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Interleukin-8 (IL-8) is a potent proinflammatory chemokine that plays an important role in inflammation by activating and recruiting neutrophils, lymphocytes, and eosinophils. To demonstrate the effect of intracellular Ca2+ on IL-8 production and related signaling, we stimulated human mast cell line HMC-1 with either calcium ionophore A23187 or thapsigargin. Increase of intracellular Ca2+ resulted in inducing IL-8 gene expression and protein secretion, and addition of EGTA or BAPTA/AM before Ca2+ stimulation inhibited the induction of IL-8 production. Intracellular Ca2+ triggered the activation of mitogen-activated protein kinase (MAPK) in HMC-1, especially p42 and p44 isoforms of extracellular signal-regulated kinase (ERK) and p38 MAPK, but not c-Jun N-terminal kinase (JNK). Pretreatment of MAPK inhibitors (PD98059 and SB203580) markedly blocked Ca2+-induced IL-8 production from cells, and anti-inflammatory drugs, such as dexamethasone and cyclosporin A, partially inhibited the activation of ERK1/2. We determined that increased Ca2+ activates the nuclear translocation of the transcription factor NF-κB. NF-κB inhibitors blocked the ability of Ca2+ to induce IL-8 production, and the activation of NF-κB was required for intracellular Ca2+-induced up-regulation of IL-8. These results suggest that increased intracellular Ca2+ stimulated p38 and ERK1/2 MAPK signaling cascades result in NF-κB activation and IL-8 production in HMC-1 cells. This study is the first to identify the intracellular signaling pathways involved in the Ca2+-mediated up-regulation of IL-8 synthesis and release from HMC-1 cells.