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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032035-15
Application #
2684760
Study Section
General Medicine B Study Section (GMB)
Project Start
1984-04-01
Project End
2000-06-30
Budget Start
1998-04-01
Budget End
2000-06-30
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Trujillo, Glenda; Habiel, David M; Ge, Lingyin et al. (2013) Neutrophil recruitment to the lung in both C5a- and CXCL1-induced alveolitis is impaired in vitamin D-binding protein-deficient mice. J Immunol 191:848-56
Hiroki, Tomoko; Liebhaber, Stephen A; Cooke, Nancy E (2007) An intronic locus control region plays an essential role in the establishment of an autonomous hepatic chromatin domain for the human vitamin D-binding protein gene. Mol Cell Biol 27:7365-80
Hiroki, Tomoko; Song, Young-Han; Liebhaber, Stephen A et al. (2006) The human vitamin D-binding protein gene contains locus control determinants sufficient for autonomous activation in hepatic chromatin. Nucleic Acids Res 34:2154-65
White, Peter; Liebhaber, Stephen A; Cooke, Nancy E (2002) 129X1/SvJ mouse strain has a novel defect in inflammatory cell recruitment. J Immunol 168:869-74
Song, Y H; Naumova, A K; Liebhaber, S A et al. (1999) Physical and meiotic mapping of the region of human chromosome 4q11-q13 encompassing the vitamin D binding protein DBP/Gc-globulin and albumin multigene cluster. Genome Res 9:581-7
Safadi, F F; Thornton, P; Magiera, H et al. (1999) Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. J Clin Invest 103:239-51
Song, Y H; Ray, K; Liebhaber, S A et al. (1998) Vitamin D-binding protein gene transcription is regulated by the relative abundance of hepatocyte nuclear factors 1alpha and 1beta. J Biol Chem 273:28408-18
Tang, W X; Bazaraa, H M; Magiera, H et al. (1996) Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera. Anal Biochem 237:245-51
Wang, X; Ray, K; Szpirer, J et al. (1992) Analysis of the human cysteine-rich protein gene (CSRP), assignment to chromosome 1q24-1q32, and identification of an associated MspI polymorphism. Genomics 14:391-7
Wang, X; Lee, G; Liebhaber, S A et al. (1992) Human cysteine-rich protein. A member of the LIM/double-finger family displaying coordinate serum induction with c-myc. J Biol Chem 267:9176-84

Showing the most recent 10 out of 21 publications