Role of NFAT in the Skeleton

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 DESCRIPTION (provided by applicant): Investigations from our laboratory revealed that skeletal cells synthesize Nuclear factor of activated T- cells (NFAT)c1 through c4. Characterization of cells expressing constitutively active (ca) Nfatc1 or Nfatc2 indicates an inhibitory role of Nfat in osteoblastic differentiation and function. This was confirmed in vivo, ad transgenics expressing caNfatc2 in osteoblasts under the control of the type I α1 collagen promoter (Col3.6- Nfatc2) are osteopenic and have impaired bone formation. Studies in mouse models of global Nfatc2 inactivation are difficult to interpret because they have non-specific inflammatory responses and upregulation of various cytokines. To understand the function of Nfatc2 in osteoblasts, we obtained conditional Nfatc1loxP/loxP and created conditional Nfatc2loxP/loxP mice placing us in a unique position to establish its function, singly and in the context of the Nfatc1 inactivation. In accordance with the osteopenic phenotype of the Col3.6-Nfatc2 transgenics, inactivation of Nfatc1 or Nfatc2 in Osterix expressing cells resulted in an increase in cancellous bone volume. In independent studies, we discovered that Notch induces Nfatc2 expression in osteoblasts selectively and acts by post-transcriptional mechanisms. In a Notch2 gain-of-function model, Notch2Q2319X mutant mice exhibited osteopenia due to increased bone resorption and lack of a compensatory bone forming response. In this model, Nfatc2 is induced in osteoblasts and may account for the uncoupling of the two remodeling events. The goal of this project is to understand the function of Nfatc2 in cells of the osteoblastc lineage. For this purpose, we will study mouse and cellular models misexpressing Nfatc2 in cells of the osteoblastic lineage. Our specific aims are: 1) To establish the function of Nfatc2 in osteoblasts. To this end, the skeletal phenotype of conditional deletion mutants of Nfatc2, singly and in combination with Nfatc1, will be established in cells of the osteoblastic lineage using microcomputed tomography and bone histomorphometric analysis. In addition, we will examine the biomechanical properties of bone and study mechanisms responsible for the effects; 2) To establish the post-transcriptional control of Nfatc2 by Notch in osteoblasts. This Aim will define the control of Nfatc2 expression by Notch and identify RNA sequences, binding proteins and microRNAs responsible for Nfatc2 post-transcriptional regulation; and 3) To establish the contribution of Nfatc2 to Notch actions in osteoblasts and determine whether the Nfatc2 induction by Notch is responsible for the failure of Notch2Q2319X mutants to manifest a bone forming response to enhance bone resorption. To this end, the Notch2Q2319X phenotype will be examined in the context of the Nfatc2 inactivation. These investigations should clarify the function of Nfatc2 in cells of the osteoblastic lineage, and identify novel mechanisms by which the skeleton integrates distinct signals to regulate bone remodeling.