No TNF-α Secretion Without ERK Activation

Abstract

When stimulated by the microbial product lipopolysaccharide (LPS), macrophages secrete the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). LPS stimulation of macrophages results in the sequential activation of the mitogen-activated protein kinase (MAPK) kinase kinase TPL2; the MAPK kinases MKK1 and MKK2; and the MAPKs extracellular signal-regulated kinase (ERK) 1 and 2. Macrophages from mice deficient in TPL2 fail to secrete TNF-α when stimulated with LPS, which suggests that TPL2 might be targeted in the treatment of inflammatory disorders. Rousseau et al . studied LPS-stimulated secretion of TNF-α in a mouse macrophage cell line (RAW24.7 cells). Although inhibition of MKK1 blocked secretion of TNF-α from LPS-stimulated RAW24.7 cells, as measured by enzyme-linked immunosorbent assay, production of the precursor form of TNF-α, as detected by analyses of Western blots of cell lysates, was barely affected. The abundance of pre-TNF-α in lysates of LPS-treated TPL2-deficient macrophages was similar to that detected in LPS-treated wild-type macrophages, consistent with a role for ERK activation in the processing of pre-TNF-α. Immunogold electron microscopy demonstrated that inhibition of ERK activity did not interfere with the intracellular trafficking of pre-TNF-α from the Golgi through the secretory pathway; however, immunofluorescence staining showed that inhibition of ERK activation prevented LPS-induced localization of pre-TNF-α to the cell surface. TNF-α-converting enzyme (TACE) is thought to form a complex with pre-TNF-α that localizes it to the surface of the cell, where TACE processes pre-TNF-α to produce mature TNF-α. Using a phosphospecific antibody, the authors showed that Thr 735 of TACE was phosphorylated by ERK1/2 in LPS-treated wild-type, but not TPL2-deficient, macrophages. This study shows that rather than interfering with TNF-α production at the level of gene expression, as had been previously thought, the TPL2-MKK1-ERK1/2 pathway regulates the processing of pre-TNF-α instead. S. Rousseau, M. Papoutsopoulou, A. Symons, D. Cook, J. M. Lucocq, A. R. Prescott, A. O’Garra, S. C. Ley, P. Cohen, TPL2-mediated activation of ERK1 and ERK2 regulates the processing of pre-TNFα in LPS-stimulated macrophages. J. Cell Sci. 121 , 149-154 (2008). [Abstract] [Full Text]