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Role of mitogen-activated protein kinases in inducible nitric oxide synthase and TNFα expression in human fetal astrocytes
Journal of Neuroimmunology 126 (2002) 180 – 189 www.elsevier.com/locate/jneuroim
Role of mitogen-activated protein kinases in inducible nitric oxide synthase and TNFa expression in human fetal astrocytes Liwei L. Hua, Meng-Liang Zhao, Melissa Cosenza, Mee-Ohk Kim, Huan Huang, Herbert B. Tanowitz, Celia F. Brosnan, Sunhee C. Lee * Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA Received 28 August 2001; received in revised form 5 February 2002; accepted 22 February 2002
Abstract Astrocytes are important sources of proinflammatory mediators such as iNOS and TNFa in the diseased central nervous system. In previous studies, we showed that the cytokine IL-1 plays a critical role in the activation of human astrocytes to express TNFa and the inducible form of nitric oxide synthase (iNOS). In the present study, we have addressed the role of the MAP-kinase pathway in the signaling events leading to the induction of these genes. Treatment with SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), potently inhibited IL-1-mediated induction of iNOS and TNFa in cultures of human fetal astrocytes. In contrast, PD98059, an upstream inhibitor of the extracellular regulated kinase (ERK)1/2 pathway, had little or no effect. Interestingly, SB203580 reduced the mRNA expression for iNOS, TNFa, and IL-6, indicating inhibition prior to translation. Transfection of astrocytes with a dominant-negative JunNH2-terminal kinase (JNK) construct also reduced iNOS expression. Western blot analysis showed phosphorylated p38 and JNK in IL-1activated astrocytes, and phosphorylated ERK in both resting and activated cells. Electrophoretic mobility shift assay (EMSA) showed that IL-1 induced NF-nB and AP-1 DNA complex formation in astrocytes, and that SB203580 inhibited AP-1 complex formation. Taken together, these results demonstrate the differential roles played by the three MAP kinases in human astrocyte inflammatory gene activation and point to a crucial function of p38 and JNK MAP kinases in IL-1-mediated astrocyte activation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Astrocytes; Kinase; Nitric oxide synthase
1. Introduction Astrocytes play an important role in maintaining normal homeostasis as well as regulation of inflammatory responses in the CNS (Mucke and Eddleston, 1993; Lee and Brosnan, 1997; Benveniste, 1998). Astrocytes express key proinflammatory mediators such as TNFa and iNOS in response to bacterial/viral products or cytokines produced by inflammatory cells and activated microglial cells. Among these inflammatory mediators, IL-1 has potent astrocyte activating capacity. IL-1 induces functional and phenotypic changes in astrocytes, including cytoskeletal remodeling and production of cytokines, chemokines, and nitric oxide (Liu et al., 1994; Lee et al., 1995b; Liu et al., 1996; Hua and Lee, 2000). Recently, IL-1 was also found to affect astrocyte
*
Corresponding author. Tel.: +1-718-430-2666; fax: +1-718-430-8867. E-mail address: [email protected] (S.C. Lee).
communication pathways by gap junctions and purinergic receptors (John et al., 1999; Liu et al., 2000). Mitogen-activated protein kinases (MAPK) have been revealed to be an important group of regulators of a broad range of genes involved in cellular responses to inflammatory and stress signals (Cobb and Goldsmith, 1995; Han et al., 1994; Lee et al., 1994). Three mammalian MAPK pathways have been identified: the extracellular regulated kinase (ERK) pathway, the Jun-NH2-terminal kinase (JNK) pathway, and the p38 MAPK pathway. The activation of MAPK is effected by dual threonine and tyrosine phosphorylation that is catalyzed by specific upstream MAPK kinases and MAPK kinases upstream of those. The ERK pathway is primarily regulated by growth factors and tumor promoters, and the JNK and p38 pathways are activated by stress and inflammatory agents including IL-1 (Cobb and Goldsmith, 1995; Davis, 1994). Specific inhibitors of the p38 MAPK and ERK pathways are available: PD98059 inhibits MEK1, the upstream kinase of ERK1/2, and SB203580 inhibits the p38 pathway by competitive binding
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 ( 0 2 ) 0 0 0 5 5 - 3
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to the ATP pocket of p38 MAPK (Davies et al., 2000). Commercial inhibitors of the JNK pathway are not available, but JNK dominant-negative constructs can be used to study its role in gene expression. A number of factors, including proinflammatory cytokines such as IL-1, induce the activation of both NF-nB and MAP-kinase pathways. This overlapping pattern of activation led us to hypothesize that IL-1-induced expression of iNOS and TNFa in human fetal astrocytes may be mediated by MAP kinases, as well as NF-nB. In this study, we report that SB203580 markedly inhibited the expression of iNOS and TNFa, while PD98059 had little or no effect. In addition, a dominant negative mutant for JNK also inhibited iNOS expression. These results indicate a relative importance of p38 and JNK MAP-kinase pathways over the ERK pathway in IL-1-mediated inflammatory gene activation in primary human astrocytes.
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described (Lee et al., 1993a). OD was measured at 540 nm in a plate reader. 2.4. TNFa ELISA Supernatants from astrocyte cell cultures were measured in triplicate for TNFa using TNFa ELISA kits (Immunotech), according to the manufacturer’s instructions. 2.5. Ribonuclease protection assay Total RNA from astrocytes was harvested using Trizol (Molecular Research Center, Cincinnati, OH) at 16 h, and mRNA was examined using a custom-made RPA template from B.D. Pharmingen (San Diego, CA), as described (Hua and Lee, 2000). Blots were exposed on autoradiographic film at multiple time points, and densitometry was performed using Ambis Imagequant software. Values were normalized to the GAPDH housekeeping gene.
2. Materials and methods 2.6. Transfection of astrocytes 2.1. Cell culture Human fetal astrocytes were isolated and cultured as previously described (Lee et al., 1992). Briefly, cerebral tissue from second trimester human fetal abortuses was dissociated by trituration and enzymatic digestion, and plated as mixed cultures consisting of astrocytes, neurons, and microglia. After 2 weeks, microglia were shaken off, and the cells adhering to the plate were trypsinized and replated. This process was repeated every 2 weeks, eventually resulting in highly enriched cultures of astrocytes ( > 99% GFAP+). Astrocytes were seeded in 96-well plates at 4104 cells per well then further grown until confluent monolayers are obtained, then stimulated with cytokines for nitrite measurement and ELISA. Cells were plated in 100-mm dishes for RNA and nuclear extraction. Culture medium was Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Bethesda, MD), supplemented with 5% fetal calf serum (FCS) (Gemini, Calabasas, CA) and penicillin (100 U/ml)/ streptomycin (100 Ag/ml)/fungizone (0.25 Ag/ml).
Purified plasmids containing JNK (RK) dominant-negative constructs (pJNK) (Karin, 1995; Chu et al., 1999) were mixed with PLUS reagent (Lipofectamine: Life Technology) in serum- and antibiotic-free DMEM for 15 min at RT. Lipofectamine was diluted with serum- and antibiotic-free DMEM and then combined with the DNA mixture for 15 min at RT. Astrocytes were seeded in 96-well plates at 2104 cells per well (half of the normal density) and used 1 day after plating to obtain subconfluent culture. Astrocytes in 50 Al of serum- and antibiotics-free medium were then incubated with the transfection mixture at 0.1 Ag of DNA per well for 3 h at 37 jC. Control astrocytes were transfected with empty vector (pa3). At the end of the incubation, 30 Al of 15% FCS-containing DMEM was added to the well then further incubated for 24 h. Culture medium was changed to 5% FCS- and antibiotic-containing DMEM, and astrocytes were then treated with IL-1/IFNg for 48 h for nitrite and 24 h for TNFa. Transfection efficiency of human fetal astrocytes indicated by a vector (CMV-driven GFP expression) was approximately 30% (Liu et al., 2000).
2.2. Cytokines and other reagents 2.7. Western blot analysis and antibodies Cultures of astrocytes were stimulated with IL-1h (National Cancer Institute, Frederick, MD), IFNg (Peprotech, Rocky Hill, NJ), or TNFa (Peprotech), all at 10 ng/ml. Cells were pretreated for an hour with MAPK inhibitors SB203580 (Calbiochem) and/or PD98059 (New England Biolabs) at the indicated concentrations. 2.3. Measurement of nitrite Nitrite production was measured in triplicate, using the Griess reaction by mixing 95 Al of astrocyte culture supernatant (harvested at 72 h) with 95 Al of Griess reagent, as
Confluent astrocyte cultures in 100-mm dishes were scraped and pelleted in PBS, and resuspended in 300 Al of 8 M urea. Either 10 Ag (for ERK or JNK) or 100 Ag (for p38) of total protein was separated in 12.5% SDSPAGE, then transferred to PVDF membrane. The blots were blocked in TBS containing 5% nonfat milk and 0.1% Tween-20, and then incubated with antibodies to p38, JNK, or ERK1/2 (total or phospho-specific, all from New England Biolabs) at 1:1000 dilution in 5% BSA/ TTBS. Following overnight incubation at 4 jC, the blots were washed in TTBS and then further incubated with
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concentration using the Bradford assay, aliquotted, and stored at 80 jC until use. 2.9. Electrophoretic mobility shift assay NF-nB binding consensus oligonucleotides (5V AGT TGA GGG GAC TTT CCC AGG C 3V) or AP-1 binding oligonucleotides (5V CCA GCT TGA GTC ACA CTC 3V) from the human iNOS promoter (Marks-Konczalik et al., 1998) were labeled with g-32P ATP (Amersham) according to Promega Gel Shift Assay Core System kit instructions (Promega: Madison, WI). The 3 Ag of protein was incubated with binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris – HCl, 50 Ag/ml poly(dI –dC)], and probe for 20 min at RT. Specific and nonspecific competitor oligonucleotides at 2.5 pmol were used to assure the specificity of binding. These were incubated with the sample for 15 min at RT, prior to the addition of probe. Supershifting antibodies (p50 and p65 for NF-nB gel shifts and ATF-2, c-jun, and c-fos for AP-1 gel shifts, all from Santa Cruz) were preincubated with the sample for 40 min at RT before the addition of probe. Samples were then loaded and run on 5.5% polyacrylamide gels containing 5% glycerol and 1 TGE at 150 V for 2.5 h. Gels were then dried and exposed to film at 80 jC. Fig. 1. (A) MAP kinase involvement in astrocyte nitrite production. (B) Dose – response of p38 inhibitor. Nitrite levels were measured in human fetal astrocyte cultures treated with either SB203580 or PD98059 or both at 10 AM (or as indicated in B), 1 h prior to stimulation with cytokines. Cytokines were added simultaneously at 10 ng/ml. Data from cultures 72-h poststimulation are shown. MeanFS.D. from triplicate wells. Results are representative of six independent experiments.
goat anti-rabbit IgG at 1:2000 in 5% nonfat milk/TTBS for 1 h at RT. The reaction was developed using ECL (Pierce).
3. Results 3.1. Effects of SB203580 and PD98059 on astrocyte nitrite production Initial screening was performed with 10 AM of drugs (Fig. 1A). As our previous data showed that IL-1 is essential for human astrocyte iNOS expression and that either IFNg
2.8. Preparation of astrocyte nuclear extracts Astrocytes were serum starved for 48 h prior to pretreatment with MAPK inhibitors for 1 h and stimulation with IL-1 or IL-1/IFNg for 1 – 6 h. Nuclear extracts were then harvested. Briefly, astrocytes were scraped off dishes in PBS/1 mM PMSF. Cells were centrifuged and pellets resuspended in low salt buffer (10 mM Hepes, pH 7.9/1.5 mM MgCl2/10 mM KCl, supplemented with 1 mM PMSF, 1 mM DTT, and a cocktail of protease inhibitors). Samples were allowed to sit on ice for 10 min before the addition of 10% Nonidet P-40, vortexed, and centrifuged. Nuclear pellets were resuspended in high salt buffer [20 mM Hepes, pH 7.9/25% glycerol/420 mM NaCl/1.5 mM MgCl2, supplemented with 1 mM PMSF, 1 mM DTT, and a cocktail of protease inhibitors (Boerhinger-Mannheim)] and rocked gently at 4 jC for 30 min. Samples were then centrifuged for 15 min. The supernatant was quantitated for protein
Fig. 2. Effect of MAP kinase inhibitor on astrocyte TNFa production. Astrocytes were treated with SB203580, PD98059, or both at 10 AM, then stimulated with IL-1 or IL-1/IFNg for 24 h. TNFa protein levels were measured by ELISA. MeanFS.D. from triplicate wells. Similar results were obtained in four independent experiments.
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inhibitor were different depending on the stimulating conditions. In both IL-1- or IL-1/TNFa-stimulated cultures, SB203580 (5 –50 AM) completely inhibited nitrite production, whereas a dose-dependent inhibition up to 70% was achieved with the same dose range in IL-1/ IFNg-stimulated cultures (Fig. 1B). Subsequent experiments showed that SB203580 at 1 AM also completely inhibited IL-1-induced nitrite (not shown), consistent with the reported IC50 (f0.5 AM) of this inhibitor for p38 kinase (Davies et al., 2000). These results indicate that p38 MAP kinase plays a specific and potent stimulatory role in IL-1-induced iNOS expression in human astrocytes. They
Fig. 3. Effect of MAP kinase inhibitor on astrocyte inflammatory gene expression. RPA was performed using a custom-ordered multiprimer probe set (Pharmingen). Total RNA from approximately 106 astrocytes stimulated with cytokines and drugs for 16 h were analyzed as described in Materials and methods and also in Fig. 1 legend. Data represent one of four independent experiments with identical results. SB203580 but not PD98059 inhibited mRNA expression for TNFa, iNOS, and IL-6. ICAM and IFNgR mRNA were not changed.
or TNFa could serve as a priming agent (Lee et al., 1993a; Liu et al., 1996), cells were stimulated in three different settings (IL-1 alone, IL-1/IFNg, and IL-1/TNFa). The results showed that SB203580 and PD98059 had quite different effects on astrocyte iNOS. Whereas the p38 inhibitor potently inhibited iNOS, the MEK inhibitor had little or no effect (Fig. 1A). A combination of the two inhibitors (SB203580+PD98059) had the same effect as SB203580 alone. Although the p38 inhibitor dramatically reduced nitrite, the potency and the maximal inhibition achieved with this
Table 1 Effect of JNK inhibition on astrocyte nitrite production
Case Case Case Case Case
1 2 3 4 5
Lipofectamine (AM)
pa3 (AM)*
pJNK (AM)**
11.0F0.7 10.5F0.4 14.7F0.4 8.6F0.3 3.6F0.2
2.6F0.7 5.4F0.6 9.7F0.6 5.0F0.6 1.8F0.4
1.2F0.3 1.9F0.2 5.8F0.2 2.2F0.3 1.2F0.8
Subconfluent astrocyte cultures from five different cases were treated with lipofectamine alone, a dominant negative JNK construct (pJNK) or an empty vector (pa3), as described in Materials and methods. Astrocytes were stimulated with IL-1/IFNg, then 48-h culture supernatants were examined for nitrite concentration. MeanFS.D. (AM) from triplicate wells. * p<0.05 vs. lipofectamine. ** p<0.05 vs. pa3 by one-way ANOVA followed by Student – Newman – Keuls Test.
Fig. 4. Effect of JNK inhibition: (A – C) astrocytes were transfected with 10 Ag of dominant negative JNK (pJNK) or with control vector (pa3), as described in Materials and methods. Cultures were then stimulated with cytokines for 48 h, then immunocytochemistry was performed for iNOS, as described elsewhere (Liu et al., 1996; Hua et al., 1998). Results show comparable culture viability in pa3- and pJNK-transfected cultures (also confirmed by trypan blue exclusion), but fewer iNOS-positive cells in pJNK-transfected wells. Results are representative of five independent experiments.
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Table 2 Effect of JNK inhibition on astrocyte TNFa production
Case Case Case Case Case
1 2 2 3 3
(IL-1) (IL-1) (IL-1/IFNg) (IL-1) (IL-1/IFNg)
3.3. SB203580 inhibits iNOS, TNFa, and IL-6 mRNA expression
Lipofectamine (pg/ml)
pa3 (pg/ml)
pJNK (pg/ml)
144F6 264F48 290F62 67F3 589F2
52F8 89F22 192F21 49F8 252F24
78F6 157F59 188F72 72F13 286F43
Subconfluent astrocyte cultures from three different cases were treated with lipofectamine alone, a dominant negative JNK construct (pJNK) or an empty vector (pa3), as described in Materials and methods. Astrocytes were stimulated with IL-1 or IL-1/IFNg as indicated, then 24-h culture supernatants were examined for TNFa levels by ELISA. MeanFS.D. (pg/ ml) from triplicate wells. No significant differences were observed ( p>0.05) between any pairs (lipofectamine vs. pa3 vs. pJNK) by oneway ANOVA.
also indicate that TNFa and IL-1 activated similar MAP kinase signaling pathways to induce iNOS in human astrocytes unlike rat cardiac muscle cells in which TNFa induces iNOS through ERK {1330}. In IL-1/IFNg-stimulated astrocytes, SB203580 was less potent (IC50f20 AM: n=3), suggesting that p38 plays differential roles in the two (IL-1 vs. IL-1/IFNg) stimulating conditions (see Discussion). 3.2. Effect of MAPK inhibitors on astrocyte TNFa production SB203580’s effect on p38 MAP kinase was first discovered by virtue of its suppressive effects on cytokine biosynthesis (Lee et al., 1994). Furthermore, in primary human astrocytes, iNOS and TNFa expression are regulated similarly in many respects, including the requirement for IL-1 and the priming effect of IFNg (Lee et al., 1993a,b; Downen et al., 1999). Thus, we asked whether MAP kinase inhibitors had similar effects on astrocyte TNFa production. Results illustrated in Fig. 2 show that SB203580 potently inhibited both IL-1 and IL-1/IFNginduced TNFa, while PD98059 had a minor effect on IL-1/ IFNg-induced TNFa production. These results show both the similarity and the difference in the regulation of astrocyte iNOS and TNFa.
For cytokine biosynthesis, both transcriptional and posttranscriptional mechanisms have been demonstrated for MAP kinase involvement. To determine where in the iNOS/TNFa induction pathway the MAP kinases were exerting their effects, we performed ribonuclease protection assay using a commercially available cytokine RPA template. RNA harvested from IL-1 or IL-1/IFNg-stimulated astrocytes was analyzed and showed similar results at 6 h (n=2, not shown) or 16-h poststimulation (n=2; Fig. 3). SB203580 potently inhibited both iNOS and TNFa mRNA, whereas PD98059 had little or no effect. Little change was noted in the expression of ICAM-1 and IFNg receptor h chain mRNA, demonstrating the specificity of the p38 inhibitor effect. IL-6 mRNA, which is induced by IL-1 or IL-1/IFNg in similar quantities, was potently inhibited by SB203580 and not by PD98059. In sum, these results show a high degree of co-regulation in the induction of TNFa, IL6, and iNOS in astrocytes, and that the p38 MAP kinase plays an important role in the mRNA induction of these genes. 3.4. Effect of transfection with dominant-negative JNK plasmid (pJNK) Because specific pharmacological inhibitors of JNK MAP-kinase pathway are not available, we employed an alternative approach to examine its role in iNOS expression. We transfected primary astrocytes with plasmids containing an inactivating (dominant negative) JNK sequence (pJNK) or an empty vector (pa3), both originated from Dr. M. Karin’s laboratory. Astrocyte nitrite production was measured after stimulation with IL-1/IFNg. The results are summarized in Table 1. Data from five different cases show that significantly less nitrite was produced following transfection (pa3 or pJNK vs. Lipofectamine), and also following transfection with pJNK compared with control vector (pJNK vs. pa3). Immunostaining for iNOS showed that whereas iNOS-positive astrocytes were frequent in pa3-transfected cultures, they were fewer in pJNK-transfected cultures (Fig. 4). Although
Fig. 5. Western blot analysis of MAP kinase phosphorylation: astrocyte protein was extracted at indicated time points following stimulation with IL-1h and analyzed using antibodies specific to phosphorylated or total MAP kinases. Approximately 100, 10, and 10 Ag of total protein were loaded in each lane for p38, ERK, and JNK immunoblots, respectively. The two bands in ERK immunoblot represent the 44 kDa (ERK1) and 42 kDa (ERK2) proteins.
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some degree of cell degeneration was observed following transfection, there was no difference in cell death between pa3 and pJNK-transfected cultures (trypan blue exclusion; not shown). These results collectively support that inhibition of JNK specifically suppressed iNOS expression in astrocytes. TNFa production was examined in three different cases with both IL-1 and IL-1/IFNg stimulation in two of them. The results are summarized in Table 2. They show that the levels of TNFa between control (pa3) and pJNK-transfected cultures were not different ( p>0.05 by one-way ANOVA followed by Student – Newman – Keuls Test). These results do not support a role for JNK MAP kinase in TNFa production in human astrocytes. 3.5. MAP kinase phosphorylation in astrocytes Because our data suggest that MAP kinases are active in IL-1-stimulated astrocytes, we directly examined this by Western blot analysis. The amounts of unphosphorylated MAP kinases are known to be constant, while activating signals (phosphorylation of specific upstream MAPK kinases) induce dual phosphorylation of specific MAP kinases. Results illustrated in Fig. 5 show that p38 and JNK kinases were phosphorylated by IL-1 with different kinetics. By contrast, ERK1/2 were phosphorylated in resting cells, even in serum-starved conditions, and the phosphorylation remained unchanged following cytokine stimulation (Fig.
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5). These results demonstrate rather dramatic differences in the way MAP kinases are activated in human fetal astrocytes, and may offer a partial explanation for the differential effects of p38 and ERK inhibitors on astrocyte activation (Figs. 1 – 4). 3.6. Role of MAPK in astrocyte NF-nB and AP-1 activation Cytokines such as IL-1 induce gene expression via activation of several well-known transcription factors. Among these are the NF-nB and AP-1 family of transcription factors, which are also known to be important in the induction of iNOS and TNFa genes (Marks-Konczalik et al., 1998; Means et al., 2000). To test whether MAPK are involved in the cytokine-mediated activation of these transcription factors in human fetal astrocytes, electrophoretic mobility shift assays (EMSAs) were performed. Using NFnB consensus sequence oligonucleotides, we found that IL1 or IL-1/IFNg induced specific shifted complexes composed of p65/p50 (Liu et al., 2000) (Fig. 6). Neither SB203580 nor PD98059 reduced the amounts of NF-nB complex formed at 1-, 3-, 4-, and 6-h poststimulation (Fig. 6 and data not shown). EMSA using a human iNOS-specific AP-1 sequence showed a single AP-1 complex in astrocytes stimulated with IL-1 or IL-1/IFNg (Fig. 7A and B). The induction was minimal at 1 h, but clearly visible at 4 h. The AP-1 complex was completely displaced by anti-c-jun antibody, but minimally by anti-ATF-2 antibody. Pretreatment
Fig. 6. Electrophoretic mobility shift assay (EMSA) for NF-nB. Astrocytes were pretreated with SB203580 or PD98059 at 20 AM for 1 h, then stimulated with 1L-1 for 1 h. Nuclear extracts were incubated with 32P-labeled NF-nB consensus sequence probe (Promega) in the presence of 100-fold unlabeled specific (NF) or non-specific (AP) competitor probes. Supershift experiments were performed by preincubating the nuclear extracts with 10 Ag of p50 or p65 (Rel A) antibodies for 40 min prior to incubation with probes. Results are representative of three experiments with similar results.
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Fig. 7. AP-1 EMSA in cytokine-treated astrocytes (A and B). Astrocytes prepared as in Fig. 6 (except nuclear protein harvested both at 1 and 4 h) were examined with 32P-labeled AP-1 consensus nucleotides. Specificity of the reaction was determined by competing with unlabeled, excess AP-1 (+AP-1) or mutant oligonucleotide (+mt). Supershifting experiments were performed by preincubating nuclear extracts with an antibody to ATF-2, c-jun, or control IgG (A and B). The AP-1 complex was induced at 4 h, and SB203580 reduced AP-1 complex formation.
with SB203580 reduced the amount of AP-1 complex induced by IL-1/IFNg, whereas no such effect was observed with PD98059. Thus, these experiments demonstrated that
both NF-nB and AP-1 transcription factors are activated in cytokine-treated astrocytes but p38 MAP kinase is involved in AP-1 activation only.
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4. Discussion In this report, we investigated the role of MAP kinases in the cytokine-induced expression of iNOS and TNFa in human fetal astrocytes. TNFa can be expressed by both microglia and astrocytes, depending on the stimuli, but the iNOS expression has been shown to be highly species dependent. Whereas macrophages and microglia are a ready source of nitric oxide in rodents, in human macrophages and microglia, iNOS is difficult to induce (Denis, 1994; Nathan and Xie, 1994; Lee et al., 1995a). iNOS in human cells is induced in non-macrophage cells such as astrocytes, hepatocytes, and chondrocytes, and in all of these cell types, the cytokine IL-1 plays a crucial role in the transcriptional induction of this gene (Liu et al., 1996; Geller et al., 1995; Chartrain et al., 1994). The crucial role of astrocytes as the expressor of iNOS in the human CNS is demonstrated by our recent studies of multiple sclerosis and HIV-1 encephalitis (Liu et al., 2001; Zhao et al., 2001). Our results demonstrate an important role for p38 JNK and a relatively minor role for ERK in this induction. This may be, in part, related to their activation profile following IL-1 stimulation, since p38 and JNK kinases were phosphorylated by IL-1 in astrocytes, ERK1/2 were phosphorylated in resting astrocytes, even after serum deprivation. This cytokine-mediated activation of MAP kinases is consistent with the general observation that p38 and JNK MAP kinases are stress and inflammatory signal activated, whereas ERK is growth-factor activated (Cobb and Goldsmith, 1995; Davis, 1994; Herlaar and Brown, 1999). SB203580 is a member of a novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs) whose activity was originally described in the inhibition of cytokine (TNFa) synthesis in LPS-stimulated human monocytederived macrophages (Lee et al., 1994). In this and a subsequent study of rat glial cells (Bhat et al., 1998), SB203580 was shown to inhibit translation rather than the transcription of TNFa gene. Interestingly, we find that in human fetal astrocytes, p38 was involved in the mRNA expression of TNFa, as well as that of IL-6 and iNOS, suggesting that the mechanism of p38 action differs depending on the cell type. MAP kinases have been shown to be important in iNOS induction in other cells. In bovine chondrocytes (Badger et al., 1998) and in murine astrocytes (Da Silva et al., 1997), IL-1-induced iNOS mRNA production and nitrite production were inhibited by SB203580, but not by PD98059. Moreover, in rat mesangial cells, both p38 and JNK and their upstream kinases (MKK3, 4, and 6) are involved in iNOS induction (Guan et al., 1999). Our study confirms and extends these observations supporting the importance of IL1-activated p38 and JNK pathways in the induction of iNOS. Our data do not support, however, the reported prominent role of ERK kinase in iNOS expression in heart muscle cells (Kan et al., 1999) and in rat glial cells activated by cytokines or LPS (Bhat et al., 1998). LPS plays little or
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no role in the induction of iNOS in human cells including astrocytes and hepatocytes (Liu et al., 1996; Geller et al., 1995), therefore, we cannot address the role of LPS-induced ERK in iNOS expression. Interestingly, ERK inhibitor had no effect on IL-1-activated iNOS, but did show a minor effect when astrocytes were co-stimulated with IFNg. These data may indicate that ERK was involved in IFNg-mediated cell activation (see below), which played a minor or insignificant role in the case of human iNOS induction. Our study showed that p38 inhibitor SB203580 had a different potency in IL-1- and IL-1/IFNg-stimulated iNOS activation. Whereas SB203580 at 1 AM completely inhibited IL-1-mediated nitrite production, higher concentrations (IC50f20 AM) were required to block IL-1/IFNgmediated nitrite production. The significance of these observations is not clear, but the data collectively suggest a link between ERK/p38 and IFNg-activated cell signaling. In this regard, both ERK and p38 have been shown to activate Stat1, the transcription factor involved in IFNg signaling (David et al., 1995; Uddin et al., 1999). Although tyrosine phosphorylation is primarily responsible for IFN signaling via the Jak/Stat pathway, recent data show that MAP kinasedependent phosphorylation is important in the maximal transcriptional activation by Stats (Wen et al., 1995; David et al., 1995; Goh et al., 1999). Experiments are ongoing to examine these potential scenarios in astrocytes. Different IC50 may also indicate that IFNg stimulated other pathways in astrocytes and the induction of iNOS became less dependent on p38 in the presence of IFNg. Analysis of the human iNOS promoter reveals distinct features, including the number and location of important cytokine responsive elements: multiple NF-nB and two perfectly matched AP-1 sites are present in the human promoter which are not present in the murine iNOS promoter (Taylor et al., 1998; Chu et al., 1998; Marks-Konczalik et al., 1998). The AP-1 upstream site (at 5301) in the human iNOS promoter has been shown to be essential for transcriptional activation and in lung epithelial cells, Jun D, and Fra-2 were found to be complexed with the AP-1 sequence (Marks-Konczalik et al., 1998). Using the same oligonucleotide sequence as a probe, we found that AP-1 binding was induced in cytokine-activated human astrocytes, and that this induction was greatly diminished by p38 inhibition, identifying IL-1-induced AP-1 activation as the possible molecular target for p38 activity in astrocytes. Although MAP kinase-dependent NF-nB activation has been demonstrated (Schulze-Osthoff et al., 1997), it is unlikely that MAP kinases were involved in NF-nB activation in astrocytes, because we see no evidence for this. Rather, our results are consistent with the view that NF-nB and AP-1 cooperated after binding to the promoter elements of the iNOS gene. We also tested cycloheximide, a protein synthesis inhibitor, on the accumulation of astrocyte iNOS mRNA, and found that iNOS mRNA induction is dependent on new protein synthesis (not shown). This indicates an indirect
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mechanism for mRNA induction. Because AP-1 activation was a delayed response (4 h>1 h) and is known to require new protein synthesis of immediate early genes such as c-jun or cfos, these data additionally support the role of AP-1 in human astrocyte iNOS induction. In addition, posttranslational modification of AP-1 proteins also potentiates transactivation function, such as that shown for NH2-terminal phosphorylation of c-jun by JNK (Karin, 1995). These are all potential mechanisms for iNOS induction in human astrocytes, as we found that JNK inhibition, as well as p38 inhibition (through AP-1 inhibition), affected iNOS expression. The regulation of TNFa gene expression is complex. Several levels of regulation are described, including transcription and mRNA stability, as well as translational and posttranslational control. The promoter of the TNFa gene contains consensus sites for NF-nB and AP-1, but whether these elements play critical roles in transcriptional activation varies depending on the study (Means et al., 2000; Yao et al., 1997; Rhodes et al., 1992; Zheng et al., 1993; Monick et al., 1999). For example, LPS-induced TNFa transcription shows different requirements for AP-1 activation depending on the macrophage populations (Means et al., 2000), suggesting that both MAPK-dependent and -independent regulatory elements are used for transcription. Studies of cytokine-induced astrocyte TNFa production have shown an involvement of p38 as well as ERK (Bhat et al., 1998; Lee et al., 2000). Our study indicates that p38 is the dominant MAP kinase involved in IL-1-mediated TNFa biosynthesis in human astrocytes, but that ERK may also (albeit minor) play a role (see Fig. 2, for instance). In contrast to iNOS expression, which was inhibited by inactivating JNK expression, we see no consistent effect of JNK inhibition on TNFa expression. The interpretation of these data is complex because of the suboptimal transfection efficiency of primary cells using non-viral vectors. While we cannot entirely exclude the possibility of JNK involvement in TNFa induction, our data at least point to the differential roles played by JNK in iNOS and TNFa induction in astrocytes. Our study may have implications for the pathogenesis and therapy for human CNS inflammatory diseases, such as multiple sclerosis and HIV encephalitis, in which proinflammatory cytokines and iNOS are expressed (Selmaj et al., 1991; Tyor et al., 1992; Zhao et al., 2001; Liu et al., 2001). In previous and ongoing studies, we observe that for microglial chemokine gene induction, p38 plays a negative regulatory role, while ERK plays different roles depending on the stimulus involved (Kim, Si, and Song; unpublished). The cell-, stimulus-, and target promoter-dependent involvement of MAP kinases in the biosynthesis inflammatory genes thus demonstrates a possibility that a more specific blockade of glial cell activation can be achieved. Further studies directed at the molecular mechanisms of MAP kinase involvement in human glial activation will enhance the opportunity for MAP kinase-based therapeutic interventions.
Acknowledgements The authors thank Einstein Fetal Tissue Repository for supplying tissue and Wa Shen for preparing excellent tissue culture. We also thank Drs. Moon L. Shin, Gareth John, Judy Liu, and Julie Simpson for helpful discussions, and Dr. Qiusheng Si for assisting with the figure preparations. This study was supported by MH55477, AI44641, and NMMS RG2771 to SCL and NS11920 to CFB. References Badger, A.M., Cook, M.N., Lark, M.W., Newman-Tarr, T.M., Swift, B.A., Nelson, A.H., Barone, F.C., Kumar, S., 1998. SB203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes. J. Immunol. 161, 467 – 473. Benveniste, E.N., 1998. Cytokine actions in the central nervous system. Cytokine Growth Factor Rev. 9, 259 – 275. Bhat, N.R., Zhang, P., Lee, J.C., Hogan, E.L., 1998. Extracellular signalregulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-a gene expression in endotoxin-stimulated primary glial cultures. J. Neurosci. 18, 1633 – 1641. Chartrain, N.A., Geller, D.A., Koty, P.P., Sitrin, N.F., Nussler, A.K., Hoffman, E.P., Billiar, T.R., Hutchinson, N.I., Mudgett, J.S., 1994. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J. Biol. Chem. 269, 6765 – 6772. Chu, S.C., Marks-Konczalik, J., Wu, H.P., Banks, T.C., Moss, J., 1998. Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters. Biochem. Biophys. Res. Commun. 248, 871 – 878. Chu, W.M., Ostertag, D., Li, Z.W., Chang, L., Chen, Y., Hu, Y., Williams, B., Perrault, J., Karin, M., 1999. JNK2 and IKKbeta are required for activating the innate response to viral infection. Immunity 11, 721 – 731. Cobb, M.H., Goldsmith, E.J., 1995. How MAP kinases are regulated. J. Biol. Chem. 270, 14843 – 14846. Da Silva, J., Pierrat, B., Mary, J.-L., Lesslauer, W., 1997. Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J. Biol. Chem. 272, 28373 – 28380. David, M., Petricoin, E., Benjamin, C., Pine, R., Weber, M.J., Larner, A.C., 1995. Requirement for MAP kinase (ERK2) activity in interferon alphaand beta-stimulated gene expression through STAT proteins. Science 269, 1721 – 1723. Davies, S.P., Reddy, H., Caivano, M., Cohen, P., 2000. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95 – 105. Davis, R.J., 1994. MAPKs: new JNK expands the group. Trends Biochem. Sci. 19, 170 – 173. Denis, M., 1994. Human monocytes/macrophages: NO or no NO? J. Leukocyte Biol. 55, 682 – 684. Downen, M., Amaral, T.D., Hua, L.L., Zhao, M.L., Lee, S.C., 1999. Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha. Glia 28, 114 – 127. Geller, D.A., de Vera, M.E., Russell, D.A., Shapiro, R.A., Nussler, A.K., Simmons, R.L., Billiar, T.R., 1995. A central role for IL-1h in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase. J. Immunol. 155, 4890 – 4898. Goh, K.C., Haque, S.J., Williams, B.R.G., 1999. p38 MAP kinase is required for STAT1 serine phosphorylation and transcriptional activation induced by interferons. EMBO J. 18, 5601 – 5608. Guan, Z., Buckman, S.Y., Springer, L.D., Morrison, A.R., 1999. Both p38a
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Role of mitogen-activated protein kinases in inducible nitric oxide synthase and TNFa expression in human fetal astrocytes Liwei L. Hua, Meng-Liang Zhao, Melissa Cosenza, Mee-Ohk Kim, Huan Huang, Herbert B. Tanowitz, Celia F. Brosnan, Sunhee C. Lee * Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA Received 28 August 2001; received in revised form 5 February 2002; accepted 22 February 2002
Abstract Astrocytes are important sources of proinflammatory mediators such as iNOS and TNFa in the diseased central nervous system. In previous studies, we showed that the cytokine IL-1 plays a critical role in the activation of human astrocytes to express TNFa and the inducible form of nitric oxide synthase (iNOS). In the present study, we have addressed the role of the MAP-kinase pathway in the signaling events leading to the induction of these genes. Treatment with SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), potently inhibited IL-1-mediated induction of iNOS and TNFa in cultures of human fetal astrocytes. In contrast, PD98059, an upstream inhibitor of the extracellular regulated kinase (ERK)1/2 pathway, had little or no effect. Interestingly, SB203580 reduced the mRNA expression for iNOS, TNFa, and IL-6, indicating inhibition prior to translation. Transfection of astrocytes with a dominant-negative JunNH2-terminal kinase (JNK) construct also reduced iNOS expression. Western blot analysis showed phosphorylated p38 and JNK in IL-1activated astrocytes, and phosphorylated ERK in both resting and activated cells. Electrophoretic mobility shift assay (EMSA) showed that IL-1 induced NF-nB and AP-1 DNA complex formation in astrocytes, and that SB203580 inhibited AP-1 complex formation. Taken together, these results demonstrate the differential roles played by the three MAP kinases in human astrocyte inflammatory gene activation and point to a crucial function of p38 and JNK MAP kinases in IL-1-mediated astrocyte activation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Astrocytes; Kinase; Nitric oxide synthase
1. Introduction Astrocytes play an important role in maintaining normal homeostasis as well as regulation of inflammatory responses in the CNS (Mucke and Eddleston, 1993; Lee and Brosnan, 1997; Benveniste, 1998). Astrocytes express key proinflammatory mediators such as TNFa and iNOS in response to bacterial/viral products or cytokines produced by inflammatory cells and activated microglial cells. Among these inflammatory mediators, IL-1 has potent astrocyte activating capacity. IL-1 induces functional and phenotypic changes in astrocytes, including cytoskeletal remodeling and production of cytokines, chemokines, and nitric oxide (Liu et al., 1994; Lee et al., 1995b; Liu et al., 1996; Hua and Lee, 2000). Recently, IL-1 was also found to affect astrocyte
*
Corresponding author. Tel.: +1-718-430-2666; fax: +1-718-430-8867. E-mail address: [email protected] (S.C. Lee).
communication pathways by gap junctions and purinergic receptors (John et al., 1999; Liu et al., 2000). Mitogen-activated protein kinases (MAPK) have been revealed to be an important group of regulators of a broad range of genes involved in cellular responses to inflammatory and stress signals (Cobb and Goldsmith, 1995; Han et al., 1994; Lee et al., 1994). Three mammalian MAPK pathways have been identified: the extracellular regulated kinase (ERK) pathway, the Jun-NH2-terminal kinase (JNK) pathway, and the p38 MAPK pathway. The activation of MAPK is effected by dual threonine and tyrosine phosphorylation that is catalyzed by specific upstream MAPK kinases and MAPK kinases upstream of those. The ERK pathway is primarily regulated by growth factors and tumor promoters, and the JNK and p38 pathways are activated by stress and inflammatory agents including IL-1 (Cobb and Goldsmith, 1995; Davis, 1994). Specific inhibitors of the p38 MAPK and ERK pathways are available: PD98059 inhibits MEK1, the upstream kinase of ERK1/2, and SB203580 inhibits the p38 pathway by competitive binding
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 ( 0 2 ) 0 0 0 5 5 - 3
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to the ATP pocket of p38 MAPK (Davies et al., 2000). Commercial inhibitors of the JNK pathway are not available, but JNK dominant-negative constructs can be used to study its role in gene expression. A number of factors, including proinflammatory cytokines such as IL-1, induce the activation of both NF-nB and MAP-kinase pathways. This overlapping pattern of activation led us to hypothesize that IL-1-induced expression of iNOS and TNFa in human fetal astrocytes may be mediated by MAP kinases, as well as NF-nB. In this study, we report that SB203580 markedly inhibited the expression of iNOS and TNFa, while PD98059 had little or no effect. In addition, a dominant negative mutant for JNK also inhibited iNOS expression. These results indicate a relative importance of p38 and JNK MAP-kinase pathways over the ERK pathway in IL-1-mediated inflammatory gene activation in primary human astrocytes.
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described (Lee et al., 1993a). OD was measured at 540 nm in a plate reader. 2.4. TNFa ELISA Supernatants from astrocyte cell cultures were measured in triplicate for TNFa using TNFa ELISA kits (Immunotech), according to the manufacturer’s instructions. 2.5. Ribonuclease protection assay Total RNA from astrocytes was harvested using Trizol (Molecular Research Center, Cincinnati, OH) at 16 h, and mRNA was examined using a custom-made RPA template from B.D. Pharmingen (San Diego, CA), as described (Hua and Lee, 2000). Blots were exposed on autoradiographic film at multiple time points, and densitometry was performed using Ambis Imagequant software. Values were normalized to the GAPDH housekeeping gene.
2. Materials and methods 2.6. Transfection of astrocytes 2.1. Cell culture Human fetal astrocytes were isolated and cultured as previously described (Lee et al., 1992). Briefly, cerebral tissue from second trimester human fetal abortuses was dissociated by trituration and enzymatic digestion, and plated as mixed cultures consisting of astrocytes, neurons, and microglia. After 2 weeks, microglia were shaken off, and the cells adhering to the plate were trypsinized and replated. This process was repeated every 2 weeks, eventually resulting in highly enriched cultures of astrocytes ( > 99% GFAP+). Astrocytes were seeded in 96-well plates at 4104 cells per well then further grown until confluent monolayers are obtained, then stimulated with cytokines for nitrite measurement and ELISA. Cells were plated in 100-mm dishes for RNA and nuclear extraction. Culture medium was Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Bethesda, MD), supplemented with 5% fetal calf serum (FCS) (Gemini, Calabasas, CA) and penicillin (100 U/ml)/ streptomycin (100 Ag/ml)/fungizone (0.25 Ag/ml).
Purified plasmids containing JNK (RK) dominant-negative constructs (pJNK) (Karin, 1995; Chu et al., 1999) were mixed with PLUS reagent (Lipofectamine: Life Technology) in serum- and antibiotic-free DMEM for 15 min at RT. Lipofectamine was diluted with serum- and antibiotic-free DMEM and then combined with the DNA mixture for 15 min at RT. Astrocytes were seeded in 96-well plates at 2104 cells per well (half of the normal density) and used 1 day after plating to obtain subconfluent culture. Astrocytes in 50 Al of serum- and antibiotics-free medium were then incubated with the transfection mixture at 0.1 Ag of DNA per well for 3 h at 37 jC. Control astrocytes were transfected with empty vector (pa3). At the end of the incubation, 30 Al of 15% FCS-containing DMEM was added to the well then further incubated for 24 h. Culture medium was changed to 5% FCS- and antibiotic-containing DMEM, and astrocytes were then treated with IL-1/IFNg for 48 h for nitrite and 24 h for TNFa. Transfection efficiency of human fetal astrocytes indicated by a vector (CMV-driven GFP expression) was approximately 30% (Liu et al., 2000).
2.2. Cytokines and other reagents 2.7. Western blot analysis and antibodies Cultures of astrocytes were stimulated with IL-1h (National Cancer Institute, Frederick, MD), IFNg (Peprotech, Rocky Hill, NJ), or TNFa (Peprotech), all at 10 ng/ml. Cells were pretreated for an hour with MAPK inhibitors SB203580 (Calbiochem) and/or PD98059 (New England Biolabs) at the indicated concentrations. 2.3. Measurement of nitrite Nitrite production was measured in triplicate, using the Griess reaction by mixing 95 Al of astrocyte culture supernatant (harvested at 72 h) with 95 Al of Griess reagent, as
Confluent astrocyte cultures in 100-mm dishes were scraped and pelleted in PBS, and resuspended in 300 Al of 8 M urea. Either 10 Ag (for ERK or JNK) or 100 Ag (for p38) of total protein was separated in 12.5% SDSPAGE, then transferred to PVDF membrane. The blots were blocked in TBS containing 5% nonfat milk and 0.1% Tween-20, and then incubated with antibodies to p38, JNK, or ERK1/2 (total or phospho-specific, all from New England Biolabs) at 1:1000 dilution in 5% BSA/ TTBS. Following overnight incubation at 4 jC, the blots were washed in TTBS and then further incubated with
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concentration using the Bradford assay, aliquotted, and stored at 80 jC until use. 2.9. Electrophoretic mobility shift assay NF-nB binding consensus oligonucleotides (5V AGT TGA GGG GAC TTT CCC AGG C 3V) or AP-1 binding oligonucleotides (5V CCA GCT TGA GTC ACA CTC 3V) from the human iNOS promoter (Marks-Konczalik et al., 1998) were labeled with g-32P ATP (Amersham) according to Promega Gel Shift Assay Core System kit instructions (Promega: Madison, WI). The 3 Ag of protein was incubated with binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris – HCl, 50 Ag/ml poly(dI –dC)], and probe for 20 min at RT. Specific and nonspecific competitor oligonucleotides at 2.5 pmol were used to assure the specificity of binding. These were incubated with the sample for 15 min at RT, prior to the addition of probe. Supershifting antibodies (p50 and p65 for NF-nB gel shifts and ATF-2, c-jun, and c-fos for AP-1 gel shifts, all from Santa Cruz) were preincubated with the sample for 40 min at RT before the addition of probe. Samples were then loaded and run on 5.5% polyacrylamide gels containing 5% glycerol and 1 TGE at 150 V for 2.5 h. Gels were then dried and exposed to film at 80 jC. Fig. 1. (A) MAP kinase involvement in astrocyte nitrite production. (B) Dose – response of p38 inhibitor. Nitrite levels were measured in human fetal astrocyte cultures treated with either SB203580 or PD98059 or both at 10 AM (or as indicated in B), 1 h prior to stimulation with cytokines. Cytokines were added simultaneously at 10 ng/ml. Data from cultures 72-h poststimulation are shown. MeanFS.D. from triplicate wells. Results are representative of six independent experiments.
goat anti-rabbit IgG at 1:2000 in 5% nonfat milk/TTBS for 1 h at RT. The reaction was developed using ECL (Pierce).
3. Results 3.1. Effects of SB203580 and PD98059 on astrocyte nitrite production Initial screening was performed with 10 AM of drugs (Fig. 1A). As our previous data showed that IL-1 is essential for human astrocyte iNOS expression and that either IFNg
2.8. Preparation of astrocyte nuclear extracts Astrocytes were serum starved for 48 h prior to pretreatment with MAPK inhibitors for 1 h and stimulation with IL-1 or IL-1/IFNg for 1 – 6 h. Nuclear extracts were then harvested. Briefly, astrocytes were scraped off dishes in PBS/1 mM PMSF. Cells were centrifuged and pellets resuspended in low salt buffer (10 mM Hepes, pH 7.9/1.5 mM MgCl2/10 mM KCl, supplemented with 1 mM PMSF, 1 mM DTT, and a cocktail of protease inhibitors). Samples were allowed to sit on ice for 10 min before the addition of 10% Nonidet P-40, vortexed, and centrifuged. Nuclear pellets were resuspended in high salt buffer [20 mM Hepes, pH 7.9/25% glycerol/420 mM NaCl/1.5 mM MgCl2, supplemented with 1 mM PMSF, 1 mM DTT, and a cocktail of protease inhibitors (Boerhinger-Mannheim)] and rocked gently at 4 jC for 30 min. Samples were then centrifuged for 15 min. The supernatant was quantitated for protein
Fig. 2. Effect of MAP kinase inhibitor on astrocyte TNFa production. Astrocytes were treated with SB203580, PD98059, or both at 10 AM, then stimulated with IL-1 or IL-1/IFNg for 24 h. TNFa protein levels were measured by ELISA. MeanFS.D. from triplicate wells. Similar results were obtained in four independent experiments.
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inhibitor were different depending on the stimulating conditions. In both IL-1- or IL-1/TNFa-stimulated cultures, SB203580 (5 –50 AM) completely inhibited nitrite production, whereas a dose-dependent inhibition up to 70% was achieved with the same dose range in IL-1/ IFNg-stimulated cultures (Fig. 1B). Subsequent experiments showed that SB203580 at 1 AM also completely inhibited IL-1-induced nitrite (not shown), consistent with the reported IC50 (f0.5 AM) of this inhibitor for p38 kinase (Davies et al., 2000). These results indicate that p38 MAP kinase plays a specific and potent stimulatory role in IL-1-induced iNOS expression in human astrocytes. They
Fig. 3. Effect of MAP kinase inhibitor on astrocyte inflammatory gene expression. RPA was performed using a custom-ordered multiprimer probe set (Pharmingen). Total RNA from approximately 106 astrocytes stimulated with cytokines and drugs for 16 h were analyzed as described in Materials and methods and also in Fig. 1 legend. Data represent one of four independent experiments with identical results. SB203580 but not PD98059 inhibited mRNA expression for TNFa, iNOS, and IL-6. ICAM and IFNgR mRNA were not changed.
or TNFa could serve as a priming agent (Lee et al., 1993a; Liu et al., 1996), cells were stimulated in three different settings (IL-1 alone, IL-1/IFNg, and IL-1/TNFa). The results showed that SB203580 and PD98059 had quite different effects on astrocyte iNOS. Whereas the p38 inhibitor potently inhibited iNOS, the MEK inhibitor had little or no effect (Fig. 1A). A combination of the two inhibitors (SB203580+PD98059) had the same effect as SB203580 alone. Although the p38 inhibitor dramatically reduced nitrite, the potency and the maximal inhibition achieved with this
Table 1 Effect of JNK inhibition on astrocyte nitrite production
Case Case Case Case Case
1 2 3 4 5
Lipofectamine (AM)
pa3 (AM)*
pJNK (AM)**
11.0F0.7 10.5F0.4 14.7F0.4 8.6F0.3 3.6F0.2
2.6F0.7 5.4F0.6 9.7F0.6 5.0F0.6 1.8F0.4
1.2F0.3 1.9F0.2 5.8F0.2 2.2F0.3 1.2F0.8
Subconfluent astrocyte cultures from five different cases were treated with lipofectamine alone, a dominant negative JNK construct (pJNK) or an empty vector (pa3), as described in Materials and methods. Astrocytes were stimulated with IL-1/IFNg, then 48-h culture supernatants were examined for nitrite concentration. MeanFS.D. (AM) from triplicate wells. * p<0.05 vs. lipofectamine. ** p<0.05 vs. pa3 by one-way ANOVA followed by Student – Newman – Keuls Test.
Fig. 4. Effect of JNK inhibition: (A – C) astrocytes were transfected with 10 Ag of dominant negative JNK (pJNK) or with control vector (pa3), as described in Materials and methods. Cultures were then stimulated with cytokines for 48 h, then immunocytochemistry was performed for iNOS, as described elsewhere (Liu et al., 1996; Hua et al., 1998). Results show comparable culture viability in pa3- and pJNK-transfected cultures (also confirmed by trypan blue exclusion), but fewer iNOS-positive cells in pJNK-transfected wells. Results are representative of five independent experiments.
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Table 2 Effect of JNK inhibition on astrocyte TNFa production
Case Case Case Case Case
1 2 2 3 3
(IL-1) (IL-1) (IL-1/IFNg) (IL-1) (IL-1/IFNg)
3.3. SB203580 inhibits iNOS, TNFa, and IL-6 mRNA expression
Lipofectamine (pg/ml)
pa3 (pg/ml)
pJNK (pg/ml)
144F6 264F48 290F62 67F3 589F2
52F8 89F22 192F21 49F8 252F24
78F6 157F59 188F72 72F13 286F43
Subconfluent astrocyte cultures from three different cases were treated with lipofectamine alone, a dominant negative JNK construct (pJNK) or an empty vector (pa3), as described in Materials and methods. Astrocytes were stimulated with IL-1 or IL-1/IFNg as indicated, then 24-h culture supernatants were examined for TNFa levels by ELISA. MeanFS.D. (pg/ ml) from triplicate wells. No significant differences were observed ( p>0.05) between any pairs (lipofectamine vs. pa3 vs. pJNK) by oneway ANOVA.
also indicate that TNFa and IL-1 activated similar MAP kinase signaling pathways to induce iNOS in human astrocytes unlike rat cardiac muscle cells in which TNFa induces iNOS through ERK {1330}. In IL-1/IFNg-stimulated astrocytes, SB203580 was less potent (IC50f20 AM: n=3), suggesting that p38 plays differential roles in the two (IL-1 vs. IL-1/IFNg) stimulating conditions (see Discussion). 3.2. Effect of MAPK inhibitors on astrocyte TNFa production SB203580’s effect on p38 MAP kinase was first discovered by virtue of its suppressive effects on cytokine biosynthesis (Lee et al., 1994). Furthermore, in primary human astrocytes, iNOS and TNFa expression are regulated similarly in many respects, including the requirement for IL-1 and the priming effect of IFNg (Lee et al., 1993a,b; Downen et al., 1999). Thus, we asked whether MAP kinase inhibitors had similar effects on astrocyte TNFa production. Results illustrated in Fig. 2 show that SB203580 potently inhibited both IL-1 and IL-1/IFNginduced TNFa, while PD98059 had a minor effect on IL-1/ IFNg-induced TNFa production. These results show both the similarity and the difference in the regulation of astrocyte iNOS and TNFa.
For cytokine biosynthesis, both transcriptional and posttranscriptional mechanisms have been demonstrated for MAP kinase involvement. To determine where in the iNOS/TNFa induction pathway the MAP kinases were exerting their effects, we performed ribonuclease protection assay using a commercially available cytokine RPA template. RNA harvested from IL-1 or IL-1/IFNg-stimulated astrocytes was analyzed and showed similar results at 6 h (n=2, not shown) or 16-h poststimulation (n=2; Fig. 3). SB203580 potently inhibited both iNOS and TNFa mRNA, whereas PD98059 had little or no effect. Little change was noted in the expression of ICAM-1 and IFNg receptor h chain mRNA, demonstrating the specificity of the p38 inhibitor effect. IL-6 mRNA, which is induced by IL-1 or IL-1/IFNg in similar quantities, was potently inhibited by SB203580 and not by PD98059. In sum, these results show a high degree of co-regulation in the induction of TNFa, IL6, and iNOS in astrocytes, and that the p38 MAP kinase plays an important role in the mRNA induction of these genes. 3.4. Effect of transfection with dominant-negative JNK plasmid (pJNK) Because specific pharmacological inhibitors of JNK MAP-kinase pathway are not available, we employed an alternative approach to examine its role in iNOS expression. We transfected primary astrocytes with plasmids containing an inactivating (dominant negative) JNK sequence (pJNK) or an empty vector (pa3), both originated from Dr. M. Karin’s laboratory. Astrocyte nitrite production was measured after stimulation with IL-1/IFNg. The results are summarized in Table 1. Data from five different cases show that significantly less nitrite was produced following transfection (pa3 or pJNK vs. Lipofectamine), and also following transfection with pJNK compared with control vector (pJNK vs. pa3). Immunostaining for iNOS showed that whereas iNOS-positive astrocytes were frequent in pa3-transfected cultures, they were fewer in pJNK-transfected cultures (Fig. 4). Although
Fig. 5. Western blot analysis of MAP kinase phosphorylation: astrocyte protein was extracted at indicated time points following stimulation with IL-1h and analyzed using antibodies specific to phosphorylated or total MAP kinases. Approximately 100, 10, and 10 Ag of total protein were loaded in each lane for p38, ERK, and JNK immunoblots, respectively. The two bands in ERK immunoblot represent the 44 kDa (ERK1) and 42 kDa (ERK2) proteins.
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some degree of cell degeneration was observed following transfection, there was no difference in cell death between pa3 and pJNK-transfected cultures (trypan blue exclusion; not shown). These results collectively support that inhibition of JNK specifically suppressed iNOS expression in astrocytes. TNFa production was examined in three different cases with both IL-1 and IL-1/IFNg stimulation in two of them. The results are summarized in Table 2. They show that the levels of TNFa between control (pa3) and pJNK-transfected cultures were not different ( p>0.05 by one-way ANOVA followed by Student – Newman – Keuls Test). These results do not support a role for JNK MAP kinase in TNFa production in human astrocytes. 3.5. MAP kinase phosphorylation in astrocytes Because our data suggest that MAP kinases are active in IL-1-stimulated astrocytes, we directly examined this by Western blot analysis. The amounts of unphosphorylated MAP kinases are known to be constant, while activating signals (phosphorylation of specific upstream MAPK kinases) induce dual phosphorylation of specific MAP kinases. Results illustrated in Fig. 5 show that p38 and JNK kinases were phosphorylated by IL-1 with different kinetics. By contrast, ERK1/2 were phosphorylated in resting cells, even in serum-starved conditions, and the phosphorylation remained unchanged following cytokine stimulation (Fig.
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5). These results demonstrate rather dramatic differences in the way MAP kinases are activated in human fetal astrocytes, and may offer a partial explanation for the differential effects of p38 and ERK inhibitors on astrocyte activation (Figs. 1 – 4). 3.6. Role of MAPK in astrocyte NF-nB and AP-1 activation Cytokines such as IL-1 induce gene expression via activation of several well-known transcription factors. Among these are the NF-nB and AP-1 family of transcription factors, which are also known to be important in the induction of iNOS and TNFa genes (Marks-Konczalik et al., 1998; Means et al., 2000). To test whether MAPK are involved in the cytokine-mediated activation of these transcription factors in human fetal astrocytes, electrophoretic mobility shift assays (EMSAs) were performed. Using NFnB consensus sequence oligonucleotides, we found that IL1 or IL-1/IFNg induced specific shifted complexes composed of p65/p50 (Liu et al., 2000) (Fig. 6). Neither SB203580 nor PD98059 reduced the amounts of NF-nB complex formed at 1-, 3-, 4-, and 6-h poststimulation (Fig. 6 and data not shown). EMSA using a human iNOS-specific AP-1 sequence showed a single AP-1 complex in astrocytes stimulated with IL-1 or IL-1/IFNg (Fig. 7A and B). The induction was minimal at 1 h, but clearly visible at 4 h. The AP-1 complex was completely displaced by anti-c-jun antibody, but minimally by anti-ATF-2 antibody. Pretreatment
Fig. 6. Electrophoretic mobility shift assay (EMSA) for NF-nB. Astrocytes were pretreated with SB203580 or PD98059 at 20 AM for 1 h, then stimulated with 1L-1 for 1 h. Nuclear extracts were incubated with 32P-labeled NF-nB consensus sequence probe (Promega) in the presence of 100-fold unlabeled specific (NF) or non-specific (AP) competitor probes. Supershift experiments were performed by preincubating the nuclear extracts with 10 Ag of p50 or p65 (Rel A) antibodies for 40 min prior to incubation with probes. Results are representative of three experiments with similar results.
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Fig. 7. AP-1 EMSA in cytokine-treated astrocytes (A and B). Astrocytes prepared as in Fig. 6 (except nuclear protein harvested both at 1 and 4 h) were examined with 32P-labeled AP-1 consensus nucleotides. Specificity of the reaction was determined by competing with unlabeled, excess AP-1 (+AP-1) or mutant oligonucleotide (+mt). Supershifting experiments were performed by preincubating nuclear extracts with an antibody to ATF-2, c-jun, or control IgG (A and B). The AP-1 complex was induced at 4 h, and SB203580 reduced AP-1 complex formation.
with SB203580 reduced the amount of AP-1 complex induced by IL-1/IFNg, whereas no such effect was observed with PD98059. Thus, these experiments demonstrated that
both NF-nB and AP-1 transcription factors are activated in cytokine-treated astrocytes but p38 MAP kinase is involved in AP-1 activation only.
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4. Discussion In this report, we investigated the role of MAP kinases in the cytokine-induced expression of iNOS and TNFa in human fetal astrocytes. TNFa can be expressed by both microglia and astrocytes, depending on the stimuli, but the iNOS expression has been shown to be highly species dependent. Whereas macrophages and microglia are a ready source of nitric oxide in rodents, in human macrophages and microglia, iNOS is difficult to induce (Denis, 1994; Nathan and Xie, 1994; Lee et al., 1995a). iNOS in human cells is induced in non-macrophage cells such as astrocytes, hepatocytes, and chondrocytes, and in all of these cell types, the cytokine IL-1 plays a crucial role in the transcriptional induction of this gene (Liu et al., 1996; Geller et al., 1995; Chartrain et al., 1994). The crucial role of astrocytes as the expressor of iNOS in the human CNS is demonstrated by our recent studies of multiple sclerosis and HIV-1 encephalitis (Liu et al., 2001; Zhao et al., 2001). Our results demonstrate an important role for p38 JNK and a relatively minor role for ERK in this induction. This may be, in part, related to their activation profile following IL-1 stimulation, since p38 and JNK kinases were phosphorylated by IL-1 in astrocytes, ERK1/2 were phosphorylated in resting astrocytes, even after serum deprivation. This cytokine-mediated activation of MAP kinases is consistent with the general observation that p38 and JNK MAP kinases are stress and inflammatory signal activated, whereas ERK is growth-factor activated (Cobb and Goldsmith, 1995; Davis, 1994; Herlaar and Brown, 1999). SB203580 is a member of a novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs) whose activity was originally described in the inhibition of cytokine (TNFa) synthesis in LPS-stimulated human monocytederived macrophages (Lee et al., 1994). In this and a subsequent study of rat glial cells (Bhat et al., 1998), SB203580 was shown to inhibit translation rather than the transcription of TNFa gene. Interestingly, we find that in human fetal astrocytes, p38 was involved in the mRNA expression of TNFa, as well as that of IL-6 and iNOS, suggesting that the mechanism of p38 action differs depending on the cell type. MAP kinases have been shown to be important in iNOS induction in other cells. In bovine chondrocytes (Badger et al., 1998) and in murine astrocytes (Da Silva et al., 1997), IL-1-induced iNOS mRNA production and nitrite production were inhibited by SB203580, but not by PD98059. Moreover, in rat mesangial cells, both p38 and JNK and their upstream kinases (MKK3, 4, and 6) are involved in iNOS induction (Guan et al., 1999). Our study confirms and extends these observations supporting the importance of IL1-activated p38 and JNK pathways in the induction of iNOS. Our data do not support, however, the reported prominent role of ERK kinase in iNOS expression in heart muscle cells (Kan et al., 1999) and in rat glial cells activated by cytokines or LPS (Bhat et al., 1998). LPS plays little or
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no role in the induction of iNOS in human cells including astrocytes and hepatocytes (Liu et al., 1996; Geller et al., 1995), therefore, we cannot address the role of LPS-induced ERK in iNOS expression. Interestingly, ERK inhibitor had no effect on IL-1-activated iNOS, but did show a minor effect when astrocytes were co-stimulated with IFNg. These data may indicate that ERK was involved in IFNg-mediated cell activation (see below), which played a minor or insignificant role in the case of human iNOS induction. Our study showed that p38 inhibitor SB203580 had a different potency in IL-1- and IL-1/IFNg-stimulated iNOS activation. Whereas SB203580 at 1 AM completely inhibited IL-1-mediated nitrite production, higher concentrations (IC50f20 AM) were required to block IL-1/IFNgmediated nitrite production. The significance of these observations is not clear, but the data collectively suggest a link between ERK/p38 and IFNg-activated cell signaling. In this regard, both ERK and p38 have been shown to activate Stat1, the transcription factor involved in IFNg signaling (David et al., 1995; Uddin et al., 1999). Although tyrosine phosphorylation is primarily responsible for IFN signaling via the Jak/Stat pathway, recent data show that MAP kinasedependent phosphorylation is important in the maximal transcriptional activation by Stats (Wen et al., 1995; David et al., 1995; Goh et al., 1999). Experiments are ongoing to examine these potential scenarios in astrocytes. Different IC50 may also indicate that IFNg stimulated other pathways in astrocytes and the induction of iNOS became less dependent on p38 in the presence of IFNg. Analysis of the human iNOS promoter reveals distinct features, including the number and location of important cytokine responsive elements: multiple NF-nB and two perfectly matched AP-1 sites are present in the human promoter which are not present in the murine iNOS promoter (Taylor et al., 1998; Chu et al., 1998; Marks-Konczalik et al., 1998). The AP-1 upstream site (at 5301) in the human iNOS promoter has been shown to be essential for transcriptional activation and in lung epithelial cells, Jun D, and Fra-2 were found to be complexed with the AP-1 sequence (Marks-Konczalik et al., 1998). Using the same oligonucleotide sequence as a probe, we found that AP-1 binding was induced in cytokine-activated human astrocytes, and that this induction was greatly diminished by p38 inhibition, identifying IL-1-induced AP-1 activation as the possible molecular target for p38 activity in astrocytes. Although MAP kinase-dependent NF-nB activation has been demonstrated (Schulze-Osthoff et al., 1997), it is unlikely that MAP kinases were involved in NF-nB activation in astrocytes, because we see no evidence for this. Rather, our results are consistent with the view that NF-nB and AP-1 cooperated after binding to the promoter elements of the iNOS gene. We also tested cycloheximide, a protein synthesis inhibitor, on the accumulation of astrocyte iNOS mRNA, and found that iNOS mRNA induction is dependent on new protein synthesis (not shown). This indicates an indirect
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mechanism for mRNA induction. Because AP-1 activation was a delayed response (4 h>1 h) and is known to require new protein synthesis of immediate early genes such as c-jun or cfos, these data additionally support the role of AP-1 in human astrocyte iNOS induction. In addition, posttranslational modification of AP-1 proteins also potentiates transactivation function, such as that shown for NH2-terminal phosphorylation of c-jun by JNK (Karin, 1995). These are all potential mechanisms for iNOS induction in human astrocytes, as we found that JNK inhibition, as well as p38 inhibition (through AP-1 inhibition), affected iNOS expression. The regulation of TNFa gene expression is complex. Several levels of regulation are described, including transcription and mRNA stability, as well as translational and posttranslational control. The promoter of the TNFa gene contains consensus sites for NF-nB and AP-1, but whether these elements play critical roles in transcriptional activation varies depending on the study (Means et al., 2000; Yao et al., 1997; Rhodes et al., 1992; Zheng et al., 1993; Monick et al., 1999). For example, LPS-induced TNFa transcription shows different requirements for AP-1 activation depending on the macrophage populations (Means et al., 2000), suggesting that both MAPK-dependent and -independent regulatory elements are used for transcription. Studies of cytokine-induced astrocyte TNFa production have shown an involvement of p38 as well as ERK (Bhat et al., 1998; Lee et al., 2000). Our study indicates that p38 is the dominant MAP kinase involved in IL-1-mediated TNFa biosynthesis in human astrocytes, but that ERK may also (albeit minor) play a role (see Fig. 2, for instance). In contrast to iNOS expression, which was inhibited by inactivating JNK expression, we see no consistent effect of JNK inhibition on TNFa expression. The interpretation of these data is complex because of the suboptimal transfection efficiency of primary cells using non-viral vectors. While we cannot entirely exclude the possibility of JNK involvement in TNFa induction, our data at least point to the differential roles played by JNK in iNOS and TNFa induction in astrocytes. Our study may have implications for the pathogenesis and therapy for human CNS inflammatory diseases, such as multiple sclerosis and HIV encephalitis, in which proinflammatory cytokines and iNOS are expressed (Selmaj et al., 1991; Tyor et al., 1992; Zhao et al., 2001; Liu et al., 2001). In previous and ongoing studies, we observe that for microglial chemokine gene induction, p38 plays a negative regulatory role, while ERK plays different roles depending on the stimulus involved (Kim, Si, and Song; unpublished). The cell-, stimulus-, and target promoter-dependent involvement of MAP kinases in the biosynthesis inflammatory genes thus demonstrates a possibility that a more specific blockade of glial cell activation can be achieved. Further studies directed at the molecular mechanisms of MAP kinase involvement in human glial activation will enhance the opportunity for MAP kinase-based therapeutic interventions.
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