Spontaneous melanoma phenotype switching is controlled by unknown environmental factors and may determine melanoma outcome and responsiveness to anticancer therapy. We show that Orai1 and STIM2 are highly expressed and control store-operated Ca(2+) entry in human melanoma. Lower extracellular Ca(2+) or silencing of Orai1/STIM2 caused a decrease in intracellular Ca(2+) , which correlated with enhanced proliferation and increased expression of microphthalmia-associated transcription factor, a marker for proliferative melanoma phenotype. In contrast, the invasive and migratory potential of melanoma cells was reduced upon silencing of Orai1 and/or STIM2. Accordingly, markers for a non-proliferative, tumor-maintaining phenotype such as JARID1B and Brn2 decreased. Immunohistochemical staining of primary melanomas and lymph node metastases revealed a heterogeneous distribution of Orai1 and STIM2 with elevated expression in the invasive rim of the tumor. In summary, our results support a dynamic model in which Orai1 and STIM2 inversely control melanoma growth and invasion. Pharmacological tuning of Orai1 and particularly STIM2 might thus prevent metastatic spread and render melanomas more susceptible to conventional therapy.

In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

Benzoquinones (BQ) have important functions in many biological processes. In alkaline environments, BQs can be hydroxylated at quinoid ring proton positions. Very little is known about the chemical reaction leading to these structural transformations as well as about the properties of the obtained hydroxyl benzoquinones. We analyzed the behavior of the naturally occurring 2,6-dimethoxy-1,4-benzoquinone under alkaline conditions and show that upon substitution of methoxy-groups, poly-hydroxyl-derivatives (OHBQ) are formed. The emerging compounds with one or several hydroxyl-substituents on single or fused quinone-rings exist in oxidized or reduced states and are very stable under physiological conditions. In comparison with the parent BQs, OHBQs are stronger radical scavengers and redox switchable earth-alkaline metal ligands. Considering that hydroxylated quinones appear as biosynthetic intermediates or as products of enzymatic reactions, and that BQs present in food or administered as drugs can be hydroxylated by enzymatic pathways, highlights their potential importance in biological systems.

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Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half‐life and are resistant to genetic manipulation. Neutrophil‐like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER‐HoxB8 oncoprotein, but whether these cells can replace neutrophils in high‐throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER‐HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro‐inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2O2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays.