Background/Purpose:

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.

Methods:

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.

Results:

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.

Conclusion:

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.