INTERLEUKIN-1?? STIMULATES ENDOTHELIN-1 GENE TRANSCRIPTION AND ENDOTHELIN CONVERTING ENZYME ACTIVITY IN HUMAN ENDOTHELIAL CELLS

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163 We and others have identified elevated secretion of endothelin-1 (ET-1) in the vascular neointima of human allografts with transplant vasculopathy and chronic rejection. Studies in animal models of chronic rejection suggest that the potent pressor and mitogenic actions of ET-1 contribute to the pathogenesis of transplant vasculopathy. Alloreactive cytokines are likely to trigger ET-1 secretion by donor endothelium, but the molecular mechanisms underlying induction of ET-1 gene expression are unknown. We therefore investigated the mechanisms underlying ET-1 gene induction by interleukin-1β (IL-1β) in human umbilical vein endothelial cells. IL-1β stimulated a time- and dose-dependent increase in ET-1 peptide secretion that reached maximal levels (2.7-fold) 5 hrs after administration of 10 ng/ml IL-1β. IL-1β also evoked a 3.4-fold increase in steady state preproET-1 mRNA levels. The increase in ET-1 peptide secretion and preproET-1 mRNA induction by IL-1β was equivalent to that for phorbol ester, which maximally induces ET-1 gene expression by activating protein kinase C. Two lines of evidence demonstrated that the elevation in preproET-1 mRNA by IL-β was apparently due to increased transcription of the preproET-1 gene. First IL-1β increased preproET-1 promoter activity(3.1-fold) in endothelial cells transfected with a preproET-1 promoter construct (1.5 kbppET-1LUC) linked to a luciferase reported gene. Second, we showed that ET-1 peptide secretion was completely blocked by actinomycin D(10 mg/ml for 4 hr). It is unclear at present whether the activity of endothelin converting enzyme (ECE) is regulated by stimuli of ET-1 secretion, so we asked whether IL-1β might also increase ECE activity. IL-1β(10 ng/ml) stimulated a 2.2-fold increase in ECE activity that was blocked by the known ECE inhibitor phosphoramidon but not by inhibitors of other classes of proteases such as PMSF, leupeptin, and pepstatin. The time course of ECE activation by IL-1β closely paralleled that for ET-1 secretion. Taken together, these results suggest that IL-1β elevates ET-1 secretion in vascular endothelial cells by increasing transcription of the preproET-1 gene and by elevating the activity of ECE. The results also suggest that inhibitors of ECE activity might be useful in attenuating the elevation of ET-1 secretion caused by alloreactive cytokines in transplant vasculopathy and chronic rejection.