Genetic and chemical biological studies of a novel Wnt regulator Tiki2

Osteoporosis and bone fractures are global health problems. Identification of newtherapeutic targets and strategies are of paramount importance. Signaling by the Wnt family of secretedproteins has emerged as a key pathway for human bone mass regulation, as loss-of-function and ‘gain-offunction’mutations in Wnt coreceptor LRP5 are associated with familial osteoporosis and high bone massdiseases, respectively. Wnt/LRP5 signaling primarily regulates the osteoblast lineage and bone generation,providing a potential avenue for therapeutics that stimulates bone growth. Remarkably secreted Wntantagonists are expressed in bone and modulate Wnt/LRP5 signaling locally, and thus offer treatmentopportunities targeting bone specifically. Indeed Sclerostin, an osteocyte-specific secreted factor that binds toand inhibits LRP5, has become a key drug target for osteoporosis. We have identified a new family of Wntantagonists, referred to as Tiki proteins, which likely are novel enzymes that post-translationally modify andinactivate Wnt ligands. We have generated Tiki2-/- mutant mice, which surprisingly are viable but exhibit highbone mass, suggesting that Tiki2, like Sclerostin, is an important negative regulator of bone homeostasis.Because of its enzymatic activity human TIKI2 may be an ideal target for small molecule inhibitors for potentialtherapeutic intervention for osteoporosis. We propose four aims to investigate the role of Tiki2 in bone biologyand potential therapeutic implications. In Aim 1, we will characterize the high bone mass phenotype ofTiki2-/- mutant mice, employing histological, biochemical and biomechanical methods. We will also examinein details the expression of Tiki2 in bone cell types, its regulation in bone by anabolic and mechanical stimuli.In Aim 2, we will generate mutant mice with conditional Tiki2 deletion in the bone and in the adulthoodvia cell type-specific and a tamoxifen-inducible Cre lines. These studies will define the site and stage of Tiki2action in bone and in the aging process. In Aim 3, we will investigate human TIKI2 (and TIKI1) bone-relatedfunctions and biochemical properties in vitro. We will investigate TIKI2 and TIKI1 expression in bone, andstudy whether TIKI2 and TIKI1 regulates bone formation in human osteoblast-like cell lines, and characterizethe specific Wnt proteins that are modified/inactivated by TIKI proteins in bone mass regulation. In Aim 4, wewill identify small molecule inhibitors of TIKI2 via chemical compound screening and to evaluate theirpotential in bone growth stimulation in vitro and in vivo. As a new class of Wnt antagonists with anenzymatic activity, TIKI proteins are ideal targets for small molecule inhibitors for use in experimentalmanipulation and therapeutic intervention. We will perform a high throughput chemical compound screen toidentify Tiki2 inhibitors for their potential applications in stimulating bone growth. These studies togetherrepresent a comprehensive analysis of TIKI function in bone biology via genetic, biochemical and chemicalbiological methods, and may discover potential novel therapeutics for osteoporosis and bone regeneration.