Intracellular calcium dependence of gene expression in single T lymphocytes.
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 beta-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 CD3 epsilon 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 microM increased the fraction of cells expressing lacZ, with Kd approximately 1 microM. Activation of protein kinase C enhanced the [Ca2+]i sensitivity of gene expression (Kd = 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.
Negulescu
P A
PA
Department of Physiology and Biophysics, University of California, Irvine 92717.
Shastri
N
N
Cahalan
M D
MD
D000818:Animals; D017252:CD3 Complex / Q000502:physiology; D016827:CD8 Antigens; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006825:Hybridomas; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D051379:Mice; D008853:Microscopy; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D015290:Second Messenger Systems; D013601:T-Lymphocytes / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014741:Video Recording; D001616:beta-Galactosidase / Q000096:biosynthesis
D017252:CD3 Complex; D016827:CD8 Antigens; D002148:Calmodulin-Binding Proteins; D007376:Interleukin-2; D013729:Terpenes; D015759:Ionomycin; D019284:Thapsigargin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D001616:beta-Galactosidase; D000252:Calcium-Transporting ATPases; D002118:Calcium