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
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/769 |
| Date | January 2012 |
| Creators | Belcher, Joyce Yvonne |
| Contributors | Popoff, Steven N., Safadi, Fayez F., Barbe, Mary F., Monroy, Alexandra M., Yingling, Vanessa R., Owen, Thomas A. |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 135 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/751, Theses and Dissertations |
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