Carole A. MacKay

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

The osteopetrotic mutation toothless (tl) is a loss-of-function frameshift mutation in the rat Csf1 gene: Evidence of a crucial role for CSF-1 in osteoclastogenesis and endochondral ossification

The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a truncated, nonfunctional protein and an early stop codon, thus rendering the tl rat CSF-1null. All mutants were homozygous for the mutation and all carriers were heterozygous. No CSF-1 transcripts were identified in rat mRNA that would avoid the mutation via alternative splicing. The biology and actions of CSF-1 have been elucidated by many studies that use another naturally occurring mutation, the op mouse, in which a single base insertion also disrupts the reading frame. The op mouse has milder osteoclastopenia and osteopetrosis than the tl rat and recovers spontaneously over the first few months of life. Thus, the tl rat provides a second model in which the functions of CSF-1 can be studied. Understanding the similarities and differences in the phenotypes of these two models will be important to advancing our knowledge of the many actions of CSF-1.

Administration of colony stimulating factor-1 corrects some macrophage, dental, and skeletal defects in an osteopetrotic mutation (toothless, tl) in the rat

The toothless (tl/tl) mutation in the rat results in a paucity of osteoclasts and osteopetrosis that cannot be corrected by bone marrow transplantation. In the present study we demonstrate that tl/tl rats also have profound deficiencies of femoral, peritoneal, and pleural cavity macrophages. Furthermore, the macrophage colony stimulating activity of post-endotoxin sera from tl/tl rats is substantially reduced, suggesting that, as in the case of the op mutation in mice, the basis of the tl mutation is a deficiency of the macrophage growth factor, colony stimulating factor-1 (CSF-1). Consistent with this suggestion, treatment of tl/tl rats from birth for up to six weeks with CSF-1 reduced the osteopetrosis, increased body weight, and permitted tooth eruption. In addition, CSF-1 treatment induced large numbers of osteoclasts in tl/tl bones and macrophages in the peritoneal cavity and bone marrow. Persistence of metaphyseal sclerosis, however, indicated that the disease was not totally corrected by this treatment. These studies indicate that the basis of the tl mutation is most likely another CSF-1 deficiency, and further emphasize the role of this growth factor in osteoclast differentiation.

Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation

Microarray and real‐time RT‐PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC‐STAMP. Specific siRNA and antibodies were used to inhibit OC‐STAMP RNA and protein, respectively, and tartrate‐resistant acid phosphatase (TRAP)+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/− RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL‐induced differentiation was studied. OC‐STAMP was very strongly up‐regulated during osteoclast differentiation. Northern blots and sequence analysis revealed two transcripts of 2 and 3.7 kb differing only in 3′UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multipass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy‐terminal 193 amino acids, however, are significantly similar to the DC‐STAMP family consensus sequence. DC‐STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC‐STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of TRAP+, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC‐STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC‐STAMP is a previously unknown, RANKL‐induced, multipass transmembrane protein that promotes the formation of multinucleated osteoclasts. J. Cell. Physiol. 215: 497–505, 2008.

CSF-1 treatment promotes angiogenesis in the metaphysis of osteopetrotic (toothless, tl) rats

It has recently been shown that following treatment with colony-stimulating factor-1 (CSF-1) the osteopetrotic condition in toothless (tl) rats greatly improves and growth is accelerated. We have examined the effects of such treatment on the microvasculature of the distal femoral chondro-osseous junction, a site where bone growth in length is coordinated with angiogenesis. Vascular casts and ultrastructural analyses of this region showed that, compared to untreated normal rats, untreated mutants showed little bone growth or angiogenesis. When mutants were treated with CSF-1 angiogenesis was markedly accelerated. These data show a remarkable effect of this growth factor on angiogenesis in this osteopetrotic mutation. Whether this effect of CSF-1 on angiogenesis is direct or indirect is not known and indicates that its effects on the normal microvasculature deserve further study.

Histochemistry and biochemistry of tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant acid adenosine triphosphatase (TrATPase) in bone, bone marrow and spleen: implications for osteoclast ontogeny

In order to evaluate the usefulness of a recently described acid ATPase as a marker for osteoclast differentiation, we have performed histochemical and biochemical analyses of the distribution of tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant acid ATPase (TrATPase) in bone, bone marrow and spleen. Histochemical studies of bone demonstrated that multinucleated osteoclasts stained for both TRAP and TrATPase. However, staining for TRAP covered the entire cytoplasm, whereas TrATPase staining was localized primarily to cytoplasmic areas next to bone and on adjacent mineralized surfaces. Occasionally TrATPase-positive mononuclear cells were observed on excavations in the bone surface. In the spleen, mononuclear TRAP-positive cells were located in the marginal zone between the white and red pulp, whereas no staining for TrATPase was observed. Comparison of the biochemically measured TRAP and TrATPase activities showed that bone had the highest specific activity for both enzymes followed by the bone marrow and spleen. However, enzyme activity in the spleen compared to bone was about 4-fold higher for TRAP compared to TrATPase. Additional evidence for a restricted expression of TrATPase to bone relative to spleen was obtained by in vitro translation studies. These data indicate that TrATPase is a more selective marker than TRAP in histochemical and biochemical studies of osteoclast differentiation and furthermore suggest that development of TrATPase is a late acquisition in osteoclast ontogeny.

Mechanical properties in long bones of rat osteopetrotic mutations.

Osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. Experimental studies with rat osteopetrotic mutations have shown increased bone density and decreased bone strength. However, it is not known if this reduction in bone strength is only due to changes in structure and geometry or if the tissue properties of bone material itself are changed as well. We have evaluated bone tissue properties with nanoindentation in three osteopetrotic mutations in the rat (incisors-absent ia/ia, osteopetrosis op/op and toothless tl/tl) to test the hypothesis that reduced bone resorption in these mutations results in reduced tissue properties of bone material. No significant differences in elastic modulus or hardness were found between osteopetrotic mutants and their normal littermates (NLMs) in any of the three stocks. This indicates that the tissue properties of bone material are not changed significantly in osteopetrosis, even if the mechanical strength is decreased at the macroscopic level.

Experimental studies of osteopetrosis in laboratory animals

Osteopetrosis is a metabolic bone disease characterized by a systemic increase in skeletal mass. It results from a defect in the production or function of osteoclasts and is inherited in nine genetically distinct osteopetrotic animal mutations and man. Studies of these mutations have revealed that osteopetrosis is a complex, heterogeneous disorder in its expression, etiology, and response to treatment by bone marrow transplantation or by hormone/growth factor therapy. These animal mutations have been valuable tools for probing the pathogenesis and treatment of osteopetrosis, and information obtained from these studies has been used clinically for the treatment of humans with osteopetrosis. In addition, studies of these mutations have contributed significantly to understanding normal bone cell biology, including the origin of the osteoclast and the significance of colony-stimulating factor-1 in osteoclast development. The resistance of some of these mutations to cure by stem cell transplantation and hormone therapy, coupled with similar observations and experiences in the human condition, indicates that these animal mutations will continue to serve important roles in the development of alternative therapies to treat resistant forms of the disease. These studies are bound to improve the understanding of normal bone biology by providing additional insights into the regulation of osteoclasts by osteoblasts and their products or by other elements of the skeletal microenvironment.

The skeletal effects of colony-stimulating factor-1 in toothless (osteopetrotic) rats: persistent metaphyseal sclerosis and the failure to restore subepiphyseal osteoclasts

Toothless (tl), one of four osteopetrotic mutations in the rat, is characterized by few osteoclasts, undetectable bone resorption, and failure of correction by bone marrow transplantation. We recently reported that CSF-1 treatment improves these skeletal problems but that metaphyseal sclerosis persists. In the present study we demonstrate that optimal reduction of the skeletal sclerosis in tl rats following CSF-1 treatment has lower and upper dosage thresholds and that skeletal sclerosis returns after CSF-1 withdrawal. Osteoclasts increase significantly in tl rats after CSF-1 treatment, but compared to untreated normal littermates, histochemical staining for characteristic enzymes and osteoclast number is reduced and no osteoclasts appear in the subepiphyseal areas of long bones. These data are interpreted to mean that there are dosage limits to the beneficial skeletal effects of CSF-1, that persistent sclerosis is related to the failure to restore subepiphyseal osteoclasts, that osteoclast or progenitor populations may be deficient or differ in their responses to CSF-1, and that the defect in tl rats is not merely lack of a circulating, biologically active form of CSF-1.

Expression of and Role for Ovarian Cancer G-protein-coupled Receptor 1 (OGR1) during Osteoclastogenesis

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-κB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1tl/csf1tl) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.