Generation of reactive oxygen species in a human keratinocyte cell line: role of calcium

In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca(2+)-ionophore A23187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min. Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+]i elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+]i generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca2+]i has been linked to cell proliferation, differentiation, and apoptosis.