The Regulation of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase 2 (SHP-2) in Brain Microglia

SHP-2, a member of protein tyrosine phosphatases (PTPs), plays a role on the regulation of several signaling pathway such as NF-kB, MAP kinase, JAK-STAT, and PI3 kinase. However, the roles of SHP-2 in brain microglia activation are largely unknown. Here, I determined the regulation of SHP-2 on JAK-STAT signaling pathway in brain microglia. This study firstly shows that curcumin has inhibitory effects on JAK-STAT signaling, which is mediated by SHP-2 in brain microglia. Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. I firstly determined whether curcumin suppressed the gangliosdies-, LPS-, and IFN-γ induced microglial activation. In both rat primary microglia and murine BV2 microglial cells, curcumin effectively suppressed the gangliosides-, LPS-, or IFN-γ-stimulated induction of COX-2 and iNOS, important enzymes that mediate inflammatory processes. Also, curcumin consistently suppressed nuclear factor binding to GAS/ISRE sequences which regulate iNOS and COX-2 expressions. Curcumin markedly inhibited the phosphorylation of STAT1/3 as well as JAK1/2 in microglia activated with gangliosides, LPS, or IFN-γ. Curcumin inhibits the expression of inflammation-associated genes, including ICAM-1 and MCP-1, whose promoters contain STAT-binding elements. To test the inhibitory mechanism of curcumin on JAK-STAT pathway, I firstly tested the expression of SOCS1/3 by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). As a result, their expressions were not induced by curcumin. Also, I examined the phosphorylation of SHP-2 by immunoprecipitation in microglia. Treatment of microglial cells with curcumin induced phosphorylation of SHP-2. I further showed that curcumin led to an increase in association with JAK1/2 of SHP-2, which inhibit the initiation of JAK-STAT inflammatory signaling in activated microglia. Taken together, these data suggest curcumin suppresses JAK-STAT signaling via activation of SHP-2, thus attenuating inflammatory response of brain microglial cells. Secondly, this study also shows that SHP-2 is regulated by lipid rafts in gangliosides-activated microglia. It was reported that, in cultured rat brain microglia, gangliosides induce rapid and transient activation of the JAK-STAT pathway. I hypothesized that raft-mediated SHP-2 activation is involved in transient JAK-STAT signaling by gangliosides. To test this hypothesis, I first used Western blot analysis to show that SHP-2 is rapidly phosphorylated by gangliosides. This was inhibited by pretreatment with the lipid raft disrupter, filipin and was restored following filipin removal. Immunostaining using antibodies directed against p-SHP-2 and flotillin-1 revealed gangliosides-induced clustering and polarization of p-SHP-2 in membrane rafts. Raft-associated regulation of SHP-2 was further demonstrated in fractionation experiments using detergent and detergent-free sucrose gradient ultracentrifugation. Rapid SHP-2 recruitment to detergent-insoluble raft fractions by gangliosides was inhibited by filipin, further indicating the involvement of rafts. Immunoprecipitation experiments confirmed that SHP-2 rapidly binds to JAK2 in response to gangliosides and this binding could be inhibited by filipin and restored upon filipin removal. This study therefore showed that transient activation of the JAK-STAT pathway by gangliosides is accomplished by SHP-2 in a raft-dependent manner in brain microglia. In conclusion, raft-mediated regulation of SHP-2 is suggested to be an essential component of inflammatory mechanism of brain microglia.

Author dc:creator

• 김, 희영

Contributors dc:contributor

• 주, 일로
• 대학원 의학과
• 104438

• SHP-2
• JAK-STAT
• Curcumin
• Gangliosides
• Lipid raft
• Microglia
• Brain

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000692
000000000692

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1347