Interleukin (IL)-8, a prototypic human chemokine, was detected more than a decade ago as the founding member of the chemokine superfamily. One of the most remarkable properties of IL-8 is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by ten- to 100-fold in response to proinflammatory cytokines such as tumor necrosis factor or IL-1, bacterial or viral products, and cellular stress. Recently, significant advances in the understanding of signaling pathways, which coordinately regulate IL-8 transcription as well as mRNA stabilization in response to external stimuli, have been made. Maximal IL-8 amounts are generated by a combination of three different mechanisms: first, derepression of the gene promoter; second, transcriptional activation of the gene by nuclear factor-κB and JUN-N-terminal protein kinase pathways; and third, stabilization of the mRNA by the p38 mitogen-activated protein kinase pathway. In that way, cells are able to rapidly increase and at the same time, to fine-tune the amount of IL-8 secreted and thereby control the extent of leukocytes attracted to sites of tissue injury.

Phosphorylation of NF-κB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IκB kinase (IKK)-α/β (the best characterized Ser-536 kinases so far) or the IKKβ inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKα/β-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKα, IKKβ, IKKϵ, and TBK1. Overexpressed IKKϵ and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter.

A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-κB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-κB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-κB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-κB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation.

Approximately one-quarter of circulating cobalamin (vitamin B-12) binds to transcobalamin (holoTC) and is thereby available for the cells of the body

Binding sites for the dimeric transcription factor activator protein (AP)-1 are found in numerous immunoregulatory and inflammatory genes. The precise mechanisms by which AP-1 activates or represses immune response genes and in particular the roles of individual AP-1 subunits in inflammatory responses are largely unknown. We report here that c-Fos and Fos-related antigen-1 (Fra-1), two inducible components of AP-1, are recruited to the endogenous interleukin (IL)-8 promoter in an IL-1-dependent manner. c-Fos activates IL-8 transcription and synergizes in this effect with p65 NF-κB. In contrast, Fra-1 strongly inhibits inducible IL-8 transcription. Fra-1 activation involves its stabilization, ubiquitination, and interaction with histone deacetylase-1. Blockade of MEK1 by PD98059 suppresses c-Fos and Fra-1 expression and, thus, affects two counteractive signals for IL-8 mRNA synthesis simultaneously. This disturbs the inducible recruitment of TATA box-binding protein and RNA polymerase II to the IL-8 promoter. Additional experiments reveal that, in conjunction with p65 NF-κB, the MEK1-ERK-dependent synthesis of c-Fos and Fra-1 serves to adjust the overall expression level of IL-8 in response to two of its physiological inducers, IL-1 and epidermal growth factor. Relative to c-Fos, the delayed recruitment of Fra-1 to the IL-8 promoter provides an example how AP-1 subunits may dampen excessive chemokine synthesis.

Streptococcus pneumoniae is the major cause of community-acquired pneumonia and one of the most common causes of death by infectious disease in industrialized countries. Little is known concerning the mechanisms of target cell activation in this disease. The present study shows that NF-κB and p38 MAPK signaling pathways contribute to chemokine synthesis by lung epithelial cells in response to pneumococci. In infected lungs of mice pneumococci stimulate expression of the interleukin (IL)-8 homolog keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor, as well as activate p38 MAPK. Human bronchial epithelium was chosen as a cellular model, because it establishes the first barrier against pathogens, and little is known about its function in innate immunity. Pneumococci infection induces expression of IL-8 and granulocyte-macrophage colony-stimulating factor as well as activation of p38 MAPK in human bronchial epithelial cells (BEAS-2B). Inhibition of p38 MAPK activity by SB202190 and SB203580 blocks pneumococci-induced cytokine release. In mouse lungs in vivo as well as in cultured cells, pneumococci activate NF-κBinanIκB kinase-dependent manner. Inhibition of p38 MAPK by chemical inhibitors or by RNA interference targeting p38α reduces pneumococci-induced NF-κB-dependent gene transcription. Blockade of p38 activity did not affect inducible nuclear translocation and recruitment of NF-κB/RelA to the IL-8 promotor but did reduce the level of phosphorylated RelA (serine 536) at IL-8 promotor and inhibited pneumococci-mediated recruitment of RNA polymerase II to IL-8 promotor. Thus, p38 MAPK contributes to pneumococci-induced chemokine transcription by modulating p65 NF-κB-mediated transactivation.