STIMULATION OF THE CA2-MEDIATED EGR-1 AND C-FOS EXPRESSION IN MURINE ERYTHROLEUKEMIA-CELLS BY CYCLOSPORINE-A()

The Ca2+-induced expression of the primary response genes egr-1 and c- fos was investigated in the murine erythroleukaemia cell line ELM-I-1. Exposure of the cells to the Ca2+-ionophore A23187 led to a rapid tra nsient rise in egr-1 and c-fos mRNA production followed by an increase in Egr-1 and c-Fos protein levels as well as an increase in Egr-1 and activator protein 1 (AP-1) DNA-binding activity. Preincubation of the cells with KN-62, a specific inhibitor of Ca2+/calmodulin-dependent p rotein kinases, strongly decreased the Ca2+-mediated expression of egr -1 and c-fos. In contrast, treatment with cyclosporin A, which inhibit s the Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin, increased both egr-1 and c-fos mRNA production and the DNA-binding ac tivity of the Egr-1 and AP-1 transcription factors in response to the intracellular Ca+ concentration ([Ca2+](i))-increasing agents A23187 o r cyclopiazonic acid. Enhancement of the Ca2+-induced c-fos and egr-1 expression by cyclosporin A was correlated with the capability of this agent to inhibit calcineurin phosphatase activity in ELM-I-1 cells. S tudies on the phosphorylation state and DNA-binding activity of the cA MP response element-binding protein (CREB) did not demonstrate an earl y Ca2+-dependent activation of this transcription factor, suggesting t hat the regulation of c-fos and egr-I expression by Ca2+ is not linked to CREB in the haematopoietic ELM-I-I cells. The results indicate tha t calcineurin exerts negative regulatory effects on both egr-1 and c-f os expression in murine erythroleukaemia cells, in addition to the cal cineurin-mediated down-regulation of c-myb expression observed previou sly in this cell system. This study therefore emphasizes the important role of calcineurin as a negative modulator of gene expression in cer tain cell types.