No description available.

Osteoclasts polarise upon activation and form four distinct membrane domains; the basolateral domain, the sealing zone, the functional secretory domain and the ruffled border. The ruffled border is the resorptive organelle of the cell and provides a large surface area for the release of protons and enzymes into the space beneath the osteoclast. Defects in osteoclast formation or function can lead to diseases such as osteopetrosis. Ruffled border formation is a critical event in osteoclast function but the process by which it and other membrane domains form is only partially understood. Vesicular trafficking is essential for the tight regulation of the osteoclast membrane domains and it has been shown previously that treatment with pharmacological inhibitors causes disruption of trafficking. The aims of this PhD were to increase our understanding of vesicular trafficking in osteoclasts and to optimise ways of visualising osteoclast membrane domains. My studies of patients with osteoclast-poor osteopetrosis identified defects in RANKL as a cause of the defect. This in turn has identified a potential therapy of recombinant RANKL for patients with this form of the disease. Although purification of wild type or mutant RANKL was not completely successful, it did suggest that the mutant forms of RANKL were not functional. I have used pharmacological inhibitors to study osteoclast membrane domains, and found that transmission electron microscopy is an essential tool for studying membrane changes following pharmacological inhibition at the ultrastructural level. I also established that the study of vesicular trafficking to analyse formation of membrane domains can make excellent use of immuno-electron methods. Furthermore, genetic diseases associated with defective ruffled border formation such as XLA and osteopetrosis provide useful tools to further analyse the dynamics involved in the formation and maintenance of the ruffled border, as well as revealing more about the diseases themselves.

Osteopetrosis is characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit causes this disease. Modeling the R444L mutation in mouse a3 caused endoplasmic reticulum (ER) retention of a3 with attendant abrogation of maturation and trafficking of the glycoprotein and its degradation. The mutant protein also displayed altered conformation and increased degradation. Together, these data suggest that R444 is involved in protein folding or stability significant to mammalian a3, and that infantile osteopetrosis caused by the R444L mutation in the V-ATPase a3 subunit is another member of the growing class of protein folding diseases. We also ascertained that the N-Glycosylation sites of the a3 glycoprotein lie at position N484 and N504, data that help to refine the topology of the a subunit. Overall, this study sheds new light onto the role that R444 plays in a subunit structure, and refines a subunit topology.

Osteopetrosis is characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit causes this disease. Modeling the R444L mutation in mouse a3 caused endoplasmic reticulum (ER) retention of a3 with attendant abrogation of maturation and trafficking of the glycoprotein and its degradation. The mutant protein also displayed altered conformation and increased degradation. Together, these data suggest that R444 is involved in protein folding or stability significant to mammalian a3, and that infantile osteopetrosis caused by the R444L mutation in the V-ATPase a3 subunit is another member of the growing class of protein folding diseases. We also ascertained that the N-Glycosylation sites of the a3 glycoprotein lie at position N484 and N504, data that help to refine the topology of the a subunit. Overall, this study sheds new light onto the role that R444 plays in a subunit structure, and refines a subunit topology.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. To determine the role of OA in skeletal homeostasis in vivo. we utilized a mouse model with a natural mutation in the osteoactivin gene. This mutation is due to a premature stop codon, which results in the generation of a truncated 150 amino acid OA protein. This animal, which we will refer to as OA mutant, was shown by ìCT and histomorphometric analysis to have increased bone volume, trabecular thickness, and trabecular number compared to wild-type (WT) mice at 4 weeks of age, which is a time at which bone formation is most active. Histological analysis of long bones stained with TRAP (tartrate resistant acid phosphatase) and colorimetric analysis of serum TRAP 5b levels indicated that the numbers of osteoclasts are significantly increased in OA mutant samples. Interestingly, although the numbers of osteoclasts as compared to WT were higher in OA mutant mice, serum levels of C-telopeptide of type I collagen (CTX) and osteocalcin, biomarkers for bone resorption and bone formation respectively, were significantly decreased. These data suggested that in mice the presence of truncated OA protein results in increased osteoclast number, but that they are inefficient in resorbing bone and may in part contribute to the increase in bone volume in OA mutant mice in vivo. To further investigate the role of OA in osteoclast differentiation, osteoclasts were differentiated from hematopoietic stem cell progenitors ex vivo. HSCs were cultured in the presence of 50 ng/ml of M-CSF for two days and then with M-CSF and 100 ng/ml of RANKL in the presence or absence of 50 ng/ml recombinant OA. We observed a dramatic increase in multinucleated TRAP-positive osteoclasts and the number of nuclei per osteoclast in OA-treated cultures compared to control. Additionally, analysis of HSCs showed increased cell proliferation in response to exogenous OA treatment. When osteoclasts were differentiated in ex vivo cultures derived from OA mutant and WT mice, we observed decreased osteoclast number, size, and function in OA mutant compared to WT cultures. This decrease was abrogated when cultures were treated exogenously with recombinant OA. Quantitative PCR analysis of RNA isolated during osteoclast differentiation from WT and OA mutant mice reveal decreased gene expression of critical osteoclast differentiation and functional markers, which explains the osteoclast defect observed ex vivo. To investigate the role of OA in osteoblast differentiation, primary osteoblasts were derived from mesenchymal progenitors isolated from calvariae of WT and OA mutant neonatal pups. OA mutant osteoblasts were found to have decreased alkaline phosphatase (ALP) staining and activity at day 14 in culture. Furthermore when cultures were differentiated to 21 days to simulate matrix mineralization in vitro, OA mutant osteoblasts exhibited decreased Alizarin Red and Von Kossa staining. Quantitative measurement of calcium also showed decreased mineral deposition in OA mutant mice compared to WT. Electron microscopic and protein studies were able to eliminate the notion of ER stress or cell toxicity as a result of ER stress playing a role in the delayed osteoblast differentiation observed in OA mutant osteoblasts. Furthermore, OA mutant osteoblasts exhibited decreased proliferation and survival ex vivo. These data reveal an effect of osteoactivin in osteoblasts ex vivo. This study provided an in vivo tool to study the role of osteoactivin in bone cells and the regulation of bone formation and bone resorption by this molecule. Taken together, these findings suggest that the presence of truncated OA leads to increased bone volume due to defective interplay between bone-resorbing osteoclasts and bone-forming osteoblasts. Data presented here support the notion of osteoactivin as a novel molecule in modulating skeletal homeostasis in vivo. / Cell Biology

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Osteopetrosis is a metabolic bone disease in mammals characterized by a generalized skeletal sclerosis caused by reduced bone resorption. This reduced bone resorption is manifested in afflicted animals by abnormal bone shape, reduced or absent marrow cavities, extramedullary hemopoiesis, abnormal mineral homeostasis and absent or delayed tooth eruption. The available osteopetrotic animal mutations have been a constant source of fruitful investigations concerning the systemic regulation of osteoclastogenesis and bone metabolism. Tooth eruption, on the other hand, is a localized manifestation of the timely activation of bone resorption and bone formation on opposite sides of an erupting tooth. Its rate-limiting step is the speed of bone resorption to form the eruption pathway. In this dissertation, we used two osteopetrotic rat mutations, toothless (tl) and microphthalmia blanc (mib), to investigate the abnormal development of osteoclasts and tooth eruption in mutant rats with an emphasis on the role of systemic and local factors. The significant contributions to this work are listed below. 1. In the toothless rat, a mutation lacking erupted dentition due to severely reduced bone resorption, colony-stimulating factor-1 (CSF-1) promoted tooth eruption but this was delayed compared to normal rats. Eruption was accompanied by changes in the populations of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces. These cell populations were dramatically increased in treated mutants compared to untreated tl rats, but the timing of their appearance was delayed compared to normal littermates. This lag in the appearance of osteoclasts and their precursors corresponded to the delay in eruption of first molars in treated tl rats. 2. CSF-1 also accelerated the eruption of molars in normal rats. CSF-1 increased the number of TRAP+ mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces, but had no effect on the timing of their appearance in normal rats. 3. Our data revealed a differential effect on tooth eruption of the growth factors CSF-1 and epidermal growth factor (EGF). CSF-1 accelerated eruption of molars in normal rats, but had no effect on incisor eruption. On the other hand, EGF accelerated incisor eruption; but did not affect molar eruption in normal rats. 4. We have described the mechanism for the transient, mild form of osteopetrosis inherited by mib rats. Mutant animals possess a typical sclerosis at birth, which diminished--but was not resolved--during the first postnatal month. These characteristics are caused by early reductions in osteoclast number and function which improve to normal levels by 4 weeks. Osteoclast numbers were severely reduced in mib rats between birth and 2 weeks, but improved to near normal levels by 4 weeks. Neonatal abnormalities in osteoclast function included reduced staining for the functional enzymes TRAP and TrATPase, decreased levels of mRNA for both TrATPase and CAll, and inability to form a well-developed ruffled border. None of these defects were apparent after the first postnatal month. 5. Finally, we have shown that the dental abnormalities caused by the mild, transient form of osteopetrosis in mib rats are limited to incisor defects and delayed eruption of all teeth. Histologic and radiographic examination of mutant incisors revealed that, contrary to the situation in normal rats, the apex of the incisors of mib rats failed to extend past the first molar region to the third molar. The incisor apex of newborn mib rats was misshaped due to ankylosis of incisor matrices with alveolar bone. This ankylosis was temporary, being resolved by the third postnatal day. The delayed eruption of incisors in mib rats and abnormal shape and occlusion of these teeth in older animals is a consequence of the temporary ankylosis in newborn rats.