The overall goal of the proposed studies is to define the mechanisms by which MD-2, a co-receptor to TLR4 participates in LPS-induced signaling, to investigate the regulation of MD2 promoter and to characterize the expression, function and regulation of an alternatively spliced version of MD2 (MD2-S) that occurs in human but not mouse tissues. MD-2 is a secreted surface protein, which is required for LPS/TLR4 signaling. A fundamental gap in our understanding of LPS/TLR4/MD-2 signaling is the exact function of MD-2 and the molecular mechanisms, which make MD-2 a critical component of the LPS/TLR4 signaling complex. The regulatory mechanisms of the LPS/TLR4/MD-2-mediated inflammatory signaling at the level of MD-2 and the role for alternative splicing of human MD-2 in this regulation are also unknown. We have observed that an alternatively spliced version of MD-2 exists in human tissues. The studies proposed in this application will seek to characterize and define the functional role and the regulation of the alternatively spliced form of human MD-2 and will investigate the role of MD-2 as signaling molecule and the regulation of the human MD-2 promoter. The hypothesis: Based on our preliminary data, we hypothesize that altematively spliced isoform of MD-2 (short MD-2 or MD-2 S) is unable to transduce LPS/TLR4 signaling and that it may play a negative regulatory role in LSP/TLR4/MD2-induced inflammatory signaling at the level of MD-2. Based on preliminary data, we further hypothesize that MD-2 participates in LPS signaling, and is tyrosine phosphorylated upon LPS stimulation and internalization and that mutations of the tyrosine sites in MD-2 diminishes it's function to transduce LPS/TLR4 signaling. We propose that MD-2 may provide a potential regulatory step in TLR4/MD-2-mediated inflammatory signaling. We have recently cloned the promoter site of MD-2 and we propose to investigate the regulation of MD-2 expression by investigating the regulation of MD-2 promoter. Thus, MD-2 may provide an additional potential regulatory step in LPS/TLR4/MD2-induced inflammatory signaling.
The Specific Aims are: 1- To characterize and determine the tissue expression and function of alternatively-spliced MD-2 isoform (MD-2 S) and expression of recombinant MD-2 S to investigate its function in LPS/TLR4 signaling in in-vitro and in-vivo experiments. 2-To define the molecular signaling mechanisms for the involvement of MD-2 as a signaling molecule in LPS/TLR4-mediated NF-kB activation. Investigate the role of Tyrosine phosphorylation of MD-2 upon LPS signaling. 3-To investigate the regulation of MD-2 promoter, and to determine cytokine responsive elements in the promoter. Significance: Improved understanding of the molecular mechanisms by which MD-2 participates in LPS/TLR4 signaling and the understanding of the functional role of alternative splicing of MD-2 and of the regulation of MD-2 promoter may provide new targets for intervention and prevention of inflammatory diseases where TLR4/MD-2 signaling plays a major role, including gram-negative sepsis and endotoxic shock.
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