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INFLAMMATION AND IMMUNOPHARMACOLOGY

Anti-Inflammatory Actions of St. John's Wort: Inhibition of Human Inducible Nitric-Oxide Synthase Expression by Down-Regulating Signal Transducer and Activator of Transcription-1 (STAT-1) Activation

Biochemistry Section, Department of Neuroscience and Vision, University of Verona, Verona, Italy (E.T., M.M., D.M., H.S.); Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany (U.F., H.K.)

Received May 13, 2003; accepted June 20, 2003.

	Abstract

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St. John's wort (SJW) has been described to show anti-inflammatory properties due to its inhibitory effects on the expression of pro-inflammatory genes like cyclooxygenase-2, interleukin-6, and inducible nitric-oxide synthase (iNOS). Since iNOS plays a critical role in chronic inflammatory diseases, we have focused our attention on the regulation of iNOS expression by SJW in two different human epithelial cell lines, alveolar A549/8 and colon DLD-1 cells. SJW extract concentration dependently inhibited human iNOS expression evaluated by measuring the amounts of iNOS mRNA, iNOS protein, and NO production in both cell lines. This inhibitory effect resulted from transcriptional inhibition as shown in reporter gene experiments. With electrophoretic mobility shift experiments, we found a SJW-mediated down-regulation of the DNA binding activity of the transcription factor signal transducer and activator of transcription-1 (STAT-1), but not of nuclear factor-B. This down-regulation of the STAT-1 DNA binding was shown to result from reduced tyrosine phosphorylation of the STAT-1 protein. The diminished STAT-1 tyrosine phosphorylation resulted from SJW-mediated reduction of Janus kinase 2 activity. These data suggest that extracts from SJW may be a promising anti-inflammatory principle in chronic inflammatory diseases.

Nitric oxide (NO) synthase is a widely distributed enzyme catalyzing the synthesis of NO from L-arginine. There are three isoforms of NO synthase encoded by distinct genes, including the constitutively expressed neuronal and endothelial NO synthase and inducible NO synthase (iNOS). iNOS is expressed in most cell types analyzed after induction by various compounds [e.g., by cytokines in inflammatory conditions (Kleinert et al., 2000)]. NO produced by iNOS may reach high concentrations (up to millimolar concentration) around the site of inflammation. These massive amounts of NO are believed to be involved in the elimination of intruding organisms such as bacteria, parasites, and viruses (MacMicking et al., 1997). However, these large amounts of NO are also potentially toxic to host tissues. This may explain the involvement of iNOS in the pathobiology of several human diseases (Kröncke et al., 1998). In addition to its immunological role, iNOS-derived NO seems to be important for wound repair (Stallmeyer et al., 1999) and may exert protective functions especially in stress situations of the liver (Kim et al., 1997). iNOS-derived NO seems to be important for human cancer development, either as a weapon of the immune system against the tumor (Umansky and Schirrmacher, 2001) or as an antiapoptotic (Payne et al., 1999) and proangiogenetic (Jaiswal et al., 2001) factor supporting tumor growth. Therefore it is reasonable to expect that the expression of iNOS mRNA is finely controlled by a concerted action of two categories of compounds, inducers and suppressors (Kleinert et al., 2000). Pro-inflammatory cytokines such as interferon- (IFN-), tumor necrosis factor- (TNF-), and interleukin-1 (IL-1) belong to the former. The latter includes transforming growth factor-, basic fibroblast growth factor, estrogen, morphine, and dexamethasone (Kleinert et al., 2000). However, in recent years it has been shown that regulation of iNOS expression is cell- and species-specific (Kleinert et al., 2000).

Induction of human iNOS gene expression has been shown to depend on at least two major transcription factors, NF-B and the signal transducer and activator of transcription-1 (STAT-1) (Geller and Billiar, 1998; Kleinert et al., 2000). The relative importance of these two transcription factors varies with the cell type analyzed (Kleinert et al., 2000). NF-B is rapidly activated by lipopolysaccharide (LPS), TNF-, and IL-1 (Adcock, 1997), whereas the activation of STAT-1 is triggered by IFN- (Darnell et al., 1994). Activated NF-B and STAT-1 stimulate the activity of the promoter region of iNOS, thereby inducing expression of the iNOS gene (Kleinert et al., 1998; Ganster et al., 2001). Regulation of the activation of NF-B and STAT-1 involves both tyrosine and serine phosphorylation. Therefore, the modulation of this process should control the time spatial expression of iNOS and the successive NO production.

In the current study, we tested the hypothesis that the anti-inflammatory action of SJW could depend on an inhibition of the pro-inflammatory transcription factors STAT-1 and/or NF-B. Our data indicate that SJW efficiently inhibits cytokine-induced STAT-1 activation (but not NF-B activation), thereby reducing iNOS expression.

	Materials and Methods

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Reagents
Trypsin, glutamine, and pyruvate solutions, agarose, tRNA, and bovine serum albumin were purchased from Sigma Chemie (Deisenhofen, Germany). Isotopes were obtained from ICN Biomedicals (Eschwege, Germany). T3 and T7 RNA polymerase, RNase A, RNase T1, and DNase I were obtained from Roche Diagnostics (Mannheim, Germany). Human IFN-, IL-1, and TNF- were obtained from Strahtmann (Hannover, Germany). Fetal calf serum (FCS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from PAN-Systems (Nürnberg, Germany). The Dual-Luciferase reporter assay system and Passive lysis buffer were purchased from Promega (Heidelberg, Germany). Restriction enzymes, Klenow DNA polymerase, dNTPs, NTPs, and the PhosphoPlus STAT-1 (Tyr701) antibody kit were purchased from New England Biolabs (Frankfurt am Main, Germany). The monoclonal anti-Janus kinase 2 (JAK2) antibody and the monoclonal antiphosphotyrosine antibody were obtained from Upstate Biotechnology (Milton Keynes, UK). The monoclonal anti-IFN- receptor subunit antibody and the protein A-agarose were obtained from Santa Cruz Biochemicals (Heidelberg, Germany). The SJW extract was obtained from Indena (Milan, Italy).

Cell Culture, Cytokine Treatment, RNA Isolation, and Nitrite Measurement
The human alveolar epithelial A549/8 cells, human colon carcinoma DLD-1 cells, and human ECV304 cells were grown in DMEM with 5 to 10% fetal bovine serum, 2 mM L-glutamine, penicillin, and streptomycin. For RNA isolation, they were plated onto 10-cm diameter (58 cm²/well) dishes, whereas those experiments involving luciferase activity determinations or NO production studies were performed with cells plated onto 6-well plates (9.6 cm²/well) or 24-well plates (1.75 cm²/well). Eighteen hours before cytokine induction, cells were washed with phosphate-buffered saline solution and incubated with DMEM containing 2 mM L-glutamine in the absence of serum and phenol red. After this preincubation time, cells were pretreated with different concentrations of SJW extract for 1 h. A549/8 and DLD-1 cells were induced with a cytokine mixture (CM) composed of IFN- (100 U/ml), IL-1 (50 U/ml), and TNF- (10 ng/ml) for the time periods indicated. Afterward, the supernatant of the cells (300 µl) was used to measure by the Griess reaction, and cells were processed for RNA isolation by guanidinium thiocyanate/phenol/chloroform extraction as described (Kleinert et al., 1998).

RNase Protection Analysis
For the generation of radiolabeled human iNOS- and human GAPDH-antisense probes for RNase protection assays, 0.5 µg of the linearized plasmids pCR_iNOS human (Kleinert et al., 1996) or pXcm_GAPDH human (Witteck et al., 2003) were in vitro transcribed using T3 or T7 RNA polymerase and [-³²P]UTP. To quantify human iNOS mRNA levels, RNase protection experiments were performed as described (Kleinert et al., 1998). In all experiments, GAPDH mRNA expression was determined for normalization purposes. Densitometric analyses were performed using a PhosphorImager (Bio-Rad, Munich, Germany). The protected fragments of human iNOS- and human GAPDH-mRNA were 386 nt or 195 nt and 105 nt, respectively.

Analysis of the Human iNOS Promoter Activity and Human eNOS Promoter Activity
To investigate the effect of SJW extract on cytokine-induced iNOS promoter activity, pools of stably transfected A549/8 or DLD-1 cells [containing a 16-kb fragment of the human iNOS promoter cloned in front of a luciferase reporter gene (Yao et al., 2003)] were incubated for 18 h with DMEM without FCS and phenol red. Before cytokine induction, the cells were pretreated with SJW extract in the concentrations indicated. After CM incubation for 4.5 h in the presence or absence of SJW extract, cells were lysed in 1x Passive lysis buffer.

To investigate the effect of SJW extract on the constitutive human eNOS promoter activity, pools of stably transfected ECV304 cells [containing a 3.5-kb fragment of the human eNOS promoter cloned in front of a luciferase reporter gene (Yao et al., 2003)] were incubated for 24 h with DMEM without FCS and phenol red. Cells were then incubated with or without different concentrations of SJW extract for 4 to 5 h and lysed in 1x Passive lysis buffer.

Firefly luciferase activity was determined using the Dual-Luciferase assay kit. Protein concentrations of the extracts were determined by Bradford reagent using bovine serum albumin as standard. Protein content of the extracts was used for normalization of the luciferase activity.

Western Blot Experiments
Detection of STAT-1 Phosphorylation. A549/8 and DLD-1 cells incubated with or without CM in the presence or absence of SJW extract for 0.5 h were lysed on ice with 20 mM HEPES, pH 7.4, 420 mM NaCl, 1% NP40, 1 mM EGTA, and 1 mM EDTA for 15 min. After centrifugation for 15 min at 12,000 rpm, proteins (50 µg/lane) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 7.5% gel, electroblotted onto PVDF membrane (Millipore S.p.A., Rome, Italy), and reacted with anti-STAT-1 phosphotyrosine 701 antibody diluted 1:700 (New England Biolabs, Hetchin, England) and, after stripping, with anti-STAT-1 antibody diluted 1:1,000 according to standard procedures. Immune complexes were detected by using anti-rabbit horseradish peroxidase-conjugated immunoglobulin for detection of the primary antibody. The immunoreactive proteins on the blot were detected by an enhanced chemiluminescence detection system (ECL) (Amersham Biosciences AB, Uppsala, Sweden).

Detection of iNOS Protein. DLD-1 cells incubated with or without CM in the presence or absence of SJW extract for 24 h were lysed on ice with RIPA buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EGTA, and 1 mM EDTA) for 20 min. After centrifugation for 20 min at 12,000 rpm, proteins (80 µg/lane) were fractionated by SDS-PAGE in a 7.5% gel, electroblotted onto PVDF membrane (Millipore S.p.A.), and reacted with a polyclonal anti-iNOS antibody (Transduction Laboratories, Lexington, KY) diluted 1:200 according to standard procedures. Immune complexes were detected by using anti-rabbit horseradish peroxidase-conjugated immunoglobulin for detection of the primary antibody. The immunoreactive proteins on the blot were detected by ECL detection system (Amersham Biosciences AB).

Immunoprecipitation Analyses of Tyrosine-Phosphorylated JAK2 or IFN- Receptor Subunit
A549/8 and DLD-1 cells incubated with or without CM in the presence or absence of SJW extract for 0.5 h were lysed on ice with RIPA buffer for 15 min. After centrifugation for 15 min at 12,000 rpm, proteins (500 µg/sample) were incubated overnight at 4°C with a monoclonal anti-JAK2 or anti-IFN- receptor subunit antibody at the concentration recommended by the supplier. Then the immuno-complexes were incubated with protein A-agarose and precipitated by centrifugation. After three washing steps with RIPA buffer (2x) and phosphate-buffered saline (1x), the immunoprecipitated material was resuspended in Laemmli sample buffer and separated on SDS-PAGE in a 5 or 7.5% gel, electroblotted onto PVDF membrane (Millipore S.p.A.), and reacted with an anti-phosphotyrosine antibody (Upstate Biotechnology) diluted 1:1,000 according to standard procedures. Immune complexes were detected by using anti-rabbit horseradish peroxidase-conjugated immunoglobulin for detection of the primary antibody. The immunoreactive proteins on the blot were detected by an ECL detection system (Amersham Biosciences AB). After detection of tyrosine-phosphorylated JAK2 or IFN- receptor subunit , the blots were stripped and reprobed with a monoclonal anti-JAK2- or anti-IFN- receptor subunit antibody, respectively (for normalization).

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from cells treated with or without cytokines in the presence or absence of SJW extract according to Osborne et al. (1997) in the presence of 10 µg/ml leupeptin, 5 µg/ml antipain, and pepstain, and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemie). Protein concentration in the nuclear extracts was determined by using the Bradford reagent. Nuclear extracts (10 µg) were incubated at room temperature for 20 min with a ³²P-labeled double-stranded oligonucleotide containing the STAT-1 binding site (sis-inducible factor-binding recognition element, SIE/m67) from the c-Fos promoter (5'-gtcgaCATTTCCCGTAAATCg-3') or the NF-B binding sequence from the IL-6 promoter (5'-gatcCAGAGGGGACTTTCCGAGt-3') (Promega, Milan, Italy) in a 15-µl reaction mixture containing 20 mM HEPES, pH 7.9, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, 0.1 mM EDTA, 2 µg of poly(dI-dC), 1 µg of salmon sperm DNA. Products were fractioned on a nondenaturing 5% polyacrylamide gel in Tris-borate-EDTA buffer (1.08% Tris, pH 8.3, 0.55% boric acid, and 20 mM EDTA).

Calculations
All data are presented as mean ± S.E.M. Differences between means were tested for statistical significance using factorial analysis of variance followed by Fisher's protected least significant difference test as the post hoc test (StatView software; SAS Institute, Cary, NC).

	Results

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SJW Extract Inhibited Human iNOS mRNA and Protein Expression and Nitrite Production in A549/8 and DLD-1 Cells. As shown in Fig. 1, SJW extract inhibited concentration-dependent CM-elicited iNOS mRNA expression in A549/8 and DLD-1 cells. Also the cytokine-induced expression of iNOS protein was inhibited by SJW extract (Fig. 2). Inhibition of cytokine-induced NO production was also observed (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of SJW extract on cytokine-induced human iNOS mRNA expression in epithelial A549/8 and DLD-1 cells. A, representative RNase protection assay for iNOS mRNA performed with total RNA from human A549/8 cells. Cells were preincubated for 18 h in serum-free medium and for an additional 1 h with different concentrations of SJW extract (1–100 µg/ml). A cytokine mixture (100 U/ml interferon-, 100 U/ml interleukin-1, and 10 ng/ml tumor necrosis factor-) was added to some of the cultures, and the cells were incubated for an additional 8 h with or without the above concentrations of SJW. Experiments were performed using antisense RNA probes for human iNOS and GAPDH (used for normalization). The positions of the protected iNOS and GAPDH fragments are indicated. (M, molecular weight standard, X174-restricted with HinfI; I, iNOS antisense probe; G, GAPDH antisense probe). B, densitometric analyses of six different gels using RNA isolated from A549/8 cells. Columns (mean ± S.E.M.) represent relative iNOS mRNA levels at the different concentrations of SJW extract (*, p < 0.05; ***, p < 0.001 versus CM). C, data obtained using RNA isolated from human DLD-1 cells. Columns (mean ± S.E.M., n = 5) represent relative iNOS mRNA levels at the different concentrations of SJW extract (***, p < 0.001; ns, not significant versus CM).

View larger version (41K): [in this window] [in a new window]   Fig. 2. Effect of SJW extract on cytokine-induced iNOS protein expression in human DLD-1 cells. DLD-1 cells were preincubated for 18 h with serum-free DMEM. Cells were then incubated for 24 h with or without a CM in the presence or absence of SJW extract (50 µg/ml). Total cell extracts (80 µg of total proteins) were analyzed for iNOS and (for normalization) for STAT-1 protein expression by Western blotting using a polyclonal anti-iNOS antibody and a monoclonal anti-STAT-1 antibody, respectively. The blot shown is representative of three experiments showing the same results.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Effect of SJW extract on cytokine-induced nitrite production in human epithelial A549/8 and DLD-1cells. A, statistical analysis of nine different Griess assays performed with supernatants from untreated A549 cells or cells stimulated for 24 h with a CM. Cells were then preincubated for 18 h in serum-free medium, and some cultures then received SJW extract (10–50 µg/ml) for an additional 1 h. The extract remained present for the subsequent 24-h incubation (see above). Columns (mean ± S.E.M.) represent the relative nitrite levels determined (100% corresponds to 400 pmol/ml/24 h; ***, p < 0.001 versus CM). B, data generated with DLD-1 cells using 10 to 100 µg/ml SJW extract. Columns (mean ± S.E.M.) represent the relative nitrite levels determined under the conditions indicated (100% corresponds to 400 pmol/ml/24 h; ***, p < 0.001 versus CM).

SJW Extract Inhibited Human iNOS Promoter Activity in Stably Transfected A549/8 and DLD-1 Cells. To analyze the effect of SJW extract on human iNOS promoter activity, we incubated A549/8 and DLD-1 cells and stably transfected with pNOSII(16)Luc (Yao et al., 2003) with different concentrations of SJW extract. The plasmid pNOSII(16)Luc contains a 16-kb fragment of the human iNOS promoter cloned in front of a luciferase reporter gene. In line with previous reports (Hausding et al., 2000; Witteck et al., 2003; Yao et al., 2003), CM incubation of these pools of stably transfected A549/8 and DLD-1 cells resulted in an up to 5-fold enhancement of the luciferase expression (Fig. 4). SJW extract concentration dependently inhibited on the CM-induced iNOS promoter activity in these cells (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect of SJW extract cytokine-induced human iNOS promoter activity in stably transfected A549/8 and DLD-1 cells. A549/8 and DLD-1 cells, stably transfected with pNOSII(16)Luc, a construct containing a 16-kb fragment of the human iNOS promoter in front of a luciferase reporter gene (Yao et al., 2003), were preincubated in serum-free medium for 18 h and then with SJW extracts for an additional 1 h. Cells were then incubated with a CM for another 4 h in the presence or absence of the SJW extract. Luciferase activity and protein content were determined in cell extracts. A, columns (mean ± S.E.M., n = 18) represent the relative luciferase activities (percentage of the CM effect) in extracts of stably transfected A549/8 cells incubated with SJW extract (1–50 µg/ml; **, p < 0.01; ***, p < 0.001 versus CM). B, columns (mean ± S.E.M., n = 16) represent the relative luciferase activities (percentage of the CM effect) in extracts of stably transfected DLD-1 cells incubated with SJW extract (1–100 µg/ml; **, p < 0.01; ***, p < 0.001; ns, not significant versus CM).

Only Moderate Inhibition by SJW Extract of a Human 3.5-kb eNOS Promoter. To determine whether the inhibition of iNOS expression was a result of the inhibition of general transcription factors, the effect of SJW extract on a constitutively active promoter was analyzed. For this purpose, ECV-pNOS III-Hu-3500-Luc-neo cells (Yao et al., 2003) were used. These cells derive from human ECV304 cells transfected with the plasmid pNOS III-Hu-3500-Luc-neo. This plasmid contains a 3.5-kb fragment of the human eNOS promoter cloned in front of a luciferase reporter gene. This promoter displayed a significant constitutive activity in the stably transfected cells (see Fig. 5). Incubation of ECV-pNOS III-Hu-3500-Luc-neo cells with the highest concentrations of SJW extract only resulted in a moderate reduction of eNOS promoter activity (control cells 100% SJW extract, 50 µg/ml, 65.1% ± 4.3%, n = 14).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Effect of SJW extract on the activity of the constitutive human eNOS promoter in stably transfected ECV-pNOS III-Hu-3500-Luc-neo cells. ECV-pNOS III-Hu-3500-Luc-neo cells (Yao et al., 2003) stably transfected with pNOS III-Hu-3500-Luc-neo, a construct containing a 3.5-kb human eNOS promoter fragment in front of a luciferase reporter gene were preincubated with serum-free medium for 18 h. Cells were then incubated with or without SJW extract (1–50 µg/ml) for an additional 6 h in serum-free medium. Cell extracts were prepared, and luciferase activity and protein content of the extracts were determined. Columns (mean ± S.E.M., n = 12) represent relative luciferase activities (percentage of control cells) in the presence of the different concentrations of the SJW extract (*, p < 0.05; ns, not significant versus control).

SJW Extract Inhibited STAT-1 DNA Binding, but Did Not Effect NF-B DNA Binding. To study the effect of SJW extract on the DNA binding activity of transcription factors in A549/8 and DLD-1 cells, we performed an electrophoretic mobility shift assay for STAT-1 and NF-B, known to play a critical role in human iNOS expression. As shown in Figs. 6 and 7, cytokine treatment rapidly induced both STAT-1 and NF-B DNA binding in both cell lines. However, only STAT-1 DNA binding was reduced concentration dependently by co-incubation of cytokine-treated cells with SJW extract (Fig. 6). In contrast, cytokine-induced NF-B DNA binding remained unchanged (Fig. 7).

View larger version (79K): [in this window] [in a new window]   Fig. 6. Effect on SJW extracts on cytokine-induced STAT-1 DNA binding activity in human A549/8 and DLD-1 cells. Gel shift experiments using a radiolabeled oligonucleotide containing a consensus STAT-1 binding site and nuclear extracts from untreated A549/8 and DLD-1 cells or A549/8 and DLD-1 cells stimulated with a CM in the presence or absence of SJW extract (1–50 µg/ml). The position of the DNA-protein complex (STAT) and the free oligonucleotide is indicated. The gels are representative of four gels each showing similar results.

View larger version (88K): [in this window] [in a new window]   Fig. 7. Effect on SJW extracts on cytokine-induced NF-B DNA binding activity in human A549/8 and DLD-1 cells. Gel shift experiments using a radiolabeled oligonucleotide containing a consensus NF-B binding site and nuclear extracts from untreated A549/8 and DLD-1 cells or A549/8 and DLD-1 cells stimulated with a CM in the presence or absence of SJW extract (1–50 µg/ml). The position of the DNA-protein complex (NF-B) and the free oligonucleotide is indicated. The gels are representative of three gels each showing similar results.

SJW Extract Inhibited the Activation of STAT-1 in A549/8 Cells and DLD-1 Cells. To further analyze the mechanism of SJW-mediated inhibition of STAT-1 activity in DLD-1 and A548/9 cells, we studied STAT-1 activation by immunoblot analyses using an antibody that specifically detects the tyrosine (Tyr701)-phosphorylated form of STAT-1 (Ihle, 1995). This phosphorylation is critical for the activation of the STAT-1 protein and enables it to dimerize, migrate into the nucleus, and bind to specific STAT-1 binding sites on the DNA (the gamma-activated site elements). Incubation of A549/8 and DLD-1 cells for 30 min with CM resulted in a marked enhancement of STAT-1 tyrosine phosphorylation (Fig. 8). Co-incubation of CM-treated A549/8 and DLD-1 cells with SJW extract (50 µg/ml) completely inhibited the Tyr701 phosphorylation, in line with the inhibition of DNA binding activity seen upon treatment with SJW (Fig. 6).

View larger version (47K): [in this window] [in a new window]   Fig. 8. Effect of SJW extract on cytokine-induced activation of STAT-1 in human A549/8 and DLD-1 cells. A, A549/8 cells were preincubated for 18 h with serum-free medium and then for 1 h with SJW extract (50 µg/ml). Subsequently, cells were incubated for 30 min with or without a CM in the presence or absence of SJW extract. Nuclear extracts were prepared, and 50 µg of nuclear proteins were analyzed for STAT-1 tyrosine (Tyr701) phosphorylation by Western blotting using the PhosphoPlus STAT-1 (Tyr701) antibody kit. The blots shown are representative of four experiments yielding the same results. The top of each panel shows Western blots using a STAT-1-tyrosine-(Tyr701)-phosphate-specific antibody. The bottom of each panel shows the same blot (after stripping) using an anti-STAT-1 antibody, which detects phosphorylated and nonphosphorylated STAT-1. B, experiments similar to panel A generated with nuclear extracts from DLD-1 cells.

SJW Extract Inhibited the Activity of JAK2 in DLD-1 Cells. Finally, we analyzed the mechanism of inhibition of STAT-1 activation by testing the effect of SJW extract on JAK2 activity. JAK2 activation has been described to be an essential step for STAT-1 activation in several cell types (Darnell et al., 1994). Specific inhibition of JAK2 activation by tyrphostin AG490 has been described to inhibit cytokine-induced iNOS expression in human cells (Kleinert et al., 1998). Therefore, we performed immunoprecipitation analyses for JAK2 tyrosine autophosphorylation (Fig. 9A) and for tyrosine phosphorylation of the IFN- receptor subunit (Fig. 9B). Both assays indicated that SJW extract (50 µg/ml) inhibited cytokine-mediated JAK2 activation in A549/8 and DLD-1 cells.

View larger version (47K): [in this window] [in a new window]   Fig. 9. Effect of SJW extract on cytokine-induced JAK2 activity in human A549/8 and DLD-1 cells. A, A549/8 cells were preincubated for 18 h with serum-free DMEM and then for 1 h with SJW extract (50 µg/ml). Cells were then incubated for 30 min with or without a CM in the presence or absence of SJW extract. Total protein extracts were prepared and immunoprecipitated with a monoclonal anti-JAK2 antibody using protein Aagarose. The immunoprecipitated protein was loaded on a SDS-PAGE, and after blotting, tyrosine phosphorylation of JAK2 was analyzed using an anti-phosphotyrosine antibody. The blots shown are representative of four experiments showing the same results. Western blot using the anti-phosphotyrosine-specific antibody is shown (top). The bottom shows the same blot (after stripping) using an anti-JAK2 antibody, which detects phosphorylated and nonphosphorylated JAK2. B, DLD-1 cells were preincubated for 18 h with serum-free DMEM and then for 1 h with SJW extract (50 µg/ml). Cells were then incubated for 30 min with or without a CM in the presence or absence of SJW extract. Total protein extracts were prepared and immunoprecipitated with a monoclonal anti-IFN- receptor subunit antibody using protein A-agarose. The immunoprecipitated protein was loaded on a SDS-PAGE, and after blotting, tyrosine phosphorylation of IFN- receptor subunit was analyzed using an anti-phosphotyrosine antibody. The blots shown are representative of three experiments showing the same results. The top shows a Western blot using the anti-phosphotyrosine-specific antibody. The bottom shows the same blot (after stripping) using an anti-IFN- receptor subunit antibody, which detects phosphorylated and nonphosphorylated IFN- receptor subunit .

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Induction of iNOS expression is a hallmark of inflammatory diseases, especially at the early phase of inflammation. Under certain conditions, time spatially well controlled production of NO by iNOS are beneficial for the body because of their involvement in the elimination of intruding organisms causing inflammation (MacMicking et al., 1997). However, deregulation of iNOS expression, leading to either prolonged or spatially abnormal production of massive amounts of NO, can trigger severe tissue damage and is often observed in chronic inflammation (Kröncke et al., 1998; Kleinert et al., 2000). Studies in which cells were treated with iNOS enzyme inhibitors, in an attempt to decrease overproduction of NO, often produced conflicting results. This may be due in part to toxic secondary effect of these inhibitors or other nonspecific effects (Alderton et al., 2001). Also the use of iNOS enzyme inhibitors can cause unwanted effects such as hypertension (Husain, 2002).

An alternative approach to block iNOS-dependent NO production is the suppression of iNOS induction (Yao et al., 2003). In contrast to eNOS and neuronal NOS, which are constitutively expressed, expression of iNOS has to be induced by cytokines and other compounds (Kleinert et al., 2000). Also the transcription machinery involved in the expression of the three NOS isoforms differs markedly (Kleinert et al., 2000). Therefore, there is a good probability that specific inhibitors of iNOS expression can be generated.

At least two distinct transcription factors, NF-B and (probably more important) STAT-1, are necessary for the induction of human iNOS expression (Kleinert et al., 2000). The transcription factor NF-B, activated by LPS, TNF-, and IL-1, has been considered a promising target of drugs that reduce the expression of pro-inflammatory genes (Makarov, 2000). However, in some cases, inhibition of NF-B failed to reduce iNOS-derived NO production (Kleinert et al., 1998; Leonard et al., 1998). Glucocorticoids, which are widely used as anti-inflammatory drugs, are effective NF-B inhibitors; however, prolonged clinical use is associated with serious side effects (Schacke et al., 2002).

More recently, several authors have described the dependence of iNOS induction on the IFN--JAK-STAT pathway (Kleinert et al., 1998, 2000; Dell'Albani et al., 2001; Ganster et al., 2001; Ohmori and Hamilton, 2001). Therefore, the IFN--JAK-STAT pathway seems to be a reasonable target for the development of inhibitors of iNOS expression.

However, most inhibitors of STAT-1 activation are associated with severe cell toxicity. Recently some compounds like the green tea polyphenol epigallocatechin-3-gallate (Menegazzi et al., 2001), statins (Sadeghi et al., 2001), and the fungal metabolites S-curvularin, S14–95, and sporogen (Yao et al., 2003) have been reported as inhibitors of STAT-1 activation with low toxicity.

SJW, a plant extract widely used in phytomedicine, has been reported to exert some anti-inflammatory effects. Since the molecular mechanism of these anti-inflammatory actions are unknown, we tested the hypothesis that SJW could interfere with pro-inflammatory transcription factors thereby inhibiting iNOS expression. We chose two epithelial-derived human cell lines as cellular models, DLD-1 colon carcinoma and A548/9 alveolar carcinoma cells, because several chronic inflammatory diseases of the colon and the lung correlate with an aberrant expression of iNOS (Guslandi, 1998; Kröncke et al., 1998).

Data presented in this work clearly show that SJW extract inhibited concentration-dependent iNOS synthesis (mRNA and protein; Figs. 1 and 2) and NO production (Fig. 3) in these cell lines. This inhibition of iNOS expression seemed to result from SJW-mediated inhibition of human iNOS promoter activity (Fig. 4). In all cases, the concentrations required for half-maximal inhibition were below the concentrations showing an effect on an unrelated promoter (eNOS) used for control purposes (Fig. 5). Thus the inhibitory effect of SJW extract on iNOS expression is unlikely to result from the inhibition of general transcription factors or cell viability.

Because both NF-B and STAT-1 have been reported as regulators of human iNOS promoter activity, we analyzed the effect of SJW extract on these two transcription factors. As shown in Figs. 6 and 7, SJW extract inhibited concentration-dependent STAT-1 but not NF-B DNA binding activity in human epithelial A549/8 and DLD-1 cells. Therefore, the SJW-mediated inhibition of iNOS expression is likely to result from SJW-related inhibition of STAT-1 activation. To analyze the mechanism of SJW-related inhibition of STAT-1 activation, we determined the effect of SJW on the tyrosine phosphorylation of STAT-1 and on the tyrosine kinase JAK2, described to be essential for IFN--mediated STAT-1 activation (Briscoe et al., 1996). As shown in Fig. 8, SJW extract inhibited STAT-1 tyrosine phosphorylation, which had been described to be essential for STAT-1 DNA binding and activation of promoter activity of STAT-1-dependent genes. This down-regulation of STAT-1 tyrosine phosphorylation resulted from SJW extract-mediated inhibition of cytokine-induced JAK2 activity (Fig. 9).

Our data show that inhibition of the IFN--JAK-STAT-1 pathway by SJW extract may be an efficient way to downregulate iNOS-related NO production. Therefore, in treating inflammatory diseases, the use of SJW may be an alternative or adjuvant to the use of anti-inflammatory steroids or may help to reduce the steroid dose. This can be clinically relevant, because the long-term use of anti-inflammatory steroids is associated with significant side effects (Shanley et al., 2002), and glucocorticoids failed to exert a substantial effect on intestinal NO synthesis in inflammatory bowel disease (Leonard et al., 1998).

Interestingly, in contrast to the murine system (Raso et al., 2002), we did not observe any inhibitory effect of SJW extract on the LPS/cytokine-induced NF-B binding activity in human epithelial A549/8 or DLD-1 cells. Therefore in human cells, unlike murine cells, inhibition of NF-B is unlikely to be involved in the reduction of iNOS expression by SJW. This is in agreement with our previous report, showing that induction of iNOS depends only partially on NF-B activation in human cells (Kleinert et al., 1998).

In conclusion, SJW extract represents an effective inhibitory principle of human iNOS induction. The major mechanism of action seems to be suppression of IFN--elicited STAT-1 activation.

	Acknowledgements

 
The expert technical assistance of I. Ihrig-Biedert and K. Masch is gratefully acknowledged.

	Footnotes

 
This work was supported by Grant 8312-38 62 61/322a,b from the Innovation Foundation of the State of Rhineland-Palatinate (to H.K. and U.F.), by the Collaborative Research Center SFB 553 (Project A7 to H.K.), and Italian COFIN Grants 2000–2002 (to H.S.).

E.T. is supported by a doctoral fellowship in Biochemical Science from the University of Verona.

DOI: 10.1124/jpet.103.054460.

ABBREVIATIONS:: SJW, St. John's wort; NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; eNOS, endothelial NOS; NF-B, nuclear factor-B; CM, cytokine mixture; IFN-, interferon-; IL-1, interleukin-1; JAK2, Janus kinase 2; STAT-1, signal transducer and activator of transcription-1; TNF-, tumor necrosis factor-; LPS, lipopolysaccharide; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotide; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; ECL, enhanced chemiluminescence.

Address correspondence to: Dr. Hartmut Kleinert, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55101 Mainz, Germany. E-mail: Kleinert{at}mail.uni-mainz.de

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