Apoptosis-induced proteinase 3 membrane expression is independent from degranulation.

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.

Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils.

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.

Cleavage of p21/WAF1/CIP1 by proteinase 3 modulates differentiation of a monocytic cell line. Molecular analysis of the cleavage site.

Proteinase 3 (PR3), also called myeloblastin, is involved in the control of myeloid cell growth, but the underlying molecular mechanisms have not been elucidated. In U937/PR3, stably transfected with PRCRSV/PR3 to overexpress PR3, PMA-induced p21 expression was significantly decreased as compared with control U937, and this phenomenon was reversed in the presence of the serine proteinase inhibitor, pefabloc. Conversely, when PR3 was inactivated by small interfering RNA, p21 protein was increased, and PMA-induced monocytic differentiation was potentiated. Mass spectrometry analysis identified Ala45 as the primary cleavage site on p21, and the recombinant mutated p21A45R, generated by site-directed mutagenesis and expressed in Escherichia coli, was resistant to in vitro PR3 cleavage. The U937 cells were then stably transfected with either PRCRSV/p21 or PRCRSV/p21A45R, to ectopically express wild type p21 or PR3-resistant p21, respectively. In U937/p21A45R treated with PS-341, a selective proteasome inhibitor, a significant decrease in the S phase and a blockade in the G0-G1 phase of cell cycle were observed when compared with U937/p21 or control U937. This suggested that both PR3 and the proteasome are efficiently involved in the proteolytic regulation of p21 expression in myeloid cells. Moreover, PMA-induced p21 expression was more pronounced in U937/p21A45R compared with U937/p21 and was concomitant with the morphological features of early differentiation. Our data demonstrated that p21 is one specific target of PR3 and that PR3-mediated p21 cleavage prevents monocytic differentiation.