Nitric oxide (NO) and its reactive derivatives are widely known for regulating vascular tone. We have demonstrated that NO is a modulator of uterine infections due to Escherichia coli expressing Dr fimbriae (Dr+). E. coli through its Dr+ binds to decay-accelerating factor (CD55), a complement regulatory protein that protects cells from autologous complement-mediated damage. Elevated NO significantly decreased CD55 protein and mRNA in endometrial Ishikawa cells in a time and dose-dependent manner and consequently reduced bacterial invasion. We, therefore, hypothesize that NO inhibits CD55 expression and cellular distribution through direct actions at transcriptional level (Aim 1), indirectly through the modulation of sphingolipid metabolism and, thus altering P13K/Akt path-way (Aim 2) or locally at the membrane through altering the components of lipid rafts and their distribution (Aim 3). Further, NO modulates CD55 expression in rat uterus similar to the cell lines. These hypotheses will be tested in three specific aims.
AIM 1. We will investigate the effect of NO on the transcription of CD55 DNA and on the stability of CD55 mRNA. 1.1. We will determine, by site directed mutations, the role of CREB, AP1, AP2, and SP1 binding sites at -74 to -43 region of CD55 promoter in the NO modulated transcription of CD55. 1.2. We will examine the effect of NO on CD55 mRNA stability by using actinomycin D assay.
AIM 2. We will determine the involvement of sphingolipid metabolism and PI3K/Akt pathway signaling in NO-induced downregulation of CD55 expression. We will assess if NO 2.1. inhibits generation of ceramide in endometrial cells through decreasing hydrolysis of sphingomyelin, 2.2. alters intracellular distribution of ceramiide. 2.3. causes activation of PI3K/Akt pathway in down-regulating CD55 expression.
AIM 3. We will examine the local effects of NO on the interaction of CD55 molecules with the components of lipid rafts in the membrane and their distribution. We will determine if NO 3.1. increases distance between lipid raft-associated molecules: GPI anchored CD55 and caveolin-1 and increase CD55 lateral mobility in the plasma membrane, 3.2. will alter dissociation of CD55 molecule from annexin II and actin cytoskeleton.
AIM 4. We will demonstrate the effects of NO manipulations in vivo and ex-vivo on CD55 expression in the rat uterus, and assess the mechanisms of action. 4.1. We will determine the effects of in vivo manipulation of NO and its effects on the CD55 expression, PI3K/Akt pathway activation and ceramide levels in the uterus of rats. 4.2. We will investigate the in vitro effects of NO modifiers on CD55 expression in the rat uterus, and the involvement of PI3K/Akt pathway and ceramide in this process. 4.3. We will investigate the effects of NO modifiers on CD55 expression in human endometriosis and the involvement of PI3K/Akt pathway and ceramide in this process. These studies will provide mechanistic insights into the NO-induced down-regulation of CD55 expression and implications for implantation failure, pregnancy loss and severity of infection.

Public Health Relevance

We hypothesize that NO inhibits CD55 expression and cellular distribution through direct actions at the transcriptional level, indirectly through the modulation of spingolipid metabolism and, thus altering P13K/Akt pathway, or locally at the membrane through altering the components of lipid rafts and their distribution.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD057013-04
Application #
8206846
Study Section
Pregnancy and Neonatology Study Section (PN)
Program Officer
Ilekis, John V
Project Start
2009-01-15
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$297,784
Indirect Cost
$100,576
Name
University of Texas Medical Br Galveston
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
Banadakoppa, M; Chauhan, M S; Havemann, D et al. (2014) Spontaneous abortion is associated with elevated systemic C5a and reduced mRNA of complement inhibitory proteins in placenta. Clin Exp Immunol 177:743-9
Banadakoppa, Manu; Goluszko, Pawel; Liebenthal, Daniel et al. (2014) PI3K/Akt pathway restricts epithelial adhesion of Dr + Escherichia coli by down-regulating the expression of decay accelerating factor. Exp Biol Med (Maywood) 239:581-94
Yallampalli, Chandra; Chauhan, Madhu; Sathishkumar, K (2013) Calcitonin gene-related family peptides in vascular adaptations, uteroplacental circulation, and fetal growth. Curr Vasc Pharmacol 11:641-54
Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E et al. (2013) Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+ Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide. FEBS J 280:840-54
Sathishkumar, Kunju; Balakrishnan, Meena; Chinnathambi, Vijayakumar et al. (2012) Temporal alterations in vascular angiotensin receptors and vasomotor responses in offspring of protein-restricted rat dams. Am J Obstet Gynecol 206:507.e1-10