Intracellular calcium mobilization suppresses the TNF-alpha-stimulated synthesis of PAI-1 in human endothelial cells - Indications that calcium acts at a translational level

We investigated in human umbilical vein endothelial cells (HUVECs) the interaction between the signaling pathways triggered by calcium mobilization and those affected by human recombinant tumor necrosis factor-alpha (TNF) on the expression of type-1 plasminogen activator inhibitor (PAI-1). Calcium ionophore A23187 alone exerted a modest increase (50%) on PAI-1 synthesis. TNF alone increased PAI-1 accumulation in the culture medium in a time-and dose-dependent fashion, but this increase was abolished vr hen A23187 was added simultaneously with TNF. The downregulating effect of A23187 was not the result of impaired protein secretion, proteolysis, cytotoxicity, or an apoptotic process. A23187 did not decrease the TNF-enhanced PAI-1 mRNA level but did provoke a significant shift in the distribution pattern of PAI-1 transcripts by increasing the 2.3-kb, relative to the 3.2-kb form. Comparable inhibitory effects on PAI-1 protein synthesis were observed when A23187 was added 7 hours after the onset of TNF stimulation, strongly suggesting a posttranscriptional inhibitory action of calcium signaling on TNF-stimulated PAI-1 synthesis, However, treatment with actinomycin D showed that PAI-1 mRNA stability was not altered by the various treatments. Chelation of extracellular calcium by EGTA did not prevent the A23187-induced inhibition of TNF-stimulated PAI-1 protein synthesis, emphasizing the role of internal calcium stores in the inhibition of PAI-1 synthesis. Sucrose gradient fractionation of cell lysates revealed that regardless of which treatment was used, both PAI-1 mRNA transcripts exhibited similar sedimentation profiles in the actively translating polysomal pool, suggesting that the A23187-induced shift had no functional consequence on translation. However, in TNF-stimulated cells, A23187 induced a higher proportion of PAI-1 mRNAs that sedimented in fractions corresponding to less dense polysomes, a phenomenon that usually reflects a slower initiation rate during mRNA translation. A23187 also abolished the increase in PAI-1 synthesis induced by recombinant human interleukin 1 beta, and thapsigargin exerted effects comparable to those of A23187 on PAI-1 synthesis in TNF stimulated cells. It is proposed that in HUVECs, the A23187-induced release of calcium from endoplasmic stores suppresses at the translational level the increase in PAI-1 synthesis triggered by proinflammatory cytokines.