Proteinase 3 (PR3) and human neutrophil elastase (HNE) are serine proteinases stored in the azurophilic granules of neutrophils. In contrast to HNE, PR3 is the target of antineutrophil cytoplasm antibodies (ANCA) in Wegener's granulomatosis. The mechanisms leading to the membrane expression of PR3 and HNE are still unclear and appear to be critical to understand the pathophysiological role of ANCA. Stably transfected rat basophilic cell lines (RBL) with PR3 or HNE were used to analyze the PR3 and HNE secretion mechanisms and differentiate between them. RBL cells were lacking endogenous PR3 and HNE. They were stably transfected with HNE or PR3 or an inactive mutant of PR3 (PR3S203A). Using the calcium ionophore A23187 as a secretagogue, higher serine proteinase activity was secreted in the supernatant of RBL/HNE than in RBL/PR3. It is interesting that PR3 and PR3/S203A were also expressed at the plasma membrane, thus demonstrating that serine protease activity was not required for plasma membrane expression. In contrast, no expression of plasma membrane HNE could be detected in RBL/HNE. Apoptosis induced by etoposide was evaluated by DNA fragmentation, the presence of cytoplasmic histone-associated DNA fragments, and annexin V labeling. No membrane HNE was detected in RBL/HNE. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 and PR3S203A increased with etoposide concentrations and appeared closely related to annexin V labeling. Our data suggest that membrane PR3 originates from two distinct pools, the granular pool mobilized following degranulation or a plasma membrane pool mobilized upon apoptosis.

In this study, we present evidence for the critical role of proteinase-3 (PR3) in the proliferation of myeloid cells via the proteolytic regulation of the cyclin-dependent kinase inhibitor p21 waf1 . Expression of recombinant PR3 in rat (RBL) or human (HMC1) mast cell lines increased bromodeoxyuridine incorporation and CDK2 activity compared with RBL and HMC1 cells transfected with an enzymatically inactive PR3 mutant (PR3(S203A)) or with human neutrophil elastase. Western blot analysis of p21 waf1 showed an absence of detectable protein, despite normal levels of p21 mRNA. Ectopic overexpression of p21 restored normal levels of p21 in the RBL/PR3/p21 double transfectants and reverted the proliferative effect of PR3. Inhibition of the 26 S proteasome by lactacystin or of caspases by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not inhibit p21 proteolysis. p21 cleavage correlated with PR3 expression in HMC1 cells infected with recombinant adenoviral vector Ad/PR3. During in vitro studies, purified p21 was cleaved by PR3, resulting in a 10-kDa p21 fragment. Employing double immunofluorescence confocal microscopy, subcellular fractionation, and coimmunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-␣, which induces PR3 reexpression, we observed that p21 disappeared and was reversed by Pefabloc, a serine proteinase inhibitor. The physiopathological implications of the cleavage of p21 by PR3 have to be determined.Myeloid cells express several lineage-specific proteinases in the course of their differentiation and store them in granular pools. As a result, mature phagocytes are equipped with a large assortment of proteinases that play a key role in the noxious potential to pathogens or host tissues (1, 2). We hypothesized that a redundancy in serine proteinase activities might be important for mediating various regulatory functions in myeloid cell differentiation as well as in mature phagocytes, including, but not restricted to, microbicidal activity. We also assumed that each serine proteinase could have a unique substrate specificity. Proteinase-3 (PR3) 1 and human neutrophil elastase (HNE) belong to the myeloid serine proteinase family, which also includes cathepsin G and azurocidin, which are implicated in the destruction of microorganisms and extracellular matrix degradation (3). Although PR3 shares the highest sequence homology with HNE (60%) and has a similar substrate specificity, PR3 has some very special properties that are distinct from those of HNE (4). First, the subcellular localization of PR3 is not restricted to the azurophil granule compartment, but its membrane-associated form is also localized in secretory vesicles, thus leading to plasma membrane expression upon very mild neutrophil stimulation (5). Second, in contrast to all other proteins from azurophil granules whose biosynthesis is restricted to the promyelocytic stage, PR3 mRNA is re-expressed in vitro in both mature neutrophils and monocytes after tumor necr...