Pilot--FGD1/Cdc42 signaling and osteoblast function

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

This proposal is directed towards understanding the role of FGD1/Cdc42 signaling in osteoblast function. FGD1 is a guanine nucleotide exchange factor (GEF) and a specific activator of Cdc42, a p21 Rho GTPase, Cdc42 is involved in the regulation of several key cellular processes including cytoskeletal remodeling, cellular spreading and motility, cell proliferation and survival , and cellular differentiation. FGD1 is composed of (in order) a proline-rich N-terminal region, adjacent GEF and pleckstrin homology (PH) domains, a FYVE domain, a second C-terminal PH (PH2) domain. FGD1 mutations are responsible for the inherited disease Faciogenital Dysplasia (FGDY; Aaarskog-Scott syndrome), a skeletal dysplasia that is manifested, in part, by delayed bone formation, generalized short stature and progressive osteoarthritis. Most FGD1 mutations are predicted null alleles; thus, the FGDY phenotype is typically due to the absence of gene product in affected patients. Fgd1, the mouse FGD1 ortholog, exhibits a restricted pattern of expression in osteoblasts and in regions of active ossification including skeletal elements involved in the FGDY phenotype. Thus, the amassed data suggests that FGD1/Cdc42 significant plays an important role in normal osteoblast function and bone formation. This project is designed to systematically analyze the role that FGD1/cDC42 signaling plays in osteoblast function. Immunocytochemical and microinjection assays will be used to molecularly dissect the FGD1 protein and identify domains and residues involved in the regulations of targeting and GEF activity. Nuclear microinjection and transient transfection will be used to express deletion constructs that remove specific targeting FGD1 domains. After initial studies identify potential regions of interest, reconstitution and targeted missense mutation constructs will be used to further define sequences involved in targeting and GEF activity. To study how FGD1/Cdc42 signaling is involved in the transmission of the ECM signal to regulate osteoblast function, we propose using dominant negative molecules and anti-sense strategies to study the consequences of down-regulated FGD1. In addition, we will use dominant negative forms of Cdc42 to disrupt FGD1/Cdc42 signaling. Expression constructs will be expressed in osteoblasts by microinjection and transient transfection analysis. Osteoblasts will be analyzed to identify changes in cellular processes regulated by Cdc42 including cytoskeletal organization, cell spreading, proliferation and survival. We will perform a cell-spreading assay to study changes in integrin-mediated spreading and adhesion; immunocytochemistry assays will e used to study changes in cytoskeletal reorganization. Cell proliferation will be determined by counting cells; TUNEL and Hoechst staining will be used to examine apoptosis and survival. The proposed analysis should lead to a better understanding of the role that FGD1/Cdc42 signaling plays in osteoblast function and human disease. Furthermore, the studies proposed in this study will provide insight into how the ECM signals are transduced by integrins to regulate osteoblast biology.