Presentation: Differential Regulation of Cytokines by MKK3 and MKK6 in Synoviocytes: Complementary Signaling Pathways (2007)

131 Differential Regulation of Cytokines by MKK3 and MKK6 in Synoviocytes: Complementary Signaling Pathways

Purpose: The crucial role of p38 MAP kinase in cytokine production suggests that might serve as a therapeutic target in RA. Because p38 participates in many functions, systemic inhibition can suppress host defense or interfere with homeostasis. Upstream kinases MKK3 and MKK6, which independently regulate p38, could potentially regulate a subset of inflammatory responses while leaving others intact. Because deficiency of either kinase suppresses passive K/BxN arthritis, we examined functional consequences of MKK3 or 6 deficiency on fibroblast-like synoviocyte (FLS) function.
Methods: Wild type (WT), MKK3-/-, and MKK6-/- FLS lines (n=3 each) were stimulated for 15 min (Western blot) or 18 hours (cytokine analysis of supernatants) with IL-1β (2ng/ml), TNFα (50ng/ml) or LPS (1μg/ml). Cell culture supernatants were analyzed by Luminex multiplex assay to measure MCP-1, G-CSF, GM-CSF, IL-15, IP-10, RANTES, MIP1α, KC (IL-8), and IL-6.
Results: IL-1- and TNF-induced P-p38 expression was decreased 45±6% in MKK3-/- FLS compared to WT FLS (n=3 each). Surprisingly, P-p38 induction in MKK6-/- FLS was the same as WT. Despite differences in P-p38 regulation in the two knockout strains, IL-1-induced P-MK2 (a p38 substrate) was significantly decreased in both MKK3-/- and MKK6-/- FLS compared with WT (54±3% and 48±7% inhibition, respectively; n=3 each, p=0.02). To determine functional consequences of MKK deficiency, MKK3-/- and MKK6-/- FLS were stimulated with IL-1β, TNFα or LPS. Cytokine production was determined by multiplex analysis. The table below lists the cytokines that were inhibited by ≥40% in response to IL-1β, TNFα or LPS in MKK deficient FLS. Note that the cytokine profiles regulated by the two MKKs are complementary.
IL-1βTNFαLPS
MKK3-/-
n=3
MIP1α, G-CSF, MCP-1IP-10None
MKK6-/-
n=3
NoneIL-6, MIP1α, KCIL-6, MIP1α, KC, GM-CSF

Conclusion: MKK3 and MKK6 independently activate p38 and MK2 with normal phosphorylation of the p38 by despite MKK6 deficiency. While MKK3 and MKK6 both converge on p38 and MK2, they regulate distinct, complementary groups of cytokines because IL-1-induced cytokine expression requires MKK3 while TNF and LPS responses require MKK6. Based on the differing cytokine and signaling profiles of MKK3 and MKK6, selective targeting of an individual MKK could provide benefit of p38 inhibition while limiting potential for systemic effects.

 D.R. Hammaker, None; D.L. Boyle, None; G.S. Firestein, None.