More than 300 rare genetic bone diseases have been identified but treatment for these disorders is usually limited because little of their pathogeneses is known. Studies of rare diseases have been plagued by the unavailability of primary cells/tissues and lack of suitable animal models. Recent advance in patient-specific induced pluripotent stem (iPS) cell biology opened new avenues for studying bone cells from patients. In this application, i plan to use patient-specific IPS cells to study disease mechanisms of craniometaphyseal dysplasia (CMD) with a focus on the role of osteoclasts (OCs), the bone resorbing cells. The onset of CMD begins in childhood with thickening of craniofacial bones and abnormally shaped long bones. Its lifelong progression leads to life-threatening consequences in some patients. To date, there is no treatment other than repetitive surgery. Previous studies in a knock-in (Kl) mouse model carrying a CMD-causing Ank mutation revealed OC defects in Ank[Ki/Ki] mice. Similar results were found in human peripheral blood cultures of CMD patients. Ank[Ki/Ki] OCs also showed slower movement with abnormal actin organization.
Two specific aims are proposed to test the hypothesis that CMD-causing ANK mutations reduce individual osteoclast activity by negatively affecting the actin cytoskeleton.
In Aim 1 the applicant will compare iPS-derived OCs from healthy controls and CMD patients to identify differences in OC formation, matrix and mineral resorption, expression of OC marker genes, adhesion and migration by adhesion assays and live-cell time-lapse imaging, respectively.
In Aim 2 the applicant will study the organization and dynamics of the actin cytoskeleton in CMD and control OCs by confocal microscopy. Two critical regulatory mechanisms in actin biology, the activation of GTPase family members Rac, Rho and Cdc42, and tyrosine phosphorylation in iPS-derived OCs of controls and CMD patients will be examined by active GTPase pull-down assays, immunostaining and immunoblots. Alternatively, we will use a FRET (fluorescent resonance energy transfer)-based analysis to determine the dynamic regulation of Rac, Rho and Cdc42 in life osteoclasts from CMD patients and controls. The long-term goal of this study is to establish CMD as a paradigm for studying mechanisms of rare genetic skeletal disease.