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.

The interleukin-1 (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 distinct genes in humans and mice. IL-1–type cytokines are major mediators of innate immune reactions, and blockade of the founding members IL-1α or IL-1β by the interleukin-1 receptor antagonist (IL-1RA) has demonstrated a central role of IL-1 in a number of human autoinflammatory diseases. IL-1α or IL-1β rapidly increase messenger RNA expression of hundreds of genes in multiple different cell types. The potent proinflammatory activities of IL-1α and IL-1β are restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signal transduction. This pathway summarizes extracellular and intracellular signaling of IL-1α or IL-1β, including positive- and negative-feedback mechanisms that amplify or terminate the IL-1 response. In response to ligand binding of the receptor, a complex sequence of combinatorial phosphorylation and ubiquitination events results in activation of nuclear factor κB signaling and the JNK and p38 mitogen-activated protein kinase pathways, which, cooperatively, induce the expression of canonical IL-1 target genes (such as IL-6, IL-8, MCP-1, COX-2, IκBα, IL-1α, IL-1β, MKP-1) by transcriptional and posttranscriptional mechanisms. Of note, most intracellular components that participate in the cellular response to IL-1 also mediate responses to other cytokines (IL-18 and IL-33), Toll-like-receptors (TLRs), and many forms of cytotoxic stresses.

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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.