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Role of MAPK Kinase 6 in Arthritis: Distinct Mechanism of Action in Inflammation and Cytokine Expression¹

Toshio Yoshizawa^2,*, Deepa Hammaker^2,3,*, David L. Boyle^*, Maripat Corr^*, Richard Flavell, Roger Davis, Georg Schett and Gary S. Firestein^3,*

^* Division of Rheumatology, Allergy and Immunology, University of California San Diego, School of Medicine, La Jolla, CA 92093; Department of Immunobiology, Yale University, New Haven, CT 06520; Department of Molecular Medicine, University of Massachusetts, Worcester, MA 01605; and Friedrich-Alexander-University, Erlangen, Germany

Development of p38 inhibitors for rheumatoid arthritis has been hindered by toxicity and limited efficacy. Therefore, we evaluated whether MKK6, an upstream kinase that regulates multiple p38 isoforms, might be an alternative therapeutic target in inflammatory arthritis. Wild-type (WT), MKK6^–/–, and MKK3^–/– mice were administered K/BxN serum to induce arthritis. Articular expression of activated kinases and cytokines was determined by Western blot, qPCR, ELISA, and multiplex analysis. Immunoprecipitation and confocal microscopy experiments were performed to determine the subcellular location of MKK6, P-p38, and MAPKAPK2 (MK2). Arthritis scores were significantly lower in MKK6^–/– mice compared with WT mice. Joint destruction and osteoclast differentiation were lower in MKK6^–/–, as were articular IL-6 and matrix metalloproteinase-3 expression. Phospho-p38 levels were modestly decreased in the joints of arthritic MKK6^–/– mice compared with WT but were significantly higher than MKK3^–/– mice. P-MK2 was low in MKK6^–/– and MKK3^–/– mice. Uncoupled p38 and MK2 activation was also observed in cultured, MKK6^–/– FLS and confirmed using kinase assays. Immunoprecipitation assays and confocal microscopy showed that P-p38 and MK2 colocalized in activated WT but not MKK6^–/– FLS. Distinct patterns of cytokine production were observed in MKK6^–/– and MKK3^–/– cells. MKK6 deficiency suppresses inflammatory arthritis and joint destruction, suggesting it might be a therapeutic target for inflammation. Although MKK3 and MKK6 activate the p38 pathway, they regulate distinct subsets of proinflammatory cytokines. MKK6 appears mainly to facilitate p38 and MK2 colocalization in the nucleus rather than to phosphorylate p38.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant AI070555 and AI067752.

² T.Y. and D.H. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Deepa Hammaker and Dr. Gary S. Firestein, University of California San Diego, 9500 Gilman Drive, Mail Code 0656, La Jolla, CA 92093. E-mail address: dhammaker{at}ucsd.edu

⁴ Abbreviations used in this paper: RA, rheumatoid arthritis; MMP, matrix metalloproteinase; MKK, MAPK kinase; MAPKAPK2 (MK2), MAPK activated protein kinase 2; RIPA, radioimmunoprecipitation assay; FLS, fibroblast-like synoviocytes; WT, wild type; P, phospho; P-MSK1, stress-activated protein kinase; MIP1, macrophage inflammatory protein 1; KC, mouse IL-8; IP-10, interferon induced protein-10.