Calcium-dependent interleukin-8 gene expression in T84 human colonic epithelial cells

Objective and Design: IL-8 is a chemokine that activates and recruits neutr ophils and plays a major role in intestinal inflammation. Signal transducti on pathways mediated by protein kinases are central in regulating IL-8 gene expression, however, little is known about the role of Ca2+ in this event. In this study, we characterize the effect of intracellular Ca2+ on interle ukin-8 gene expression in T84 human colonic epithelial cells. Materials and methods. Cells were stimulated with Ca2- ionophore, A23187 or thapsigargin, a Ca2+-ATPase inhibitor. Semi-quantitative RT-PCR was used t o examine IL-8 mRNA and ELISA for protein quantification. Reporter gene tec hniques were used to determine transcription rate. Results: A23187 and thapsigargin caused a dose- and time-dependent accumula tion of IL-8 mRNA and protein production which was dependent on the release of Ca2+ from intracellular stores. FK506, a specific inhibitor of calcineu rin, inhibited A23187- and thapsigargin-induced IL-8 mRNA expression in a d ose dependent manner. Reporter gene studies and actinomycin D chase experim ents showed that A23187 and thapsigargin enhanced IL-8 gene transcription a nd stabilized IL-8 mRNA transcripts, respectively. Conclusion: Intracellular Ca2+ plays an important role in regulating IL-8 t ranscriptionally and posttranscriptionally through calcium/calmodulin-depen dent calcineurin.