Signal transduction pathways of membrane expression of proteinase 3 (PR-3) in human endothelial cells.

Abstract

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-alpha)-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 PKA 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-alpha stimulation, i.e. TNF-alpha-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-alpha which induces PR-3 expression at a transcriptional level.

• Authors: W J Mayet, A Schwarting, T Orth, U Sibelius, K Hattar, K H Meyer zum Büschenfelde
• Journal: European journal of clinical investigation (Eur J Clin Invest) Vol. 27 Issue 11 Pg. 893-9 (Nov 1997) ISSN: 0014-2972 [Print] England
• PMID: 9395784 (Publication Type: Journal Article)

Chemical References

• Tumor Necrosis Factor-alpha
• Dactinomycin
• Protein Kinase C
• Serine Endopeptidases
• Myeloblastin
• Tetradecanoylphorbol Acetate

Topics

• Cell Membrane (enzymology)
• Dactinomycin (pharmacology)
• Endothelium, Vascular (enzymology)
• Humans
• Myeloblastin
• Protein Kinase C (physiology)
• Serine Endopeptidases (metabolism)
• Signal Transduction
• Tetradecanoylphorbol Acetate (pharmacology)
• Tumor Necrosis Factor-alpha (pharmacology)