Carbonic anhydrase II and H+ -ATPase in osteoclasts of four osteopetrotic mutations in the rat.

Osteopetrosis in laboratory animals is a metabolic bone disease characterized by increased skeletal mass. It is inherited as an autosomal recessive and results from a defect in the development and/or function of osteoclasts. We studied two enzymes essential for bone resorption, carbonic anhydrase II isoenzyme (CA II) and H+ -ATPase, in osteoclasts from four osteopetrotic mutations in the rat; namely incisors-absent (ia), osteopetrosis (op), toothless (tl), and microphthalmia (mib), to test the hypothesis that reduced bone resorption in one or more of these mutations results from defects in the synthesis or activity of one of these enzymes. CA II was present in most osteoclasts from normal, tl, op, and mib littermates and was homogeneously distributed in cytoplasm. CA II staining in ia osteoclasts was more variable and less intense than in the other mutations. H+-ATPase was also present in osteoclasts from normal animals and mutants and immunostaining showed clear polarization to the ruffled border region in all normal rats and mutants except ia, which showed diffuse distribution of staining in the cytoplasm. H+-ATPase activity (proton transport) in a related tissue, kidney, was normal in tl and ia rats but increased in op and mib rats compared to their normal littermates. These results suggest that the osteoclasts in osteopetrotic rat mutations are not abnormal with respect to the distribution of CA II and H+ -ATPase and that the function of these enzymes in the skeleton, while likely normal, needs to be tested directly in bone.

Bone surface morphology reflects local skeletal metabolism.

The metabolic activity of bone cells is faithfully reflected in the surface topography of mineralized bone surfaces, and this can be easily detected by scanning electron microscopy (SEM). Forming bone surfaces exhibit knobby projections which represent foci of mineralization, resorbing surfaces are scalloped, and resting surfaces undergoing neither activity are smooth, as shown by Boyde and Hobdell 25 years ago. These phenomena are illustrated in vivo by tooth eruption, a local activity in alveolar bone where resorption and formation are polarized around an erupting tooth, and osteopetrosis, a metabolic bone disease characterized by a congenital reduction or absence of bone resorption. The ability to analyze bone metabolism over large areas of the skeleton by SEM offers a convenient and powerful microscopic technique to assess regional and global bone cell activity in an era where the investigative focus is increasingly molecular.

Use of bone cell cultures to study skeletal pathology.

We describe procedures for the isolation, culture, and analysis of neonatal osteoblasts from osteopetrotic (toothless (tl) and osteopetrosis [op]) rats and normal littermates. Normal osteoblasts produce and mineralize an extracellular matrix indistinguishable from that of well-characterized fetal rat osteoblasts in vitro. Mutant (tl and op) cultures show an early abnormal pattern of cell proliferation and a later premature, extensive mineralization which mimic the mutant phenotype in vivo. In cocultures with normal osteoclasts, mutant (tl) osteoblasts also show a greatly reduced ability to orchestrate bone resorption, as revealed by pit formation in bone slices, in response to physiologic mediators. These phenomena in vitro are consistent with the behavior of mutant osteoblasts and osteoclasts in vivo and suggest that more definitive microscopic analyses of osteoblasts from each mutation in vitro will provide insights on the roles of osteoblasts in the compromised bone resorption which characterizes the osteopetroses as well as their role in osteoclast ontogeny. This study shows that when their behavior is confirmed in vivo, bone cell cultures offer rigorous systems for understanding skeletal cell dysfunction in normal and pathological development.

Osteoblasts from the toothless (osteopetrotic) mutation in the rat are unable to direct bone resorption by normal osteoclasts in response to 1,25-dihydroxyvitamin D.

Osteopetrosis describes a diversified group of metabolic bone disorders characterized by a generalized, skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption. The toothless (tl) osteopetrotic mutation in the rat is characterized by few osteoclasts and the inability to be cured by transplants of hemopoietic stem cells. This implies that the defect(s) responsible for reduced osteoclast activity in tl rats is within the skeletal microenvironment (cells or matrices). Osteoblasts and their products are known to play a role in regulating bone resorption and abnormalities in the osteoblast population in tl rats have been reported. The purpose of this study was to determine whether osteoblasts isolated from tl mutant rats, when cultured with normal osteoclasts, could increase bone resorption (pit formation) in response to stimulation by 1,25 dihydroxyvitamin D (1,25(OH)2D). The addition of 1,25(OH)2D produced a highly significant response in normal osteoblast cocultures but no response in mutant cultures. A dose response study with 1,25(OH)2D (10(-6) to 10(-9)M) revealed that mutant osteoblasts are unable to increase osteoclast activity. These data indicate that the vitamin D receptor-signal transduction pathway in tl rats needs to be examined.

Bafilomycin A1 in bone resorption and tooth eruption in dogs.

Tooth eruption depends on bone resorption to form an eruption pathway. We have previously shown that a 2-wk local infusion of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPases in osteoclasts, into the crypts of erupting mandibular premolars in dogs blocks bone resorption during this period and eruption of these teeth is delayed for 8 wk. Here we report the limits of inhibition of resorption that still permit eruption of these teeth. In 3 dogs 10(-6) M bafilomycin was delivered by osmotic minipumps early (18 wk) in eruption to the fourth premolar for 1, 3 or 4 wk. Radiographs taken at weekly intervals thereafter showed that bafilomycin delivery for 1 wk delayed eruption for 3 wk, delivery for 3 wk delayed eruption 9 wk and delivery for 4 wk prevented eruption. These data show that tooth eruption is delayed in direct proportion to the time resorption is blocked, and that this process for dog premolars cannot be blocked for more than 3 wk with 10(-6) M bafilomycin without blocking eruption itself.

Colony-stimulating factor-1 injections improve but do not cure skeletal sclerosis in osteopetrotic (op) mice.

The osteopetrotic (op) mutation in mice is characterized by general skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Contrary to previous reports, we have noted persistent metaphyseal sclerosis in op mice treated with CSF-1 at doses above physiological concentrations of circulating CSF-1. We pursued this observation by quantitating osteoclasts and macrophages in the first 500 microns (area A) and the subsequent 1000 microns (area B) in the proximal tibial metaphysis using tartrate-resistant acid phosphatase and F4/80 as cell markers. In untreated normal mice, osteoclasts and macrophages were found in areas A (9.1 and 13.8 cells/1000 microns2) and B (4.1 and 9.4 cells/1000 microns2), respectively. In untreated mutants, osteoclasts and macrophages as percentages of normal were, respectively, 0% and 2% (area A) and 30% and 13% (area B). After CSF-1 treatment (0.15, 0.3, 0.5, and 1.0 x 10(6) U/day) for 28 days, marrow cavity size and numbers of osteoclasts and macrophages increased significantly in area B. However, area A remained sclerotic, with few macrophages (3% to 20%), and although osteoclast numbers were normal, their distribution was not, being absent in subepiphyseal sites. High CSF-1 gene expression occurs at bone modeling sites, co-localizes with osteoblasts, and temporally correlates with their differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bafilomycin A1 inhibits bone resorption and tooth eruption in vivo.

It has been shown that a specific inhibitor of vacuolar H(+)-ATPases, bafilomycin A1, inhibits bone resorption by isolated chicken osteoclasts by blocking the proton pump in the ruffled border membrane. We report here the effects of bafilomycin A1 on bone resorption in vivo. Using a cannulated osmotic minipump delivery system, we infused bafilomycin locally to the eruption pathway of permanent premolars of beagle dogs. We used pit formation by osteoclasts in vitro to estimate the concentrations and heat stability of bafilomycin to be used in vivo. In this model, osteoclasts were cultured on thin bone slices, in which they form pits indicative of resorption. After 2 weeks preincubation at 37 degrees C, bafilomycin concentrations of 10(-6) and 10(-7) M but not 10(-8) M completely inhibited the resorptive activity of cultured osteoclasts, and the two larger doses were chosen for use in vivo. Local delivery of 10(-6) M bafilomycin to the eruption pathway of the fourth permanent mandibular premolar during mideruption inhibited tooth eruption by blocking bone resorption as assayed by radiography, light microscopy, and scanning electron microscopy. Bafilomycin at 10(-7) M had similar but less intensive effects. Moreover, osteoclasts in the alveolar bone of crypts treated with 10(-7) M bafilomycin A1 stained very weakly for tartrate-resistant acid phosphatase. The effect of bafilomycin on bone resorption was shown to be very local, and no side effects of treatment with bafilomycin were observed in adjacent teeth or the behavior of dogs. We report here, for the first time, inhibition of tooth eruption caused by inhibited bone resorption using bafilomycin A1 in vivo.

Carbonic anhydrase isoenzymes in isolated rat peripheral monocytes, tissue macrophages, and osteoclasts.

The presence of carbonic anhydrase isoenzymes I and II in rat monocytes and macrophagelike cells was studied using monospecific antisera against rat carbonic anhydrase I and II purified from red blood cells. CA II was strongly stained immunohistochemically in osteoclasts and macrophagelike cells in the umbilical cord. Foreign body giant cells, peritoneal macrophages, lung macrophages, and cultured peripheral monocytes, the presumed progenitor cells for osteoclasts, were negative with both antisera. Radioimmunoassay and immunoblotting similarly failed to demonstrate CA II in peripheral monocytes. The lack of CA in monocytes adds a new aspect to the discussion concerning the origin of osteoclasts and monocyte-mediated bone resorption.