Expression of TRAIL in human astrocytes induced by IFN-γ is modulated by p38 MAPK.
성상세포는 중추신경계에서 가장 많은 수를 차지하고 있는 세포로서 다른 조직에 비해 상대적으로 결체조직이 적은 중추신경계의 물리적인 지지를 담당한다. 성상세포는 이러한 수동적인 역할 이외에도 다양한 생리적, 면역학적인 기능을 하고 있어 중추신경계 내에서 세포고사와 신경계 질환의 면역반응 그리고 중추신경계의 면역특권에 중요한 역할을 한다고 알려져 있다. 세포고사는 다양한 종류의 자극에 의해 일어날 수 있으며, 여러 리간드-수용체의 결합에 의해서도 유도된다. 본 연구에서는 사람 성상세포를 대상으로 세포고사를 유도할 수 있는 TRAIL과 TRAIL 수용체의 발현 그리고 IFN-g에의한 TRAIL 발현조절기전에 대하여 연구하였다. 그 결과, 아무 자극을 주지 않은 일차배양 태아 성상세포에서는 TRAIL의 발현이 거의 관찰되지 않았다. 그러나, 태아 성상세포에 IFN-g를 처치하였을 때 TRAIL의 mRNA 전사가 증가하였으며, TRAIL 단백의 발현도 증가하였다. 이와 같이 IFN-에 의해 유도된 TRAIL이 Peer 세포의 세포고사를 유도하였기 때문에 성상세포에서 발현된 TRAIL이 적절하게 기능함을 관찰하였다. 성상세포에서 IFN-g 신호전달체계를파악하기위하여STAT1과 MAPK 활성화를 측정하였다. 그결과 IFN-g에 의해 STAT1의 Tyr701과 Ser727의 인산화가 유도되었으며 p38 MAPK 그리고 p38 MAPK의 상위 kinase인 MKK3/MKK6의 인산화도 일어남을 알 수 있었다. p38 MAPK의 억제제인 SB203580을 전처치하고 IFN-g를 처치했을 때, STAT1의 Tyr701 인산화는 억제되지 않았지만 Ser727의 인산화가 억제되었으며 IFN-g에 의한 TRAIL 발현의 증가가 억제되었다. 태아 성상세포에서 p38 MAPK의 isoform인 p38a, p38b2뿐만 아니라, p38 MAPK의 상위 kinase인 MKK6와 MKK3도 발현되고 있는 것을 관찰하였다. 이상의 결과로 MKK3/6-p38 MAPK 경로를 통한 신호 전달이 사람 성상세포에서 IFN-에의한TRAIL 발현조절 기전으로 작용한다는 것을 관찰하였고, 이런 기전이 중추 신경계 내에서 성상세포가 면역학적 기능을 수행하거나 면역특권을 유지하는데 관여하리라 생각된다.
Astrocytes represent the major population of central nervous system (CNS) cells. They function mainly as a physical protective support for neurons. Recently, the immunological activity of astrocytes has been studied. TRAIL (tumor necrosis factor-related apoptosis inducing ligand), a member of the death ligand/receptor pairs, is independent of Fas-mediated apoptosis and expressed in most human tissues. To identify the functional role of astrocytes in modulating apoptosis in the brain, the expression of TRAIL induced by IFN-g and the involvement of p38 MAPK (mitogen-activated protein kinase) in IFN-g signaling were investigated in this study. RT-PCR, RPA and western blotting were performed to determine the expression of the TRAIL and TRAIL receptors in the primary astrocytes. The results show that the TRAIL mRNA transcript was not detected in fetal astrocytes, and was expressed at a low level in adult astrocytes. To characterize the effect of cytokines, astrocytes were treated with IL-1, TNF-a or IFN-g. The TRAIL expression level increased after being stimulated with IL-1, TNF-a or IFN-g both in fetal and adult astrocytes. Among three cytokines, the effect of IFN-g was prominent. The TRAIL protein expression level was similar to the result of RT-PCR or RPA. The astrocytes were reacted with Peer cells which are sensitive to TRAIL-mediated apoptosis in order to address the biological function of TRAIL up-regulation in astrocytes. The astrocytes pretreated with IFN-g induced apoptosis in Peer cells, which suggests that IFN-g induces the functional TRAIL molecule in astrocytes. The TRAIL receptors, DR4, DR5, DcR1 and DcR2, were detected constitutively in astrocytes. DR4, DR5 and DcR1 expression were not modulated by IFN-g treatment. Only DcR2 was slightly down-regulated by IFN-g. To investigate the signaling pathway in the astrocytes after IFN-g stimulation, STAT1 or p38 MAPK phosphorylation was evaluated. STAT1 is an essential transcription factor for IFN-g signaling. IFN-g activates STAT1 by phosphorylating at two sites. Ser727 phosphorylation is important for the transcriptional activity, whereas, Tyr701 is necessary for the homodimerization of STAT1. The p38 MAPKs, (p38a, p38b, p38b2, p38g, p38d) belong to the third family of MAPK, which are widely expressed in many tissues including the brain. Among these isoforms, p38b2 is reportedly the functionally active form of the p38 MAPK family in the brain. To examine the effect of p38 MAPK on TRAIL expression in astrocytes, SB203580, a specific p38a and p38b2 MAPK inhibitor, was treated prior to IFN-g stimulation. SB203580 inhibited ser727 phosphorylation of STAT1, but not tyr701 phosphorylation. TRAIL expression induced by IFN-g was also inhibited by SB203580. Therefore, p38 MAPK may play a key role in the ser727 phosphorylation of STAT1 and the transcriptional activation of TRAIL expression by IFN-g in the brain astrocytes. MKK6, which is responsible for phosphorylating p38 MAPK, is highly expressed in many tissues, but is absent from brain. The fact that p38b2 is only activated by MKK6 raises the question as to the presence of MKK6 in the activation of p38b2 in astrocytes. In this study, MKK6 was detected in human astrocytes and phosphorylated after IFN-g treatment. This suggests that IFN-g signaling in the brain astrocytes is mediated at least in part by MKK6 and p38 MAPK. These findings suggest that IFN-g up-regulates TRAIL expression during an immune reaction in brain, and that up-regulation of TRAIL may contribute to the immune privilege during fetal brain development or the apoptosis of neighboring cells in the pathological condition of the brain