Astrocytes -Stimulated Rat Γ STAT1 and STAT3 in IFN- Rac1

Rac1 Contributes to Maximal Activation of STAT1 and STAT3 in IFN- γ-Stimulated Rat Astrocytes

This information is current as Eun Jung Park, Kyung-Ae Ji, Sae-Bom Jeon, Woo-Hyuck of September 24, 2021. Choi, Inn-oc Han, Hye-Jin You, Jae-Hong Kim, Ilo Jou and Eun-Hye Joe J Immunol 2004; 173:5697-5703; ; doi: 10.4049/jimmunol.173.9.5697

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Rac1 Contributes to Maximal Activation of STAT1 and STAT3 in IFN-␥-Stimulated Rat Astrocytes1

Eun Jung Park,2* Kyung-Ae Ji,2† Sae-Bom Jeon,†‡ Woo-Hyuck Choi,† Inn-oc Han,§ Hye- Jin You,¶ Jae-Hong Kim,¶ Ilo Jou,* and Eun-Hye Joe3*†‡

Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-␥-induced inflammatory responses in rat astrocytes. We revealed that IFN-␥ rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN- ␥-responsive transcriptional responses. Compared with levels in control cells, IFN-␥-induced IFN-␥-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-␥-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-␥-induced phosphorylation of STAT1 and STAT3 was lower in C6 Downloaded from cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-␥R␣ subunit and IFN-␥-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-␥R␣ and augment the interaction of IFN-␥R with either STAT1 or STAT3 in response to IFN-␥. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-␥-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-␥-responsive inflammatory signaling in rat astrocytes. The Journal of Immunology, 2004, 173: 5697–5703. http://www.jimmunol.org/

rain inflammation is closely associated with the patho- protein (6–8). Generally, binding of IFN-␥ to its receptors induces genesis of various neurodegenerative diseases. Com- assembly of an active receptor complex, and consequent B pared with inflammation in peripheral tissues, inflamma- transphosphorylation of the receptor-associated JAK1 and JAK2 tion in brain appears to follow distinct pathways and time-courses. (4, 5). Phosphorylated JAK1 and JAK2 lead to phosphorylation of The major immune cells that respond to inflammatory stimuli in tyrosine residues in the cytoplasmic tail of the receptor, which the brain are astrocytes and microglia. The inflammatory responses provide the docking sites for STAT. After being recruited to the in these cells are coordinated by the subsequent production of cy- receptor complex, STAT becomes phosphorylated on both Tyr701 by guest on September 24, 2021 tokines, chemokines, and reactive oxygen species (1–3). These and Ser727. Phosphorylated STAT is released from the receptor molecules function in synergistic and/or antagonistic manner, complex and forms dimers. These dimers translocate to the nucleus eventually leading to neurodegeneration via inflammatory cascade. where they directly bind to IFN-␥-activated sequences (GAS),4 Thus, it is important to understand the molecular mechanisms gov- thereby regulating transcription of IFN-␥-responsive genes (9, 10). erning immune reactions in the brain. There are increasing reports that JAK/STAT signaling is finely IFN-␥ is an inflammatory cytokine that exhibits antiviral, anti- regulated by cross-talk and convergence between different signal- microbial, antitumor, and immunomodulatory properties (for re- ing pathways. G protein-coupled receptors (GPCR) have been views, see Refs. 4, 5). In the brain, IFN-␥ can induce reactive shown to closely associate with JAK/STAT signaling (11–13). For astrogliosis. Upon exposure to IFN-␥, astrocytes proliferate and instance, GPCR agonist angiotensin II can stimulate the tyrosine increase expression of various inflammation-associated molecules, phosphorylation of JAKs and STAT by angiotensin II AT1 recep- including ICAM-1 and TNF-␣, as well as glial fibrillary acidic tor (11). Recently, it has been reported that small GTPase Rho and Rac are necessary for GPCR-induced activation of JAK and STAT (14). In line with these findings, several reports have shown that *Department of Pharmacology, †Department of Neuroscience, ‡Brain Disease Re- Rac1 directly interacts with STAT3 and stimulates the phosphor- search Center, School of Medicine, Ajou University, Suwon, Korea; §Research In- stitute, National Cancer Center, Goyang, Gyeonggi, Korea; and ¶School of Life Sci- ylation of STAT3 in response to growth factors (15, 16). ences and Biotechnology, Korea University, Seoul, Korea In immune responses, functional importance of Rac has been Received for publication February 23, 2004. Accepted for publication August emerging (17–19). Studies by Rac-deficient mice and patients 10, 2004. showed that Rac is closely associated with immunodeficiency syn- The costs of publication of this article were defrayed in part by the payment of page drome and abnormalities in host defense against pathogenic infec- charges. This article must therefore be hereby marked advertisement in accordance tion (20, 21). Based on these evidences, we questioned whether with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Rac1 could contribute to the activation of JAK/STAT pathways This work was supported by the interdisciplinary research program of the Korean ␥ Science and Engineering Foundation (KOSEF 1999-2-207-004-5), KOSEF through triggered by IFN- in rat astrocytes. In this study, we show that the Brain Disease Research Center at Ajou University, and Grant M103KV010006 IFN-␥ rapidly activates Rac1, and that Rac1 deficiency leads to 03K2201 00650 from the Brain Research Center of the 21st Century Frontier Re- attenuation of STAT activation and subsequent inflammatory research Program funded by the Ministry of Science and Technology, Republic of Korea (to E.-H.J.). sponses by IFN-␥. The results in this study suggest that Rac1 may 2 E.J.P. and K.-A.J. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Eun-Hye Joe, Department of 4 Abbreviations used in this paper: GAS, ␥-IFN-activated sequence; GPCR, G pro- Pharmacology, School of Medicine, Ajou University, Suwon, 442-721, Korea. E-mail tein-coupled receptor; PAK, p21-activated kinase; PBD, p21-binding domain; SOCS, address: [email protected] suppressor of cytokine signaling; Sos, son of sevenless.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 5698 Rac1 IN IFN-␥ SIGNALING participate as an auxiliary component of IFN-␥-induced inflam- oxidase-conjugated secondary Abs, and then visualized using an ECL matory signaling in rat brain astrocytes. system. Immunoprecipitation Materials and Methods The cells grown on the 100-mm dishes were washed with PBS twice before Reagents lysis. The modified RIPA buffer containing the protease inhibitors were Rat IFN-␥ and Ab against phosphorylated JAK1 were from Calbiochem added for cell lysis. The cell lysate was collected, and centrifuged at ϫ (San Diego, CA). MEM, Lipofectamine Plus, and G418 antibiotics were 13,000 g for 20 min. The supernatants were incubated for overnight with from Invitrogen Life Technologies (Carlsbad, CA. DMEM and FBS were the primary Abs with rocking, and the immune complexes were collected from HyClone (Logan, UT). Abs against STAT1, Tyr701-phosphorylated on protein G beads for 2 h. The immunoprecipitates were washed three STAT1, and STAT3 were from Cell Signaling Technology (Beverly, MA). times with modified RIPA buffer and collected. The resulting precipitates Abs against JAK2, phosphorylated JAK2, and rac1 were from Upstate were then examined by SDS-PAGE and immunoblot analysis. ␥ ␣ Biotechnology (Lake Placid, NY). Ab against IFN- R was purchased RT-PCR from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated secondary Abs were from Vector Laboratories (Burlingame, CA). Total RNA was extracted using RNAzol B (Tel-Test, Friendswood, TX) and cDNA was prepared using reverse transcriptase that originated from Avian Cell culture Myeloblastosis Virus (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. PCR was performed with 30 cycles of sequential reactions: 94°C C6 rat astroglioma cells were obtained from the American Type Culture for 30 s, 55°C for 30 s, and 72°C for 90 s. Oligonucleotide primers were Collection (Manassas, VA; ATCC CCL-107). Cells were grown in DMEM purchased from Bioneer (Seoul, Korea). The sequences of PCR primers were supplemented with 10% (v/v) FBS. Primary astrocytes were cultured from as follows: reverse, 5Ј-AAGGCCGCAGAGAGCAAAAGAAGC-3Ј, and for- the cerebral cortices of 1- to 3-day-old Sprague Dawley rats as previously

ward, 5Ј–CTGGAGAGCACAAACAGCAGAG-3Ј, for ICAM-1; reverse, 5Ј– Downloaded from described (22). Briefly, the cortices were triturated into single cells in ATGCAGGTCTCTGTCACGCT-3Ј, and forward, 5Ј–CTAGTTCTCTGTCA MEM containing 10% FBS and plated into 75 cm2 T-flasks (0.5 hemi- TACTGG-3Ј, for MCP-1; reverse, 5Ј–AGATCCACAACGGATACATT-3Ј, sphere/flask) for 2–3 wk. To prepare pure astrocytes, microglia were de- and forward, 5Ј–TCCCTCAAGATTGTCAGCAA-3Ј, for GAPDH. tached from the flasks by mild shaking and the cells remaining in the flask were harvested with 0.1% trypsin. Astrocytes were plated in dishes and Flow cytometric analysis cultured in MEM supplemented with 10% FBS. Cells were incubated in serum-free medium for 24 h, and then treated with ␥ GST-PAK-PBD-binding assays 10 U/ml IFN- for the indicated times. The cells were washed twice with http://www.jimmunol.org/ PBS containing 1% FBS, collected, and then stained with FITC-conjugated The activation of Rac1 was determined as previously reported (23). Briefly, anti-rat CD54 (ICAM-1) mAb (BD Pharmingen, San Diego, CA) for 30 the pGEX construct encoding the GTPase-binding domain of PAK1 was min at 4°C. After washing, the cells were analyzed with a FACSVantage expressed in Escherichia coli as a GST fusion protein (GST-PBD). C6 cells (BD Biosciences, San Jose, CA), and the data were processed using the were incubated in serum-free medium for 24 h, and then treated with 10 CellQuest program (BD Biosciences) and WinMDI. U/ml IFN-␥. Cells were lysed and incubated with 3 ␮g of GST-PBD. To detect GTP-bound Rac1, proteins were separated by SDS-PAGE, and Western blot analysis was performed using Abs against Rac1. Results IFN-␥ stimulates the activation of Rac1 Plasmids In an effort to investigate whether Rac1 mediates the action of Constitutively active mutant, and pEXV-RacN17, a dominant-negative IFN-␥ in astrocytes, we examined whether IFN-␥ could stimulate by guest on September 24, 2021 mutant of Rac1, were gifts from Dr. A. Hall (University College London, the activation of Rac1 in C6 astroglioma cells. The activation of London, U.K.). The 8-GAS luciferase reporter construct was a gift from Rac1 was defined as its binding ability to the GTPase-binding Dr. M.-h. Shong (Chungnam National University, Daejon, Korea). domain of p21-activated kinase (PAK-PBD) (23). C6 cells were Generation of cells stably expressing RacN17 stimulated with 10 U/ml IFN-␥, and GST-PAK-PBD-bound active For the generation of C6 clonal cell lines stably expressing RacN17, the Rac1 was detected by Western blot analysis using anti-Rac1 Ab. pEXV-RacN17 and pcDNA-NeoR plasmid constructs were cotransfected Interestingly, binding of Rac1 to GST-PAK-PBD significantly in- into C6 cells by Lipofectamine Plus. The transfected cells were selected creased within 5 min of IFN-␥ treatment, reached the peak at and maintained in the presence of 400 ␮g/ml G418 antibiotic in DMEM 15–30 min, and then gradually decreased (Fig. 1A, and data not supplemented with 10% FBS. Individual G418-resistant colonies were iso- shown). To further examine the IFN-␥-induced activation of Rac1, lated 2–3 wk later and expanded into cell lines. Expression of RacN17 ␥ protein in the clones was confirmed by Western blot analysis using an C6 cells were treated with various concentrations of IFN- for 15 anti-c-Myc epitope Ab (24). min, and then binding ability of Rac1 to GST-PAK-PBD was examined. Binding of Rac1 to GST-PAK-PBD was slightly detected Luciferase assay in cells treated with 0.1 U/ml IFN-␥, and clearly observed in cells Transient transfections were performed in duplicate on 35-mm dishes using treated with 1–10 U/ml IFN-␥ (Fig. 1B). These results indicate that Lipofectamine Plus reagents as instructed by the manufacturer (Invitrogen IFN-␥ rapidly stimulates the activation of Rac1, suggesting the Life Technologies). To normalize the variations in cell number and trans- possibility that Rac1 may contribute to the function of IFN-␥ in fection efficiency, all clones were cotransfected with pCMV-␤-galactosidase for 24 h. Luciferase assay was performed according to the manufac- astrocytes. turer’s instruction (Promega, Madison, WI). Luciferase activity was ␥ measured using 20 ␮l of cell extract in 100 ␮l of assay buffer. The light Dominant-negative Rac1 reduces IFN- -induced GAS promoter intensity was measured for 30 s on a luminometer (Berthold Lumat activity in rat astrocytes LB9501; Berthold Technologies, Oak Ridge, TN). Luciferase activity was ␥ normalized by measurement of the ␤-galactosidase activity (in OD ). Because IFN- rapidly activated Rac1 in astrocytes, we investi- 420 gated whether Rac1 could participate in IFN-␥-induced signaling. Western blot analysis For this, we first examined the effect of Rac1 dominant-negative Cells were washed twice with cold PBS, and then lysed in ice-cold mod- mutant (pEXV-RacN17) or constantly active mutant (pEXV- ified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% RacV12) on transcriptional activation of GAS. C6 cells were tran- sodium deoxycholate, 150 mM NaCl, 1 mM Na3VO4, and 1 mM NaF) siently transfected with luciferase reporter plasmids containing containing protease inhibitors (2 mM PMSF, 100 ␮g/ml leupeptin, 10 8-GAS elements and either pEXV-RacN17 or pEXV-RacV12, or ␮g/ml pepstatin, 1 ␮g/ml aprotinin, and 2 mM EDTA). The lysate was centrifuged for 20 min at 13,000 ϫ g at 4°C, and the supernatant was control vector. Twenty-four hours after transfection, the cells were collected. Proteins were separated by SDS-PAGE and transferred to nitro- stimulated with 10 U/ml IFN-␥ for 24 h. Luciferase activity was cellulose membrane. The membrane was incubated with primary Abs, per- then determined and the result was normalized for transfection The Journal of Immunology 5699

FIGURE 1. IFN-␥ induces activation of Rac1. A, C6 cells were incubated in serum-free medium for 24 h and treated with 10 U/ml IFN-␥ for the indicated times. The amount of Rac1 bound to GST-PAK1-PBD fusion protein was analyzed by Western blot as described in Materials and Meth- ods. B, C6 cells were treated with various concentration of IFN-␥ for 15 min, and then GST-PAK-PBD-bound active Rac1 was detected by Western Downloaded from blot analysis using anti-Rac1 Ab. GST band was shown as a loading control. efﬁciency by comparing ␤-galactosidase activity. IFN-␥ dramati-

cally enhanced GAS luciferase activity in control cells transfected http://www.jimmunol.org/ with vector alone. By contrast, IFN-␥-induced GAS luciferase activity was significantly reduced in cells transfected with RacN17 (Fig. 2A). There was no significant difference in luciferase activity between cells transfected with RacV12 and control vector (Fig. 2A). Intriguingly, the basal level of GAS luciferase activity was also lowered in cells transfected with RacN17 compared with that in cells transfected with control vector (Fig. 2A). To extend upon these studies, C6 cells were transiently transfected with different concentrations of RacN17. Consistent with the above result, rela- by guest on September 24, 2021 tive luciferase activity was diminished with increasing the concentration of RacN17 (Fig. 2B). We next examined whether the effect of RacN17 on IFN-␥- induced GAS activity was also observed in primary astrocytes. Primary rat astrocytes were cotransfected with RacN17 and GAS luciferase reporter genes, and then treated with various concentra- ␥ FIGURE 2. Expression of RacN17 markedly attenuates GAS promoter tions of IFN- for 24 h. In primary astrocytes, as well as C6 glioma activity in rat astrocytes. A, The 8-GAS luciferase construct was cotrans- ␥ cells, the expression of RacN17 significantly reduced IFN- -in- fected with pEXV-RacN17, pEXV-RacV12, or vector alone into C6 cells duced GAS luciferase activity (Fig. 2C). These findings support for 24 h. The cells were untreated or treated with 10 U/ml IFN-␥ for 24 h the hypothesis that Rac1 plays a role in IFN-␥ signaling in rat and then assayed for luciferase activity. B, C6 cells were cotransfected with astrocytes. the 8-GAS luciferase construct and various concentrations of pEXV- RacN17 for 24 h. Cells were then treated with 10 U/ml IFN-␥ for 24 h and The expression of IFN-␥-responsive genes is attenuated in C6 harvested for luciferase assay. C, The 8-GAS luciferase construct was co- cells expressing RacN17 transfected with pEXV-RacN17 or vector alone into primary rat astrocytes, and the cells were treated with the indicated concentration of IFN-␥ for To further evaluate the contribution of Rac1 to IFN-␥-induced cel- 24 h. The luciferase activities shown in this study were calculated follow- lular responses, we established the C6-RacN17 cell lines, domi- ing measurement of ␤-galactosidase activity to normalize for transfection nant-negative mutant cells stably expressing RacN17 (referred to efficiency. Values are mean Ϯ SD of duplicates. Data shown are represen- as C6-RacN17) (24). After examining the expression level of tative of at least four independent experiments. RacN17 using anti-c-Myc epitope Ab (Fig. 3A), we first investigated whether IFN-␥-induced GAS luciferase activity was also reduced in C6-RacN17 stable cell lines. Cells were incubated with mRNA levels of MCP-1 and ICAM-1, which are previously re- 10 U/ml IFN-␥ for the indicated times, after which luciferase ac- ported to have functional GAS elements (25, 26). Cells were stim- tivity was measured. We found that IFN-␥-induced GAS luciferase ulated with 10 U/ml IFN-␥ for the indicated times, and total RNA activities were significantly reduced in C6-RacN17 cells, com- was extracted for RT-PCR analysis. As shown in Fig. 3B, IFN-␥- pared with those in C6 control cells (Fig. 3A). These results are in induced transcript levels of MCP-1 and ICAM-1 were relatively agreement with the data from C6 cells transiently transfected with low down in C6-RacN17 cells compared with C6 control cells. We RacN17. observed similar results in primary astrocytes transiently trans- The decline of IFN-␥-enhanced GAS promoter activity in Rac1- fected with RacN17 (data not shown). deficient cells led us to investigate the effect of Rac1 deficiency on In addition, we investigated the cell surface expression of the transcript level of IFN-␥-responsive genes. We examined the ICAM-1 by flow cytometric analysis using FITC-conjugated anti- 5700 Rac1 IN IFN-␥ SIGNALING

FIGURE 4. The expression level of IFN-␥R is not changed by expres-

sion of RacN17. A, Cells were untreated or treated with 10 U/ml IFN-␥ for Downloaded from 12 h, and then assayed for Western blot analysis using Ab against IFN- ␥R␣. B, After C6 cells were transiently transfected with pEXV-RacN17 or vector alone for 24 h, the expression level of IFN-␥R␣ was assessed by Western blot analysis. Data shown are representative of four independent experiments. http://www.jimmunol.org/

(Fig. 4B). We also found that IFN-␥ did not change the expression level of IFN-␥R␣ in C6 cells and rat primary astrocytes (Fig. 4, A and B, and data not shown). These results indicate that inhibitory effect of dominant-negative Rac1 on IFN-␥-induced transcriptional FIGURE 3. IFN-␥-enhanced GAS luciferase activity and gene expres- regulation is not due to down-regulation of IFN-␥R expression. sion are attenuated in cells expressing RacN17. A, The difference of GAS luciferase activity between C6 control cells (a) and C6-RacN17 cells (b) Rac1 plays a role in IFN-␥-stimulated phosphorylations of by guest on September 24, 2021 increased in a time-dependent fashion. The expression level of RacN17 STAT1 and STAT3 but not JAK1 was determined by Western blotting using an anti-c-Myc Ab in C6- RacN17 stable clone. C6 and C6-RacN17 cells were treated with 10 U/ml The activation of STAT1 and STAT3 is a prerequisite for GAS IFN-␥ for the indicated times, and then assayed for luciferase activity. B, promoter activation, and both tyrosine and serine phosphorylation The transcriptions of MCP-1 and ICAM-1 were low down in the C6- are required for maximal STAT activity (5, 14). Given the critical ␥ RacN17 cells. Cells were treated with 10 U/ml IFN- for the indicated role of STAT1 and STAT3 in IFN-␥ signaling, we examined ac- times, and then RT-PCR analysis was performed to detect mRNA expres- tivation of these molecules in cells expressing RacN17. Cells were sion of MCP-1 and ICAM-1. The expression of GAPDH was detected as stimulated with IFN-␥ for the indicated times, and the levels of an internal control. C, Cell surface expression of ICAM-1 was low in C6-RacN17 cells compared with that in C6 control cells. Cells were treated phosphorylated STAT1 and STAT3 were determined by Western with 10 U/ml IFN-␥ for the indicated times, and then FACS analysis was blot analysis using Abs against tyrosine-phosphorylated STAT1 performed with FITC-conjugated anti-rat CD54 (ICAM-1) mAb. Data and STAT3 (Fig. 5A). In C6 control cells, strong phosphorylations shown are representative of more than three independent experiments. of STAT1 and STAT3 were observed within 2.5 min of IFN-␥ addition, and the levels were sustained up to 1 h. However, IFN- ␥-stimulated phosphorylation of STAT1 and STAT3 in C6- rat CD54 (ICAM-1) mAb. Consistent with above results, the basal RacN17 cells was significantly weak (Fig. 5A). The expression expression level of ICAM-1 was significantly low down in C6- level of total STAT1 and STAT3 was not different in C6 control RacN17 cells compared with that in C6 control cells, and IFN-␥- cells and C6-RacN17 cells. Taken together, these results showed induced enhancement of ICAM-1 expression was also weak in that the activation of STAT1 and STAT3 differed between C6 C6-RacN17 cells (Fig. 3C). These results further support the no- control and C6-RacN17 cells, which is consistent with the differ- tion that Rac1 contributes to IFN-␥-induced transcriptional acti- ences in GAS activity and mRNA expression of MCP-1 and vation in astrocytes. ICAM-1 between C6 control and C6-RacN17 cells. Next, we investigated whether RacN17 affected the activation of ␥ ␣ RacN17 does not affect the expression level of IFN- R subunit JAK because both phosphorylations of STAT1 and STAT3 depend Given that the expression of the dominant-negative Rac1 signifi- on the activation of JAK. Cells were stimulated with IFN-␥ for the cantly attenuated GAS-driven gene expression, we questioned indicated times, and cell lysates were Western blotted using Ab whether RacN17 caused down-regulation of IFN-␥R. Using Ab against Tyr-phosphorylated JAK1 (Fig. 5B). Interestingly, in C6- against IFN-␥R␣ subunit, we tested this possibility. Western blot RacN17 cells, JAK1 phosphorylation was not attenuated: in both analysis showed that the expression level of IFN-␥R␣ was not C6 control cells and C6-RacN17 cells, JAK1 phosphorylation was different in C6 control and C6-RacN17 cells (Fig. 4A). In cells detected within 2.5 min of IFN-␥ addition, and sustained up to 60 transiently expressing RacN17, we did not detect the reduced ex- min (Fig. 5B). These results suggest that Rac1 deficiency lead to pression of IFN-␥R␣, compared with cells expressing vector alone blocking of IFN-␥-signaling at the level below JAK1. To further The Journal of Immunology 5701

ern blot analysis was performed with Abs against Rac1, JAK1, STAT1, and IFN-␥R␣, respectively. Interestingly, coimmunopre- cipitation assay showed that IFN-␥R␣ interacted with Rac1 as well as JAK1 and STAT1 (Fig. 6A). Furthermore, the association of IFN-␥R␣ with Rac1 was detected in the absence of IFN-␥, while association of IFN-␥R␣ with either JAK1 or STAT1 was detected in the presence of IFN-␥ (Fig. 6A). To conﬁrm this notion, we performed immunoprecipitation assay with Rac1 Ab. Consistently, the association of Rac1 with IFN-␥R was also detected in immunoprecipitated complex with Rac1 (Fig. 6B). However, we did not observe the difference in the association of Rac1 with IFN-␥R␣ in C6-RacN17 cells compared with that in C6 control cells (Fig. 6B). These results suggest that Rac1 may interact with IFN-␥R␣, but their interaction is not regulated by IFN-␥-induced activation of Rac1. Given the report that Rac1 bind to and regulate STAT3 activity in response to epidermal growth factor (15), we investigated whether Rac1 could interact with STAT1 and STAT3 in IFN-␥-

treated C6 cells. C6 cells were stimulated with IFN-␥ and immu- Downloaded from noprecipitated with Rac1 Ab. Then, Western blot analysis was performed with STAT1 or STAT3. As shown in Fig. 6C, both STAT1 and STAT3 were coimmunoprecipitated with Rac1 in C6 cells (Fig. 6C, a and b). Although STAT1 and STAT3 were faintly detected in the absence of IFN-␥, they were clearly detected in the

presence of IFN-␥ (Fig. 6C, a and b). Thus, we examined whether http://www.jimmunol.org/ Rac1 deﬁciency could affect the IFN-␥-stimulated Rac1 association with STAT1 or STAT3. Interestingly, though we overexposed the ﬁlm, we did not observe the enhanced association of Rac1 with either STAT1 or STAT3 in the presence of IFN-␥ (Fig. 6C, a and FIGURE 5. Expression of RacN17 attenuates the IFN-␥-induced phos- c). These results imply that Rac1 may associate with either STAT1 phorylation of STAT1 and STAT3, but not JAK1. A, Cells were serum- or STAT3 in response to IFN-␥, and their association may con- ␥ starved for 24 h and then stimulated with 10 U/ml IFN- for the indicated tribute to maximal activation of STAT1 and STAT3 in C6 cells. times. Cell lysates were separated by 10% SDS-PAGE and Western blot

analysis was performed using Abs specific for phospho-Tyr-STAT1 by guest on September 24, 2021 (pSTAT1) or phospho-Tyr STAT3 (pSTAT3). The membrane was then Discussion stripped and analyzed sequentially with Abs specific for total STAT1 or Rac GTPase has been shown to play a key regulatory role in var- STAT3, and actin, respectively. B, RacN17 does not affect IFN-␥-induced ious cellular events, such as cytoskeletal organization, cell growth, phosphorylation of JAK1. The phosphorylation of JAK1 was detected by development, apoptosis, transcriptional regulation, and superoxide Western blot analysis using Ab specific for phospho-Tyr-JAK1 (pJAK1). production (for reviews, see Refs. 27–29). Recently, several re- The membrane was stripped and probed with actin Ab to show the amount ports have suggested that Rac GTPase is important in immune of loaded protein. Ca, C6 cells were treated with 10 U/ml IFN-␥ for 2.5 responses (17–21). However, little is known about the specific ␮ min and 5 min. Cb, C6 cells were pretreated with AG490 (5–10 M) for function of Rac in IFN-␥-induced immune responses. In this study, ␥ 1 h and then stimulated with 10 U/ml IFN- for 30 min. The amount of we show that Rac1 acts as a mediator in IFN-␥-induced activation Rac1 bound to GST-PAK-PBD fusion protein was analyzed by Western of STAT inflammatory signaling pathway, thereby contributing blot using Ab against Rac1. GST band was shown as a loading control. ␥ Data shown are representative of four independent experiments. to full activation of IFN- -induced signaling responses in rat astrocytes. The involvement of Rac1 in IFN-␥-signaling was obtained from delineate this notion, we finely examine the kinetics of Rac1 ac- measurement of Rac1 activity. Rac1 is a binary switch that cycles tivation in relation to JAK activation. Binding of Rac1 to GST- between active GTP-bound and inactive GDP-bound states to reg- PAK-PBD was observed at 2.5 min of IFN-␥ treatment as rapid as ulate various cellular processes. Rac1, in its GTP-bound active phosphorylation of JAK1 (Fig. 5Ca). Thus, we investigated forms, has been reported to bind to PAK via interaction with PBD whether IFN-␥-induced activation of Rac1 is affected by inhibition (23). In C6 cells, the binding of Rac1 to the GST-PAK-PBD was of JAK activity in C6 cells. In agreement with the above data (Fig. significantly enhanced by treatment of IFN-␥ (Fig. 1). Guanine 5B), treatment of AG490, a specific inhibitor of JAK1 and JAK2, nucleotide exchange factors, such as Vav and son of sevenless significantly diminished the binding of Rac1 to GST-PAK-PBD in (Sos)-1 activate Rac1 by exchanging GDP into GTP (30, 31). Sev- C6 cells (Fig. 5Cb). These results indicate that IFN-␥ may activate eral studies have reported the possible involvement of Vav in Rac1 through activation of JAK, and Rac1 deficiency has no effect IFN-␥ and -␣ signaling. In murine RAW 264.7 macrophages, in IFN-␥-induced activation of JAK in astrocytes. IFN-␥ triggers the rapid tyrosine phosphorylation of Vav (32). In megakaryocytic cells, Vav is physically associated with both ␣ and ␥ ␣ Rac1 associates with IFN- R , STAT1, and STAT3 ␤ IFN-␥R chains (33). In addition, receptor tyrosine kinases acti- Our above results raised the question of how Rac1 could modulate vate Sos-1 through interaction with the adaptor molecule, Grb2 the phosphorylation of STAT1 and STAT3 in astrocytes. In an (34), though it has not been clearly defined whether Sos-1 is ac- attempt to understand it, we examined whether Rac1 could interact tivated by IFN-␥. In our experiments, activation of Rac1 was abol- with IFN-␥R. C6 cells were treated with 10 U/ml IFN-␥, and the ished in the presence of JAK inhibitor, AG490, while Rac1 did not cell lysates were immunoprecipitated with IFN-␥R␣. Then, West- affect the IFN-␥-induced phosphorylation of JAK1 (Fig. 5, B and 5702 Rac1 IN IFN-␥ SIGNALING

FIGURE 6. Rac1 interacts with IFN- ␥R␣, JAK, and STAT. A, C6 cells were incubated in serum-free medium for 24 h and treated with 10 U/ml IFN-␥ for the indicated times. Lysates were immunoprecipitated with Ab against IFN-␥R␣ subunit, and were subjected to Western blot analysis. The membrane was serially blotted using Abs against JAK1, STAT1, and Rac1. B,C6 and C6-RacN17 cells were incubated in serum-free medium for 24 h and treated with IFN-␥ for the indicated times. Lysates were immunoprecipitated with Ab against Rac1, and subjected to Western blot analysis. The membrane was serially blotted using Abs against IFN-␥R␣ and Rac1. C, C6 and C6- RacN17 cells were incubated in serum-free medium for 24 h and treated with 10 U/ml Downloaded from IFN-␥ for the indicated times. Lysates were immunoprecipitated with Ab against Rac1, and were subjected to Western blot. The membrane was serially blotted using Abs against STAT1, STAT3, and Rac1. Data shown are representative of three independent experiments. http://www.jimmunol.org/

C). These results imply that IFN-␥ activates Rac1 in a JAK-de- tokine signaling (SOCS), in C6-RacN17 cells. To prevent detri- pendent manner. In this regard, it is possible that IFN-␥ activates mental effects, the intensity and duration of JAK-STAT activation JAK and then a guanine nucleotide exchange factor, thus leading are tightly regulated. Generally, SOCS are present in cells at very to activation of Rac1 in astrocytes. The detailed mechanism will be low levels, but are rapidly transcribed after exposure of cells to by guest on September 24, 2021 clarified in the future studies. stimulus. SOCS has been reported to bind to the JAKs and inhibits Our findings raised the question that Rac1 plays a role in IFN- their tyrosine kinase activity (35, 36). That is, Rac1 deficiency may ␥-induced inflammatory signaling. In an effort to examine the role reduce the phosphorylation of STATs, and then diminish the in- of Rac1 in IFN-␥ signaling, we used the well-characterized Rac1 duction of IFN-␥-induced inhibitory molecules for JAK1. Conse- mutant, RacN17, which lacks GTPase activity. IFN-␥-enhanced quently, reduced action of JAK1 inhibitors may lead to sustained GAS promoter activity was significantly attenuated in C6 astro- phosphorylation of JAK1 in C6-RacN17 cells. Taken together, glioma cells expressing RacN17, and similar effects were observed these findings convincingly show that Rac1 contributes to IFN-␥ in primary rat astrocytes (Figs. 2 and 3). However, transient trans- signaling at least at the level of STAT phosphorylation. fection with the constitutively active mutant, RacV12, did not Next question is how Rac1 regulates STAT phosphorylation. cause a significant change in the GAS activity. These findings We considered the possibility that Rac1 could interact with suggest Rac1 may participate as a mediator in IFN-␥-signaling and IFN-␥R complex and augment its activity. It has been reported that that the endogenous expression level of Rac1 may be sufficient to Rac1 directly bind to several proteins, including p67phox, p21-ac- perform this role in rat astrocytes. The contribution of Rac1 in tivated kinase 1, and STAT3, and then stimulate their activity by IFN-␥-inducible transcriptional regulation was further demon- constitution of active complex (15, 37). The results in this study strated by measuring the transcription of MCP-1 and ICAM-1, showed several interesting findings about the roles of Rac1 in which have STAT-binding motifs in their promoter regions (25, IFN-␥ signaling. First, Rac1 was coimmunoprecipitated with 26). The basal level of these transcripts in cells expressing RacN17 IFN-␥R complex, including IFN-␥R␣, STAT1, and STAT3. Sec- was significantly lower than in control cells. Moreover, the induc- ond, the association of Rac1 with IFN-␥R␣ was detected in the tion of these transcripts by IFN-␥ in cells expressing RacN17 was absence of IFN-␥. Third, the association of Rac1 with IFN-␥R␣ markedly weaker compared with control cells (Fig. 3B). Consistent was also detected in C6-RacN17 cells as well as C6 cells. Fourth, with these results, cell surface expression of ICAM was also ap- IFN-␥ significantly increased the association of Rac1 with either parently low down in cells expressing RacN17 (Fig. 3C). In addi- STAT1 or STAT3 in C6 cells while not in C6-RacN17 cells. These tion, the results in the present study showed that Rac1 deficiency results suggest that Rac1 may augment the formation of active attenuated IFN-␥-stimulated tyrosine phosphorylation of not only IFN-␥R complex including IFN-␥R␣ and STATs in response to STAT1 but also STAT3. However, it did not reduce the expression IFN-␥, thereby contributing to maximal activation of STAT1 and level of IFN-␥R␣ and the phosphorylated level of JAK1 (Figs. 4 STAT3. Previously, Simon et al. (15) suggested that Rac1 could and 5). The phosphorylation of JAK1 in C6-RacN17 was rather directly bind to STAT3 by growth factors and regulate STAT3 elevated and sustained compared with that in C6 control cells (Fig. phosphorylation in COS-1 cells. However, interaction between 5B). One possible explanation for this is decrease of IFN-␥-in- Rac1 and IFN-␥R␣ has not yet been reported. Although intensive duced inhibitory molecules for JAK1, such as suppressors of cy- experiments are required for explaining the questions about the The Journal of Immunology 5703 association of Rac1 with IFN-␥R complex, our findings suggest a 17. Arrieumerlou, C., C. Randriamampita, G. Bismuth, and A. Trautmann. 2000. Rac novel cross-talk between Rac1 and IFN-␥-induced inflammatory is involved in early TCR signaling. J. Immunol. 165:3182. 18. Woo, C. H., H. J. You, S. H. Cho, Y. W. Eom, J. S. Chun, Y. J. Yoo, and signaling in rat astrocytes. In summary, the present study suggests J. H. Kim. 2002. Leukotriene B4 stimulates Rac-ERK cascade to generate reac- that Rac1 is rapidly activated by IFN-␥, and then contributes to tive oxygen species that mediates chemotaxis. J. Biol. Chem. 277:8572. 19. Li, B., H. Yu, W. Zheng, R. Voll, S. Na, A. W. Roberts, D. A. Williams, activation of STAT phosphorylation as an auxiliary mediator, R. J. Davis, S. Ghosh, and R. A. Flavell. 2000. Role of the guanosine triphos- thereby resulting in maximal activation of inflammatory signaling phatase Rac2 in T helper 1 cell differentiation. Science 288:2219. responses in rat astrocytes. 20. Ambruso, D. R., C. Knall, A. N. Abell, J. Panepinto, A. Kurkchubasche, G. Thurman, C. Gonzalez-Aller, A. Hiester, M. deBoer, R. J. 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