Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

https://www.pnas.org/content/pnas/95/7/3597.full.pdf

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

Macrophages, the most plastic cells of the haematopoietic system, are found in all tissues and show great functional diversity. They have roles in development, homeostasis, tissue repair and immunity. Although tissue macrophages are anatomically distinct from one another, and have different transcriptional profiles and functional capabilities, they are all required for the maintenance of homeostasis. However, these reparative and homeostatic functions can be subverted by chronic insults, resulting in a causal association of macrophages with disease states. In this Review, we discuss how macrophages regulate normal physiology and development, and provide several examples of their pathophysiological roles in disease. We define the 'hallmarks' of macrophages according to the states that they adopt during the performance of their various roles, taking into account new insights into the diversity of their lineages, identities and regulation. It is essential to understand this diversity because macrophages have emerged as important therapeutic targets in many human diseases.

/pdf/macrophage-biology-in-development-homeostasis-and-disease-2nq2u94n8n.pdf

Macrophage biology in development, homeostasis and disease

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

Left ventricular remodeling is a major cause of progressive heart failure and death after myocardial infarction. Although neoangiogenesis within the infarcted tissue is an integral component of the remodeling process, the capillary network is unable to support the greater demands of the hypertrophied myocardium, resulting in progressive loss of viable tissue, infarct extension and fibrous replacement. Here we show that bone marrow from adult humans contains endothelial precursors with phenotypic and functional characteristics of embryonic hemangioblasts, and that these can be used to directly induce new blood vessel formation in the infarct-bed (vasculogenesis) and proliferation of preexisting vasculature (angiogenesis) after experimental myocardial infarction. The neoangiogenesis resulted in decreased apoptosis of hypertrophied myocytes in the peri-infarct region, long-term salvage and survival of viable myocardium, reduction in collagen deposition and sustained improvement in cardiac function. The use of cytokine-mobilized autologous human bone-marrow-derived angioblasts for revascularization of infarcted myocardium (alone or in conjunction with currently used therapies) has the potential to significantly reduce morbidity and mortality associated with left ventricular remodeling.

Neovascularization of ischemic myocardium by human bone-marrow–derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid, myeloid, and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required, and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells, this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells.

https://science.sciencemag.org/content/sci/265/5175/1098.full.pdf

Generation of lymphohematopoietic cells from embryonic stem cells in culture

We demonstrated previously that a single injection of recombinant human macrophage colony-stimulating factor (rhM-CSF) is sufficient for osteoclast recruitment and survival in osteopetrotic (op/op) mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene. In this study, we show that a single injection of recombinant human vascular endothelial growth factor (rhVEGF) can similarly induce osteoclast recruitment in op/op mice. Osteoclasts predominantly expressed VEGF receptor 1 (VEGFR-1), and activity of recombinant human placenta growth factor 1 on osteoclast recruitment was comparable to that of rhVEGF, showing that the VEGF signal is mediated through VEGFR-1. The rhM-CSF–induced osteoclasts died after injections of VEGFR-1/Fc chimeric protein, and its effect was abrogated by concomitant injections of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF showed no significant difference in the bone-resorbing activity. op/op mice undergo an age-related resolution of osteopetrosis accompanied by an increase in osteoclast number. Most of the osteoclasts disappeared after injections of anti-VEGF antibody, demonstrating that endogenously produced VEGF is responsible for the appearance of osteoclasts in the mutant mice. In addition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclast differentiation. These results demonstrate that M-CSF and VEGF have overlapping functions in the support of osteoclastic bone resorption.

/pdf/vascular-endothelial-growth-factor-can-substitute-for-2zyvy3dtgo.pdf

Vascular Endothelial Growth Factor Can Substitute for Macrophage Colony-Stimulating Factor in the Support of Osteoclastic Bone Resorption

Osteopetrotic (op/op) mice have a severe deficiency of osteoclasts, monocytes, and peritoneal macrophages because of a defect in the production of functional macrophage colony-stimulating factor (M-CSF) resulting from a mutation within the M-CSF gene. In this study, we examined whether daily 5-microgram injections of purified recombinant human M-CSF (rhM-CSF) for 14 d would cure these deficiencies in the mutant mice. Monocytes in the peripheral blood of the op/op mice were significantly increased in number after subcutaneous injections of the factor two or three times a day. In contrast, osteopetrosis in the long bones of op/op mice was completely cured by only one injection of rhM-CSF per day. Bone trabeculae in the diaphyses were removed. Many osteoclasts were detected on the surface of bone trabeculae in the metaphyses. Although development of tooth germs of uninjected op/op mice was impaired, rhM-CSF injection restored the development of molar tooth germs and led to tooth eruption as a consequence of the recovery of bone-resorbing activity. These results demonstrate that M-CSF is one of the factors responsible for the differentiation of osteoclasts and monocyte/macrophages under physiological conditions.

/pdf/congenital-osteoclast-deficiency-in-osteopetrotic-op-op-mice-4rt6vyznc8.pdf

Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor

Severe deficiency of osteoclasts, monocytes, and peritoneal macrophages in osteopetrotic (op/op) mutant mice is caused by the absence of functional macrophage colony-stimulating factor (M-CSF). To clarify the role of M-CSF in the osteoclast differentiation, we established a clonal stromal cell line OP6L7 capable of supporting hemopoiesis from newborn op/op mouse calvaria. Although very few macrophages appeared in the cocultures of bone marrow cells and OP6L7 cells, a 50-fold larger number of macrophages was detected in the day 7 cocultures when purified recombinant human M-CSF (rhM-CSF) was exogenously supplied. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive cells appeared only when bone marrow cells were cultured in contact with OP6L7 cells and both rhM-CSF and 1 alpha, 25 (OH)2D3 were added. The TRACP-positive cells became multinucleated with increasing time in culture and expressed the c-fms/M-CSF receptor. These results indicate that both contact with stromal cells and M-CSF are requisite for osteoclast differentiation under physiological conditions.

https://rupress.org/jem/article-pdf/173/5/1291/1394762/1291.pdf

Hiroaki Kodama

Papers

Generation of lymphohematopoietic cells from embryonic stem cells in culture

Vascular Endothelial Growth Factor Can Substitute for Macrophage Colony-Stimulating Factor in the Support of Osteoclastic Bone Resorption

Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor

Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells.

Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells.