Signal Transduction Mechanisms of Gouty Inflammation.

Gout is an inflammatory disease characterized by episodic bouts of attacks. However, its mechanism of inflammation is elusive. It is well-known that monosodium urate (MSU) can stimulate various tissues to evoke intense inflammation and its role as a danger signal and possibly immunomodulator is also recently discovered. The signaling pathways between MSU stimulation and cytokine production are poorly defined. There may be 3 putative pathways: First, IgG-coated MSU crystal may be recognized by Fcγ receptor. The internal signaling probably involves src family protein tyrosine kinases (PTK) and ERK1/ 2 pathways, as reported in other phagocytosis-induced signaling. However, little study can be found for MSU phagocytosis-related signaling pathways and this would be the focus of our project. Second, studies have demonstrated the importance of toll-like receptor (TLR) 2/4 and its intracellular adaptor protein, MyD88, in the signaling of crystal-induced inflammation. TLRs also activate ERK1/2 and subsdequently activation of AP-1 and NF-κB, resulting in increased production of pro-IL-1β and pro-IL-18. Third, the recognition of MSU crystal by NALP3 activates inflammasome and starts processing of pro-IL-1β and pro-IL-18 to mature IL-1 and IL-18 and exerts their pro-inflammatory effect. The crosstalk between TLR and inflammasome is obvious but the relationship between FcγR, TLR and inflammasome remains to be clarified. The clinical manifestation of gout cannot be explained by any single pathway alone. The elucidation of crosstalks can provide more insight into molecular mechanism of gout and its treatment. We propose to investigate the mechanism of gouty inflammation in three years. We will clarify the involved signal transduction pathways as well as its upstream and downstream regulation and crosstalks between different signaling pathways. We also hope to find out the mechanisms that colchicines plays in treatment of gout and prevention of gouty inflammation. In the first year, we will prepare MSU crystals by chemical method and also coat it with immunoglobulin. The monocyte cell line will be used to demonstrate the stimulation of cytokines production through FcR and TLR pathways by coated or naked MSU. Confocal microscopy and in vivo immunofluorescence will assist in the colocalization of MSU crystals with relevant signaling molecules. In the second year, we』ll investigate crosstalk between FcγR and TLR signaling pathways. Not only TLR 2/4, we would like to study if TLR 7/9 is involved in the recognition of MSU crystals and take part in the crystal-induced inflammation. In the third year, we will investigate the crosstalk between FcR, TLR and NALP3/inflammasome. We also want to find out mechanisms of colchicines in treatment and prevention of gout. The specific aims will be First year 1. demonstration of MSU stimulates cytokine production , IL-8 and IL-1 etc. through FcR and TLR pathways and the involved signaling cascade. 2. Confocal microscopy to delineate the colocalization of MSU crystals and signaling molecules, Syn,Fyn, Lyn, Vav, Cbl-b 3. Confocal microscopy to demonstrate fusion of MSU-containing phagosome and TLR-containing lysosome Second year 4. To investigate the crosstalk between FcγR and TLR 2/4. A. Use MyD88 inhibition to establish the role of TLR in MSU inflammation B. Define the interaction between Syk and MyD88 C. IL-8 secretion under the blockage of NF-κB and non-specific tyrosine kinase inhibition D. Find the MyD88-independent TLR signaling in response to MSU stimulation E. Define the separate roles of FcR and TLR in gouty inflammation Third year 5. To investigate the 2 signal theory (TLR and P2X7 ) of gouty inflammation 6. To investigate the crosstalk between FcγR and NALP. A. Confocal microscopy: define the localization of intracellular MSU and inflammasome B. Define the interaction between syk and NALP family 7. To delineate the mechanism of colchicine, with emphasis on the blocking of inflammasome pathway

Project ID:PC9706-0488
External Project ID:NSC96-2314-B182-012-MY3