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Abstract: Ca2+ is a highly versatile intracellular second messenger in many cell types, and regulates many complicated cellular processes, including cell activation, proliferation and apoptosis. Influx of Ca2+ from the extracellular fluid is required for sustained elevation of the cytosolic Ca2+ concentration and full activation of Ca2+-dependent processes. It is widely accepted that Ca2+ release-activated Ca2+ channels are the major routes of Ca2+ influx in electrically non-excitable cells, including hematopoietic cells, whereas voltage-gated Ca2+ channels such as L-type Ca2+ channels (LTCCs) serve as the principal routes of Ca2+ entry into electrically excitable cells such as neurons and myocytes. However, recent pharmacological and molecular genetic studies have revealed the existence of functional LTCCs and/or LTCC-like channels in a variety of immune cells including mast cells. In this article, we review recent advances in our understanding of Ca2+ signaling in immune cells with a special interest in mast cells. We highlight roles for LTCCs in antigen receptor-mediated mast cell activation and survival. [Copyright &y& Elsevier]

Abstract: Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (FcɛRI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca2+ channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca2+ channel (SOC) activity is regulated by superoxide (O2 •−) and hydrogen peroxide (H2O2) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O2 •− and H2O2, respectively, in antigen-stimulated cells. The H2O2 generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca2+, and the FcɛRI β-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O2 •− generation was strictly dependent on extracellular Ca2+, but negatively regulated by the SFK and PI3K activities. Inhibition of O2 •− generation resulted in increased H2O2 generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca2+ store depletion. On the contrary, inhibition of H2O2 generation resulted in increased intracellular O2 •− generation and augmented SOC activity. The findings suggest that O2 •− and H2O2, which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca2+ signaling in mast cells. [Copyright &y& Elsevier]

Abstract: Store-operated Ca2+ channels (SOCs) are considered to be the principal route of Ca2+ influx in non-excitable cells. We have previously shown that in mast cells IgE+antigen (Ag) induces a dihydropyridine (DHP)-sensitive Ca2+ influx independently of Ca2+ store depletion. Since the DHP receptor is the α subunit of L-type Ca2+ channels (LTCCs), we examined the possible role of LTCCs in mast cell activation. Mast cells exhibited substantial expression of the α1C (CaV1.2) subunit mRNA and protein on their cell surface. IgE+Ag-induced Ca2+ influx was substantially reduced by the LTCC inhibitor nifedipine, and enhanced by the LTCC activator (S)-BayK8644, whereas these agents had minimal effects on thapsigargin (TG)-induced Ca2+ influx. These LTCC-modulating agents regulated IgE+Ag-induced cell activation but not TG-induced cell activation. Inhibition of SOCs by 2-aminoethoxydiphenyl borate reduced both degranulation and production of cytokines, including interleukin-13 and tumor necrosis factor-α, whereas LTCC modulation reciprocally regulated degranulation and cytokine production. IgE+Ag, but not TG, induced substantial plasma membrane depolarization, which stimulated a DHP-sensitive Ca2+ response. Moreover, IgE+Ag-, but not TG-induced mitochondrial Ca2+ increase was regulated by LTCC modulators. Finally, gene silencing analyses using small interfering RNA revealed that the α1C (CaV1.2) LTCC mediated the pharmacological effects of the LTCC-modulating agents. These results demonstrate that mast cells express LTCCs, which becomes activated by membrane depolarization to regulate cytosolic and mitochondrial Ca2+, thereby controlling mast cell activation in a distinct manner from SOCs. [Copyright &y& Elsevier]

Transcriptional regulation of the gene encoding human high affinity IgE receptor (Fc epsilon RI) alpha-chain was analyzed. Previously, we reported that GATA-1 and Elf-1 recognition sites were necessary for cell type-specific activation of the alpha-chain gene promoter. More detailed analysis revealed that other transcription factors bound the regions close to the Elf-1 recognition site and there was a more complex mechanism for the regulation of the promoter activity. On the other hand, during a course of studies to find cis-elements over this gene, CAGCTG sequence in the first intron was revealed to serve as an enhancer. A complex composed of USF1 and USF2 activated the human alpha-chain gene expression via this intronic element. Furthermore, we found two novel exons at 18.4 and 12.6kb upstream from the reported first exon and discovered an additional distal promoter.