HaCaT Keratinocytes Overexpressing the S100 Proteins S100A8 and S100A9 Show Increased NADPH Oxidase and NF-κB Activities

Abstract

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.

S100A8 and S100A9 Are Novel Nuclear Factor Kappa B Target Genes During Malignant Progression of Murine and Human Liver Carcinogenesis†

“…20 In line with this observation, we found that S100A8 and S100A9 co-expression enhances ROS levels in HCC cells that were even more pronounced in TNF-␣-stimulated cells. Numerous studies demonstrate a causal link between ROS and molecular mechanisms of liver carcinogenesis and defined ROS as a common denominator of signaling pathways induced by different HCC-promoting agents.…”

“…There is evidence that enhanced production of ROS is a common response to all described HCCinducing causes. Because S100A8 and S100A9 overexpression in HaCaT keratinocytes (immortalized human keratinocytes) increases nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and enhances ROS levels, 20 we asked whether elevated S100A8 and S100A9 protein levels induce ROS production also in HCC cells. Hep3B cells were transfected with S100A8 and S100A9 expression plasmids, and ROS levels were measured by DCFDA fluorescence staining using flow cytometry analysis.…”

Psoriasin (S100A7) increases the expression of ROS and VEGF and acts through RAGE to promote endothelial cell proliferation

“…Low levels of ROS may induce the proliferation of different cell types specifically endothelial cells [39]. In HaCaT keratinocytes overexpression of S100A8 and S100A9 promoted NADPH oxidase activation followed by higher levels of ROS generation [40]. We hypothesized that psoriasin may induce low levels of ROS by a similar mechanism, leading to a further increase in ROS levels, VEGF expression and cell growth.…”

Double‐stranded RNA induces S100 gene expression by a cycloheximide‐sensitive factor

“…S100A8 and S100A9 are predominately expressed in cells of the myeloid lineage. Beside their constitutive expression in myeloid cells, they are expressed in epithelial cells in response to stress [14,15]. Their expression is transiently induced in keratinocytes after epidermal injury [16,17], after UVB irradiation [18], and in response to proinflammatory cytokines such as TNFa and IL1b [14].…”

“…Beside their constitutive expression in myeloid cells, they are expressed in epithelial cells in response to stress [14,15]. Their expression is transiently induced in keratinocytes after epidermal injury [16,17], after UVB irradiation [18], and in response to proinflammatory cytokines such as TNFa and IL1b [14]. Data showing that the two S100 proteins are up regulated by the putative TLR ligand LPS are conflicting [19,20].…”