The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

The calcium- and arachidonic acid (AA)-binding proteins S100A8 and S100A9 are involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in phagocytes. They are specifically expressed in myeloid cells, and are also found in epithelial cells in various (patho)physiological conditions. We have investigated the consequences of S100A8/A9 overexpression in epithelial cell lines on reactive oxygen species (ROS) generation and downstream signaling. Epithelial carcinoma HeLa cells, which exclusively express Nox2, showed dramatically increased activation of NADPH oxidase by phorbol 12-myristate 13-acetate after S100A8/A9 gene transfection. HaCaT keratinocytes overexpressing S100A8/A9 showed enhanced, transient ROS generation in response to the calcium ionophore A23187 compared to mock-transfected cells. Polymerase chain reaction analysis revealed mRNA transcripts for Nox1, Nox2, and Nox5 in HaCaT keratinocytes. Detailed transfection studies confirmed that NADPH oxidase activities in Nox1- and Nox5-transfected HeLa cells were enhanced after S100A8/A9 gene complementation. Furthermore, mutational analysis revealed that AA binding and Thr113 phosphorylation are important for S100A8/A9-enhanced activation of NADPH oxidase. Nuclear factor-kappaB (NF-kappaB) activation and interleukin-8 mRNA levels were increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kappaB is a redox-sensitive transcription factor. Because they are expressed in epithelia under specific conditions, S100A8 and S100A9 might be involved in skin pathogenesis by modulating aspects of downstream signaling.

The necrotrophic pathogen Gibberella pulicaris infects potato tubers through wounds that contain fungitoxic secondary metabolites such as the phytoalexins rishitin and lubimin. In order to colonize tuber tissue, the fungus must possess a mechanism to tolerate potato defense compounds. In this paper, we show that a gene, Gpabc1, that codes an ATP-binding cassette (ABC) transporter is required for tolerance to these phytoalexins and for virulence on potato. The Gpabc1 gene, isolated in the course of a differential cDNA screen, shares high sequence homology with the ABC1 gene of Magnaporthe grisea. G. pulicaris mutants deficient in Gpabc1 were still able to metabolize rishitin but lost their tolerance to this phytoalexin as well as their virulence on potato. These results strongly suggest that the Gpabc1-encoded ABC transporter is necessary for tolerance of G. pulicaris to rishitin and that this tolerance is required for virulence on potato.

Analysis of the calcium-induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF-hand. induce different conformational changes thereby affecting the calcium-induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn 2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.z 1999 Federation of European Biochemical Societies.

a b s t r a c t S100A8/A9 promotes NADPH oxidase in HaCaT keratinocytes and subsequently increases NFjB activation, which plays important roles in the balance between epidermal growth and differentiation. S100A8/A9-positive HaCaT cells present with a significantly reduced rate of cell division and greater expression of two keratinocyte differentiation markers, involucrin and filaggrin, than control cells. S100A8/A9 mutants fail to enhance NFjB activation, TNFa-induced IL-8 gene expression and NFjB p65 phosphorylation, and S100A8/A9-positive cells demonstrate better cell survival in forced suspension culture than mutant cells. S100A8/A9 is induced in epithelial cells in response to stress. Therefore, S100A8/A9-mediated growth arrest could have implications for tissue remodeling and repair.