Kinase inhibitors abrogate IFN-γ-induced class II transactivator and class II MHC gene expression in astroglioma cell lines

Journal of Neuroimmunology 85 Ž1998. 174–185

Kinase inhibitors abrogate IFN-g-induced class II transactivator and class II MHC gene expression in astroglioma cell lines Nicholas J. Van Wagoner, George M. O’Keefe, Etty N. Benveniste

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Department of Cell Biology, UniÕersity of Alabama at Birmingham, 1918 UniÕersity BouleÕard, Birmingham, AL 35294-0005, USA Received 16 July 1997; revised 10 November 1997; accepted 26 December 1997

Abstract Multiple kinase events, involving both tyrosine Žtyr. kinase and serinerthreonine Žserrthr. kinase activity, are required for IFN-g-induced class II MHC mRNA and protein expression in primary rat astrocytes. In this study, we examined the necessity of serrthr and tyr kinase activity for IFN-g-induced stimulation of class II MHC gene expression in the human astroglioma cell lines CRT and CH235, as well as the involvement of these kinases in IFN-g-induced expression of the class II transactivator ŽCIITA., a protein critical for IFN-g-induced transcription of class II MHC genes. We show that general serrthr kinase inhibitors, inhibitors of the serrthr kinase mitogen-activated protein kinase ŽMAPK., and tyr kinase inhibitors reduce IFN-g-induced class II MHC mRNA and protein expression in a dose-dependent manner. As well, these inhibitors abrogate IFN-g-induced CIITA mRNA expression in the astroglioma cell lines. We have further demonstrated that cells constitutively expressing the CIITA protein Ž2fTGH.CIITA. show no decrease in CIITA or class II MHC mRNA expression in the presence of serrthr and tyr kinase inhibitors. Collectively, these data indicate that serrthr kinase activity, possibly MAPK, and tyr kinase activity are required for IFN-g-induced expression of CIITA mRNA, and the subsequent expression of class II MHC genes. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Glial cells; CIITA; Class II MHC; Kinases; Cytokines

1. Introduction The class II major histocompatibility complex ŽMHC. genes encode a group of polymorphic cell surface glycoproteins involved in antigen presentation to T cells, leading to their subsequent activation and differentiation ŽBenacerraf, 1981.. Constitutive class II MHC expression is limited to professional antigen presenting cells ŽAPCs. such as B cells, dendritic cells, thymic epithelium, and macrophages. As well, cytokines such as IFN-g and IL-4 can induce expression of class II MHC molecules in a wide variety of cells including pancreatic b-cells, keratinocytes, brain endothelial cells, microglia, and astrocytes Žfor review see Glimcher and Kara, 1992; Rohn et al., 1996.. Because of the critical role that class II MHC antigens play in T-cell activation, abnormal expression greatly impacts on immune responsiveness. A complete absence of class II MHC antigen is associated with a severe combined immunodeficiency disease, bare lymphocyte syndrome ŽBLS.. This )

Corresponding author. Tel.: q1 205 9347667; fax: q1 205 9756748; e-mail: [email protected] 0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 0 1 0 - 1

disease, marked by an inability to activate T-cells, results in recurring viral and microbial infection Žfor review see Mach et al., 1996.. Conversely, aberrant overexpression of class II MHC antigens in various tissues has been associated with the pathogenesis of autoimmune disorders including multiple sclerosis, rheumatoid arthritis, autoimmune nephritis, and others Žfor review see Grusby and Glimcher, 1995.. Because of its critical role in immune response induction, class II MHC expression must be tightly controlled. Regulation of class II MHC genes occurs primarily at the level of transcription. The proximal promoters of the class II MHC genes contain a number of conserved sequences including the W, X, and Y boxes. Trans-acting factors interact with these DNA sequences to either activate or inhibit both constitutive and inducible class II MHC expression Žfor review see Glimcher and Kara, 1992; Rohn et al., 1996.. In addition, the class II transactivator ŽCIITA., a non-DNA binding factor, has been shown to be involved in class II MHC regulation ŽSteimle et al., 1993.. Since its discovery, several reports have provided evidence for the requirement of CIITA in constitutive and inducible class II

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MHC expression ŽChang et al., 1994, 1995; Steimle et al., 1994.. CIITA functions as a transcriptional activator by interacting with proteins bound to the X box of the class II MHC promoter, specifically the RFX5 protein, as well as the general transcription complex ŽRiley et al., 1995; Zhou and Glimcher, 1995; Mahanta et al., 1997; Scholl et al., 1997.. IFN-g is the most potent inducer of class II MHC expression in many cell types. In brief, it signals expression of class II MHC genes by binding to its receptor. Ligandrreceptor interaction leads to receptor dimerization and the phosphorylation and activation of two non-receptor tyrosine kinases ŽJanus kinases. Jak1 and Jak2. These kinases, in turn, mediate tyrosine Žtyr. phosphorylation of Signal Transducer and Activator of Transcription ŽSTAT.1 a Žfor review see Bach et al., 1997.. Once phosphorylated, STAT-1 a homodimerizes and translocates to the nucleus where it binds to the g-activation sequence ŽGAS. of IFN-g inducible genes and induces transcription Žfor review see Darnell, 1997.. STAT-1 a is critical for IFN-ginduced CIITA expression and subsequent class II MHC expression, as determined by the use of STAT-1 a deficient mice and STAT-1 a antisense oligonucleotides ŽLee and Benveniste, 1996; Meraz et al., 1996.. It is hypothesized that phosphorylated STAT-1 a stimulates CIITA expression by directly binding to the CIITA promoter or indirectly by activating the expression of other IFN-g-inducible transcription factors that can then bind and activate the CIITA promoter. In this regard, the partial sequence of the CIITA type IV promoter Žthe IFN-g responsive promoter. has been recently published ŽMuhlethaler-Mottet et al., 1997., and the promoter contains GAS and IRF-1r2 elements that are likely to be responsible for IFN-g induction of CIITA expression via the transcription factors STAT-1 a and IRF-1, respectively. As well, other kinases have been implicated in IFN-g signaling. Protein kinase C ŽPKC., a serinerthreonine Žserrthr. kinase, has been shown to be activated and involved in HLA-DR transcription in response to IFN-g treatment of U937 cells and THP-1 monocyte-like cells. ŽFan et al., 1988.. In murine peritoneal macrophages and primary rat astrocytes, PKC activation has also been shown as necessary for IFN-g-induced class II MHC expression ŽBenveniste et al., 1991; Lee et al., 1995; Prpic et al., 1989.. These results suggest that multiple kinase events, involving both tyr kinase and serrthr kinase activity, are required for ultimate class II MHC expression in response to IFN-g stimulation. In this study, we have examined the signaling mechanisms involved in IFN-g-induced CIITA and class II MHC expression in two human astroglioma cell lines, CRT and CH235. Specifically, we have investigated the involvement of serrthr and tyr kinases in IFN-g-induced CIITA mRNA expression and class II MHC mRNA and cell surface protein expression. IFN-g-induced class II MHC mRNA and protein expression are inhibited in a dose-dependent

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manner by the general serrthr kinase inhibitors H7 and H8, while no inhibition was seen in the presence of HA1004, an inhibitor of cAMP-dependent kinases. Two specific inhibitors of mitogen-activated protein kinases ŽMAPK., which are serrthr kinases, abrogated IFN-g-induced class II MHC expression, suggesting an involvement of the MAPK pathway in this response. Similar inhibition was seen in the presence of the tyr kinase inhibitors genistein, herbimycin A and tyrphostin B42. We have also shown that IFN-g-induced CIITA mRNA levels are decreased in the presence of serrthr, MAPK, and tyr kinase inhibitors in a dose-dependent manner. Overexpression of CIITA in the fibrosarcoma cell line, 2fTGH.CIITA, abrogates the inhibitory effects of both H7, a serrthr kinase inhibitor, and genistein, a tyr kinase inhibitor. Together these data demonstrate that serrthr kinases, possibly MAPK, and tyr kinase activity are involved in IFN-g induction of CIITA expression, and subsequent class II MHC expression.

2. Materials and methods 2.1. Cell lines The CRT astroglioma cell line, derived from a neoplastic frontal lobe lesion, was grown in RPMI 1640 medium supplemented with 10 mM Hepes ŽpH 7.2., 2 mM Lglutamine, 100 Urml penicillin, 100 m grml streptomycin, and 10% fetal bovine serum ŽFBS. as described previously ŽRosenman et al., 1995.. The CH235 glioblastoma multiform cell line, was grown in DMEMrHAMS F12 medium supplemented with 10 mM Hepes ŽpH 7.2., 2 mM Lglutamine, 100 Urml penicillin, 100 m grml streptomycin, and 10% FBS, as described previously ŽBethea et al., 1992.. Stable 2fTGH cells, derived from the fibrosarcoma cell line HT1080, expressing CIITA Ž2fTGH.CIITA., have been previously described ŽLee et al., 1997; Lu et al., 1995.. Construction of the plasmid pCIITA.2.11 and its use to direct expression of functional CIITA have also been described previously ŽRiley et al., 1995.. The 2fTGH.CIITA cell line was grown in DMEM medium supplemented with 10 mM Hepes ŽpH 7.2., 2 mM Lglutamine, 100 Urml penicillin, 100 m grml streptomycin, 500 m grml G418 Žselective marker., and 10% FBS. 2.2. Reagents Human recombinant IFN-g was the generous gift of Biogen ŽCambridge, MA.. mAb to human class II MHC Žclone FMC14. was purchased from Harlan Sera-Lab ŽSussex, England.. Affinity-purified goat anti-mouse IgG1 conjugated to FITC was purchased from Southern Biotechnology ŽBirmingham, AL.. The kinase inhibitors H7, H8, and HA1004 were purchased from Seikagaku America ŽRockville, MD.. The tyr kinase inhibitors genistein and tyr-

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phostin B42 were obtained from Calbiochem ŽLa Jolla, CA., and herbimycin A was purchased from Life Technologies ŽGrand Island, NY.. The MAPK inhibitors apigenin and olomoucine were purchased from Calbiochem. Cells were treated with each inhibitor for 48 h after which viability was determined by trypan blue staining. The highest concentration of each inhibitor used in the following studies did not significantly affect cell viability, which generally remained above 80% of control cells, i.e., no inhibitor added Ždata not shown.. In addition, some of the inhibitors were diluted in dimethyl sulfoxide, which at the concentrations used had no detrimental effect on astroglioma cell viability ŽLee et al., 1995. Ždata not shown.. 2.3. QuantitatiÕe analysis of class II MHC antigen expression by immunofluorescence flow cytometry The human cell lines CRT and CH235 were resuspended in their respective media containing 10% fetal bovine serum and were plated at 1–1.5 = 10 5 cellsrwell in six-well plates ŽCostar, Cambridge, MA.. Plates were incubated until ; 80% confluent Žapproximately 2 days., and then fresh media was added to each well. All experiments involving CH235 cells were done in the absence of serum, while experiments utilizing CRT cells were done in the presence of serum. The presence or absence of serum did not affect the experimental results, however, CRT cells required serum for increased survival during longer incubations. Duplicate wells were treated with 100 Urml of recombinant human IFN-g in the presence or absence of serrthr or tyr kinase inhibitors, and incubated for 48 h. Cells were then trypsinized and stained for class II MHC antigens as described previously ŽBenveniste et al., 1991.. Briefly, cells were incubated with 35 m l of monoclonal mouse anti-HLA-DR for 60 min at 48C, washed twice, and then incubated with 40 m l of goat anti-mouse IgG1-FITC Ž1:20. for 60 min at 48C. Cells were again washed twice and fixed in 300 m l of 1% paraformaldehyde. Class II MHC antigen expression was analyzed using the FACStar ŽBecton-Dickson, Mountain View, CA.. Negative controls were incubated in 35 m l of phosphate buffered saline ŽPBS. instead of primary antibody. Analysis was performed on 10 4 cellsrsample. Results are expressed as a percentage of values obtained with IFN-g alone Ž100%.. Values represent the % of class II MHC positive cells multiplied by the mean fluorescence intensity ŽMFI.. 2.4. RNA isolation Total RNA was isolated from confluent monolayers of astroglioma cell lines that had been incubated in the presence or absence of pharmacological inhibitors, followed by treatment with IFN-g Ž100 Urml. for various time periods Ž6 or 24 h. as previously described ŽLee et al., 1997..

2.5. Riboprobes and RNase protection assay (RPA) A pGEM-T plasmid containing a 626-bp fragment corresponding to 2908–3545 bp of the human CIITA cDNA was linearized by AÕaII and used to generate an antisense RNA 498 nucleotides in length. The DR a 120 plasmid, which contains the HLA-DRA cDNA Žno. 57392, from American Type Culture Collection, Rockville, MD., was digested with BamHI and RsaI, subcloned into the BamHIrHincII polylinker site of pGEM-4Z, linearized by BamHI, and a 454-nucleotide antisense RNA probe was synthesized from this construct as previously described ŽLee and Benveniste, 1996.. A pAMP-1 vector containing a fragment of the human GAPDH cDNA Žcorresponding to 43–531 bp. was linearized with NcoI, which digests within the GAPDH cDNA insert. In vitro transcription of this linearized plasmid with T7 RNA polymerase generates a 290-bp antisense RNA probe. In vitro transcription of riboprobes and RPA were conducted as previously described ŽLee and Benveniste, 1996.. Fifteen m g of total RNA was hybridized with HLA-DR a or CIITA Ž30 = 10 3 cpm. riboprobes and GAPDH riboprobe Ž25 = 10 3 cpm. at 428C overnight. The hybridized mixture was then treated with RNase ArT1 Ž1r200 dilution. at room temperature for 1 h, analyzed by 5% denaturing Ž8 M urea. PAGE, and the gels were exposed to X-ray film. The protected fragments of the CIITA, HLA-DR a , and GAPDH riboprobes are 452, 413, and 230 nucleotides in length, respectively. Quantitation of protected RNA fragments was performed by a PhosphorImager ŽMolecular Dynamics, Sunnyvale, CA.. Values for CIITA and HLADR a mRNA expression were normalized to GAPDH mRNA levels for each experimental condition. GAPDH mRNA was used as a control gene as its levels are not affected by cytokine or inhibitor treatment. The RPAs shown throughout this article are overexposed for GAPDH because the signals for CIITA and class II MHC are weaker. However, quantitation of the original gels was performed on a PhosphorImager to arrive at accurate values. Table 1 IFN-g induction of class II MHC protein expression by human astroglioma cell lines MFI a

Total class II MHC b

Cell treatment

% Positive cells

CH235 CONTROLc IFN-g d

1.8"0.3 e 58.3"10.0

26.7"7.9 48.3"12.3

46.5"6.0 2,823.0"947.4

CRT CONTROLc IFN-g d

1.8"0.9 96.0"1.9

65.4"18.0 630.0"72.4

130.5"85.2 60,505.3"7937.1

a

Mean fluorescence intensity. Total class II MHC expression is calculated as the average percent positive cells=the average MFI. c Control medium for 48 h. d IFN-g Ž100 Urml. for 48 h. e Mean"S.D. of three experiments performed in duplicate. b

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3. Results 3.1. Effect of ser r thr kinase inhibitors on IFN-g-induced class II MHC protein and mRNA expression We have previously demonstrated that the general serrthr kinase inhibitors H7 and H8 reduce class II MHC cell surface expression in primary rat astrocytes ŽBenveniste et al., 1991; Lee et al., 1995.. To extend these findings, we examined the effects of these serrthr kinase inhibitors on IFN-g-induced class II MHC expression in the human astroglioma cell lines CH235 and CRT. As shown in Table 1, both cell lines are constitutively negative for class II MHC antigens, and strongly inducible by IFN-g , with the CRT line being the most responsive to

Fig. 2. The MAPK inhibitors apigenin and olomoucine suppress IFN-g induced class II MHC protein expression. CRT cells were pretreated with apigenin Ž0.1–10 m M. ŽA. or olomoucine Ž1–50 m M. ŽB. for 1 h, then IFN-g Ž100 Urml. was added for an additional 48 h. Cells were stained for class II MHC protein expression. Results are expressed as a percentage of values obtained with IFN-g alone Ž100%.. These data represent the mean"S.D. of two experiments.

Fig. 1. Effect of seriner threonine kinase inhibitors on IFN-g -induced class II MHC protein expression. CH235 and CRT cells were preincubated with H7 ŽA., H8 ŽB., and HA1004 ŽC. Ž5–50 m M. for 1 h; IFN-g Ž100 Urml. was added, and cultures were harvested after 48 h. Cells were stained for class II MHC antigen expression. Results are expressed as a percentage of values obtained with IFN-g alone Ž100%.. Values represent percent positive cells multiplied by the mean fluorescence intensity. These data represent the mean"S.D. of three experiments.

IFN-g stimulation. To determine the effect of serrthr kinase inhibitors on IFN-g-induced class II MHC protein expression, cells were pretreated with 5, 25, or 50 m M of H7 or H8 for 1 h. IFN-g Ž100 Urml. was then added and after 48 h, cells were stained for class II MHC protein expression and analyzed by FACS. Results are expressed as a percentage of the value obtained with IFN-g alone Ž100%. to account for differences in inducible expression of class II MHC in the two cell lines Žsee Table 1.. The serrthr kinase inhibitors H7 and H8 reduced IFN-g-induced class II MHC cell surface expression in a dose-dependent manner ŽFig. 1A and B.. Although the two cell lines showed slightly different sensitivities to H7 and H8, the 50 m M concentration of each inhibitor reduced IFN-ginduced class II MHC expression by ) 75% in both cell lines. Because both H7 and H8 have modest inhibitory effects on cGMP- and cAMP-dependent protein kinases ŽPK., we examined the effect of HA1004 on IFN-g-induced class II MHC surface expression. HA1004 is a more selective inhibitor of cGMP- and cAMP-dependent PKs and is a very weak inhibitor of other serrthr kinases, thus serving as a control for H7 and H8 ŽHidaka et al., 1984..

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No inhibition of class II MHC expression by HA1004 was seen at any of the concentrations used ŽFig. 1C., ruling out the involvement of cAMP- and cGMP-PKs as the target of H7 and H8 inhibition. We have previously determined that HA1004 is able to reverse cAMP-induced inhibition of adhesion molecules in astroglioma cells ŽBallestas and Benveniste, unpublished observation., indicating that this compound is effective at inhibiting cAMP-driven responses. This suggests that the reduction of IFN-g-induced class II MHC protein expression resulted from inhibition of other serrthr kinases, possibly PKCs. We attempted to test the effects of more specific PKC inhibitors on each cell line. Bisindolylmaleimide I, a specific PKC inhibitor ŽToullec et al., 1991., was used, but had no effect on IFN-g-induced class II MHC surface expression at any concentration tested Ždata not shown.. Previously, we had

shown that calphostin C, another specific PKC inhibitor, blocked IFN-g-induced class II MHC gene expression in primary rat glial cell cultures ŽLee et al., 1995.. Therefore, attempts were made to treat CRT and CH235 cells with calphostin C. However, because of its high toxicity to both cell lines, definitive results were not obtained. Another serrthr kinase recently implicated in IFN-g signaling is MAPK ŽLiu et al., 1994; Wen et al., 1995.. Therefore, we tested the effect of two specific MAPK inhibitors, apigenin and olomoucine, on IFN-g-induced class II MHC expression. As shown in Fig. 2, the MAPK inhibitors reduced IFN-g-induced class II MHC cell surface expression in a dose-dependent manner in CRT astroglioma cells. At the highest concentrations tested, apigenin and olomoucine inhibited IFN-g-induced class II protein expression by ; 58 and ; 52%, respectively.

Fig. 3. Dose-dependent inhibition of class II MHC mRNA expression by H7, H8, and apigenin. CRT cells were preincubated with H7 ŽA. and H8 ŽB. Ž5–50 m M. for 1 h, or apigenin ŽC. Ž0.1–10 m M. for 1 h; IFN-g Ž100 Urml. was added and RNA was harvested after 24 h for class II MHC mRNA. RPA was performed using labeled class II MHC and GAPDH cDNA probes. Representative of three experiments.

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To determine if the inhibition of class II MHC protein resulted from a decrease in class II MHC mRNA levels, RPAs were performed on RNA isolated from both cell lines in the presence of each of the inhibitors. Results for both cell lines were similar, therefore only data from the CRT line will be presented. CRT cells were pretreated with increasing concentrations Ž5–50 m M. of the serrthr kinase inhibitors H7 and H8 or the MAPK inhibitor apigenin Ž0.1–10 m M., followed by IFN-g stimulation for 24 h, then class II MHC mRNA expression was assessed. Inclusion of the serrthr and MAPK inhibitors resulted in a dose-dependent decrease in IFN-g-induced class II MHC mRNA ŽFig. 3A, B and C.. GAPDH mRNA expression was unaffected by the inhibitors, and acted as a control for normalization and quantitation. H7, at a concentration of 50 m M, inhibited IFN-g-induced class II MHC mRNA expression by ; 93% ŽFig. 3A, lane 5., while H8 inhibited expression by ; 82% ŽFig. 3B, lane 5. as determined using Imagequant. Apigenin, at a concentration of 10 m M, inhibited class II expression by ; 77% ŽFig. 3C, lane 5.. HA1004, the serrthr kinase inhibitor that was without effect on class II MHC protein expression, also had no inhibitory effect on IFN-g-induced class II MHC mRNA expression Ždata not shown.. The correlative decrease in class II mRNA and protein expression by H7, H8, and apigenin Žcompare Figs. 1–3. suggests serrthr kinase involvement in IFN-g-induced transcription and translation of class II MHC genes in human astroglioma cell lines. 3.2. Effect of tyr kinase inhibitors on IFN-g-induced class II MHC protein and mRNA expression The involvement of tyrosine phosphorylation in IFN-g induction of class II MHC expression has been previously documented ŽLee and Benveniste, 1996; Lee et al., 1995.. To better understand the role of tyr kinases in IFN-g signaling of class II MHC expression, CH235 and CRT cells were treated with genistein Ž10–50 m grml., herbimycin A Ž50–500 ngrml., and tyrphostin B42 Ž10–50 m grml., followed by IFN-g treatment, and assessed for class II MHC surface protein expression as described for the serrthr inhibitors. Inhibitory dose response effects were seen in the presence of all three tyr kinase inhibitors in both cell lines ŽFig. 4A, B and C.. While tyrphostin B42 clearly reduced IFN-g-induced class II MHC expression in both CRT and CH235 cells, greatest inhibition in CH235 cells occurred at a concentration of 25 m grml, with slightly less inhibition at the 50 m grml concentration ŽFig. 4C.. Treatment with both herbimycin A and genistein resulted in a dose-dependent decrease of IFN-g-induced class II MHC expression in both cell lines ŽFig. 4A and B.. The most potent inhibition occurred after treatment with herbimycin A, reducing IFN-g-induced class II MHC expression by ; 90% in both cell lines ŽFig. 4B.. To examine class II MHC mRNA levels in the presence of the tyr kinase inhibitors, cells were pretreated with

Fig. 4. Effect of tyrosine kinase inhibitors on IFN-g -induced class II MHC protein expression. CH235 and CRT cells were preincubated with genistein Ž10–50 m grml. for 1 h ŽA., herbimycin A Ž50–500 ngrml. for 16 h ŽB., and tyrphostin B42 Ž10–50 m grml. ŽC. for 1 h; IFN-g Ž100 Urml. was added, and cultures were harvested after 48 h. Cells were stained for class II MHC antigens. Results are expressed as a percentage of values obtained with IFN-g alone Ž100%.. Values represent percent positive cells multiplied by the mean fluorescence intensity. These data represent the mean"S.D. of three experiments.

genistein Ž10–50 m grml., herbimycin A Ž50–500 ngrml., or tyrphostin B42 Ž10–60 m grml., followed by IFN-g treatment for 24 h. Again, RPA was used to analyze class II MHC mRNA levels after inhibitor and IFN-g treatment. Genistein reduced IFN-g-induced class II MHC mRNA expression in a dose-dependent manner, with inhibition reaching ; 66% at the highest concentration tested ŽFig. 5A, lane 6.. H7 at 50 m M was included as a control for inhibition Žlane 3.. Pretreatment with herbimycin A resulted in complete inhibition Ž; 99%. of class II MHC mRNA at the highest concentration tested ŽFig. 5B, lane 5.. Tyrphostin B42 was a more effective inhibitor at the mRNA level than at the protein level, resulting in a complete loss of IFN-g-induced class II MHC mRNA Ž; 99%. ŽFig. 5C, lane 5.. The inhibition of both class II mRNA and protein expression by genistein, herbimycin A,

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Fig. 5. Dose-dependent inhibition of class II MHC mRNA expression by tyrosine kinase inhibitors. CRT cells were preincubated with genistein for 1 h Ž10–50 m grml. ŽA., herbimycin A for 16 h Ž50–500 ngrml. ŽB. or tyrphostin B42 for 1 h Ž10–60 m grml. ŽC.. IFN-g Ž100 Urml. was added and RNA was harvested after 24 h. In addition, cells were pretreated with H7 Ž50 m M. for 1 h, then exposed to IFN-g for 24 h ŽA, lane 3.. RPA was performed using labeled class II MHC and GAPDH cDNA probes. Representative of two experiments.

and tyrphostin B42 suggests tyr kinase involvement in IFN-g-induced transcription and translation of class II MHC genes in human astroglioma cell lines. 3.3. Kinase inhibitors reduce CIITA mRNA leÕels CIITA plays a critical role in the regulation of both constitutive and IFN-g inducible expression of class II MHC genes ŽChang et al., 1994, 1995; Steimle et al., 1994.. IFN-g induction of CIITA has been shown to

precede and be necessary for subsequent IFN-g induction of class II MHC mRNA and protein expression ŽLee et al., 1997.. Therefore, we desired to look at the effect of the inhibitors on IFN-g-induced CIITA mRNA expression. RPAs were performed on mRNA isolated from cells pretreated with the kinase inhibitors and then stimulated with IFN-g for 6 h, which is optimal for CIITA mRNA expression ŽLee et al., 1997.. Again, only data from CRT cells is presented since comparable results were obtained in both cell lines. In all cases, an inhibitory dose response effect

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was seen on IFN-g-induced CIITA mRNA. IFN-g induction of CIITA mRNA expression decreased significantly in the presence of increasing concentrations of H7 and apigenin ŽFig. 6A and B., as well as H8 Ždata not shown.. The tyr kinase inhibitors also decreased CIITA mRNA levels in a dose responsive manner ŽFig. 6C and D.. At the highest concentrations tested, herbimycin A inhibition was

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; 84% ŽFig. 6C, lane 5., while tyrphostin B42 inhibition reached ; 95% ŽFig. 6D, lane 5.. Similar results were obtained with genistein; ; 95% inhibition was achieved using a 50 m grml concentration Ždata not shown.. Taken together, these data suggest that serrthr kinases, possibly MAPK, and tyr kinases are involved in IFN-g-induced CIITA transcription.

Fig. 6. Dose-dependent inhibition of CIITA mRNA expression by kinase inhibitors. CRT cells were preincubated with H7 Ž5–50 m M. for 1 h ŽA., apigenin Ž0.1–10 m M. for 1 h ŽB., herbimycin A Ž50–500 ngrml. for 16 h ŽC., or tyrphostin B42 Ž10–60 m grml. for 1 h ŽD.. IFN-g Ž100 Urml. was added and RNA was harvested after 6 h. RPA was performed using labeled CIITA and GAPDH cDNA probes. Representative of two experiments.

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3.4. Effect of ser r thr and tyr kinase inhibitors on class II MHC and CIITA mRNA expression in the 2fTGH.CIITA cell line The data described thus far suggest that serrthr and tyr kinase activities are involved in IFN-g-induced activation

of CIITA transcription, and further suggest that inhibition of CIITA expression by serrthr and tyr kinase inhibitors is responsible for the subsequent reduction of class II MHC mRNA and protein expression. If this is the case, we would expect overexpression of CIITA protein to bypass the inhibitory effects of the kinase inhibitors in IFN-g-in-

Fig. 7. 2fTGH.CIITA cells show no inhibition of class II MHC mRNA or CIITA mRNA in the presence of H7 and genistein. 2fTGH.CIITA cells were treated with H7 Ž5–50 m M. or genistein Ž10–50 m grml. for 1 h, then incubated for 25 h for class II mRNA ŽA. and 7 h for CIITA mRNA ŽB.. IFN-g Ž100 Urml. was also added for either 25 h ŽA; lane 2. or 7 h ŽB; lane 2.. RPA was performed using labeled class II MHC, CIITA and GAPDH cDNA probes. Representative of two experiments.

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duced class II MHC signaling. 2fTGH.CIITA cells, a fibrosarcoma cell line stably transfected with human CIITA cDNA ŽRiley et al., 1995., were used to test this hypothesis. 2fTGH.CIITA cells constitutively express CIITA and are positive for class II MHC mRNA and protein expression in the absence of IFN-g stimulation ŽLee et al., 1997.. The parental cell line 2fTGH does not constitutively express CIITA or class II MHC; however, IFN-g treatment induces CIITA mRNA and subsequent class II MHC mRNA and protein expression ŽLee et al., 1997.. Treatment with serrthr and tyr kinase inhibitors reduces IFNg-induced expression of CIITA mRNA, class II mRNA, and class II protein in the parental cell line 2fTGH similar to what is seen in CRT and CH235-MG cells Ždata not shown.. To determine the effect of the inhibitors on CIITA and class II mRNA levels in cells overexpressing CIITA, RPAs were performed on RNA isolated from 2fTGH.CIITA cells in the presence or absence of the serrthr kinase inhibitor H7 and the tyr kinase inhibitor genistein. As shown in Fig. 7A and B Žlanes 1 and 2., 2fTGH.CIITA cells constitutively express CIITA mRNA and class II MHC mRNA, with IFN-g treatment having no further effect on CIITA or class II MHC mRNA expression. Neither H7 Žlanes 3–5. nor genistein Žlanes 6–8. treatment had any inhibitory effect on CIITA mRNA or class II MHC mRNA expression at any concentration tested, with mRNA levels remaining comparable to control levels as determined by quantitation using the Imagequant program. This suggests that overexpression of CIITA bypasses the requirement of serrthr and tyr kinase activities involved in IFN-g-induced class II MHC gene expression.

4. Discussion In this study, we examined the necessity of serrthr and tyr kinases for IFN-g-induced stimulation of CIITA mRNA and class II MHC mRNA and protein expression in the human astroglioma cell lines CRT and CH235. We have found that the general serrthr kinase inhibitors H7 and H8 decreased surface expression of class II MHC protein by more than 75% in both cell lines. As well, H7 and H8 reduced class II MHC mRNA in IFN-g-treated CRT and CH235 cells by ; 93 and ; 82%, respectively, when compared to cells treated with IFN-g alone. When probed for CIITA mRNA, IFN-g-treated cells in the presence of H7 and H8 showed a comparative decrease in CIITA mRNA. Furthermore, experiments performed in the presence of the tyr kinase inhibitors genistein, herbimycin A, and tyrphostin B42 reduced class II MHC protein, class II MHC mRNA, and CIITA mRNA expression to levels similar to those seen in the presence of the serrthr kinase inhibitors. These data suggest that serrthr and tyr kinase activities are necessary for IFN-g-induced class II MHC expression, and provide evidence for their obligatory role in IFN-g-induced activation of CIITA expression.

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Our present results, which show inhibition of IFN-g-induced CIITA mRNA and class II MHC protein and mRNA in the presence of H7 and H8, indicate that serrthr kinases are involved in the signaling events in our system. We attempted to use more specific inhibitors to determine if PKC was responsible for the serrthr kinase activity observed in the human astroglioma cell lines, as we had previously determined that PKC activity was involved in IFN-g-induced class II MHC expression in primary astrocytes ŽLee et al., 1995.. CRT and CH235 cells were treated with the PKC inhibitor calphostin C, however, calphostin C was highly toxic to both cell lines. Bisindolylmaleimide I, another specific PKC inhibitor, was also tested to determine the involvement of PKC in IFN-g-induced class II MHC expression. No inhibition of class II MHC surface expression was seen at any concentration of bisindolylmaleimide I tested Ždata not shown.. Since bisindolylmaleimide I inhibits most isoforms of PKC at the concentrations used Ž0.01–10 m M., this would suggest that IFNg-induced class II MHC expression in CH235 and CRT cells may not involve PKC, but may act through another serrthr kinase. Possible candidates include MAPK family members. Wen et al. Ž1995. have shown that maximal transcription of IFN-g-activated genes occurs only after phosphorylation of serine-727 of each subunit of the promoter bound STAT-1 a homodimer. They suggest that MAPK may be responsible for this phosphorylation event ŽWen et al., 1995.. In addition, IFN-g has been shown to activate the MAPK pathway in monocytes ŽLiu et al., 1994.. Utilizing MAPK inhibitors, we have demonstrated a dose-dependent inhibition of IFN-g-induced class II MHC protein and mRNA as well as CIITA mRNA in human astroglioma cells. These results suggest that MAPK may be the specific serrthr kinase involved in IFN-g induction of class II MHC gene expression in astroglioma cells. Further studies will be necessary to determine if IFN-g-induced MAPK activation, leading to phosphorylation of serine-727 in the STAT-1 a protein, is involved in this response. Tyrosine phosphorylation is an important event in the intracellular signal transduction pathway from the IFN-g receptor. It has been shown that the tyr kinases Jak1 and Jak2 become phosphorylated upon IFN-g receptorrligand interactions and oligomerization. The kinases are then responsible for STAT-1 a phosphorylation, a requirement for both primary and secondary responses to IFN-g Žfor review see Bach et al., 1997; Darnell, 1997.. Previous results from our laboratory, using the tyr kinase inhibitors genistein, herbimycin A, and tyrphostin B42, support the role of tyr kinases in IFN-g-induced class II MHC expression in primary astrocytes ŽLee et al., 1995.. This prompted us to study the effect of these three tyr kinase inhibitors on IFN-g-induced class II MHC protein and mRNA expression and CIITA mRNA expression in the two human astroglioma cell lines CRT and CH235. Consistent with previous results, all three inhibitors reduced IFN-g-induced

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class II MHC mRNA and protein expression. As well, each inhibited IFN-g-induced CIITA mRNA levels. These data suggest the necessity of tyr kinase activity in this pathway. As mentioned for the serrthr kinase studies, inhibition of IFN-g-induced class II MHC mRNA and protein expression likely results from inhibition of CIITA expression by these tyr kinase inhibitors. To further understand the IFN-g-induced class II MHC signaling pathway, we have shown that constitutive expression of CIITA protein abrogates the requirement for IFN-g stimulation for class II MHC expression in 2fTGH.CIITA cells ŽLee et al., 1997.. In this study, we observe that 2fTGH.CIITA cells are refractory to the inhibitory effects of H7 and genistein on CIITA and class II MHC mRNA expression. The lack of inhibition using both a serrthr and tyr kinase inhibitor is suggestive that kinase activity regulates the expression of CIITA, and that once CIITA is expressed, no further kinase event is required for expression of class II MHC mRNA. This information also offers clues as to how IFN-g-induced class II MHC expression may be modulated by cytokines. Previously, we have shown that TGF-b suppresses IFN-g-induced class II MHC gene expression by blocking CIITA mRNA transcription in astroglioma and fibrosarcoma cells ŽLee et al., 1997.. The suppressive effect of TGF-b on IFN-g-induced class II MHC gene expression could be completely abrogated in cells constitutively expressing CIITA. We have previously shown that TGF-b does not affect IFN-g-induced tyr phosphorylation of Jak1, Jak2, or STAT-1 a ŽPanek et al., 1995.. Therefore, we speculate that TGF-b may function by decreasing serrthr kinase activity Žpossibly MAPK., or the activity of a yet unidentified IFN-gactivated tyr kinase ŽNandan and Reiner, 1997., thereby modulating CIITA transcription. Alternatively, it may function by suppressing factors activated by serrthr andror tyr phosphorylation. IFN-b has also been shown to downregulate IFN-g-induced class II MHC gene expression ŽJoseph et al., 1988; Ransohoff et al., 1991.. However, constitutive CIITA protein expression does not appear to abrogate the suppressive effect of IFN-b on IFN-g-induced class II MHC gene expression, suggesting that the CIITA protein is not fully functional in IFN-b-treated cells, or that other proteins involved in the activation of the class II MHC promoter are affected by IFN-b ŽLu et al., 1995.. These results would suggest that IFN-b does not inhibit class II MHC expression by modulation of kinase activity, which is distinct from what we have observed in this study. Although advances have been made in understanding the integral function of CIITA in constitutive and inducible expression of class II MHC, little is known about the regulation of CIITA gene expression. Here we offer insight into the kinase activity preceding and activating CIITA mRNA expression by showing the involvement of serrthr kinases, possibly MAPK, as well as tyr kinases. At this time, we do not know which molecules of the IFN-g

signaling cascade bind and activate transcription of CIITA. However, we speculate that as with other IFN-g inducible genes, tyr phosphorylation and homodimerization of STAT-1 a is required for binding to putative GAS elementŽs. in the CIITA promoter. With the recent identification of the CIITA promoter, and the presence of a GAS element in the CIITA type IV ŽIFN-g responsive. promoter, this is a likely scenario ŽMuhlethaler-Mottet et al., 1997.. Once bound, STAT-1 a may be further phosphorylated on ser residues by MAPK, thereby maximally activating transcription. This would account for both of the kinase activities we observe in IFN-g-induced class II MHC expression in astrocytes. As well, the transcription factor IRF-1 has been shown to be involved in IFN-g-induced CIITA expression ŽHobart et al., 1997., and may also be a target for kinase activity. Future studies will focus on the identification of the specific kinasesrsubstrates involved in the regulation of CIITA expression. Acknowledgements We thank Sue B. Wade for excellent secretarial assistance, and Dr. Richard Ransohoff ŽCleveland Clinic Foundation. for the 2fTGH.CIITA cell line. This work was supported by National Multiple Sclerosis Society Grant RG-2205-B-9 Žto E.N.B.., and National Institutes of Health Grants MH-55795 and NS-36765 Žto E.N.B... N.J.V. and G.M.O. are supported by a National Institutes of Health Predoctoral Fellowship Ž5-T32 GM08111.. We acknowledge the support of the University of Alabama at Birmingham Flow Cytometry Core Facility ŽGrant AM-20614.. References Bach, E.A., Aguet, M., Schreiber, R.D., 1997. The IFNg receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15, 563–591. Benacerraf, B., 1981. Role of MHC gene products in immune regulation. Science 212, 1229–1238. Benveniste, E.N., Vidovic, M., Panek, R.B., Norris, J.G., Reddy, A.T., Benos, D.J., 1991. Interferon-g-induced astrocyte class II major histocompatiblity complex gene expression is associated with both protein kinase C activation and Naq entry. J. Biol. Chem. 266, 18119–18126. Bethea, J.R., Chung, I.Y., Sparacio, S.M., Gillespie, G.Y., Benveniste, E.N., 1992. Interleukin-1 b induction of tumor necrosis factor-alpha gene expression in human astroglioma cells. J. Neuroimmunol. 36, 179–191. Chang, C.-H., Fontes, J.D., Peterlin, M., Flavell, R.A., 1994. Class II transactivator ŽCIITA. is sufficient for the inducible expression of major histocompatibility complex class II genes. J. Exp. Med. 180, 1367–1374. Chang, C.-H., Hong, S.-C., Hughes, C.C.W., Janeway, C.A. Jr., Flavell, R.A., 1995. CIITA activates the expression of MHC class II genes in mouse T cells. Int. Immunol. 7, 1515–1518. Darnell, J.E. Jr., 1997. STATs and gene regulation. Science 277, 1630– 1635. Fan, X.-D., Goldberg, M., Bloom, B.R., 1988. Interferon-g-induced transcriptional activation is mediated by protein kinase C. Proc. Natl. Acad. Sci. USA 85, 5122–5125.

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