Calcium is a pervasive second messenger and transduces endocrine signaling pathways initiated by binding of many hormones and growth factors to membrane receptors. The primary Ca2+ receptor is calmodulin (CaM) and Ca2+/CaM activates several CaM kinases in endocrine cells. Our long-term objective is to elucidate how a recently discovered CaMKIV cascade participates in Ca2+/CaM-mediated gene expression and regulates cell function. Analysis of Camk4~'~ mice identified a CaMKIV cascade in hematopoeitic stem cells (HSC) and cerebellar granule cells that regulates CREB phosphorylation and CBP levels leading to increased Bcl-2 and BDNF mRNA, respectively. We will determine how hormonal activation of the CaMKIV cascade regulates hematopoiesis by influencing maintenance of HSCs. HSCs isolated from CaMKIV- deficient mice cannot reconstitute bone marrow when transplanted into irradiated recipient host mice in a manner suggesting that CaMKIV regulates stem cell homeostatis by suppressing inappropriate proliferation and exhaustion of HSCs. To evaluate this hypothesis we will: 1) elucidate the importance of the CaMKIV kinase cascade in HSC maintenance in vivo. HSCs will be isolated from mice null for one or more of the CaMKIV cascade proteins and evaluated in bone marrow reconstitution assays; 2) address the idea that the CaMKIV cascade in HSCs is regulated by specific members of the Wnt family of growth factors. The Wnt proteins are required for embryonic development and Wnt 3A and Wnt 5A, have been implicated in HSC maintenance. Wnt 5A has been shown to initiate a Ca2+ signal and we will use isolated HSCs to investigate if Ca2+ activates a CaMKIV cascade that includes CREB and CBP and leads to upregulation of pro-survival genes such as Bcl-2; 3) evaluate the mechanism by which the CaMKIV cascade regulates CBP and identify key target genes by which the cascade influences HSC maintenance. We will use cultured cells to determine the molecular mechanism by which CaMKIV regulates CBP levels. We will use cultured cells and bone marrow transplantation to determine if Bcl-2 is a primary target of the CaMKIV cascade in HSCs and, if not, evaluate the importance of other target genes identified by previous RNA microarray analysis. Identification of such downstream genes may suggest efficacious drug targets for achieving better ex vivo expansion of HSCs for bone marrow transplantation patients. ? ?
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