Hereditary angioedema develops in individuals heterozygous for C1 inhibitor (C1INH) deficiency. The product of a single allele is insufficient to control activation of the proteolytic systems normally regulated by C1INH. A logical approach to therapy is to enhance inhibitor synthesis from this single gene. This proposal will examine three aspects of C1INH synthesis regulation that have not been studied extensively in any gene and that remain poorly understood.
Specific Aim 1 will examine the role of the polypurine-polypyrimidine (Pu-Py) segment of the C1INH promoter. We will test the hypothesis that enhanced transcription mediated by this region results from interaction of transcription factors with specific sequences within the Pu-Py segment rather than via H-DNA (triple helix hinged) formation. Preliminary electrophoretic mobility shift assays and supershift experiments have demonstrated interaction of HNF-1alpha (hepatocyte nuclear factor) with one site in addition to several other, as yet unidentified, nuclear proteins that bind to sites within the region containing the Pu-Py sequence. Further studies will identify and characterize these proteins and their function. Other studies suggest cooperativity between the Pu-Py region and the HNF-1alpha site. We will test the hypothesis that this cooperativity results from interaction between transcription factors that bind to these regions by co-immunoprecipitation and by direct isolation. DNAse hypersensitivity experiments will be performed to provide support for the hypothesis that H-DNA formation takes place in vivo, as will experiments to analyze induction of transcription using mutated promoter constructs that are incapable of H-DNA formation. Lastly, nucleosomal reconstitution experiments will test the hypothesis that triplex formation creates a nucleosomal barrier during replication.
Specific Aim 2 will examine the role of phosphatases in down-regulating interferon (IFN)-gamma-mediated induction of C1INH in hepatocytes in comparison with the role of proteosome degradation or binding of STAT1 by specific inhibitory proteins. Preliminary data suggest that phosphatases play a major role in this down-regulation. This hypothesis is unexamined in hepatocytes, which are the primary source of most plasma proteins, many of which are IFN-responsive.
Specific Aim 3 will test the hypothesis that estrogens suppress C1INH transcription. Furthermore, we hypothesize that the therapeutic effect of androgens is a result of down-regulation of estrogen receptor expression, which reverses the estrogenic effect. The studies proposed here will contribute both to knowledge of the regulation of C1INH itself and to the role of hormones, nuclear phosphatases and Pu-Py sequences on gene regulation in general.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD033727-09
Application #
6637255
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Lock, Allan
Project Start
1996-02-01
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
9
Fiscal Year
2003
Total Cost
$304,727
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
059709394
City
Boston
State
MA
Country
United States
Zip Code
02115
Davis 3rd, Alvin E (2006) Mechanism of angioedema in first complement component inhibitor deficiency. Immunol Allergy Clin North Am 26:633-51
Cai, Shenghe; Dole, Vandana S; Bergmeier, Wolfgang et al. (2005) A direct role for C1 inhibitor in regulation of leukocyte adhesion. J Immunol 174:6462-6
Liu, Dongxu; Zhang, Dong; Scafidi, Jennifer et al. (2005) C1 inhibitor prevents Gram-negative bacterial lipopolysaccharide-induced vascular permeability. Blood 105:2350-5
Liu, Dongxu; Cramer, Cort C; Scafidi, Jennifer et al. (2005) N-linked glycosylation at Asn3 and the positively charged residues within the amino-terminal domain of the c1 inhibitor are required for interaction of the C1 Inhibitor with Salmonella enterica serovar typhimurium lipopolysaccharide and lipid A. Infect Immun 73:4478-87
Liu, Dongxu; Gu, Xiaogang; Scafidi, Jennifer et al. (2004) N-linked glycosylation is required for c1 inhibitor-mediated protection from endotoxin shock in mice. Infect Immun 72:1946-55
Cai, Shenghe; Davis 3rd, Alvin E (2003) Complement regulatory protein C1 inhibitor binds to selectins and interferes with endothelial-leukocyte adhesion. J Immunol 171:4786-91
Liu, Dongxu; Cai, Shenghe; Gu, Xiaogang et al. (2003) C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide. J Immunol 171:2594-601
Han, Eun D; MacFarlane, Ryan C; Mulligan, Aideen N et al. (2002) Increased vascular permeability in C1 inhibitor-deficient mice mediated by the bradykinin type 2 receptor. J Clin Invest 109:1057-63
Zahedi, Kamyar; Prada, Anne E; Mulligan, Aideen et al. (2002) Normal transcription of the C1 inhibitor gene is dependent upon a polypurine-polypyrimidine region within the promoter. Inflammation 26:183-91
Zahedi, R; MacFarlane, R C; Wisnieski, J J et al. (2001) C1 inhibitor: analysis of the role of amino acid residues within the reactive center loop in target protease recognition. J Immunol 167:1500-6

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