Proteinase 3 sidesteps caspases and cleaves p21Waf1/Cip1/Sdi1 to induce endothelial cell apoptosis

Pendergraft, William F.; Rudolph, Earl H.; Falk, Ronald J.; Jahn, Jennifer E.; Grimmler, Matthias; Hengst, Ludger; Jennette, J. Charles; Preston, Gloria A.

doi:10.1111/j.1523-1755.2004.00364.x

Cited by 58 publications

(41 citation statements)

References 51 publications

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“…PR3 has also been described to have proapoptotic properties using cellular models such as endothelial cells treated with extracellular purified PR3. Both active and inactivated PR3 could mediate this apoptotic effect (27). When active PR3 was used to induce apoptosis, the cleavage of several cell cycle regulatory proteins was observed including p21, thereby confirming our study and the cleavage of NF-KB, which has been described as necessary in some cell types during apoptosis.…”

Section: Discussionsupporting

confidence: 88%

Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils

Pederzoli

Kantari

Gausson

et al. 2005

The Journal of Immunology

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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.

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Section: Discussionsupporting

confidence: 88%

Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils

Pederzoli

Kantari

Gausson

et al. 2005

The Journal of Immunology

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“…Our data do not agree with a previously reported cleavage site on p21 by PR3, which was on a threonine at 80 in P1, which is a nonclassical P1 site. However, in this study, there was no mass spectrometry and no p21 peptide analyses with mutant studies to confirm this site (39). The preferred PR3 substrates usually contained an Ala in P1 and a hydrophobic residue in P2 and PЈ1 (40).…”

Section: Discussionmentioning

confidence: 70%

Cleavage of p21/WAF1/CIP1 by Proteinase 3 Modulates Differentiation of a Monocytic Cell Line

Dublet

Ruello

Pederzoli

et al. 2005

Journal of Biological Chemistry

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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 Ala 45 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 G 0 -G 1 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, PMAinduced 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. Proteinase 3 (PR3)1 is a neutrophil-derived proteinase originally described in azurophilic granules along with its homologues elastase, cathepsin G, and azurocidin (1-3). Despite the strong homology between PR3 and elastase, PR3 appears to display some functional and structural features of its own (4, 5). Interestingly, PR3 was cloned in HL-60 myeloid cells and was called myeloblastin, because its mRNA was down-regulated during myeloid differentiation (6). PR3 has been involved in the control of proliferation in normal hematopoietic progenitors and in early steps in their transformation. PR3 is overexpressed in myeloid leukemia (7,8). Further studies have shown that PR3 is a granulocyte-macrophage colony-stimulating factor-responsive gene, which can confer serum independence to precursor cells (9). Although several molecular targets have been postulated, such as the transcription factor Sp1 (10) or the 28-kDa heat shock protein (11), no clear demonstration has identified a specific PR3 target involved in the regulation of proliferation. In a model of mast cell lines (RBL and HMC1) stably transfected with either PR3 or elastase or an inactive PR3 mutant PR3S203A, we have provided evidence that PR3, but not elastase or its inactive mutant PR3S203A, had a proliferative activity and that the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 was a molecular target of PR3 (12). p21 belongs to the family of cyclin-dependent kinase in...

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“…Mechanistically, PR3 could have a proliferative function, because data indicate that proteolytically active PR3 cleaves p21 (cyclindependent kinase inhibitor). 20,21 However, Sköld et al 22 described an S-phase inhibitory function for the secreted proform of PR3 that is distinct from p24 PR3/MBN , because it contains N-terminal amino acids not encoded by the PRTN3-002 transcript. These data suggest that the PRTN3 gene encodes proteins with either proliferative or inhibitory activities, the latter determined by the proform of PR3 and the former ascribed to MBN.…”

Section: Discussionmentioning

confidence: 99%

Dysregulation of Autoantigen Genes in ANCA-Associated Vasculitis Involves Alternative Transcripts and New Protein Synthesis

McInnis

Badhwar

Muthigi

et al. 2015

Journal of the American Society of Nephrology

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Proteinase 3 (PR3) and myeloperoxidase (MPO) are two major autoantigens in patients with vasculitis with ANCA. The genes encoding these autoantigens are abnormally expressed in peripheral granulocytes of patients with active ANCA-associated vasculitis. This study provides evidence that this transcriptional dysregulation results in a variety of mRNA processing events from the PRTN3 gene locus. In addition to elevated levels of PR3 message, leukocyte RNA from patients contained PR3 transcripts with an alternative 39 untranslated region. Furthermore, we detected usage of an alternative transcription start site within intron 1 of the PRTN3 gene locus that coincided with active disease (odds ratio, 3.3; 95% confidence interval, 1.3 to 8.4; P=0.01). This promoter may be developmentally regulated, because it was active in normal human bone marrow, multiple leukemia cell lines, MCF-7 cells, and subjects after GM-CSF treatment but not subjects with a neutrophil left shift. This transcript, which lacks exon 1 of PRTN3, encodes a 24-kD protein (p24 PR3/MBN ) with a sequence similar to that previously described for myeloblastin. Notably, PR3, p24 PR3/MBN , and MPO were synthesized in cultured neutrophils from patients with active ANCA-associated vasculitis, indicating that increased transcription results in newly synthesized autoantigens in peripheral neutrophils of patients. The synthesis of p24 PR3/MBN seems to expand the autoantigen repertoire, because immunoblots showed that sera from patients recognized p24 PR3/MBN .These findings emphasize the importance of transcriptional dysregulation of the autoantigen in autoimmune disease.

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Proteinase 3 sidesteps caspases and cleaves p21Waf1/Cip1/Sdi1 to induce endothelial cell apoptosis

Abstract: A theme is developing that the granulocyte protease, PR3, is an exogenous caspase-like molecule that can sidestep intracellular caspase functions at sites of inflammation.

Cited by 58 publications

References 51 publications

Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils

Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils

Cleavage of p21/WAF1/CIP1 by Proteinase 3 Modulates Differentiation of a Monocytic Cell Line

Dysregulation of Autoantigen Genes in ANCA-Associated Vasculitis Involves Alternative Transcripts and New Protein Synthesis

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