INTRACELLULAR CA2-EXPRESSION IN HL-60 MYELOID-LEUKEMIA CELLS( AND THEREGULATION OF EARLY RESPONSE GENE)

To gain direct insight into the action of the second messenger Ca2+ on transcriptional regulation, we have developed an intact cell model in which the intracellular free Ca2+ concentration (Ca2+!i) can be meas ured, set, and varied at any level within the physiological range and in which the expression of early response genes is assayed in parallel . Using promyelocytic HL-60 cells, we have observed an exquisite sensi tivity to Ca2+ of c-fos, c-jun, and zif268 mRNA accumulation, since ea rly and maximal inductions were observed at 200-300 nm Ca2+!i. At ear ly times (10-20 min), the Ca2+!i dose dependence of c-fos transcripti on and mRNA accumulation displayed a bell shape since c-fos expression was barely modified at high (700-1,250 nM) Ca2+!i. The threshold Ca 2+!i concentration for prolonged (60 min) c-fos mRNA accumulation was greater than 200 nM. This indicates that the quantitative effects of C a2+ on a given gene can vary markedly as a function of both the Ca2+! i concentration and the duration of stimulation. Strikingly, a Ca2+!i perturbation of only 1 min was sufficient for full induction of c-fos and zif268 transcripts. This demonstrates that a transient perturbati on of Ca2+!i has long term effects on gene expression. The half-life of c-fos mRNA (16 min) was unaltered by Ca2+. Nuclear run-on analysis of the distribution of RNA polymerase II along the c-fos locus indicat ed that Ca2+ promotes a small increase in transcriptional initiation a nd a pronounced relief of a block to transcriptional elongation beyond intron 1. The extreme sensitivity to Ca2+!i, in terms of both the le ngth of time and the dose of Ca2+!i required for maximal gene inducti on, demonstrates that Ca2+ is a major physiological regulator of early response gene expression. In addition, the results indicate that a c- fos intragenic element is the main target of Ca2+-regulated transcript ional activation.