PKA-Wnt Crosstalk in Bone is Mediated by beta-Catenin Nuclear Complex Formation

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DESCRIPTION (provided by applicant): The Wnt signaling pathway is a well-conserved developmental paradigm with roles in cellular differentiation, proliferation, and homeostasis. This pathway has been shown to be particularly important for bone physiology, as inactivation of Wnt signaling causes a loss of bone, whereas excess signaling causes increased bone mass. Protein Kinase A (PKA, cAMP-dependent protein kinase) signaling is also important in bone physiology, as osteoblast proliferation and differentiation can be controlled by PKA signaling mediated by activation of the parathyroid hormone receptor. We have previously demonstrated that mice exhibiting global PKA activation due to mutation of the PKA regulatory subunit Prkar1a frequently develop bone tumors. These tumors are derived from the osteoblast lineage and exhibit excess responsiveness to the growth promoting effects of PKA. Analysis of tumors using global expression profiling identified upregulation of members of the Wnt signaling pathway in Prkar1a+/- mouse bone tumors. We have subsequently determined that these tumors exhibited enhanced Wnt-dependent transcription, although no alterations in ¿-catenin abundance or nuclear-cytoplasmic localization pattern were observed. However, immunofluorescence analysis of ¿-catenin subcellular localization in the tumors revealed that ¿-catenin underwent a nuclear re-distribution which could be recapitulated in wild-type cells by stimulation of the PKA pathway with forskolin. Co-localization experiments indicated that ¿-catenin associated with promyelocytic leukemia (PML) bodies in response to PKA activation. Furthermore, analysis of the promoters of genes upregulated in the Prkar1a+/- tumors demonstrated that most of these genes contain both Wnt- and cAMP-response elements in their promoters. Based on this preliminary data, we hypothesize that PKA phosphorylation of ¿-catenin enhances its binding to transcriptionally active multi-protein nuclear complexes that also contain phosphorylated CREB. In this grant application, we propose 1) to study the requirement of PKA phosphorylation sites within ¿-catenin for this intranuclear redistribution; 2) to determine how PKA and/or Wnt activation alters the distribution of ¿-catenin and CREB-containing nuclear complexes at the promoters of genes with altered transcription, and 3) to determine the full nature of these ¿-catenin-PML containing complexes in order to identify other proteins (including CREB) present. These studies will provide new insights into the mechanism by which ¿-catenin exerts its biological effects in bone cells and provide new mechanistic details describing the crosstalk of the ¿-catenin and PKA signaling pathways. These studies not only have implications for bone physiology, but may provide larger insights into the formation of multiprotein transcriptional complexes mediating complex signaling cascades. PUBLIC HEALTH RELEVANCE: The Protein Kinase A (PKA) and Wnt/¿-catenin signaling pathways are both essential for the development and maintenance of normal bone, as they are involved in the control of cell function and growth. Using a new model of bone tumors, we have found that PKA stimulates the function of the Wnt/¿-catenin pathway and propose to characterize an unusual mechanism by which this interaction occurs. The results of these studies may be important not only for identifying new mean by which bone mass can be modulated (e.g., treatment for osteoporosis), but may also have larger implications for understanding how PKA may modulate the activity of Wnt/¿-catenin in tumors.