Doctor of Philosophy
Matthew J. Beckman
Secreted Phosphoprotein-24 (Spp24) was initially isolated and characterized as a component of bovine cortical bone matrix. Subsequent characterization has shown it is multiply phosphorylated and homologous to cystatin and TGF-β receptor type II. Spp24 is a minor component of the serum fetuin mineral complex that binds calcium-phosphate minerals and prevents their deposition. The TGF-β receptor homology domain binds BMP-2 weakly in vitro and enhances BMP-2’s osteogenic effects in vivo. The ability of Spp24 to affect BMP activity suggests an important role for Spp24 as a native, bioactive componentof bone that regulates bone development.
Spp24 was highly up-regulated in rat cortical kidneys following a low calcium diet regime. Tissue distribution of both Spp24 protein and RNA showed that while Spp24 accumulates in bone, a majority is produced at distant sites, namely the liver and kidney. Additionally, Spp24 was present in more tissues than previously believed. Spp24 migrates to a number of different molecular weights, suggesting multiple, alternative posttranslational modifications may generate subtly different forms of the protein. Theexpression of Spp24 in the kidney may be regulated to counteract changes in serum mineral levels. Additionally, homology in the Spp24 sequence suggests that it, like other bone and dentine matrix proteins, may interact with mineral as an important influencer of mineral calcification.
Utilizing microarray analysis of primary bone marrow-derived mesenchymal stem cells transduced with Spp24 and control viruses we examined changes elicited by the overexpression of Spp24. A change in overall morphology was observed for cellstransduced with the Spp24 similar to changes described in cells undergoing osteoblasticdifferentiation. Nodule formation was also seen in the Spp24 transduced cells. Microarray results showed key markers of osteoblast differentiation, CBFA1/RUNX2 and osterix(OSX), were not up-regulated although there were distinguishable changes in the gene expression profile of mesenchymal stem cells. The cells appeared to be blocked from differentiation into a number of mesenchymal lineages: adipocytes, myocytes andchondrocytes. The changes appeared to prime cells for signals that activate osteoblastdifferentiation by blocking other pathways and altering internal signaling response pathways to those signals.
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