Aberrant gene expression in cultured mammalian bone cells demonstrates an osteoblast defect in osteopetrosis

Osteopetrosis is a skeletal condition in which a generalized radioopacity of bone is caused by reduced resorption of bone by osteoclasts. However, it has recently been shown that during skeletal development in several osteopetrotic rat mutations specific aberrations occur in gene expression reflecting the activity of the bone forming cells, osteoblasts, and the development of tissue organization. To evaluate their pathogenetic significance, progressive osteoblast differentiation was studied in vitro. Primary cultures of normal osteoblasts undergo a sequential expression a cell growth and tissue-related genes associated with development of skeletal tissue. We report that osteoblast cultures can be established from one of these mutants, toothless; that these cells in vitro exhibit similar aberrations in gene expression during cell proliferation and extracellular matrix formation and mineralization observed in vivo; and that an accelerated maturation sequence by mutant osteoblasts mimics the characteristic skeletal sclerosis of this disease. These data are the first direct evidence for an intrinsic osteoblast defect in osteopetrosis and establish an in vitro model for the study of heritable skeletal disorders. © 1994 Wiley-Liss, Inc.     

Congenitally osteosclerotic (os/os) rabbits are not cured by bone marrow transplantation from normal littermates.

The osteopetrotic (os) rabbit is a lethal mutation of autosomal recessive inheritance characterized by hypocalcemia, hypophosphatemia, fibrosis of marrow spaces, and ultrastructural abnormalities in both osteoclasts and osteoblasts. Procedures involving the transplantation of cells from normal hemopoietic tissues, which are sources of osteoclast precursors, are known to cure osteopetrosis in several mutations including some children. We tested the ability of transplanted bone marrow and/or spleen from normal littermates to reverse the skeletal sclerosis in os rabbits. Treatment of 15 neonatal mutants consisted of immunosuppression by whole-body irradiation followed by transplantation of normal bone marrow and/or spleen cell suspensions. This treatment failed to prolong life span or to cure osteopetrosis judged radiographically and histologically for up to 3 weeks posttreatment, the longest time of survival. These data indicate that transplantation of stem cells from multiple hemopoietic tissues, procedures known to cure osteopetrosis in other mutations, is not effective in the os rabbit. These results support the hypothesis that the skeletal microenvironment is not capable of supporting the development and function of normal osteoclasts in this mutation.     

The osteopetrotic rabbit: skeletal cytology and ultrastructure

The lethal, autosomal recessive osteopetrotic mutation in the rabbit, osteosclerosis (os/os), has recently been made available for experimental investigation. We have examined the cytology and ultrastructure of skeletal cells in mutants and report abnormalities in osteoblasts, osteocytes, and osteoclasts. Mutant osteoclasts lack a well-defined ruffled border and show few morphological signs of bone resorption. Osteoblasts in mutants form bone in neonatal life but show signs of degeneration by 2 weeks after birth. Mutant osteoblasts and osteocytes contain large, electron-dense cytoplasmic inclusions. External surfaces of mutant long bones show no evidence of bone resorption by scanning electron microscopy, and fibrosis of intertrabecular spaces is a prominent feature in mutants. These data, considered with recent evidence that the functions of osteoblasts and osteoclasts are interrelated, suggest that reduced bone resorption, a characteristic feature of osteopetrosis, may be related to osteoblast incompetence in this mutation.     

Defective osteoclast differentiation and function in the osteopetrotic (os) rabbit

We tested the ability of normal osteoclast progenitors found in neonatal liver and bone marrow to develop into functional osteoclasts when co-cultured with metatarsals from newborn osteopetrotic rabbits; the latter inherit an osteoclast incompetence resistant to cure by bone marrow transplantation. This system, developed by Burger and colleagues, has been shown to produce normal, functional osteoclasts when used with normal metatarsals. Our study tested the competence of the mutant skeletal microenvironment for differentiation of normal osteoclasts. Mutant and normal metatarsals were cultured alone or with normal liver, spleen, or bone marrow for up to 14 days. All normal cultures possessed a marrow cavity and contained numerous osteoclasts with cytochemical characteristics (tartrate-resistant acid phosphatase) of active cells. Mutant metatarsals co-cultured with normal spleen, liver, or bone marrow failed to develop a marrow cavity (evidence in itself of reduced bone resorption) and had osteoclasts reduced in both numbers and cytochemically detectable activity. Similar metatarsal cultures of an osteopetrotic rat mutation (incisors--absent) curable by bone-marrow transplantation exhibited marrow cavity development in mutant metatarsals co-cultured with normal spleen. These data suggest that the skeletal environment of osteopetrotic rabbits contains an inhibitor or lacks a promoter of osteoclast differentiation and function.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are characteristics of the osteosclerotic mutation which suggest that it is a unique osteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

Osteopetrosis, from mouse to man

The osteoclast is the main effector of bone resorption. Failure in osteoclast differentiation or function leads to osteopetrosis, a bone disease characterized by an impaired bone resorption. Analysis of mouse models developing osteopetrosis as a consequence of naturally occurring mutations or gene knockouts allowed to establish the osteoclast differentiation pathway. Among these models, the oc/oc, the gl/gl and the Clcn7(-/-) mice present a phenotype similar to the one displayed by patients with infantile malignant osteopetrosis, the most severe form of osteopetrosis in human. Analysis of these models led to the identification of different mutations in the corresponding human genes TCIRG1, GL and CLCN7, in osteopetrotic patients. Mutations in the TCIRG1 gene seem the most frequent cause of malignant osteopetrosis and mutations in the CLCN7 gene seem the most frequent cause of type II osteopetrosis. Therefore, these three mouse models appear to be particularly well suited for the study of the osteoclast function in order to provide new insights in the therapy of osteopetrosis.     

OSTM1 bone defect reveals an intercellular hematopoietic crosstalk

The most severe form of bone autosomal recessive osteopetrosis both in humans and in the gray-lethal (gl/gl) mouse is caused by mutations in the Ostm1 gene. Although osteopetrosis is usually associated with a defect in the hematopoietic-derived osteoclast cells, this study determined that Ostm1 is expressed in many hematopoietic cells of the myeloid and lymphoid B- and T-lineages. Hematopoiesis in gl/gl mice is characterized by a marked expansion of the osteoclast lineage but also by deregulation of the lymphoid lineages with a decrease in B-lymphoid cell populations and altered distribution in T-lymphoid double and single CD4 CD8-positive cells. In committed gl/gl osteoclasts, specific Ostm1 transgene targeting showed a requirement of additional factors and/or cells for normal osteoclast function, and importantly, defined the gl osteopetrotic defect as non-cell autonomous. By contrast, gl/gl osteoclast, B- and T-lymphoid lineage phenotypes were rescued when Ostm1 is expressed under PU.1 regulation from a bacterial artificial chromosome transgene, which established an essential role for Ostm1 in hematopoietic cells in addition to osteoclasts. Together these experiments are the first to demonstrate the existence of hematopoietic crosstalk for the production of functional and active osteoclasts.     

Osteoclast biology in the osteopetrotic (op) rat

Osteopetrosis is a metabolic bone disease characterized by reduced bone resorption. From experimental studies of various osteopetrotic mutations has emerged the hypothesis that each is unique with respect to mechanisms whereby osteoclast development and/or function are reduced. The osteopetrotic (op) mutation in the rat was discovered in Fatty/ORL stock over a decade ago. The paucity of data about osteoclast biology in this mutation prompted this study of cytological, cytochemical, and ultrastructural features of osteoclasts. In op rats, osteoclasts are significantly reduced in number, but are larger and more vacuolated than in normal littermates. Mutant osteoclasts can form ruffled borders and clear zones, but their ability to fragment and excavate bone surfaces is greatly impaired. Cytoplasmic vacuoles in op osteoclasts are randomly distributed and greatly enlarged, and they stain weakly for two cytochemical characteristics of osteoclasts, tartrate-resistant acid phosphatase and acid ATPase. These findings suggest that an abnormality in the lysosomal/vacuolar system, an important component of the resorptive mechanism, may be involved in the interception of osteoclast function in this mutation.     

Osteoclasts express high levels of pp60c-src in association with intracellular membranes

Deletion of the c-src gene in transgenic mice by homologous recombination leads to osteopetrosis, a skeletal defect characterized by markedly deficient bone resorption (Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Cell. 64:693-702), demonstrating a critical functional role of pp60c-src in osteoclast activity. Since decreased bone resorption could result from a defect either within the osteoclast or within other cells present in its environment, indirectly affecting osteoclast functions, we determined which cell(s) in bone expressed high levels of pp60c-src Measuring pp60c-src protein and kinase activities in osteoclasts and immunolocalizing pp60c-src in bone, we find that expression of pp60c-src is nearly as high in osteoclasts as in brain and platelets. In contrast, other bone cells contain only very low levels of the protein. In addition, expression of the c-src gene product increases when bone marrow cells are induced to express an osteoclast-like phenotype by 1,25-dihydroxy-vitamin D3, further suggesting that high expression of pp60c-src is part of the osteoclast phenotype. Three other src-like kinases, c-fyn, c-yes, and c-lyn, are also expressed in osteoclasts at ratios to pp60c-src similar to what is found in platelets. These src-related proteins do not, however, compensate for the absence of pp60c-src in the src- mice, thereby suggesting that pp60c-src may have a specific function in osteoclasts. Although further work is necessary to elucidate what the critical role of pp60c-src in osteoclasts is, our observation that the protein is associated mostly with the membranes of intracellular organelles suggests the possibility that this role might be at least in part related to the targeting or fusion of membrane vesicles.     

The hematogenous origin of osteoclasts: experimental evidence from osteopetrotic (microphthalmic) mice treated with spleen cells from beige mouse donors

The excessive skeletal mass and reduced bone resorption characteristic of osteopetrosis in microphthalmic (mi) mice can be corrected by irradiation and transfer of spleen cells from a normal littermate. Osteoclasts in beige (bg) mice, a mutation without osteopetrosis, have giant lysosomal granules. These two facts were exploited to trace osteoclast lineage. Microphthalmic mice treated with whole-body irradiation and spleen cells from a beige donor resorbed the excessive skeletal mass and recovered from osteopetrosis. Furthermore, osteoclasts in treated mi mice had giant lysosomal granules and resembled those found in bg donors when examined by light and transmission electron microscopy. These data provide direct evidence for a hematogenous origin of osteoclasts in mammals.     

The synergistic role of Pu.1 and Fms in zebrafish osteoclast-reducing osteopetrosis and possible therapeutic strategies

Osteoclasts are bone resorption cells of myeloid origin. Osteoclast defects can lead to osteopetrosis, a genetic disorder characterized by bone sclerosis for which there is no effective drug treatment. It is known that Pu.1 and Fms are key regulators in myelopoiesis, and their defects in mice can lead to reduced osteoclast numbers and consequent osteopetrosis. Yet how Pu.1 and Fms genetically interact in the development of osteoclasts and the pathogenesis of osteopetrosis is still unclear. Here, we characterized pu.1^G242D;fms^j4e1 double-deficient zebrafish, which exhibited a greater deficiency of functional osteoclasts and displayed more severe osteopetrotic symptoms than the pu.1^G242D or fms^j4e1 single mutants, suggesting a synergistic function of Pu.1 and Fms in the regulation of osteoclast development. We further demonstrated that Pu.1 plays a dominant role in osteoclastogenesis, whereas Fms plays a dominant role in osteoclast maturation. Importantly, treatment with the drug retinoic acid significantly relieved the different degrees of osteopetrosis symptoms in these models by increasing the number of functional osteoclasts. Thus, we report the development of valuable animal models of osteopetrosis, and our results shed light on drug development for antiosteopetrosis therapy.     

Chemokine receptor 3 is a negative regulator of trabecular bone mass in female mice

Chemokines are secreted by a wide variety of cells; their functions are dependent on the binding to their chemokine receptors (CCRs) which induce directed chemotaxis in nearby responsive cells. Chemokines and their receptors can be induced under several different conditions. Based on data from clinical studies showing an increased expression of chemokine receptor 3 (CCR3) in circulating monocytes of human subjects with lower bone mineral density (BMD) as compared to those with high BMD, we predicted a role for CCR3 in the development of peak bone mass. We, therefore, first evaluated the expression pattern of Ccr3 in bone cells, in comparison to other CCRs, that have common ligands with CCR3. While Ccr1 and Ccr3 messenger RNA (mRNA) levels increased during both RANKL-induced osteoclast differentiation and AA-induced osteoblast differentiation, the levels of Ccr5 mRNA only increased during osteoblast differentiation. To examine if CCR3 influences osteoclast and/or osteoblast differentiation, we evaluated the consequence of blocking CCR3 function using neutralizing antibody on the expression of osteoclast and osteoblast differentiation markers. Treatment with CCR3 neutralizing antibody increased mRNA levels of Trap and cathepsin K in osteoclasts and osteocalcin in osteoblasts compared to cells treated with control IgG. Based on these in vitro findings, we next assessed the role of CCR3 in vivo by evaluating the skeletal phenotypes of Ccr3 knockout and corresponding control littermate mice. Disruption of CCR3 resulted in a significant increase in femur areal BMD at 5 and 8 weeks of age by dual-energy X-ray absorptiometry. Micro-CT analysis revealed a 25% increase in trabecular bone mass at 10 weeks of age caused by corresponding changes in trabecular number and thickness compared to wild type mice. Based on our findings, we conclude that disruption of CCR3 function favors bone mass accumulation, in part via enhancement of bone metabolism. Understanding the molecular pathways through which CCR3 acts to regulate osteoclast and osteoblast functions could lead to new therapeutic approaches to prevent inflammation-induced bone loss.     

Evidence that the rat osteopetrotic mutation toothless (tl) is not in the TNFSF11 (TRANCE, RANKL, ODF, OPGL) gene.

The toothless (tl) osteopetrotic mutation in the rat affects an osteoblast-derived factor that is required for normal osteoclast differentiation. Although the genetic locus remains unknown, the phenotypic impact of the tl mutation on multiple systems has been well characterized. Some of its actions are similar to tumornecrosis factor superfamily member 11(TNFSF11; also called TRANCE, RANKL, ODF and OPGL) null mice. TNFSF11 is a recently described member of the tumor necrosis factor superfamily which, when expressed by activated T cells, enhances the survival of antigen-presenting dendritic cells, and when expressed by osteoblasts, promotes the differentiation and activation of osteoclasts. The skeletal similarities between tl rats and TNFSF11(-/-) mice include 1) profound osteoclastopenia (TNFSF11-null mice, 0% and tl rats 0-1% of normal); 2) persistent, non-resolving osteopetrosis that results from 3) a defect not in the osteoclast lineage itself, but in an osteoblast-derived, osteoclastogenic signal; and 4) a severe chondrodysplasia of the growth plates of long bones not seen in other osteopetrotic mutations. The latter includes thickening of the growth plate with age, disorganization of chondrocyte columns, and disturbances of chondrocyte maturation. These striking similarities prompted us to undertake studies to rule in or out a TNFSF11 mutation in the tl rat. We looked for expression of TNFSF11 mRNA in tl long bones and found it to be over-expressed and of the correct size. We also tested TNFSF11 protein function in the tl rat. This was shown to be normal by flow cytometry experiments in which activated, spleen-derived T-cells from tl rats exhibited normal receptor binding competence, as measured by a recombinant receptor assay. We also found that tl rats develop histologically normal mesenteric and peripheral lymph nodes, which are absent from TNFSF11-null mice. Next, we found that injections of recombinant TNFSF11, which restores bone resorption in null mice, had no therapeutic effect in tl rats. Finally, gene mapping studies using co-segregation of polymorphic markers excluded the chromosomal region containing the TNFSF11 gene as harboring the mutation responsible for the tl phenotype. We conclude that, despite substantial phenotypic similarities to TNFSF11(-/-) mice, the tl rat mutation is not in the TNFSF11 locus, and that its identification must await the results of further studies.     

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.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Osteoclasts secrete non-bone derived signals that induce bone formation

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption, in face of normal or even increased bone formation. This suggests that osteoclasts, not their resorptive activity, are important for sustaining bone formation. To investigate whether osteoclasts mediate control of bone formation by production of bone anabolic signals, we collected conditioned media (CM) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts secrete non-bone derived factors, which induce preosteoblasts to form bone-like nodules, potentially explaining the imbalanced coupling seen in osteopetrotic patients.     

PPAR-gamma regulates osteoclastogenesis in mice

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Regulation of osteoclast function is central to the understanding of bone diseases such as osteoporosis, rheumatoid arthritis and osteopetrosis. Although peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been shown to inhibit osteoblast differentiation, its role, if any, in osteoclasts is unknown. This is a clinically crucial question because PPAR-gamma agonists, "such as thiazolidinediones-" a class of insulin-sensitizing drugs, have been reported to cause a higher rate of fractures in human patients. Here we have uncovered a pro-osteoclastogenic effect of PPAR-gamma by using a Tie2Cre/flox mouse model in which PPAR-gamma is deleted in osteoclasts but not in osteoblasts. These mice develop osteopetrosis characterized by increased bone mass, reduced medullary cavity space and extramedullary hematopoiesis in the spleen. These defects are the result of impaired osteoclast differentiation and compromised receptor activator of nuclear factor-kappaB ligand signaling and can be rescued by bone marrow transplantation. Moreover, ligand activation of PPAR-gamma by rosiglitazone exacerbates osteoclast differentiation in a receptor-dependent manner. Our examination of the underlying mechanisms suggested that PPAR-gamma functions as a direct regulator of c-fos expression, an essential mediator of osteoclastogenesis. Therefore, PPAR-gamma and its ligands have a previously unrecognized role in promoting osteoclast differentiation and bone resorption.