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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 (HUVECs) may play a key role. Recently we were able to show that ANCAs recognize their target antigen, PR‐3, translocated into the membrane of HUVECs. The objective of this study was to investigate regulation, i.e. signal transduction pathways, of PR‐3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe et al. and cultured under standard conditions. A cyto‐enzyme‐linked immunosorbent assay (ELISA) with unfixed cells was performed. Membrane‐expressed PR‐3 was detected by affinity‐purified and monoclonal anti‐PR‐3 Ab. Tumour necrosis factor alpha (TNF‐α)‐induced membrane expression of PR‐3 could be blocked with the RNA synthesis inhibitor actinomycin D, the protein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, the specific PKC inhibitor calphostin C, the c‐AMP‐dependent PKA inhibitor KT5720 and the tyrosine kinase inhibitor genistein in a dose‐dependent manner. The effect of calphostin C was the most significant. In addition, the effect of phorbol 12‐myristate 13‐acetate (PMA), a mediator 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‐α stimulation, i.e. TNF‐α‐induced membrane expression of PR‐3 was resistant to down‐regulation of PKC. In conclusion, our data suggest that translocation of PR‐3 in HUVECs is an active process depending on protein synthesis. PR‐3 expression by HUVECs may involve a PKC reactive to cytokines such as TNF‐α which induces PR‐3 expression at a transcriptional level.