Regulation of inflammatory gene expression and acute inflammatory response by MKP-1

Riku Korhonen1,2, Tuija Hömmö1,2, Noora Huotari1,2, Ville Taimi1,2, Mirka Laavola1,2, Riina Nieminen1,2, Tiina Leppänen1,2, Eeva Moilanen1,2. 1The Immunopharmacology Research Group, University of Tampere School of Medicine, Finland, 2Tampere University Hospital, Tampere, Finland

MAP kinase phosphatase-1 (MKP-1) is an endogenous inhibitor of the stress-activated MAP kinases p38 and JNK. In the present study, we investigated the effect of MKP-1 on inflammatory gene expression in J774 mouse macrophages and A549 human lung epithelial cells transfected with MKP-1 siRNA, and in peritoneal macrophages (PMs) or bone marrow-derived macrophages (BMMs) from WT and MKP-1 KO mice. The effect of MKP-1 on inflammatory response in vivo was investigated in acute carrageenan-induced paw inflammation in WT and MKP-1 KO mice. MKP-1 protein levels and p38 phosphorylation was detected by Western blot. IL-6, IL-8, TNF, IL-12p40 were measured by ELISA, and COX-2 protein was detected by Western blot after 24 h incubation with LPS or CM. IRF1 mRNA levels were measured by quantitative RT-PCR. Statistical significance was tested with One-way or repeated Two-way ANOVA with Bonferroni’s or Dunnett’s post-test.

The expression of inflammatory genes IL-6, TNF, IL-8 and COX-2 was induced by cytokine mixture (CM: TNF, IFNγ and IL-1β, 10 ng/mL each) in A549 cells, or by LPS (10 ng/mL in J774 cells and 100 ng/mL inPMs) in 24 h incubation. p38 MAPK inhibitors SB202190 (1 µM) and BIRB 796 (100 nM) inhibited inflammatory gene expression in A549 cells (40-78% inhibition in IL-6 and IL-8), J774 cells (48-76% inhibition in IL-6 and COX-2) and in PMs (62% inhibition in TNF). Silencing of MKP-1 by siRNA enhanced p38 phosphorylation at 1h induced by CM or LPS in A549 and J774 cells, respectively. Silencing of MKP-1 by siRNA increased IL-6 (42 ± 2 ng/mL vs. 24 ± 1 ng/mL in MKP-1 siRNA vs. control siRNA treated cells, p<0.001, n=6) and IL-8 (159 ± 27 ng/mL vs. 78 ± 5 ng/mL in MKP-1 siRNA vs.control siRNA treated cells, p<0.01, n=6) production in A549 cells. Also TNF (4201 ± 201 pg/mL vs. 2494 ± 159 pg/mL in MKP-1 siRNA vs. control siRNA treated cells, p<0.01, n=6) and IL-6 (2648 ± 355 pg/mL vs. 1675 ± 80 pg/mL in MKP-1 siRNA vs. control siRNA treated cells, p<0.01, n=6) production was increased by MKP-1 siRNA in J774 cells. TNF (1785 ± 178 pg/mL vs. 949 ± 95 pg/mL, p<0.001, n=3) and IL-6 (3126 ± 586 pg/mL vs. 1263 ± 233 pg/mL, p<0.001, n=3) production as well as COX-2 levels (418% vs. 100%, p<0.001, n=3) were increased in BMMs from MKP-1 KO mice as compared to those from WT mice. Further, carrageenan-induced paw edema response was increased by 50% in MKP-1 KO mice as compared to that in WT mice at 3 h (p<0.05, n=6). IL-12 is a fundamental cytokine to induce cell-mediated antimicrobial responses. Interestingly, LPS-induced IL-12p40 expression was suppressed in PMs from MKP-1 KO mice (990 ± 52 pg/mL vs. 1828 ± 86 pg/mL in PMs from MKP-1 KO and WT mice, p<0.001, n=4). This was related to suppressed IRF1 mRNA levels induced by LPS at 3h (65 ± 4% vs. 100% in PMs from MKP-1 KO and WT mice, p<0.001, n=6), which is interesting as IRF1 is a key transcription factor for IL-12.

In conclusion, our result suggests that MKP-1 suppresses inflammatory gene expression and acute inflammatory response, and supports appropriate antimicrobial defense by regulating p38 MAPK activity. Compounds that increase MKP-1 expression or enhance its function may hold potential as anti-inflammatory drugs.