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. 2006;2006(2):16161.
doi: 10.1155/MI/2006/16161.

JAK inhibitors AG-490 and WHI-P154 decrease IFN-gamma-induced iNOS expression and NO production in macrophages

Outi Sareila  1 , Riku Korhonen, Outi Kärpänniemi, Riina Nieminen, Hannu Kankaanranta, Eeva Moilanen
Affiliations

JAK inhibitors AG-490 and WHI-P154 decrease IFN-gamma-induced iNOS expression and NO production in macrophages

Outi Sareila et al. Mediators Inflamm. 2006.

Abstract

In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-gamma (IFN-gamma). JAK inhibitors AG-490 and WHI-P154 decreased IFN-gamma-induced nuclear levels of signal transducer and activator of transcription 1alpha (STAT1alpha). JAK inhibitors AG-490 and WHI-P154 decreased also iNOS protein and mRNA expression and NO production in a concentration-dependent manner. Neither of the JAK inhibitors affected the decay of iNOS mRNA when determined by actinomycin D assay. Our results suggest that the inhibition of JAK-STAT1-pathway by AG-490 or WHI-P154 leads to the attenuation of iNOS expression and NO production in IFN-gamma-stimulated macrophages.

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Figures

Figure 1
Figure 1
(a) Time-dependent activation (Tyr701 phosphorylation) of signal transducer and activator of transcription 1 (STAT1) by interferon-γ (IFN-γ) in J774 macrophages. Cells were treated with IFN-γ (5 ng/mL) for different times as indicated. Proteins were extracted with modified RIPA-buffer, and the protein contents were measured. Equal amounts of lysates (20 μg protein) were subjected to immunoblot analysis with antibody specific for STAT1 phosphorylated at the tyrosine residue 701. Similar results were obtained in three independent experiments. (b) Time-dependent nuclear translocation of STAT1α in IFN-γ-stimulated J774 macrophages. Cells were treated with IFN-γ (5 ng/mL) for different times as indicated. The nuclear proteins were extracted as described in materials and methods. The protein content of the samples was measured, and equal amounts of proteins (20 μg) were subjected to immunoblot analysis with antibody against STAT1α. Similar results were obtained in two independent experiments.
Figure 1
Figure 1
(a) Time-dependent activation (Tyr701 phosphorylation) of signal transducer and activator of transcription 1 (STAT1) by interferon-γ (IFN-γ) in J774 macrophages. Cells were treated with IFN-γ (5 ng/mL) for different times as indicated. Proteins were extracted with modified RIPA-buffer, and the protein contents were measured. Equal amounts of lysates (20 μg protein) were subjected to immunoblot analysis with antibody specific for STAT1 phosphorylated at the tyrosine residue 701. Similar results were obtained in three independent experiments. (b) Time-dependent nuclear translocation of STAT1α in IFN-γ-stimulated J774 macrophages. Cells were treated with IFN-γ (5 ng/mL) for different times as indicated. The nuclear proteins were extracted as described in materials and methods. The protein content of the samples was measured, and equal amounts of proteins (20 μg) were subjected to immunoblot analysis with antibody against STAT1α. Similar results were obtained in two independent experiments.
Figure 2
Figure 2
Effects of AG-490 and WHI-P154 on nuclear translocation of STAT1α in IFN-γ-stimulated J774 macrophages. The cells were pretreated with (a) AG-490 or (b) WHI-P154 for 30 minutes. Thereafter, the medium was replaced with fresh medium containing the combination of the inhibitor and IFN-γ (5 ng/mL). The cells were incubated for another 30 minutes, and the nuclear proteins were extracted as described in materials and methods. The protein content of the samples was measured and equal amounts (20 μg) were subjected to immunoblot analysis with antibody against STAT1α. The results are expressed as mean ± SEM (n = 2–3 for AG-490 and n = 4 for WHI-P154).
Figure 3
Figure 3
Effects of (a) AG-490 and (b) WHI-P154 on IFN-γ-induced nitric oxide (NO) generation in J774 macrophages. After 24-hour incubation with IFN-γ (5 ng/mL), the supernatants were collected and nitrite was measured in the culture medium as an indicator of NO production by Griess reaction. The values are mean ± SEM (n = 6), *P < .05, and **P < .01 when compared to cells treated with IFN-γ alone.
Figure 4
Figure 4
Effects of (a) AG-490 and (b) WHI-P154 on iNOS protein expression in J774 macrophages. Cells were incubated with IFN-γ (5 ng/mL) in the presence or in the absence of the tested compound for 24 hours. Cells were lysed and the protein content of the lysates was measured, and equal amounts of proteins (20 μg) were subjected to immunoblot analysis with an antibody against iNOS. The results are shown as mean ± SEM (n = 6 for AG-490 and n = 4 for WHI-P154), **P < .01 when compared to cells treated with IFN-γ alone.
Figure 5
Figure 5
Effects of JAK inhibitors, AG-490 and WHI-P154, on IFN-γ-induced iNOS mRNA expression and degradation in J774 macrophages. (a) Cells were incubated with IFN-γ (5 ng/mL) in the absence or in the presence of AG-490 and WHI-P154 (10 μM). The cells were disrupted after 4-hour incubation and total RNA was collected. Isolated RNA was converted to cDNA. iNOS and GAPDH mRNAs were measured by quantitative PCR, and iNOS mRNA levels were normalized against GAPDH mRNA. The results are expressed as mean ± SEM, n = 3. **P < .01 when compared to cells treated with IFN-γ alone. (b) Cells were incubated as in (a) except that actinomycin D (actD) (0.1 μg/mL) was added after 6-hour incubation to stop transcription. Incubations were terminated at the indicated time points after addition of actD into the culture medium. Total RNA was isolated and converted to cDNA. iNOS and GAPDH mRNA were measured by quantitative PCR. iNOS mRNA levels were normalized against GAPDH mRNA. The results are expressed as mean ± SEM, n = 3.
Figure 5
Figure 5
Effects of JAK inhibitors, AG-490 and WHI-P154, on IFN-γ-induced iNOS mRNA expression and degradation in J774 macrophages. (a) Cells were incubated with IFN-γ (5 ng/mL) in the absence or in the presence of AG-490 and WHI-P154 (10 μM). The cells were disrupted after 4-hour incubation and total RNA was collected. Isolated RNA was converted to cDNA. iNOS and GAPDH mRNAs were measured by quantitative PCR, and iNOS mRNA levels were normalized against GAPDH mRNA. The results are expressed as mean ± SEM, n = 3. **P < .01 when compared to cells treated with IFN-γ alone. (b) Cells were incubated as in (a) except that actinomycin D (actD) (0.1 μg/mL) was added after 6-hour incubation to stop transcription. Incubations were terminated at the indicated time points after addition of actD into the culture medium. Total RNA was isolated and converted to cDNA. iNOS and GAPDH mRNA were measured by quantitative PCR. iNOS mRNA levels were normalized against GAPDH mRNA. The results are expressed as mean ± SEM, n = 3.
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