CONCLUSION

Microglia and astrocytes play an important role in CNS inflammation. In this report, we provide the evidences that PKR mediates LPS-induced activation of STAT signaling pathway in microglia and astrocyte cell. By using 2-AP and PKR-siRNA, we conclude that LPS-induced IFN-â expression, NF-êB and STATs activation and their inflammatory downstream signaling are mediated via PKR.

BIBLIOGRAPHY

1. Stoll G, Jander S, Schroeter M: Inflammation and glial responses in ischemic brain lesions. Prog. Neurobiol. Prog 56: 149-71, 1998

2. Kreutzberg GW: Microglia: a sensor for pathological events in the CNS.

Trends Neurosci. 19: 312-8, 1996

3. Fischer, H., and Reichmann, G.: Brai n Dendritic Cells and Macrophages/Microglia in Central Nervous System Inflammation J. Immunol.

166: 2717-26, 2001

4. Pyo H, Jou I, Jung S, Hong S, Joe EH: Mitogen-activated protein kinases activated by lipopolysaccharide and beta-amyloid in cultured rat microglia.

Neuroreport. 9: 871-4, 1998

5. Schindler C, Darnell JE Jr: Transcriptional responses to polypeptide ligands:

the JAK-STAT pathway. Annu Rev Biochem 64: 621-51, 1995

6. Darnell,J.E.,Jr: STATs and gene regulation. Science, 277: 1630–1635, 1997 7. Kishimoto T, Taga T, Akira S: Cytokine signal transduction. Cell. 76: 253-62,

1994

8. Wong, A. H., Tam, N. W., Yang, Y. L., Cuddihy, A. R., Li, S., Kirchhoff, S., Hauser, H., Decker, T., and Koromilas, A. E: Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways EMBO J. 16:

1291-9. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, Schreiber RD: Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84: 431-42, 1996

10. Durbin, J. E., Hackenmiller, R., Simon, M. C., and Levy, D. E: Targeted disrupt ion of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84: 443-50, 1996

11. Linda A. Kobierski, Smita Srivastava, David Borsook: Systemic lipopolysaccharide and interleukin-1β activate the interleukin 6: STAT intracellular signaling pathway in neurons of mouse trigeminal ganglion.

Neurosci Lett. 281: 61-4, 2000

12. P. Dell’Albani, R. Santangelo, L. Torrisi, V. G. Nicoletti, J. Vellis, and A. M.

Giuffrida Stella: JAK/STAT Signaling Pathway Mediates Cytokine-Induced iNOS Expression in Primary Astroglial Cell Cultures. J Neurosci Res. 65:

417-424, 2001

13. Deng W, Ohmori Y, and Hamilton TA: LPS does not directly induce STAT activity in mouse macrophages. Cell Immunol. 170: 20-24, 1996

14. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, Major J, Hamilton TA, Fenton MJ, Vogel SN: TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene

15. Aaron T. Jacobs and Louis J. Ignarro: Lipopolysaccharide-induced Expression of Interferon-β Mediates the Timing of Inducible Nitric-oxide Synthase Induction in RAW264.7 Macrophage. J Biol Chem. 276: 47950-57, 2001 16. Hovanessian,A.G.: Interferon-induced and double-stranded RNA-activated

proteins as key enzymes regulating protein synthesis. In Ilan,J. (ed.), Translational Regulation of Gene Expression. Plenum Press, New York, NY,

163–185, 1993

17. Romano,P.R., Green,S.R., Barber,G.N., Mathews,M.B. and Hinnebusch,A.G.:

Structural requirements for double-stranded RNA binding, dimerization and activation of the human eIF-2 kinase DAI in Saccharomyces cerevisiae. Mol.

Cell. Biol. 15: 365–378, 1995

18. Gale,M.,Jr and Katze,M.G: Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78: 29–46, 1998

19. Kaufman, R. J: Double-stranded RNA-activated protein kinase mediates virus-induced apoptosis: a new role for an old actor. Proc. Natl. Acad. Sci.

USA. 96: 11693, 1999

20. G. Luca Gusella, Tiziana Musso, Sandra E. Rottschafer, Kari Pulkki, and Luigi Varesio: Potential requirement of functional double-stranded RNA-dependent protein kinase (PKR) for the tumoricidal activation of macrophages by lipopolysaccharide or IFN-αβ, but not IFN-γ. J. Immunol. 154: 345-354,

21. Williams, B. R: Signal integration via PKR. Sci. STKE 2001:RE2

22. Maggi, L. B., Jr, M. R. Heitmeier, D. Scheuner, R. J. Kaufman, R. M. Buller, J.

A. Corbett: Potential role of PKR in double-stranded RNA-induced macrophage activation. EMBO J. 19: 3630-8, 2000

23. Heitmeier, M. R., A. L. Scarim, J. A. Corbett: Double-stranded RNA-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine macrophages requires NF- B activation. J. Biol. Chem. 273: 15301-7, 1998 24. Bandyopadhyay, S. K., C. A. De la Motte, and B. R. G. Williams: Induction

of E-selectin expression by double-stranded RNA and TNF- is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice. J. Immunol. 164: 2077-83, 2000

25. Yang, Y., L. F. L. Reis, J. Pavlovic, A. Aguzzi, R. Schafer, A. Kumar, B. R. G.

Williams, M. Aguet, C. Weissman: Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14: 6095-106, 1995

26. Kumar, A., Y. L. Yang, V. Flati, S. Der, S. Kadereit, A. Deb, J. Haque, L. Reis, C. Weissmann, B. R. G. Williams: Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF- B. EMBO J. 16: 406-16, 1997

27. Der, S. D., Y. Yang, C. Weissmann, B. R. G. Williams: A double-stranded RNA-activated protein kinase-dependent pathway mediating stress induced

28. Z a m a n i a n- D a r y o us h , M . , M o g e n s e n , T . H . , D i D o n a t o , J . A . a n d Williams,B.R.G.: NF- B activation by the dsRNA-activated protein PKR is mediated through the NF- B inducing kinase and I B kinase. Mol. Cell. Biol 20: 1278-90, 2000

29. Hu, Y. and T.W. Conway: 2-Aminopurine inhibits the double-stranded RNA-dependent protein kinase both in vitro and in vivo. J. Interferon Res. 13: 323-8, 1993

30. Sandy D. Der and Allan S. Lau: Involvement of double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediaated antiviral activity. Proc. Natl. Acad. Sci. USA. 92: 8841-5, 1995

31. Wen-Ming Chu, Derek Ostertag, Zhi-Wei Li, Lufen Chang, Yi Chen, Yinling Hu, Bryan Williams, and Jacques Perrault: JNK2 and IKKβ are required for activating the innate response to viral infection. Immunity. 11: 721-31, 1999 32. Eric Billy, Vincent Brondani, Haidi Zhang, Ulrich Müller, and Witold

Filipowicz: Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc.

Natl. Acad. Sci. USA. 98:14428~33, 2001

33. Olivi er Donzé and Didier Picard: RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res. 30:2~4, 2002

34. Gil J, Rullas J, Garcia MA, Alcami J, Esteban M.: The catalytic activity of

Oncogene. 20:385-94, 2001

35. Jacobs AT, Ignarro LJ: Lipopolysaccharide-induced expression of interferon-beta mediates the timing of inducible nitric-oxide synthase induction in RAW 264.7 macrophages. J Biol Chem.276: 47950-7, 2001

-국 문 요 약-

LPS 자극 신 경 교 세 포 활 성 에 서 PKR 을 통한 신 호 전 달 에 관 한 연구

아 주 대 학 교 대 학 원 의 학 과 이 지 훈

( 지도교수: 주 일 로)

dsRNA activated protein kinase (PKR)은 세포 내 선천성 항 바이러스 면역 기전에 작용하는 물질로 알려져 있다. 최근 연구에서 PKR은 세포 외부 자 극에 반응하여 세포 내 신호전달에 관여하는 중요한 역할을 가진다고 보 고 되었으나, 쥐 뇌의 신경 교세포에 가해지는 외부 자극에 대하여 활성화 되는 signal transducer and activator of transcription (STAT)과 PKR이 어떤 연관 성을 가지고 있는가에 대한 연구는 많이 보고 되어 있지 않다. 따라서 본 연구에서는 PKR이 STAT 활성을 조절하는 기작을 제시하고 그 활성화 기 전을 밝히고자 하였다.

본 연구에서 Primary microglia와 astrocyte 세포를 LPS로 자극하면 PKR이 매우 빠른 시간에 활성화되는 것을 관찰하였으며 뒤이어 STAT1/3, IRF-1이 활성화됨을 western blot analysis로 확인할 수 있었다. 또한, PKR의 저해제인 2-aminopurine (2-AP)과 PKR에 특이적인 short interfering RNA (siRNA)를 이

용한 실험에서 LPS에 의한 STAT1/3 활성화와 NO 분비가 현저하게 감소 함을 확인 하였다. 그리고 STATs 활성의 조절자로 알려진 IFN-â와 IFN-â 발현에 관여하는 NF-κB 역시 PKR을 저해함에 따라 그 발현정도가 감소 함을 관찰 할 수 있었다. 지금까지 결과를 모두 종합하여, LPS가 PKR통하 여 NF-κB와 IFN- â를 활성 시키고 이것이 STAT1/3, IRF-1등을 활성 시켜 쥐 뇌 신경 교세포의 염증반응을 일으키게 된다는 결론을 얻을 수 있었다.

핵심되는 말: PKR, LPS, STAT, IRF-1, NO, IFN- â