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

Abstract:

Objective and Design: IL-8 is a chemokine that activates and recruits neutrophils and plays a major role in intestinal inflammation. Signal transduction 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 interleukin-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 to examine IL-8 mRNA and ELISA for protein quantification. Reporter gene techniques were used to determine transcription rate.¶Results: A23187 and thapsigargin caused a dose- and time-dependent accumulation of IL-8 mRNA and protein production which was dependent on the release of Ca²⁺ from intracellular stores. FK506, a specific inhibitor of calcineurin, inhibited A23187- and thapsigargin-induced IL-8 mRNA expression in a dose dependent manner. Reporter gene studies and actinomycin D chase experiments showed that A23187 and thapsigargin enhanced IL-8 gene transcription and stabilized IL-8 mRNA transcripts, respectively.¶Conclusion: Intracellular Ca²⁺ plays an important role in regulating IL-8 transcriptionally and posttranscriptionally through calcium/calmodulin-dependent calcineurin.¶