A Study on the PKR-mediated signaling
in LPS-induced inflammation of
Rat Glial Cells
Department of Medical Sciences
The Graduated School, Ajou University
LPS 자극 신경 교세포 활성에서
PKR을 통한 신호전달에 관한 연구
지도교수 주 일 로
이 논문을 의학 석사학위 논문으로 제출함.
2004 년 2 월
아 주 대 학 교 대 학 원
의 학 과
이 지 훈
이지훈의 의학 석사학위 논문을
심사위원장 주 일 로 인
심사위원 조 은 혜 인
심사위원 최 경 숙 인
아 주 대 학 교 대 학 원
2003 년 1 2 월 1 9 일
Acknowledgement감 사 드 려 야 할 분이 너무 많 지 만 먼저 부 족 한 저를 2 년 동안 지 도 해 주 신 주 일 로 교 수 님 과 조 은 혜 교 수 님, 최 경 숙 교 수 님, 박은정 선 생 님 께 깊 은 감 사 의 말 씀 을 드 립 니 다. 그리고 처 음 부 터 지 금 까 지 실 험 적 으 로 많 은 도 움 을 주 었 던 우리 팀 박 사 과 정 의 희 영 언 니 와 온 순 언 니, 항상 인 생 상 담 잘 해 주 던 수 정 언 니 와 현 실 언 니, 경진언니, 앞날이 기 대 되 는 우 리 희 정 이 와 지 영 이, 큰 언 니 같 은 명 순 언 니 와 경 애 언 니, 지 형 언 니, 예쁜 경 진 언 니, 귀 여 운 새 봄 이, 든 든 한 우 혁 오 빠, 항상 덤 벙 대 기 만 하 던 저를 감 싸 주 셔 서 모 두 모 두 감 사 드 립 니 다. 그리고 저 와 실 험 실 에 서 2 년 간 고 락 을 같이 해온 동기, 수 영 이 에 게 특 별 히 고 맙 단 말을 하고 싶 습 니 다. 보 잘 것 없는 저를 위해 마 음 으 로 부 터 도 와 주 셨 던 여 러 친지 여 러 분 과 이 제 까 지 저를 믿고 끝 까 지 지 켜 봐 주신 사 랑 하 는 부 모 님 과 동 생 에 게, 그 리 고 마 지 막 으 로 올해 작 고 하 신 외 할 아 버 지 께 이 논 문 을 바 칩 니 다.
A Study on the PKR-mediated signaling in
LPS-induced inflammation of Rat Glial Cells
A Dissertation Submitted to The Graduate School of Ajou University
in Partial Fulfillment of the Requirements for the Degree of
MASTER OF MEDICAL SCIENCES
Ilo Jou, M.D., Ph.D.
Department of Medical Sciences
The Graduate School, Ajou University
A Study on the PKR-mediated signaling in
LPS-induced inflammation of Rat Glial Cells
Double-stranded RNA (dsRNA) activated protein kinase (PKR) is an established component of innate antiviral immunity. Recent ly, PKR has been shown to be essential for signal transduction in response to cellular stress. However, the relationship between PKR and the stress-activated protein, such as signal transducer and activator of transcription (STAT), is not clear in rat glial cells. Here we show that primary microglia and astrocytes stimulated with LPS rapid activation of PKR, followed by activation of STAT1 and induction of IRF1 was observed. Treatment with PKR inhibitor 2-aminopurine (2-AP) and transfection with short interfering RNA of PKR markedly diminished LPS-induced activation of STAT1, up-regulation of IRF1, NF-êB and IFN-beta, known as a mediator of STATs actvation, and NO release. EMSA results demonstrate that LPS-induced binding of a nuclear factor to a consensus GAS/ISRE, which are known to be STAT-binding sites, was also markedly reduced by inhibition of PKR activity. Thus, LPS-induced IFN-â expression, NF-êB and STATs activation and their inflammatory downstream signaling are mediated via in a PKR-dependent pathway.
TABLE OF CONTENTSTITLE PAGE ---1 ABSTRACT ---2 TABLE OF CONTENTS ---3 LIST OF FIGURES ---5 Ⅰ. INTRODUCTION --- 7
Ⅱ. MATERIAL AND METHOD A. Reagents ---10
B. Cell cultures ---10
C. Western Blot Analysis ---11
D. RNA isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR) ---11
E. Determination of NO release ---12
F. Electrophoresis Mobility Shift Assay (EMSA) ---12
G. Short interference RNA (siRNA) transfection ---14
Ⅲ. RESULTS A. LPS induces the phosphorylation of PKR in primary rat glial cells. ---15
B. Inhibition of PKR phosphorylation suppresses the LPS- induced phosphorylation of STATs. ---17
D. 2-AP suppresses the STAT-mediated transcriptional responses. ---19
E. Inhibition of PKR phosphorylation suppresses the LPS-induced NO release. ---20
F. siRNA-mediated suppression of PKR level reduces the LPS-induced activation of STATs and IRF-1 in primary astrocyte cell. ---24
G. PKR is required for LPS-induced expression of IFN-β in rat primary astrocytes. ---28
H. Activation of PKR is followed by activation of NF-κB in LPS-stimulated primary astrocytes. ---30
Ⅳ. DISCUSSION ---33
Ⅴ. CONCLUSION ---38
LIST OF FIGURES
Figure 1. PKR and STAT phosphorylation in rat glial cells. ---15 Figure 2. 2-AP suppressed LPS-induced STAT 1 / 3 phsphorylation and IRF-1
expression.---17 Figure 3. PKR is required for LPS -induced nuclear factor binding to
GAS/ISRE elements. ---20 Figure 4. 2-AP suppresses transcription of STAT-responsive inflammatory
cytokines. ---21 Figure 5. 2-AP reduces NO release and iNOS expression from rat microglial
cells. ---22 Figure 6. PKR activation was suppressed in PKR-siRNA transfected rat
astrocyte cells. ---24 Figure 7. STATs activation was suppressed in PKR-siRNA transfected rat
astrocyte cells. ---25 Figure 8. PKR-siRNA inhibits transcription of STAT-responsive inflammatory
cytokines. ---26 Figure 9. IFN-â gene expression in PKR-siRNA transfected rat primary
astrocyte cells. ---28 Figure 10. NF-êB-independent activation of PKR in LPS-stimulated astrocyte
Figure 12. A model for the LPS-induced STAT inflammatory signaling pathway in rat glial cells. ---36
Microglia and astrocyte are the major immune effector cells in the brain, and their activation is an early event in central nervoussystem inflammation. 1-3 Previous data showed that LPS could activate microglia, inducing release ofinflammatory mediators such as TNF-α and NO, which have STAT-binding elements in their promoter regions . 4
STATs (signal transducers and activators of transcription) mediate diverse biological functions in response to different ligands . 5-7 One of the major STATs intimately involved in both the innate and acquired immune responses is STAT1. Moreover, dsRNA, an intermediate produced during virus replication, can also activate STAT1 DNA binding. 8, 9 The non-redundant role of STAT1 in the antiviral response is further appreciated by findings that stat1 null mice(STAT1-/-) are highly susceptible to microbial infection. 10, 11
Recent studies show that STATs are the representative cytokine signaling molecules and rapidly activated when cytokine or growth factors bind to their receptors. But activation of STAT 1, 3 appeared 2 hours later following treatment
with LPS. This result suggests that LPS does not directly induce phosphorylation of STAT1 and STAT3 . 12-15Thus we hypothesized that LPS-induced STATs activation is mediated by PKR, which is known as inflammatory signaling transducer.
PKR, the dsRNA-activated protein kinase, is a ubiquitously expressed serine/threonine protein kinase that is induced by interferon and activated by dsRNA, cytokine, growth factor and stress signals. 16-18 It is essential for cells to respond adequately to different stresses including growth factor deprivation, products of the inflammatory response (TNF-α) and bacterial (lipopolysaccharide) and viral (dsRNA) products. In activated state PKR plays a central role in the regulation of transcription and translation. The phosphorylation of serine 51 of eukaryotic translation initiation factor eIF2α by PKR results in the suppression of protein synthesis. 19, 20 PKR also participates in the activation of several transcription factors, including NF-κB and IFN regulatory factor-1 (IRF-1) as well as in the expression of proinflammatory genes, including NO synthase (iNOS), IL-1, and IL-6. 21-23 In PKR-null cells thereis also a deficiency in the activation of IRF-1 and the activationand phosphorylation of activating transcription factor-2. 24-28 Indeed, the inhibition of
PKR by 2-aminopurine (2-AP, an inhibitor of PKR) prevented the expression of specific genes in the various stimuli such as LPS, polyinosinic acid: polycytidylic acid (poly [I:C]), or virus infection, which suggests that the activation of PKR might be a common step in the different biochemical pathways triggered by these agents. 29
In this report, we provide evidence that PKR mediates the STAT pathway by LPS and their association with IRF-1 expression in microglia and astrocytes. We observe that primary microglia and astrocytes stimulated with LPS leads to rapid activation of PKR, followed by activation of signaling components including STAT1 , -3 and IRF-1. By using the PKR inhibitor 2-AP and short interfering RNA transfection, IRF-1, STAT1 activation and NO release are markedly diminished and IFN-β, known as mediator of STATs , 14, 30, 31 mRNA expression was also impaired. These results suggest that PKR is upstream of IFN-β and subsequently mediates STAT1 and IRF-1 expression, thus LPS-induced STAT1 and IRF-1 expression is mediated via PKR-dependent pathway.
Ⅱ. MATERIAL AND METHOD
Lipopolysaccharide and 2-aminopurine was purchased from Sigma, and IFN- was
from Calbiochem. Bovine brain gangliosides mixture was purchased from Matreya (Pleasant Gap, PA). Antibodies against 701-phosphorylated STAT1, and Tyr-705-phosphorylated STAT3were from Cell Signaling Technology (Beverly, MA). An antibody against Thr-446-phosphorylated PKR and PKR were from Upstate biotechnology. Antibodies against Actin (I-19) and IRF-1 (M-20) were from Santa Cruz Biotechnology, Inc.
B. Cell cultures
Primary microglia was cultured from the cerebral cortices of 1-3 day Sprague-Dawley rats as previously describe d. Briefly, the cortices were triturated into single cells in minimal essential media containing 10 % fetal bovine serum (Hyclone, Logna, UT) and plated into 75cm2 T-flasks (0.5 hemisphere/flask) for 2 weeks. Then, microglia were detached from the flasks by mild shaking and applied to a nylon mesh to remove astrocytes and cell clumps. Cells were plated in 6-well plates (5x105 cells/well) or 60mm dishes (8x105 cells/dish) or 100mm dishes (2x106cells/dish). One hour later, the cells were washed to remove unattached cells before being used in experiments. Primary astrocytes were prepared using trypsin after microglia were
removed. Detached astrocytes were seeded in 60 mm or 35 mm dishes.
C. Western Blot Analysis
Cells were washed twice with cold phosphate-buffered saline, and then lysed in ice-cold modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP -40, 0.25% Na-deoxycholate, 150 mM NaCl, 1% NP-40, 10 mM Na2HPO4 , pH 7.2) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 0.5 mM Na3VO4 , 0.5 M NaF and 2 mM EDTA). The lysate was centrifuged for 20 min at 13000 rpm at 4 °C and the supernatant collected. Proteins were separated by 8% SDS-PAGE gel and transfered to nitrocellulose membrane. The membrane was incubated with primary antibodies and peroxidase-conjugated secondary antibodies and then visualized using an enhanced chemiluminescence system (Sigma).
D. RNA isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using RNAzolTMB (TEL-TEST Inc., friendwood, TX), and cDNA was prepared using reverse transcriptase that originated from Avian Myeloblastosis Virus (TaKaRa, Japan) according to the manufacturer’s instructions. PCR was performed with 30 cycles of sequential reactions: 94C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec. Oligonucleotide primers were purchased from Bioneer
AAG ATT GTC AGC AA-3’ and (R) 5’-AGA TCC ACA ACG GAT ACA TT-3’ for GAPDH; (F) 5’-TGA TGT TCC CAT TAG ACA GC-3’ and (R) 5’-GAG GTG CTG ATG TAC CAG TT-3’ for IL-1β; (F) 5’-GTA GCC CAC GTC GTA GCA AA-3’ and (R) 5’-CCC TTC TCC AGC TGG GAG AC-3’ for TNF-α; (F) 5’-TCC AGA AGC ACC ATG AAC C-3’ and (R) 5’-GCT GAA GAG ATT AGT ACC T-3’ for IP-10; (F) 5’-ATG CAG GTC TCT GTC ACG CT-3’ and (R) 5’-CTA GTT CTC TGT CAT ACT GG-3’ for MCP-1; (F) 5’-ATG GCC AAC AGG TGG ACC CT-3’ and (R) 5’-TCAGTT CTG GAA GTT TCT AT-3’ for IFN-β; (F) 5’-AAA GGT CTT TCG TGT CAA AA-3’ and (R) 5’-GAC AGG CTT AAA ATC GCT AA-3’ for PKR PCR products were separated by electrophoresis in a 1.5% agarose gel and detected under UV light.
E. Determination of NO release
Media nitrite concentration was measured as an indication of NO release. Following the indicated cell incubations, 50 µl ofculture medium was removed and mixed with an equal volume of Griessreagent (0.1% naphthylethylenediamine, 1% sulfanilamide, 2.5% H3PO4), and absorbance of the mixture at 540 nm was measured.
Cells were harvested and suspended in 9 times packaged cell volume of a hypotonic solution (10 mM HEPES, pH 7.9, 10 mM KCl,0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride) including 0.5% Nonidet P-40. Cells were centrifugedat 5000 rpm for 10 min at 4 °C, and the pellet (nuclear fraction)was saved. The nuclear fractions were resuspended in a buffercontaining 20 mM HEPES, pH 7.9, 20% glycerol, 0.4 M NaCl, 1 mMEDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride, incubated on ice for 60 min with occasional gentle shaking,and centrifuged at 13,000 rpm for 20 min. The crude nuclear proteinsin the supernatant were collected and stored at 70 °C for EMSA. EMSA was performed for 30 min on ice in a volume of 20 µl, containing2 µg of nuclear protein extract in a reaction buffer containing8.5 mM EDTA, 8.5 mM EGTA, 8% glycerol, 0.1 mM ZnSO4, 50 µg/mlpoly (dI-dC), 1 mM dithiothreitol, 0.3 mg/ml
bovine serum albumin,6 mM MgCl2, and 32P-radiolabeled oligonucleotide probe,
with or without 20-50-fold excess unlabeled probe. DNA-protein complexes were separated on 6% polyacrylamide gels in Tris/glycine buffer. The dried gels were exposed to x-rayfilm. The following double-stranded oligo nucleotide was used in these studies: GAS/ISRE,5-AAGTACTTTCAGTTTCATATTACTCTA-3', 27 bp (Santa Cruz Biotechnology, Inc., sc-2537). 5'-End-labeled probeswere prepared with 40 µCi of [ -32P] ATP using T4 polynucleotide kinase (Promega) and were purified on Quick Spin Columns Sephadex G-25 (Roche Molecular Biochemicals).
G. Short interference RNA (siRNA) transfection
Chemically synthesized, double stranded siRNAs, with 19-nt duplex RNA and 2-nt 3′ dTdT overhangs, were purchased from Dharmacon Research (Lafayette, CO) in deprotected and desalted form. To design PKR-specific siRNA duplexes, the mRNA sequences for PKR were screened for unique 21-nt sequences in the National Center for Biotechnology Information database using the BLAST search algorithm, yielding the PKR siRNA, 5'-GGUAGAUCAAAGCAGGAGGTT-3'. Forty to fifty percent of confluent cells were transfected with siRNA oligonucleotides using oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. All assays were performed at least 48 h after RNA transfection.
A. LPS induces the phosphorylation of PKR in primary rat glial cells.
Previous studies have shown that PKR, the dsRNA-activated protein kinase, is essential for cells to respo nd adequately to stresses, such as LPS. 20, 21 PKR is ubiquitously expressed in many cell types, but the presence or the roles of PKR in the inflammatory signaling pathway in rat glial cells are largely unknown. To confirm this, we tested whether PKR phosphorylation was induced by LPS in primary rat glial cells. When microglia from rat brain were stimulated with 100 ng/ml LPS, PKR phosphorylation increased at 5 min, peaked at 30 min, and declined thereafter in microglia cells (Fig. 1A). In astrocyte cells, phosphorylated-PKR level increased at 5 min, was sustained by 2 h (Fig. 1B). We also examined whether PKR is activated by IFN-γ, which is a well-known PKR activator. 18 But we could not observe PKR activation in IFN-ã-stimulated rat glial cells. (data not shown). These results show that LPS rather than IFN-γ is an effective activator of PKR and LPS-induced PKR activation may affect the inflammatory signaling pathway in rat glial cells.
Figure 1. PKR and STAT phosphorylation in rat glial cells. Rat primary
microglia cells (A) and astrocyte cells (B) were serum -starved for 12 h or 48 h respectively and then stimulated with 100 ng/ml LPS for the indicated times. Cell lysates were separated by 8% SDS-PAGE and Western bloting was performed using phospho PKR (thr446), pY-STAT1, PKR and anti-ACTIN. A. Primary microglia ACTIN pPKR PKR LPS 0 5 30 120 (min) pY-STAT1 B. Primary astrocytes LPS 0 5 30 120 (min) ACTIN pPKR PKR pY-STAT1
B. Inhibition of PKR phosphorylation suppresses the LPS-induced phosphorylation of STATs.
Recent studies suggesting the important roles of STAT signaling in brain inflammatory response have been reported. 5-7 Previously, we and others observed that phosphorylation of STAT 1 and STAT 3 appeared at 2 hours later following treatment with LPS (Fig. 1), which suggest that LPS does not directly induce phosphorylation of STAT 1 and STAT 3. 12-15 Because LPS rapidly induced the activation of PKR (Fig. 1), we hypothesized that LPS-induced STATs activation was mediated by PKR. In an experiment to confirm this, PKR phosphorylat ion was dose-dependently reduced by 2-aminopurine (2-AP, an inhibitor of PKR) in microglia and astrocyte cells (Fig. 2A). And 2-AP dramatically suppressed the LPS-stimulated phosphorylation of STAT 1 and STAT 3 and induction of IRF-1 protein, which isa regulator of host defense and has binding sites for STAT inits promoter (Fig. 2B). These results provide that STATs activations are mediated by PKR.
LPS - 100 100 100 (ng/ml) 2-AP - - 0.2 0.5 (mM) ACTIN pPKR pPKR ACTIN
(a) Primary microglia (b) Primary astrocytes
LPS - 100 100 100 (ng/ml) 2-AP - - 0.5 1 (mM) A B pY-STAT3 ACTIN IRF-1 pPKR pY-STAT1 ACTIN 30 min LPS - 100 100 - (ng/ml) 2-AP - - 0.5 0.5 (mM) 30 min LPS - 100 100 - (ng/ml) 2-AP - - 1 1 (mM) pPKR 120 min LPS - 100 100 - (ng/ml) 2-AP - - 0.5 0.5 (mM) 120 min LPS - 100 100 - (ng/ml) 2-AP - - 1 1 (mM) pY-STAT3 ACTIN IRF-1 pY-STAT1
(a) Primary microglia (b) Primary astrocytes
Figure 2. 2-AP suppressed LPS-induced STAT 1 / 3 phsphorylation and IRF-1 expression. A, Primary microglia (a) and astrocyte cells (b) were
pretreated with the indicated dose of 2-aminopurine (2-AP) for 30 min then stimulated with 100 ng/ml LPS for 30 min. Total cell lysates prepared for Western blot analysis. B, Primary microglia (a) and astrocyte (b) cells were pretreated 2-AP same as (A) and then stimulated with LPS for indicated times. Cell lysates were separated by 8% SDS-PAGE and Western blots probed with
C. 2-AP also reduces the LPS-induced nuclear factor binding activity to GAS/ISRE elements.
Functional GAS/ISRE elements are found in the promoter regions of several inflammation-related genes, such as IRF-1 and many other inflammatory cytokines, and these elements are known to bind the phosphorylated-STAT dimmer. Our data show that phosphorylation of STATs depends on the PKR activation (Fig. 2). Therefore, we investigated whether 2-AP reduced DNA-binding activity of STATs. After cells were stimulated with 100 ng/ml LPS for 2 h in the presence or absence of 2-AP, nuclear extracts were prepared and then analyzed by EMSA using a γ-32 P-labled consensus GAS/ISRE oligonucleotides probe. The specific binding complex was detected in nuclear extracts from LPS-treated rat astrocyte and microglia cells (Fig. 3A lane 2, 3B lane 2). And 2-AP inhibited the nuclear factor binding activity of STATs in LPS-treated rat glial cells (Fig. 3A, lane 3-4, 3B, lane 3). These results indicate that PKR also mediates LPS-induced nuclear factor binding activity to GAS/ISRE elements in the inflammation related genes.
D. 2-AP suppresses the STAT-mediated transcriptional responses.
Brain inflammatory responses are coordinated by the production of inflammatory cytokines and chemokines. The above data indicate that LPS-induced PKR activation mediates phosphorylation of STATs and their binding activity to GAS/ISRE elements (Fig. 2, 3). Therefore, we examined the transcript level of genes that have functional GAS elements and act as mediators of inflammation, such as monocyte
chemoattractant protein-1 (MCP-1) and interferon-inducibleprotein-10 (IP-10). LPS incresed the transcript ion of both genes, but this induction was inhibited by pretreatment with 2-AP. Similar inhibitory effects by 2-AP were observed for pro-inflammatory cytokines such astumor necrosis factor alpha (TNF-α) and interleukin-1-beta (IL-1β) (Fig. 4A, B). These findings demonstrate that LPS-induced PKR activation triggers STAT-dependent transcriptional activation of inflammatory genes in microglia and astrocyte cells.
E. Inhibition of PKR phosphorylation suppresses the LPS-induced NO release.
NO plays an important role in pathophysiology of inflammatory neurological diseases and the production of pro-inflammatory cytokines. So, we investigated whether LPS-induced NO production was also reduced by PKR inhibitor. We measured NO concentrations in LPS-stimulated rat microglia cells maintained up to 36 h. When cells were stimulated with 100 ng/ml LPS in the presence or absence of 2-AP, NO was detected in supernatants of the cultured microglia cells and increased thereafter by 36 h. However, when 2-AP was added before the addition of LPS, production of NO was significantly reduced (Fig 5). In our studies, 2-AP dose-dependently suppressed NO release up to 1 mM.
LPS - 100 100 - 100 (ng/ml) 2-AP - - 1 1 - (mM) Cold - - - - 20X A. Primary astrocytes LPS - 100 100 100 (ng/ml) 2-AP - - 0.5 - (mM) Cold - - - 20X B. Primary microglia
Figure 3. PKR is required for LPS-induced nuclear factor binding to
GAS/ISRE elements. Rat primary astrocyte cells (A) and microglia cells (B)
were pretreated with or without 2-AP for 30 min and stimulated with 100 ng/ml LPS for 2 h, after which nuclear extracts were prepared and assayed for the amount of binding activity to GAS/ISRE oligonucletides using EMSA.
Figure 4. 2-AP suppresses transcription of STAT -responsive inflammatory
cytokines. Rat primary microglia cells (A) and astrocyte cells (B) were
pretreated with or without 2 -AP for 30 min and then stimulated with 100 ng/ml LPS for 3 h. Total RNA was isolated and analyzed for levels of MCP-1,
TNF-α, IL-1β and IP-10 using an RT-PCR assay. MCP-1, monocyte chemoattractant protein-1; IL-1β, interleukin-1-beta; TNF-α, tumor necrosis factor-alpha;
IP-10, interferon-inducible protein-10. The transcription of glyceraldehyde
-3-posphate dehydrogenase (GAPDH) was measured for normalization.
MCP-1 TNF-α IL-1β IP-10 GAPDH A. Primary microglia LPS - 100 100 - (ng/ml) 2-AP - - 0.5 0.5 (mM) B. Primary astrocytes LPS - 100 100 - (ng/ml) 2-AP - - 1 1 (mM) MCP-1 TNF-α IL-1β IP-10 GAPDH
Figure 5. 2-AP reduces NO release and iNOS expression from rat microglial
cells. A, Cells were pretreated with the indicated dose of 2-AP for 30 min then
stimulated with 100 ng/ml LPS for 30 min. Total cell lysates prepared for Western blot analysis. B, Cells were treated with 100 ng/ml LPS for 36 h in the presence or absence of 2-AP. The amount of NO was determined by measuring the amount of nitrite in the media, as described under ‘Meterial and Method’ Data represent the mean ± S.E.M of three independent experiments.
0 20 40 60 80 100 120 con LPS 2AP 0.1mM 2AP 0.5mM 2AP 1mM NO(%) B ACTIN iNOS LPS - 100 100 100 100 (ng/ml) 2-AP - - 0.1 0.5 1 (mM) A
F. siRNA-mediated suppression of PKR level reduces the LPS-induced activation of STATs and IRF-1 in primary astrocyte cell.
Our data have shown that LPS-induced STATs activation was reduced by PKR inhibitor (Fig. 2). These findings indicate that PKR acts as a mediator of STATs signaling pathway. Similarly, we attempted to confirm that STATs activation was dependent of PKR activation by using small interference RNA (siRNA) for PKR gene. First, to investigate RNA interference (RNAi) could be used to target endogenous gene, we transfected primary rat astrocyte with a siRNA corresponding to the 21 nucleotides of PKR. PKR mRNA levels were suppressed by siRNA (Fig. 6A) and LPS-stimulated PKR phosphorylation reduced in dose-dependent manner (Fig. 6B). Next, we also examined whether STATs signaling was blocked by siRNA of PKR. As expected, we observed that phosphorylation of STATs and IRF-1 protein expression was also inhibited by siRNA (Fig. 7). In EMSA and RT-PCR experiments, STATs binding activity and inflammatory cytokines expression was also impaired by using siRNA transfection (Fig. 8A, B). These data coincide with our above results (Fig. 2~4), so we conclude that PKR certainly mediate LPS-induced STATs activation and their signal transduction in rat glial cells.
Figure 6. PKR activation was suppressed in PKR-siRNA transfected rat
astrocyte cells. A, Inhibition effects of siRNA on PKR gene expression in rat
primary astrocyte cells. Astrocyte cells were transfected with 21 base paired siRNA duplex using Oligofectamine. After 48 h incubation, cells were serum-starved for 36 h and then stimulated with 100 ng/ml LPS for 3 h. mRNA expression of PKR was detected by RT-PCR analysis. B, After PKR-siRNA transfection, cells were stimulated with 100 ng/ml LPS for 30 min to 2 h. Cell lysates were separated by 8% SDS-PAGE and Western blots probed with
B LPS - 100 100 100 100 (ng/ml) PKR siRNA - - 0.05 0.1 0.3 (µM) Oligofectamine - + + + + PKR ACTIN pPKR LPS - 100 100 100 (ng/ml) PKR siRNA - - 0.3 - (µM) Control siRNA - - - 0.3 (µM) Oligofectamine + + + + PKR → A 500 bp→
Figure 7. STATs activation was suppressed in PKR-siRNA transfected rat
astrocyte cells. PKR-siRNA transfected rat astrocyte cells were stimulated with
LPS same as Fig. 6 and analyzed by using Western blots was probed with antibodies as indicated. pY STAT1 ACTIN IRF-1 pY STAT3 LPS - - 100 100 - (ng/ml) siRNA oligo - - - 0.3 0.3 (ìM) oligofectamine - + + + + 120 min pPKR ACTIN 30 min LPS siRNA oligo oligofectamine - - + + - (ng/ml) - - - + + (ìM) - + - + + PKR
TNF-á GAPDH IP-10 IL-1â LPS - - 100 100 - (ng/ml) siRNA oligo - - - 0.3 0.3 (ìM) oligofectamine - + + + + B A LPS - - 100 100 - 100 (ng/ml) PKR siRNA - - - 0.3 0.3 - (ìM) oligofectamine - + + + + 20X
Figure 8. PKR-siRNA inhibits transcription of STAT-responsive
inflammatory cytokines. Rat primary astrocyte cells were transfected with
PKR-siRNA duplex. A, Cells were stimulated with LPS for 2 h, after which nuclear extracts were prepared and assayed for the binding activity to GAS/ISRE oligonucletides using EMSA. B, Total RNA was isolated and analyzed for levels of MCP-1, TNF-á, IL-1â and IP-10 using an RT-PCR assay. The transcription of
G. PKR is required for LPS-induced expression of IFN-β in rat primary astrocytes.
There are several reports showing that LPS-induced STATs activation is mediated by IFN-β, 14, 15 and some articles suggest that PKR is associated with IFN-β expression. 30, 31 Therefore, we have questioned whether PKR affects IFN-β expression or not. First, we examined the effects of LPS on IFN-β mRNA expression in primary astrocyte cells by using RT-PCR analysis. IFN-β mRNA level slightly increased at 1 h, peaked at 3 h in LPS-stimulated rat astrocyte cells (Fig. 9A). We further tested whether IFN-β mRNA expression level reduced by using 2-AP or siRNA transfection. As expected, LPS-induced IFN-β mRNA expression was strongly reduced by both 2-AP treatment and siRNA transfection in astrocyte cells (Fig. 9B, C). These results indicate that PKR locates upstream of IFN-β signaling and mediates the IFN-β expression and subsequently, it affects the STATs signaling pathway.
LPS - 100 100 - (ng/ml ) 2-AP - - 1 1 (mM) B A LPS 0 5 30 60 180 (min) IFN-â GAPDH LPS - - 100 100 - (ng/ml) siRNA oligo - - - 0.3 0.3 (ìM) oligofectamine - + + + + C
Figure 9. IFN-â gene expression in PKR-siRNA transfected rat primary
astrocyte cells. A, Cells were stimulated with 100ng/ml LPS for indicated times.
Then, IFN-â transcripts were analyzed by RT-PCR. In the presence of 2-AP (B) and siRNA transfection (C), IFN- â mRNA expression was also analyzed by RT-PCR.
H. Activation of PKR is followed by activation of NF-κB in LPS-stimulated primary astrocytes.
Since the IFN-β promoter has NF-κB binding sites, we questioned whether NF-κB could be involved in the sequential events of LPS-PKR-IFNβ-STAT signaling. We therefore investigated the connection between NF-κB and PKR using the pharmacological inhibitors of NF-κB, N-acetylcystein (NAC) and gliotoxin. After confirming their effects in rat primary astrocytes (Fig. 10A), the cells were treated with 100 ng/ml LPS in the presence or absence of either NAC or gliotoxin for 30 min, and the levels of phosphorylated PKR were determined by Western blot analysis using antibody against thr-446-phosphorylated PKR. Treatment with NAC or gliotoxin did not alter the phosphorylated levels of PKR (Fig. 10B, C), indicating that LPS-induced phosphorylation of PKR is independent of NF-κB activation. We also assayed the effect of the PKR inhibitor 2-AP on NF-κB activation by EMSA. Rat primary astrocytes were treated with 100 ng/ml LPS for 2 h in the presence or absence of 2-AP, and their nuclear extracts were assayed by EMSA using a γ-32 P-labeled oligonucleotide probe for consensus NF-κB binding elements. NF-κB DNA binding activity was significantly diminished in cells treated with 2-AP compared with control cells (Fig. 11), indicat ing that PKR is an upstream regulator of LPS-induced activation of NF-κB and suggesting that NF-κB is a component of LPS-induced STAT inflammatory signaling events in rat glial cells.
pPKR ACTIN LPS - + + + + + + - (100ng/ml) Gliotoxin - - 1 5 10 1 5 10 (ìM) Pretreated Co-treated pPKR ACTIN LPS - + + + + + + + - (100ng/ml) NAC - - 1 5 10 1 5 10 10 (mM) Pretreated Co-treated LPS - + + + - - + (100ng/ml) Gliotoxin - - 10 - 10 - - (ìM) NAC - - - 10 - 10 - (mM) Cold - - - - - - 20X
Figure 10. NF-êB-independent activation of PKR in LPS -stimulated astrocyte
cells. A, Cells were pretreted with NAC or gliotoxin for 30 min and treated with
100 ng/ml LPS for 30 min. To detect the effect of inhibitors on NF-êB, EMSA analysis was performed using ã-32P-labled oligonucleotide probe for consensus NF-êB binding element. (B) and (C), In pretreated- or cotreated astrocyte with LPS and NAC or gliotoxin, the levels of phosphorylated PKR were determined by western blot analysis (B)(C).
LPS - + + - + (100ng/ml) 2-AP - - + + - (1mM)
Cold - - - - 20X
Figure 11. Effects of 2-AP on NF-κB binding activity. Rat primary astrocytes
were treated for 30 min with 100 ng/ml LPS in the presence or absence of 1 mM 2-AP, and NF-κB binding activity was examined using either a γ-32P-labeled oligonucleotide probe for consensus NF-κB binding elements or unlabeled 20X cold oligonucleotide.
In this study, we found that (ⅰ) PKR mediated STATs activation and their downstream signaling in LPS-induced rat glial cells; (ⅱ) IFN-â, known as a mediator of STATs, 14, 30, 31 expression and NF-êB activation were mediated by PKR activation. These results demonstrate that the relationship between PKR activation and brain inflammation in LPS-induced rat glia cells. PKR was essential for cells to respond adequately to different stresses including growth factor deprivation, products of the inflammatory response and bacterial and viral products. It was also an established component of innate antiviral immunity. 16-18 In LPS-induced rat glial cells, however, PKR activation and its signaling pathways have not been well studied. Indeed, STATs activation was observed in late time after treatment with LPS, so, we had a question what component mediates STATs activation in LPS-induced rat glial cells. We investigated whether PKR mediated STATs activation and their inflammatory signaling in rat glial cells.
We observed that LPS activated PKR very early time (at 5 min) in primary rat glial cells (Fig. 1), but STATs activation observed in late time (at 120 min) (data not shown). Indeed, STATs activation and their signaling pathway were inhibited by 2-AP, a PKR inhibitor (Fig. 2). These results suggested that PKR mediated STATs activation and IRF-1 protein expression in LPS-treated rat glial cells. Also, these results led us to search for PKR effects on downstream of STATs. By using EMSA,
2-AP totally blocked binding activity of a nuclear factor to a consensus GAS/ISRE, which are known to be STAT-binding sites (Fig. 3). Transcription levels of inflammatory cytokine and che mokine were also blocked by 2-AP (Fig. 4). In addition, 2-AP inhibited LPS-induced NO release and iNOS protein expression in a dose-dependent manner (Fig. 5). These findings indicated that LPS activated STATs and their inflammatory down stream signaling through PKR activation. Base on these findings, we similarly confirmed that STATs activation was dependent of PKR activation by using small interference RNA (siRNA) for PKR gene. However, RNAi with long dsRNA in cultured mammalian cells generally had been less successful because PKR was activated by dsRNA. 32 But, it was controversial issue. 33 Therefore, to investigate RNA interference (RNAi) could be used to target endogenous gene, we transfected primary rat astrocyte with a siRNA corresponding to the 21 nucleotides of PKR. PKR mRNA levels were suppressed by siRNA (Fig. 6A) and LPS-stimulated PKR phosphorylation was reduced in dose-dependent manner (Fig. 6B). Next, we also examined whether STATs signaling was blocked by siRNA. As expected, we observed that phosphorylation of STATs and IRF-1 protein expression was also inhibited by siRNA (Fig. 7). In EMSA and RT-PCR experiments, STATs binding activity and inflammatory cytokines expression was also impaired by using siRNA transfection (Fig. 8A, B). These data coincide with our above results (Fig. 2~4), so we conclude that PKR certainly mediates LPS-induced STATs activation and their down stream signaling pathway in rat glial cells.
clear how IFN-â affects the STATs activation. Recently, Toshchakov V. et al. 14 suggested that IFN-â mediated STATs activation, and Sandy D. et al. 30 reported PKR is involved in â expression. So, we have questioned whether PKR affected
IFN-β expression or not. By using 2-AP and siRNA, IFN-â gene expression was impaired in LPS-treated primary rat astrocyte cell (Fig. 9). This result indicated that PKR located upstream of IFN-β signaling and mediated its expression and subsequently, IFN-â affected the STATs signaling pathway. Our experiments using pharmacological inhibitors of NF-κB and PKR suggest that PKR induces the transcription of IFN-β through the activation of NF-κB. Inhibition of NF-κB activity did not affect the phosphorylation of PKR by LPS, whereas inhibition of PKR blocked NF-κB activity. It has been reported that PKR can activate NF-κB, either by directly phosphorylating its inhibitor I-κBα 26 or indirectly by activating the I-κB kinase (IKK) complex 34, and that activation of NF-κB mediates transcription of IFN-β 35. Our findings suggest that PKR may lead to activation of STAT inflammatory signaling through activation of NF-κB and subsequent induction of IFN-β transcription.
On the basis of these data, we have formulated a model of LPS-stimulated activation of STAT inflammatory signaling (Fig. 12). According to this model, LPS activates PKR phosphorylation, leading to the induc tion and secretion of IFN-β by direct activation of NF-κB. The secreted IFN-β would then bind to interferon receptor (IFNR), thereby leading to phosphorylation of serine and tyrosine residues
promoter of inflammation associated genes, including IRF-1, iNOS, and TNF-α, leading to the secretion of newly synthesized cytokines and NO and the induction of various inflammatory responses.
Figure 12. A model for the LPS-induced STAT inflammatory signaling pathway in rat glial cells.
Our current findings demonstrate the effects of PKR on STATs activation in LPS-stimulated rat glial cells. Additional studies are needed to define the roles of this signaling pathway in LPS-induced brain inflammation. Interventions targeting PKR signaling pathway may provide a novel and improved therapeutic strategy for treating and preventing chronic brain inflammation.
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.
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-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 연구아 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.