Role of ERK in IRF3-Mediated Immune Responses

Abstract

Insulin resistance precedes and contributes to the development of type 2 diabetes mellitus, and it is now believed that chronic inflammation is a major contributor to insulin resistance. Both cellular and secreted factors participate in the pathological and physiological changes that occur to promote inflammation. However, the molecular signaling pathways that drive these processes remain elusive. The mitogen activated protein kinase, extracellular signal-regulated kinase 1 and 2 module (ERK1/2), and the transcription factor, interferon regulatory factor-3 (IRF3), are both activated downstream of Toll-like receptor 4 and associated with the development of insulin resistance. We examined whether inhibition of ERK activity blocked IRF3-mediated immune responses. ERK1 and ERK2 isoforms can have different cellular functions; thus, to determine which ERK isoform is involved in the regulation of IRF3 activity, we performed siRNA to knockdown either ERK1, ERK2, or both in RAW 264.7 macrophages and saw 70% knockdown of each ERK1 and ERK2. Knockdown of ERK1 or ERK2 or both blocked IRF3 translocation to the nucleus with LPS treatment (100 ng/ml, 1 hour), as determined by immunofluorescence using an antibody specific for nuclear IRF3. Because phosphorylated and dimerized IRF3 translocates to the nucleus to regulate the transcription of interferon (IFN)-beta expression, we investigated whether ERK1 or ERK2 inhibited IFN-beta release. Macrophages were treated with LPS (100 ng/ml, 6 hour). LPS induced an IFN-beta production of 791±18.0 pg/ml, but knockdown of either ERK1 or ERK2 decreased the release of IFN-beta to 199±12.3 and 282±32.7 pg/ml, respectively. However, double knockdown of ERK1 and ERK 2 had the greatest inhibition of IFN-beta release (121±15.2 pg/ml). In summary, both ERK1 and ERK2 regulate IRF3 nuclear translocation and signaling in macrophages. We propose that ERK positively regulates IRF3-mediated immune responses and inhibition of ERK signaling will protect against insulin resistance. Disclosure P.F. Chang: None. D. Acevedo: None. S.M. Reyna: None.