Nitric oxide (NO) and its reactive derivatives are widely known for regulating vascular tone. We havedemonstrated 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 protectscells from autologous complement-mediated damage. Elevated NO significantly decreased CD55 protein andmRNA in endometrial Ishikawa cells in a time and dose-dependent manner and consequently reducedbacterial invasion. We, therefore, hypothesize that NO inhibits CD55 expression and cellular distributionthrough direct actions at transcriptional level (Aim 1), indirectly through the modulation of sphingolipidmetabolism and, thus altering P13K/Akt path-way (Aim 2) or locally at the membrane through alteringthe components of lipid rafts and their distribution (Aim 3). Further, NO modulates CD55 expression inrat uterus similar to the cell lines. These hypotheses will be tested in three specific aims.
AIM 1. We willinvestigate 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 -43region of CD55 promoter in the NO modulated transcription of CD55. 1.2. We will examine the effect of NO onCD55 mRNA stability by using actinomycin D assay.
AIM 2. We will determine the involvement ofsphingolipid metabolism and PI3K/Akt pathway signaling in NO-induced downregulation of CD55expression. We will assess if NO 2.1. inhibits generation of ceramide in endometrial cells through decreasinghydrolysis of sphingomyelin, 2.2. alters intracellular distribution of ceramiide. 2.3. causes activation of PI3K/Aktpathway in down-regulating CD55 expression.
AIM 3. We will examine the local effects of NO on theinteraction of CD55 molecules with the components of lipid rafts in the membrane and theirdistribution. We will determine if NO 3.1. increases distance between lipid raft-associated molecules: GPIanchored CD55 and caveolin-1 and increase CD55 lateral mobility in the plasma membrane, 3.2. will alterdissociation of CD55 molecule from annexin II and actin cytoskeleton.
AIM 4. We will demonstrate theeffects of NO manipulations in vivo and ex-vivo on CD55 expression in the rat uterus, and assess themechanisms of action. 4.1. We will determine the effects of in vivo manipulation of NO and its effects on theCD55 expression, PI3K/Akt pathway activation and ceramide levels in the uterus of rats. 4.2. We willinvestigate the in vitro effects of NO modifiers on CD55 expression in the rat uterus, and the involvement ofPI3K/Akt pathway and ceramide in this process. 4.3. We will investigate the effects of NO modifiers on CD55expression 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 CD55expression and implications for implantation failure, pregnancy loss and severity of infection.
We hypothesize that NO inhibits CD55 expression and cellular distribution through direct actions at thetranscriptional level; indirectly through the modulation of spingolipid metabolism and; thus altering P13K/Aktpathway; or locally at the membrane through altering the components of lipid rafts and their distribution.
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