MLK3 Regulates Inflammatory Response via Activation of AP-1 Pathway in HEK293 and RAW264.7 Cells

Abstract

Inflammation is a critically important barrier found in innate immunity. However, severe and sustained inflammatory conditions are regarded as causes of many different serious diseases, such as cancer, atherosclerosis, and diabetes. Although numerous studies have addressed how inflammatory responses proceed and what kinds of proteins and cells are involved, the exact mechanism and protein components regulating inflammatory reactions are not fully understood. In this paper, to determine the regulatory role of mixed lineage kinase 3 (MLK3), which functions as mitogen-activated protein kinase kinase kinase (MAP3K) in cancer cells in inflammatory response to macrophages, we employed an overexpression strategy with MLK3 in HEK293 cells and used its inhibitor URMC-099 in lipopolysaccharide (LPS)-treated RAW264.7 cells. It was found that overexpressed MLK3 increased the mRNA expression of inflammatory genes (COX-2, IL-6, and TNF-α) via the activation of AP-1, according to a luciferase assay carried out with AP-1-Luc. Overexpression of MLK3 also induced phosphorylation of MAPKK (MEK1/2, MKK3/6, and MKK4/7), MAPK (ERK, p38, and JNK), and AP-1 subunits (c-Jun, c-Fos, and FRA-1). Phosphorylation of MLK3 was also observed in RAW264.7 cells stimulated by LPS, Pam3CSK, and poly(I:C). Finally, inhibition of MLK3 by URMC-099 reduced the expression of COX-2 and CCL-12, phosphorylation of c-Jun, luciferase activity mediated by AP-1, and phosphorylation of MAPK in LPS-treated RAW264.7 cells. Taken together, our findings strongly suggest that MLK3 plays a central role in controlling AP-1-mediated inflammatory responses in macrophages and that this enzyme can serve as a target molecule for treating AP-1-mediated inflammatory diseases.

Transcriptomic Analysis of Human Skeletal Muscle in Response to Aerobic Exercise and Protein Intake

“…FOS plays an important role in the gene interaction network of muscle in response to acute AE both in the YM and the OM, and it was the gene which was most upregulated in response to acute AE but was downregulated in response to PI and acute AE combined with PI. As a regulator of cell proliferation, differentiation, and transformation, FOS was involved in multiple inflammation pathways [81], which was also suggested by the results of GSEA in the present study. As the hub gene identified in response to acute AE combined with PI in the YM, DDIT3 is implicated in adipogenesis and erythropoiesis, is activated by endoplasmic reticulum stress, promotes apoptosis, and plays a crucial role in endoplasmic reticulum stress-induced apoptosis and autophagy, host innate immunity repression, and viral infection facilitation [82].…”

MIF Regulates M1 Macrophage Polarization via CD74/CXCR2/JNK Pathway and Mediates Aortic Dissection in Mice

“…After phosphorylation, c-Jun translocated into the nucleus, and the activation of the nuclear AP-1 led to the increase of mRNA expression of pro-inflammatory gene, as well as the production of pro-inflammatory cytokines and chemerins, including TNF-α, IL-6, COX-2 and CCL-12, etc. 40 The expression and secretion of MMP9 also depended on the activity of AP-1. 41 In macrophages, LPS may initiate MAPK signaling cascade via TLR4/MyD88, activate AP-1 in the nucleus, and contribute to the release of pro-inflammatory factor.…”