Osteoporosis is a significant public health problem in the U.S. and poses a substantial financial burden. The pathogenesis of osteoporosis is known to involve increased destruction of bone, not compensated by parallel increases in the synthesis of new tissue. The only approved anabolic drug for osteoporosis, PTH, also increases bone resorption, thereby limiting its long term use. Thus, there is an urgent need for development of novel anabolic therapies for the treatment of osteoporosis. In our effort to identify control molecules and their signaling pathways that contribute to the regulation of osteogenesis, we have discovered a novel role for the Claudin (Cldn) family of tight junction proteins. In this RO1 application, our focus is on elucidating the role and mechanism of action of Cldn11 in regulating functions of osteoblasts (OBs) based on our exciting new data that mice with targeted disruption of the Cldn11 gene exhibit a severe deficit in trabecular bone volume (40%). Additionally, Cldn11 expression is increased several-fold during fracture healing. While the traditional role of Cldns is to regulate paracellular transport of small molecules, we have new exciting preliminary data that suggests that Cldn11 acts on OBs non-canonically via interacting with a transmembrane protein, Tetraspanin3 (Tspan3), to regulate ADAM10-mediated Notch signaling. We will test this model of Cldn11 action as follows: 1) To test the hypothesis that the Cldn11 expressed in OBs regulates trabecular bone formation, we will characterize the skeletal phenotype of OB-specific Cldn11 transgenic (Tg) mice by micro-CT, histology, mechanical testing and gene expression and determine if the reduced bone formation in Cldn11 KO mice can be rescued by transgenic expression of Cldn11 in OBs. 2) To test the hypothesis that Cldn11 effects on OBs are mediated via its interaction with Tspan3, we will determine the functional consequence of Cldn11/Tspan3 interactions by evaluating if knockdown of Tspan3 abolishes Cldn11-mediated differentiation in OBs, in vitro and in vivo. 3) To test the hypothesis that Cldn11/Tspan3 effects on OBs are mediated via ADAM10-mediated regulation of Notch signaling, we will evaluate the consequence of disruption of the Cldn11/Tspan3 interaction on ADAM10 maturation and activity via overexpression of a dominant negative mutant Cldn11 encoding the Tspan3 binding domain and determine if the Cldn11/Tspan3 interaction regulates Notch signaling and OB differentiation via modulation of ADAM10 activity. 4) To test the hypothesis that Cldn11 promotes fracture healing via regulating Notch signaling, we will determine the fracture phenotype in Cldn11 Tg and/or Tspan3 KO and control mice using a stabilized closed femoral fracture model. We will use notch signaling reporter mice to evaluate if Notch signaling is activated at the fracture site in the Cldn11 Tg mice and determine if treatment with an ADAM10 inhibitor blocks activated Notch signaling. Successful completion of our proposed studies will provide important information on the pathway by which Cldn11 regulates OB functions and could provide novel drug targets to promote anabolic activities of OBs for treatment of osteoporosis.
Developing novel strategies to promote bone formation as an osteoporosis treatment would require a thorough understanding of the molecular pathways and the genes involved in the bone formation process since impaired bone formation is a major contributor to the pathogenesis of osteoporosis. Successful completion of the proposed studies on the role and mechanism of action of a tight junction protein, claudin11 (Cldn11) and its interacting protein, tetraspanin3 (Tspan3), should lead to elucidation of the role for these genes in regulating bone formation via a novel mechanism involving the ADAM10-mediated regulation of Notch signaling. Because Cldn11 and Tspan3 sequences are conserved between mice and humans, future confirmation of a role for these proteins in humans could provide a basis for future development of Cldn11/Tspan3/ADAM10-based novel drugs to increase bone formation for osteoporosis treatment and for skeletal healing.
