VWC2 increases bone formation through inhibiting activin signaling

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (DScD) --Boston University, Henry M. Goldman School of Dental Medicine, 2015 (Department of Molecular and Cell Biology).Includes bibliographic references: leaves 55-64.Recently, significant attention has been paid to the cysteine knot proteins (CKPs) due to their potent biological effects on transforming growth factor (TGF)-[beta] superfamily members. By a bioinformatics approach,we have identified a novel member of CKP family. VWC2 (von Willebrand factor C domain containing 2), which is previously known as Brorin. Since Brorin has been proposed to function as a bone morphogenetic protein (BMP) antagonist, we investigated the binding of Brorin/VWC2 to several BMPs; however, no binding was found. To further identify the possible target of VWC2 among TGF[beta]superfamily members, the [beta]A subunit of activin was found as a binding partner of VWC2. Our data demonstrated that Vwc2 gene expression is temporally upregulated early in osteoblast differentiation. VWC2 protein is present in bone matrix, and localized at osteoblasts/osteocytes. Activin A-induced Smad2 phosphorylation was inhibited in the presence of exogenous VWC2 in the MC3T3-E1 osteoblast cell line and in primary mouse osteoblasts. The effect of VWC2 on ex vivo cranial bone organ cultures treated with activin A was investigated, and bone morphometric parameters including total/new bone areas and width decreased by activin A were restored with VWC2 treatment. To further investigate the biological mechanism how VWC2 inhibited the effects of activin A on bone formation, osteoblast cell growth, differentiation,and mineralization were examined,and the effects of activin A on these cell functions were reversed by VWC2 treatment. Taken together, VWC2 is a novel secretory protein in bone and promotes bone formation by inhibiting Activin-Smad2 signaling pathway