Session Title: Innate Immunity and Rheumatic Disease
Session Type: Abstract Submissions (ACR)
MAPK kinases MKK3 and MKK6 regulate p38 function in inflammatory diseases like rheumatoid arthritis (RA). Targeting MKK3 or MKK6 might be superior to traditional p38 blockade because MKK-deficiency maintains p38-mediated anti-inflammatory responses. Production of IL-10 and the de-activating phosphatase Dusp1 is higher in activated MKK-deficient bone marrow-derived macrophages (BMDM) compared with p38 inhibition, especially for MKK6-/- cells. The concept that MKK6 might be the optimal target in the p38 pathway was confirmed by the observation that MKK6-/- mice have lower disease severity in collagen-induced arthritis than wild type (WT) or MKK3-/- mice (Arthritis Rheum 2012;64:2887). In this study, we explored the mechanism of protection by evaluating differential regulation of IL-10 and Dusp1 expression in MKK3-/-, MKK6-/- and p38 inhibitor-treated BMDMs.
Bone marrow isolated from WT, MKK3-/- and MKK6-/- mice was differentiated into BMDMs in vitro (n=3/group). The cells were then treated with LPS (100ng/ml) for various times. Gene expression was determined by qPCR in LPS-stimulated BMDM ( 4h). mRNA half life was measured in LPS-treated BMDM incubated with actinomycin D ± SB203580 (p38 inhibitor) by qPCR. De novo mRNA synthesis was quantified in BMDM treated with ethynyl-uridine ± LPS for 1h. The RNA was biotinylated using Click-iT Nascent RNA capture kit (Invitrogen). Nascent RNA was isolated with 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 medium-treated cells.
We first determined the effect of MKK-deficiency or p38 inhibitor on the de novo synthesis of IL-10 mRNA in response to LPS. Pre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 transcription in WT cells by 75±8% compared with control (n=3 mice/group, p<0.0001). Compared with WT, IL-10 induction in MKK3-/- was decreased by 60±7% (p<0.0001), whereas inhibition in MKK6-/- cells was minimal (16±3%, p>0.05). We also evaluated levels of Dusp1, a p38 phosphatase that is regulated transcriptionally by p38 and de-activates other MAPKs. Dusp1 expression was significantly reduced by 38±6% (p=0.002) in p38 inhibitor-treated and 46±5% (p=0.004) in MKK3-/- BMDM but not in MKK6-/- cells. We then measured the effect of MKK-deficiency and a p38 inhibitor on mRNA decay of IL-10. WT BMDM treated with p38 inhibitor showed a significantly higher IL-10 mRNA decay rate (t1/2 = 30 min, n=3, p<0.005) compared with WT control (t1/2 = 54 min). Surprisingly, IL-10 decay rates were similar in WT, MKK3-/- and MKK6-/- groups (t1/2 MKK3-/- = 45 min, MKK6-/- = 41 min), indicating that unlike p38 inhibition, MKK-deficiency does not alter IL-10 mRNA stability.
Suppressed expression of IL-10 and Dusp1 by MKK3-deficiency or p38 inhibition occurs primarily at the transcriptional level, but gene transcription is maintained in MKK6-/- cells. Preservation of Dusp1 expression in MKK6 deficiency permits de-activation of other MAPKs and terminates the inflammatory cascade. Together these data suggest that MKK6 is a potential therapeutic target in RA.
G. S. Firestein,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differential-regulation-of-il-10-and-dusp1-production-by-kinases-in-the-p38-mapk-pathway/