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.
The broad goals of the exploratory application are to establish that pigment epithelium derived factor (PEDF) reduces expression of sclerostin mRNA and protein by osteocytes in vitro and in vivo, and potential mechanisms involved in this process. Patients with a recessive form of osteogenesis imperfecta (OI) type VI, lack expression of PEDF leading to a defect in mineralization. Mechanisms that lead to defective mineralization as a result of lack of PEDF are not known. We hypothesize that PEDF suppression of sclerostin expression by osteocytes may be one of the mechanisms by which PEDF regulates matrix mineralization. The application proposes to utilize fully characterized primary human osteocytes isolated from human bone to demonstrate that PEDF suppresses Sost/sclerostin expression and the role of PEDF receptors in sclerostin suppression. Completion of the proposed studies will lead to the development of novel approaches for treating osteoporosis and other bone pathologies based on inhibition of sclerostin expression by PEDF.
| Li, Feng; Cain, Jarret D; Tombran-Tink, Joyce et al. (2018) Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling. Biochim Biophys Acta Mol Basis Dis 1864:3449-3458 |
| Li, Feng; Armstrong, Gillian B; Tombran-Tink, Joyce et al. (2016) Pigment epithelium derived factor upregulates expression of vascular endothelial growth factor by human mesenchymal stem cells: Possible role in PEDF regulated matrix mineralization. Biochem Biophys Res Commun 478:1106-10 |
