SIGNAL-TRANSDUCTION PATHWAYS OF MEMBRANE EXPRESSION OF PROTEINASE-3 (PR-3) IN HUMAN ENDOTHELIAL-CELLS

At present, the exact mechanism of the pathogenic effect of anti-PR-3 antibodies remains unknown. Interaction of anti-neutrophil cytoplasmic antibodies (ANCAs) with human umbilical vein endothelial cells (HUVEC s) may play a key role. Recently we were able to show that ANCAs recog nize their target antigen, PR-3, translocated into the membrane of HUV ECs. The objective of this study was to investigate regulation, i.e. s ignal transduction pathways, of PR-3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe ct al. and cultu red under standard conditions. A cyto-enzyme-linked immunosorbent assa y (ELISA) with unfixed cells was performed. Membrane-expressed PR-3 wa s detected by affinity-purified and monoclonal anti-PR-3 Ab. Tumour ne crosis factor alpha (TNF-alpha)-induced membrane expression of PR-3 co uld be blocked with the RNA synthesis inhibitor actinomycin D, the pro tein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, th e specific PKC inhibitor calphostin C, the c-AMP-dependent PKA inhibit or KT5720 and the tyrosine kinase inhibitor genistein in a dose-depend ent manner. The effect of calphostin C was the most significant. In ad dition, the effect of phorbol 12-myristate 13-acetate (PMA), a mediato r of intracellular second messengers, was investigated. In our study, pretreatment of cells with PMA for 48 h led to a down-regulation of PR -3 expression. This effect, however, could be overridden by TNF-alpha stimulation, i.e. TNF-alpha-induced membrane expression of PR-3 was re sistant to downregulation of PKC. In conclusion, our data suggest that translocation of PR-3 in HUVECs is an active process depending on pro tein synthesis. PR-3 expression by HUVECs may involve a PKC reactive t o cytokines such as TNF-alpha which induces PR-3 expression at a trans criptional level.