? DESCRIPTION (provided by applicant): The broad goal of the revised exploratory application is to understand mechanisms by which Pigment epithelium-derived factor (PEDF) enhances bone mineralization with present focus on effect of PEDF on sclerostin expression by osteocytes. Lack of PEDF which is encoded by Serpinf1 gene has been reported to be the cause of a recessive form of osteogenesis imperfecta type VI whose hallmark is a defect in mineralization. The mechanisms by which PEDF leads to defective mineralization are not known. We and others reported that PEDF enhances mesenchymal stem cell (MSCs) differentiation and mineralization in osteogenic cultures. Reduction or absence of PEDF expression by MSCs or osteoblastic cell line resulted in the inability of the cells to mineralize bone matrix upon differentiation. Our recent data indicate that PEDF suppresses expression of Sost/sclerostin by osteocytes differentiated from hMSCs and osteoblasts in osteogenic cultures as well as by primary osteocytes isolated from human bone. Sclerostin is a known potent inhibitor of bone formation through its inhibition of Wnt signaling. We hypothesize that reduction in Sost expression by osteocytes as a result of exposure to PEDF may be one of the mechanisms by which PEDF increases mineralization of bone matrix. In this application, we propose to establish that PEDF suppresses expression of sclerostin by osteocytes in vitro and in vivo and to examine potential mechanisms that may be involved in this process with a focus on PEDF receptors. Preliminary data suggest that PEDF may act through PEDF-R (PNPLA2) to suppress Sost expression by osteocytes. Two aims are proposed: In aim 1, we will use fully characterized primary human osteocytes to demonstrate that PEDF suppresses expression of Sost/sclerostin by osteocytes. Osteocytes will be isolated from human bone chips using established protocols and characterized for expression of osteocytic markers (E11, DMP-1, PHEX, MEPE and Sost). We will determine potential mechanisms by which PEDF suppresses expression of Sost by osteocytes with a focus on PEDF-R. We will block the receptor with siRNA or neutralizing antibodies and then assess expression of Sost by QPCR and sclerostin by western blotting. We will determine signaling pathways that may be activated (ERK, AKT, Wnt) upon PEDF binding to the receptor. In aim 2, we will use an aged rat model to determine whether PEDF suppresses expression of Sost/sclerostin in vivo. We will implant PEDF loaded microosmotic pumps in rats and they will be sacrificed at 7 and 14 days. Effect of PEDF on sclerostin expression will be assessed by QPCR, immunohistochemistry and western blotting in recipient bones. Changes in bone structure will be assessed by micro CT at day 28. Completion of the proposed studies will lead to the development of novel approaches to treat osteoporosis and other bone related pathologies based on sclerostin suppression by PEDF.
|Effective start/end date||7/17/15 → 6/30/17|
- National Institutes of Health: $170,170.00
- National Institutes of Health: $204,204.00