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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Dong, Gang
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Cedars-Sinai Medical Center
Los Angeles
United States
Zip Code
Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Jones, Heather D et al. (2015) Alternatively spliced myeloid differentiation protein-2 inhibits TLR4-mediated lung inflammation. J Immunol 194:1686-94
Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S et al. (2011) Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway. J Immunol 187:4331-7
Gray, Pearl; Michelsen, Kathrin S; Sirois, Cherilyn M et al. (2010) Identification of a novel human MD-2 splice variant that negatively regulates Lipopolysaccharide-induced TLR4 signaling. J Immunol 184:6359-66
Lowe, Emily L; Crother, Timothy R; Rabizadeh, Shervin et al. (2010) Toll-like receptor 2 signaling protects mice from tumor development in a mouse model of colitis-induced cancer. PLoS One 5:e13027
Lu, Jing; Pierce, Marissa; Franklin, Andrew et al. (2010) Dual roles of endogenous platelet-activating factor acetylhydrolase in a murine model of necrotizing enterocolitis. Pediatr Res 68:225-30
Soliman, Antoine; Michelsen, Kathrin S; Karahashi, Hisae et al. (2010) Platelet-activating factor induces TLR4 expression in intestinal epithelial cells: implication for the pathogenesis of necrotizing enterocolitis. PLoS One 5:e15044
Karahashi, Hisae; Michelsen, Kathrin S; Arditi, Moshe (2009) Lipopolysaccharide-induced apoptosis in transformed bovine brain endothelial cells and human dermal microvessel endothelial cells: the role of JNK. J Immunol 182:7280-6
Bulut, Yonca; Shimada, Kenichi; Wong, Michelle H et al. (2009) Chlamydial heat shock protein 60 induces acute pulmonary inflammation in mice via the Toll-like receptor 4- and MyD88-dependent pathway. Infect Immun 77:2683-90
Naiki, Yoshikazu; Sorrentino, Rosalinda; Wong, Michelle H et al. (2008) TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis. J Immunol 181:7176-85
Schroder, Nicolas W J; Crother, Timothy R; Naiki, Yoshikazu et al. (2008) Innate immune responses during respiratory tract infection with a bacterial pathogen induce allergic airway sensitization. J Allergy Clin Immunol 122:595-602.e5

Showing the most recent 10 out of 27 publications