The vitamin D binding protein (DBP), also known as the group-specific component (Gc-globulin), is an abundant serum protein belonging to a multigene family that includes albumin (ALB), alpha-fetoprotein (AFP), and the newly discovered alpha-albumin (alpha-ALB). It has been postulated that DBP participates in a variety of diverse biological functions. These include: vitamin D sterol binding which facilitates serum transport and serves as a circulating reservoir for vitamin D metabolites, high-affinity binding to actin monomers in the bloodstream and participation along with plasma gelsolin (GSN) in the actin scavenger pathway, neutrophil chemotaxis, macrophage activation, and blood coagulation. To study DBP's specific role in these postulated biological functions, we have generated a mouse model that is deficient in DBP by targeted homologous recombination. This DBP-/- line will be studied in Aim 1 to determine DBP's role in the metabolism of vitamin D sterols and their access to tissues, a test of the """"""""free hormone hypothesis"""""""" as it applies to DBP. The DBP-/- line will be studied alone and after mating with a GSN-/- mouse line to determine the independent and combined roles of these two proteins in protecting the animal from actin toxicity following a variety of manipulations designed to increase intravascular G- and F-actin levels. These studies are designed to test the """"""""actin scavenger hypothesis"""""""". DBP- /- mice will be injected with macrophage-activating agents to determine whether these animals have lost their ability to activate macrophages by the pathway proposed to require DBP. Chemotaxis and clotting parameters will also be studied in the DBP-/- model. Although ALB and AFP have served as major models for the study of tissue-specific and developmentally controlling gene expression, little is known about regulation of DBP gene expression.
In Aim 2 we propose to study DBP gene regulation by two tissue-specific elements in its proximal promoter that we have recently characterized. These elements will be further defined by gel retardation, footprinting and characterization of trans-acting factors associating with them. If this proximal promoter is unable to direct normal patterns of transgene expression we will localize the distal elements required to regulate the DBP gene. Transgenic mice will be generated using 100 kb P1 bacteriophage clones containing the intact DBP gene and extensive flanking regions. If appropriate patterns of expression are mimicked with this transgene, regulatory elements will be mapped by DNasel analysis of the transgene in liver chromatin. DBP overexpressing lines resulting from this study will be utilized to refine the Aim 1 functional experiments. Finally the DBP locus will be physically mapped by isolating and ordering a series of YAC clones that will link DBP to ALB, AFP, and alpha-ALB. These YACs will be useful in future experiments on controls regulating the coordinate expression of this multigene family.
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