Intracellular calcium dependence of gene expression in single T lymphocytes

Paul A. Negulescu, Nilabh Shastri, Michael D. Cahalan

Research output: Contribution to journalArticlepeer-review

Abstract

In T lymphocytes, intracellular Ca2+ concentration ([Ca2+](i)) rises within seconds of T-cell antigen-receptor stimulation and initiates the synthesis and secretion of interleukin 2, a cytokine essential for T-cell proliferation and the immune response. Using video-imaging techniques, we tracked [Ca2+](i) signals in individual T cells and measured subsequent expression of a β-galactosidase reporter gene (lacZ) controlled by the NF- AT element of the interleukin 2 enhancer. [Ca2+](i) spikes elicited by monoclonal antibody binding to the CD3ε subunit of the T-cell receptor were positively correlated with gene expression, but varied widely between individual cells and were therefore difficult to relate quantitatively to lacZ expression. The [Ca2+](i) dependence of NF-AT-regulated gene expression was determined by elevating [Ca2+](i) with either thapsigargin or ionomycin and then 'clamping' [Ca2+](i) to various, stable levels by altering either extracellular [Ca2+] or extracellular [K+]. Raising [Ca2+](i) from resting levels of 70 nM to between 200 nM and 1.6 μM increased the fraction of cells expressing lacZ, with K(d) ≃ 1 μM. Activation of protein kinase C enhanced the [Ca2+](i) sensitivity of gene expression (K(d) = 210 nM), whereas stimulation of protein kinase A inhibited [Ca2+](i)-dependent gene expression. The experiments described here provide single-cell measurements linking a second messenger to gene expression in individual cells.

Original languageEnglish (US)
Pages (from-to)2873-2877
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number7
DOIs
StatePublished - Mar 29 1994

Keywords

  • T-cell activation
  • calcium signaling
  • interleukin 2
  • lacZ
  • thapsigargin

ASJC Scopus subject areas

  • General

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