulating and bone-associated CSF-1, and the majority of CSF-1 found in bone metastases is associated with the tumor cells. These results support the idea that tumor-cell produced CSF-1 contributes to osteoclast development and survival in bone metastasis.     

Osteoclast differentiation and function in aquaglyceroporin AQP9-null mice

Background information. Osteoclasts are cells specialized for bone resorption and play important roles in bone growth and calcium homoeostasis. Differentiation of osteoclasts involves fusion of bone marrow macrophage mononuclear precursors in response to extracellular signals. A dramatic increase in osteoclast cell volume occurs during osteoclast biogenesis and is believed to be mediated by AQP9 (aquaporin 9), a membrane protein that can rapidly transport water and other small neutral solutes across cell membranes. Results. In the present study we report an increase in expression of AQP9 during differentiation of a mouse macrophage cell line into osteoclasts. Bone marrow macrophages from wild‐type and AQP9‐null mice differentiate into osteoclasts that have similar morphology, contain comparable numbers of nuclei, and digest synthetic bone to the same extent. Bones from wild‐type and AQP9‐null mice contain similar numbers of osteoclasts and have comparable density and structure as measured by X‐ray absorptiometry and microcomputed tomography. Conclusions. Our results confirm that AQP9 expression rises during osteoclast biogenesis, but indicate that AQP9 is not essential for osteoclast function or differentiation under normal physiological conditions.     

Expression of CSF-1 receptor on TRAP-positive multinuclear cells around the erupting molars in rats

Bone resorption overlying a developing tooth is a necessary event in the creation of an eruption pathway. The formation and function of osteoclasts, which play a major role in bone resorption, are controlled by several factors. Although CSF-1 and its mRNA are expressed in dental follicle cells required for eruption, little is known about the contribution of CSF-1 to osteoclast formation on the bony crypt around the tooth germ. The receptor protein of the CSF-1 encoded proto-oncogene c-fms was identified on multinucleated cells adjacent to the dental follicle, in conjunction with TRAP staining as a marker enzyme for osteoclasts in rat. c-Fms was highly expressed in TRAP-positive multinuclear cells at 3 days postnatal and the number of c-Fms-expressing cells was reduced thereafter. Administration of IL-1alpha, which enhances formation and function of osteoclasts, caused an increase in the number of c-Fms and TRAP-positive cells in rat. On the contrary, injection of calcitonin, which depresses osteoclast formation, caused a decrease in the number. It is obvious that the receptor of CSF-1 is expressed on the surface of osteoclasts around the tooth germ, on the dental follicle. These findings suggested that CSF-1 directly enhances the influx of osteoclasts adjacent to the erupting tooth, resulting in the formation of an eruption pathway.     

Biology and action of colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor, controls the survival, proliferation, and differentiation of mono-nuclear phagocytes and regulates cells of the female reproductive tract. It appears to play an autocrine and/or paracrine role in cancers of the ovary, endometrium, breast, and myeloid and lymphoid tissues. Through alternative mRNA splicing and differential post-translational proteolytic processing, CSF-1 can either be secreted into the circulation as a glycoprotein or chondroitin sulfate-containing proteoglycan or be expressed as a membrane-spanning glycoprotein on the surface of CSF-1-producing cells. Studies with the op/op mouse, which possesses an inactivating mutation in the CSF-1 gene, have established the central role of CSF-1 in directly regulating osteoclastogenesis and macrophage production. CSF-1 appears to preferentially regulate the development of macrophages found in tissues undergoing active morphogenesis and/or tissue remodeling. These CSF-1 dependent macrophages may, via putative trophic and/or scavenger functions, regulate characteristics such as dermal thickness, male fertility, and neural processing. Apart from its expression on mononuclear phagocytes and their precursors, CSF-1 receptor (CSF-1R) expression on certain nonmononuclear phagocytic cells in the female reproductive tract and studies in the op/op mouse indicate that CSF-1 plays important roles in female reproduction. Restoration of circulating CSF-1 to op/op mice has preliminarily defined target cell populations that are regulated either humorally or locally by the synthesis of cell-surface CSF-1 or by sequestration of the CSF-1 proteoglycan. The CSF-1R is a tyrosine kinase encoded by the c-fms proto-oncogene product. Studies by several groups have used cells expressing either the murine or human CSF-1R in fibroblasts to pinpoint the requirement of kinase activity and the importance of various receptor tyrosine phosphorylation sites for signaling pathways stimulated by CSF-1. To investigate post-CSF-1R signaling in the macrophage, proteins that are rapidly phosphorylated on tyrosine in response to CSF-1 have been identified, together with proteins associated with them. Studies on several of these proteins, including protein tyrosine phosphatase 1C, the c-cbl proto-oncogene product, and protein tyrosine phosphatase-phi are discussed. Mol Reprod Dev 46:4–10, 1997. © 1997 Wiley-Liss, Inc.     

Role of colony-stimulating factor-1 in reproduction and development

The CSF-1 null mouse, osteopetrotic, has provided a powerful model in which to study the biological functions of CSF-1. In this review, I will describe our studies that have used this mouse model to determine the impact of a lack of CSF-1 on developmental processes and in reproduction. A role for CSF-1 in reproduction was originally suggested by the sex steroid hormone-regulated uterine epithelial synthesis of CSF-1 and the expression of its receptor in trophoblast and decidual cells. Studies on the fertility of CSF-1 deficient osteopetrotic mice (csfm^op/csfm^op) mice confirmed this suggestion and in addition revealed an unexpected function for CSF-1 in male fertility. In both sexes, CSF-1 appears to regulate gonadal steroidogenesis, probably through its action on macrophages that are abundant throughout the ovary and testis. In the female, CSF-1 affects ovulation in vivo and in vitro, and impacts the preimplantation embryo, increasing both its rate of development and the number of trophectodermal cells in the blastocyst. CSF-1 also has a role in mammary gland development during pregnancy, since at mid-gestation in csfm^op/csfm^op mice, ductal branching is impaired, and after partiturition, there is a failure to switch to lactation. The relative failure of csfm^op/csfm^op mice to respond to external stimuli also suggested a role for CSF-1 in the brain. CSF-1 mRNA is expressed in a regional specific manner in the brain through development whilst the CSF-1 receptor is expressed throughout the brain in microglia. CSF-1 is neurotrophic in embryonic neuronal cultures and its absence in csfm^op/csfm^op mice results in severe electrophysiological abnormalities in the cortex. This suggests that CSF-1 is a neurotrophic factor acting through the microglia. The pleiotropic roles for CSF-1 in reproduction and in the brain suggest that CSF-1 exerts many of its action through the trophic activities of cells of the mononuclear phagocytic lineage. Mol Reprod Dev 46:54–61, 1997. © 1997 Wiley-Liss, Inc.     

Transformations of osteoclast phenotype in ia rats cured of congenital osteopetrosis

The reduced bone resorption characteristic of osteopetrosis is accompanied in the incisors-absent (ia) rat mutation by a significant increase in osteoclasts of inactive (mutant) phenotype. Restoration of bone resorption in ia rats by transfer of spleen cells from normal littermates is preceded by a transformation of osteoclasts from mutant to normal phenotype. In this investigation the proportions of osteoclasts of normal phenotype have been determined by light microscopy in untreated ia and normal rats and in ia rats treated with various cell populations from normal rats. Significant increases in numbers of osteoclasts of normal phenotype were seen in the mutant skeleton soon after cell treatments that eventually restored bone resorption and cured the disease. No changes in osteoclast phenotype were seen after cell transfers that did not cure the disease. These data establish transformation of osteoclast phenotype as an early event in the recovery from osteopetrosis and suggest that determination of osteoclast phenotype is a reliable predictor of the success of normal cell populations to restore bone resorption in this mutation.     

Relative role of CSF-1, MCP-1/JE,and RANTES in macrophage recruitment during successful pregnancy

It has been previously demonstrated that macrophage colony stimulating factor (CSF-1) is produced by uterine epithelial cells in response to estrogen and progesterone. Studies in normal and op/op mice demonstrated that accumulation of a portion of the uterine macrophage population could be attributed to the chemotactic properties of CSF-1. Op/op mice exhibit greatly reduced rates of fertility, but successful pregnancy is not completely blocked. Also, uteri from op/op mice are not completely macrophage deficient. There are two possible explanations for this. One is that not all tissue macrophages are recruited from the bone marrow pool; some may be derived from primitive mesenchyme. Alternatively, tissue macrophages may be recruited from the bone marrow pool through expression of other type I chemokines such as JE, RANTES, MIP-1α, MIP-1β, IP-10, and KC. Both RANTES and JE are expressed at higher levels than CSF-1 during early pregnancy. The variable expression and relative role of these various chemokines in pregnancy was addressed by measuring mRNA expression during the first 8 days of pregnancy and in a pseudopregnant model. The expression of these various genes relative to macrophage numbers and macrophage distribution will be discussed. The relative role of these various factors in preparing the uterus for blastocyst implantation will be discussed. Mol Reprod Dev 46:62–70, 1997. © 1997 Wiley-Liss, Inc.     

Bone resorption and bone remodelling in juvenile carp, Cyprinus carpio L.

The present study considers the important role of bone resorption for bone growth in general, and aims to clarify if and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte‐containing (cellular) bone. To ensure the identification of osteoclasts and sites of bone resorption independently from the morphology of the bony cells, bones were studied by histological procedures, and by demonstration of the enzymes which serve as osteoclast markers, viz. tartrate resistant acid phosphatase (TRAP), ATPase and a vacuolar proton pump. Two types of bone‐resorbing cells were observed in juvenile carp: (1) multinucleated giant cells displaying morphological and biochemical attributes which are known from mammalian osteoclasts; and (b) flat cells which lack a visible ruffled border and for which identification requires the performance of enzyme histochemical procedures. Bone resorption performed by osteoclasts mainly occurs at endosteal bone surfaces. To a lesser extent, bone resorption also takes place at periosteal bone surfaces, but without an apparent connection to bone growth. The latter observation, and the occurrence of bone remodelling, suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism. Morphological differences and biochemical similarities to bone resorption in teleosts with acellular bone are discussed.     

The dietary anthocyanin delphinidin prevents bone resorption by inhibiting Rankl-induced differentiation of osteoclasts in a medaka (Oryzias latipes) model of osteoporosis

The anthocyanin delphinidin is a natural compound found as water-soluble pigment in coloured fruits and berries. Anthocyanin-rich diets have been proposed to have bone protective effects in humans and mice, but the underlying mechanisms remain unclear. In this study, we used a medaka (Oryzias latipes) osteoporosis model to test the effects of delphinidin on bone cells in vivo. In this model, inducible transgenic expression of receptor-activator of NF-kβ ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, similar to the situation in human osteoporosis patients. Using live imaging in medaka bone reporter lines, we show that delphinidin significantly reduces the number of osteoclasts after Rankl induction and protects bone integrity in a dose-dependent manner. Our in vivo findings suggest that delphinidin primarily affects the de novo differentiation of macrophages into osteoclasts rather than the recruitment of macrophages to sites of bone resorption. For already existing osteoclasts, delphinidin treatment affected their morphology, leading to fewer protrusions and a more spherical shape. Apoptosis rates were not increased by delphinidin, suggesting that osteoclast numbers were reduced primarily by impaired differentiation from macrophage progenitors and reduced maintenance of pre-existing osteoclasts. Importantly, and in contrast to previously reported cell culture experiments, no effect of delphinidin on osteoblast differentiation and distribution was observed in medaka in vivo. Our study is the first report on the effects of delphinidin on bone cells in fish embryos, which are a unique model system for compound testing that is suitable for live imaging of bone cell behaviour in vivo.     

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases.     

Effects of prostaglandin E2 on macrophages and osteoclasts in cultured fetal long bones

Summary Bone cultures exposed to prostaglandin E₂ (PGE₂) revealed an increase in ⁴⁵Ca release from bone to medium and an increase in osteoclast number compared to control bones. In addition, PGE₂-treated osteoclasts contained a more extensive ruffled border region than control osteoclasts. These data suggest that PGE₂ activates existing osteoclasts and causes proliferation and differentiation of osteoclast precursor cells. The existence of macrophages in resorbing fetal bone explants was documented. These macrophages contain numerous phagolysosomes and lipid vacuoles and are often located adjacent to osteoclasts or closely apposed to calcified tissue surfaces. PGE₂ caused an early increase in the number of macrophages. It is postulated that fetal bone macrophages are primarily engaged in phagocytosis and digestion of cellular debris, but also play a role in the process of bone resorption.This study was supported by Grant DE-04443 from USPHS     

Regulation of new osteoclast formation by a bone cell-derived macromolecular factor

Medium conditioned by incubation with embryonic chick calvarial bones, which contain osteoblasts but not osteoclasts, stimulated new osteoclast formation in foetal long bone cultures and in adult bone marrow cultures formation of tartrate-resistant acid phosphatase (TRAP) positive cells was greatly stimulated. We have termed the factor responsible for this activity osteoclast growth/inducing factor (OGF). OGF was soluble, heat-stable and of size greater than 10kda. OGF activity was present also in mouse bone conditioned medium and in extracts of demineralized cortical diaphyseal bone of five-week-old chickens. OGF appeared to differ from the osteoblast-derived bone-resorbing factors previously observed as well as from macrophage colony stimulating factor (CSF-1). It is therefore probable that different locally secreted factors independently regulate the formation of osteoclasts and their activity.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

Human osteoclast ontogeny and pathological bone resorption

Monocytes and macrophages are capable of degrading both the mineral and organic components of bone and are known to secrete local factors which stimulate host osteoclastic bone resorption. Recent studies have shown that monocytes and macrophages, including those isolated from neoplastic and inflammatory lesions, can also be induced to differentiate into cells that show all the cytochemical and functional characteristics of mature osteoclasts, including lacunar bone resorption. Monocyte/macrophage-osteoclast differentiation occurs in the presence of osteoblasts/bone stromal cells (which express osteoclast differentiation factor) and macrophage-colony stimulating factor and is inhibited by osteoprotegerin. Various systemic hormones and local factors (e.g. cytokines, growth factors, prostaglandins) modulate osteoclast formation by controlling these cellular and humoral elements. Various pathological lesions of bone and joint (e.g. carcinomatous metastases, arthritis, aseptic loosening) are associated with osteolysis. These lesions generally contain a chronic inflammatory infiltrate in which macrophages form a significant fraction. One cellular mechanism whereby pathological bone resorption may be effected is through generation of increased numbers of bone-resorbing osteoclasts from macrophages. Production of humoral factors which stimulate mononuclear phagocyte-osteoclast differentiation and osteoclast activity is also likely to influence the extent of pathological bone resorption.     

Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (<Emphasis Type="Italic">op</Emphasis>/<Emphasis Type="Italic">op</Emphasis>) mice after macrophage depletion

We previously reported that macrophage colony-stimulating factor (M-CSF, CSF-1) played important roles in the process of the repopulation of Kupffer cells after their elimination by administration of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP). In this study, we examined the repopulation of Kupffer cells and splenic red pulp macrophages in osteopetrotic (op/op) mice defective in the production of functional M-CSF and their littermate mice by using the lipo-MDP model. In untreated op/op mice, numbers of F4/80-positive Kupffer cells in the liver and F4/80-positive splenic red pulp macrophages were reduced. Repopulation of Kupffer cells and splenic macrophages was observed in littermate (op/+) mice liver by 14 days after depletion. However, in op/op mice, repopulation of Kupffer cells was not observed in Kupffer-cell-depleted op/op mice until 56 days after depletion, whereas splenic red pulp macrophages repopulated and recovered to the level of control op/op mice by 10 days after depletion. Single injection of M-CSF was effective for the induction of the repopulation of Kupffer cells, and daily administration of M-CSF induced remarkable repopulation and maturation of Kupffer cells and proliferation of macrophage precursor cells in the liver of Kupffer-cell-depleted op/op mice. These results suggest that Kupffer cells are completely M-CSF-dependent tissue macrophages, whereas splenic red pulp macrophages are composed of M-CSF-dependent macrophages and M-CSF-independent macrophages. This mouse model provides a useful tool for the study of effects of growth factor on Kupffer cell differentiation in vivo. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, NIH grant CA20408, and a Tsukada Memorial Grant (2000).