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

Abstract

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.

SHP-2는 PTPs의 일종으로써 NF-kB, MAP kinase, Janus Kinase-Signaling Transducer and Activator of Transcription (JAK-STAT), PI3 kinase와 같은 여러 신호전달분자를 조절한다. 하지만, microglia activation에 있어서 SHP-2의 조절 기전에 대해선 많은 연구가 이루어지지 않았다. 본 연구에서는 brain microglia에서 SHP-2가 JAK-STAT 신호전달 체계를 어떻게 조절하는지 밝히고자 하였다. 먼저, microglia에서 curcumin은 Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2)를 통해 JAK-STAT 신호를 저해함을 밝혔다. Curcumin은 항산화제 및 항염증약물로 알려져 있으나 그 조절 기전은 명확하지 않다. Curcumin은 뇌염증반응에서 SHP-2의 활성을 통해 JAK-STAT 신호를 저해하였다. 먼저, microglia 염증반응에 중요한 iNOS와 COX-2의 발현을 확인하였다. Gangliosides, interferon-γ, lipopolysaccharides에 의해 활성화된 rat primary microglia와 murine BV2 microglial cell에서 curcumin은 iNOS와 COX-2의 발현을 저해하였고, iNOS와 COX-2의 발현을 조절하는 GAS/ISRE 서열에 nuclear factor binding도 또한 저하시켰다. Curcumin은 GAS/ISRE 서열에 결합하여 유전자 발현을 조절하는 STAT1/3와 STAT의 upstream인 JAK1/2의 인산화를 저해하였다. Curcumin의 JAK-STAT 신호전달체계의 저해에 대한 조절 기전을 조사하기 위해 SOCS1/3의 발현과 SHP-1/2의 활성을 조사하였다. 그 결과, curcumin은 SOCS1/3의 발현을 유도하지 않았으나 SHP-2의 인산화를 유도하였다. 그리고, curcumin은 SHP-2의 JAK1/2와의 binding을 유도하였다. 이 연구는 curcumin은 SHP-2의 활성을 통해서 JAK-STAT 신호를 저해함을 밝혔고 SHP-2가 microglia 염증반응에서 JAK-STAT 신호전달 조절기전에 negatively 작용함을 보여주었다. 두번째, 본 연구에서 gangliosides에 의해 활성화된 microglia에서 SHP-2가 lipid raft를 통해 조절됨을 보여주었다. Rat primary microglia와 murine BV2 microglial cell에서 gangliosides는 빠르고 일시적인 JAK-STAT pathway의 활성을 유도하며, 이런 활성화 과정에 lipid raft-매개 SHP-2가 관여함을 조사하였다. 먼저 gangliosides가 SHP-2의 인산화를 유도하는지 조사하였다. Rat primary microglia와 BV2 cells에서 SHP-2의 인산화가 빨리 유도되었고 lipid raft 저해제인 filipin에 의해 저해되었다. Immunostaining을 통해서 gangliosides가 p-SHP-2를 lipid raft로의 polarization을 유도함을 확인하였다. 이 결과로, SHP-2의 조절이 lipid raft에 의해서 이루어짐을 확인하였다. 또한, detergent-insoluble fractionation과 sucrose gradient ultracentrifugation을 사용하여 gangliosides에 의해 SHP-2가 lipid raft로 이동함을 확인하였다. 이 detergent-insoluble fraction으로의 이동은 filipin에 의해 저해되었고 filipin 제거로 회복되었다. Gangliosides는 JAK2도 raft로의 이동을 유도하였고, SHP-2와 JAK2와의 결합을 증가시켰다. 그리고, 이것은 filipin에 의해 저해되었다. 이 연구에서 brain microglia에서 gangliosides에 의한 일시적인 JAK-STAT pathway가 raft-dependent manner로 SHP-2에 의해 조절됨을 보여주었다. 결론적으로, 본 연구에서는 raft-매개 SHP-2가 microglia 활성을 조절함을 밝혔다.