Differential regulation of anti-inflammatory genes by p38 MAP kinase and MAP kinase kinase 6
© Hammaker et al.; licensee BioMed Central Ltd. 2014
Received: 17 December 2013
Accepted: 8 May 2014
Published: 16 May 2014
Conventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3−/−, MKK6−/−, and p38 inhibitor-treated wildtype cells.
BMDM from wild type (WT), MKK3−/−, and MKK6−/− mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.
Pre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3−/− BMDM compared with either WT or MKK6−/− cells. IL-10 mRNA expression was modestly reduced in MKK3−/− BMDM but was preserved in MKK6−/− cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3−/− and p38 inhibitor pre-treated cells, but not MKK6−/− cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6−/− cells.
Unlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA.
Keywordsp38 inhibitor Rheumatoid arthritis IL-10 MKK6 Tristetraprolin Anti-inflammatory response
Several highly specific p38α inhibitors that competitively bind the ATP binding pocket have been developed and evaluated in RA . Despite promising pre-clinical data, these compounds show little therapeutic efficacy [2, 3]. Several studies suggested that the unexpected lack of benefit in RA is due to the role of p38 in limiting inflammation, which might be blocked by the p38 inhibitor . For instance, p38α down-regulates its own activity by inducing expression of dual specificity phosphatase 1 (DUSP1), which de-phosphorylates and inactivates p38 and JNK [5, 6]. p38α also phosphorylates TAB1, leading to the inhibition of TAK1, a MAP3K that activates the p38 and JNK pathways . p38α decreases MKK6 mRNA stability under basal conditions, providing negative feedback to its own signaling cascade . Perhaps most relevant to the resolution of inflammation, p38α is required for the synthesis of IL-10, a potent anti-inflammatory cytokine that inhibits IL-6 and TNF expression  as well as Tristetraprolin (TTP), an RNA-binding protein that promotes the degradation of inflammatory cytokine mRNA [10, 11]. Blocking these anti-inflammatory roles of p38α during and after inflammation might explain the lack of long-term efficacy of p38α inhibitors.
Our previous studies demonstrated that targeting either MKK3 or MKK6, which are the primary upstream activators of p38, might be superior to p38α blockade by preserving these anti-inflammatory responses. We recently showed that p38αlysM mice, which lack p38α expression in macrophages, have increased arthritis severity in passive serum transfer and antigen-induced arthritis models . In contrast, MKK3- or MKK6-deficiency reduces arthritis severity and joint destruction [13, 14]. In addition, MKK3, but not MKK6, is required for optimal p38 activation in synovitis, whereas MKK6-deficiency is associated with lower IL-6, IL-17 and anti-collagen antibody production [14, 15]. In bone marrow-derived macrophages (BMDM), p38 inhibition blocks IL-10 and DUSP1 expression while MKK-deficiency partially spared these anti-inflammatory responses . It is not clear why the absence of p38α compared with MKK3 or MKK6 yields such divergent anti-inflammatory effects. In this study, we explored potential mechanisms by which p38 inhibition and MKK3- or MKK6-deficiency differentially regulates IL-10 production in BMDM. These data suggest that MKK6 is an attractive therapeutic target in the p38 pathway that preserves multiple p38-dependent anti-inflammatory pathways.
Bone marrow-derived macrophage culture (BMDM)
Bone marrow was isolated from the femur and tibia of DBA.1 WT, MKK3−/− and MKK6−/− mice and cultured in DMEM supplemented with 10% FCS and 20% L929-conditioned medium. After 7 days, the adherent BMDM were harvested, counted, and plated for use in experiments described below.
Gene and protein expression
For gene expression assays, BMDM were treated with p38 inhibitor SB203580 (SB, 3 μM), JNK inhibitor SP600125 (SP, 20 μM) and/or ERK inhibitor PD98059 (PD, 100 μM) (Calbiochem) for 1 h prior to LPS stimulation (100 ng/ml, Invivogen). After 4 hours, mRNA was isolated and processed for quantitative PCR. Expression of DUSP1 and TTP was evaluated 1 h after LPS stimulation and IL-1RA after 4 h. For cytokine analysis, cells were stimulated with LPS for 24 h and the cell supernatants were assayed using multiplex immunoassay (Bio-Rad).
Western blot analysis
BMDM were serum-starved overnight and stimulated with LPS (100 ng/ml) for various time points. The cells were lysed and 100 μg of lysate was subjected to SDS-PAGE. The proteins were transferred to a PVDF membrane and Western blot analysis was performed using anti-phospho p38 (Pp38) antibody (cat #9216, Cell Signaling Technology), total p38 antibody (cat #8690, Cell Signaling Technology), anti-β actin (cat #sc-1616, Santa Cruz Biotechnology) and anti-TTP antibody (a kind gift of Dr. Perry Blackshear, National Institute of Environmental Health Sciences, North Carolina, US).
After 4 h of LPS stimulation, WT (n = 6), MKK3−/− (n = 3), and MKK6−/− (n = 3) BMDM were treated with actinomycin D (10 μg/ml) for various times and IL-10 mRNA levels were analyzed by qPCR (Applied Biosystems) and normalized to β-actin. To determine the effect of p38 inhibitor on mRNA decay, WT cells were treated with SB (3 μM) after LPS stimulation (WT SB post) and mRNA expression was assayed as described above.
Nascent RNA transcription rate
De novo mRNA synthesis was measured in WT, MKK3−/−, and MKK6−/− BMDM treated with ethynyl-uridine with or without LPS for 1 h. The RNA was quantified and biotinylated using Click-iT Nascent RNA capture kit (Invitrogen). Nascent RNA was isolated using streptavidin-magnetic beads. Reverse transcription was performed using SuperScript VILO cDNA synthesis kit (Invitrogen) and IL-10 expression was measured by qPCR and presented as fold of LPS-treated WT cells.
Comparisons between WT, MKK3−/−, and MKK6−/− cells were analyzed by two-way or one-way ANOVA and Tukey or Dunnett multiple comparison tests, unless otherwise stated. Data were analyzed using GraphPad Prism 6.0 and the comparisons were considered statistically significant if p < 0.05.
Regulation of IL-10 protein and gene expression in MKK-deficient or p38 inhibitor- treated BMDM
Transcriptional regulation of IL-10 in MKK-deficient BMDM
Post-transcriptional regulation of IL-10 in MKK-deficient BMDM
IL-10 mRNA transcript stability in MKK-deficient cells was determined by comparing IL-10 decay rates in WT, MKK3−/−, and MKK6−/− cells (Figure 2b). IL-10 mRNA decay was rapid, with half-life of 40 minutes for MKK6−/−, 46 minutes for MKK3−/− and 54 minutes for WT BMDM. In WT cells treated with p38 inhibitor (WT SB post), IL-10 mRNA half-life of 30 minutes was significantly lower than in WT cells. These data suggest that unlike p38 inhibition, IL-10 expression in MKK3- or MKK6-deficient BMDM is primarily regulated at the transcriptional level but not by altering mRNA degradation.
Effect of p38 inhibitor or MKK-deficiency on the expression of other anti-inflammatory molecules
The p38 pathway regulates the resolution of inflammatory responses by increasing expression of anti-inflammatory cytokines to promote wound healing and homeostasis. Blocking p38 inhibits the expression of IL-10 and p38-dependent anti-inflammatory genes such as DUSP1, TTP, and IL-1RA. In contrast, MKK6-deficiency allows partial or full induction of IL-10 and TTP, which negatively regulate the inflammatory cascade. These data suggest that blocking MKK6 might be a potential therapeutic target for inflammatory diseases such as RA that avoids some of the limitations of a conventional p38 inhibitor.
Mitogen activated protein kinase
MAP kinase kinase
cJun N-terminal kinase
Bone marrow derived macrophages
SB203580 (p38α/β inhibitor)
SP600125 (JNK inhibitor)
PD98059 (ERK inhibitor)
Dual specificity phosphatase 1
IL-1 receptor antagonist
TAK1 binding protein 1
TGFβ-activated kinase 1
Tumor necrosis factor
MAP kinase activated protein kinase 2.
Funding: NIH Grant Numbers: AI-070555, AR047825.
- Kumar S, Boehm J, Lee JC: p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov. 2003, 2: 717-726. 10.1038/nrd1177.PubMedView ArticleGoogle Scholar
- Sweeney SE: The as-yet unfulfilled promise of p38 MAPK inhibitors. Nat Rev Rheumatol. 2009, 5: 475-477. 10.1038/nrrheum.2009.171.PubMedView ArticleGoogle Scholar
- Genovese MC: Inhibition of p38: has the fat lady sung?. Arthritis Rheum. 2009, 60: 317-320. 10.1002/art.24264.PubMedView ArticleGoogle Scholar
- Clark AR, Dean JL: The p38 MAPK pathway in rheumatoid arthritis: a sideways look. Open Rheumatol J. 2012, 6: 209-219. 10.2174/1874312901206010209.PubMedPubMed CentralView ArticleGoogle Scholar
- Chi H, Barry SP, Roth RJ, Wu JJ, Jones EA, Bennett AM, Flavell RA: Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc Natl Acad Sci U S A. 2006, 103: 2274-2279. 10.1073/pnas.0510965103.PubMedPubMed CentralView ArticleGoogle Scholar
- Hu JH, Chen T, Zhuang ZH, Kong L, Yu MC, Liu Y, Zang JW, Ge BX: Feedback control of MKP-1 expression by p38. Cell Signal. 2007, 19: 393-400. 10.1016/j.cellsig.2006.07.010.PubMedView ArticleGoogle Scholar
- Cheung PC, Campbell DG, Nebreda AR, Cohen P: Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha. EMBO J. 2003, 22: 5793-5805. 10.1093/emboj/cdg552.PubMedPubMed CentralView ArticleGoogle Scholar
- Ambrosino C, Mace G, Galban S, Fritsch C, Vintersten K, Black E, Gorospe M, Nebreda AR: Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase. Mol Cell Biol. 2003, 23: 370-381. 10.1128/MCB.23.1.370-381.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O'Garra A: IL-10 inhibits cytokine production by activated macrophages. J Immunol. 1991, 147: 3815-3822.PubMedGoogle Scholar
- Brook M, Tchen CR, Santalucia T, McIlrath J, Arthur JS, Saklatvala J, Clark AR: Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways. Mol Cell Biol. 2006, 26: 2408-2418. 10.1128/MCB.26.6.2408-2418.2006.PubMedPubMed CentralView ArticleGoogle Scholar
- Hitti E, Iakovleva T, Brook M, Deppenmeier S, Gruber AD, Radzioch D, Clark AR, Blackshear PJ, Kotlyarov A, Gaestel M: Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol Cell Biol. 2006, 26: 2399-2407. 10.1128/MCB.26.6.2399-2407.2006.PubMedPubMed CentralView ArticleGoogle Scholar
- Guma M, Hammaker D, Topolewski K, Corr M, Boyle DL, Karin M, Firestein GS: Antiinflammatory functions of p38 in mouse models of rheumatoid arthritis: advantages of targeting upstream kinases MKK-3 or MKK-6. Arthritis Rheum. 2012, 64: 2887-2895. 10.1002/art.34489.PubMedPubMed CentralView ArticleGoogle Scholar
- Inoue T, Boyle DL, Corr M, Hammaker D, Davis RJ, Flavell RA, Firestein GS: Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis. Proc Natl Acad Sci U S A. 2006, 103: 5484-5489. 10.1073/pnas.0509188103.PubMedPubMed CentralView ArticleGoogle Scholar
- Hammaker D, Topolewski K, Edgar M, Yoshizawa T, Fukushima A, Boyle DL, Burak EC, Sah RL, Firestein GS: Decreased collagen-induced arthritis severity and adaptive immunity in MKK-6-deficient mice. Arthritis Rheum. 2012, 64: 678-687. 10.1002/art.33359.PubMedPubMed CentralView ArticleGoogle Scholar
- Yoshizawa T, Hammaker D, Boyle DL, Corr M, Flavell R, Davis R, Schett G, Firestein GS: Role of MAPK kinase 6 in arthritis: distinct mechanism of action in inflammation and cytokine expression. J Immunol. 2009, 183: 1360-1367. 10.4049/jimmunol.0900483.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang N, Gates KL, Trejo H, Favoreto S, Schleimer RP, Sznajder JI, Beitel GJ, Sporn PH: Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage. FASEB J. 2010, 24: 2178-2190. 10.1096/fj.09-136895.PubMedPubMed CentralView ArticleGoogle Scholar
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