Cleavage of p21 by Proteinase-3, a Myeloid-specific Serine Protease, Potentiates Cell Proliferation
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m202789200
ISSN1083-351X
AutoresVéronique Witko‐Sarsat, Sandrine Canteloup, Stéphanie Durant, Chantal Desdouets, Romain Chabernaud, Patricia Lemarchand, Béatrice Descamps‐Latscha,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoIn 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 p21waf1. 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 p21waf1 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 co-immunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-α, which induces PR3 re-expression, 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. 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 p21waf1. 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 p21waf1 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 co-immunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-α, which induces PR3 re-expression, 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 (1Borregaard N. Cowland J.B. Blood. 1997; 89: 3503-3521Google Scholar, 2Witko-Sarsat V. Rieu P. Descamps-Latscha B. Lesavre P. Halbwachs-Mecarelli L. Lab. Invest. 2000; 80: 617-654Google Scholar). 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) 1The abbreviations used are: PR3, proteinase-3; HNE, human neutrophil elastase; TNF-α, tumor necrosis factor-α; G-CSF, granulocyte colony-stimulating factor; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; Boc, t-butoxycarbonyl; Nva, norvaline; SBzl, thiobenzyl; pfu, plaque-forming units; PMN, polymorphonuclear neutrophils; G-CSF, granulocyte colony stimulating factor 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 (3Baggiolini M. Bretz U. Dewald B. Feigenson M.E. Agents Actions. 1978; 8: 3-10Google Scholar). 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 (4Campanelli D. Melchior M. Fu Y. Nakata M. Shuman H. Nathan C. Gabay J.E. J. Exp. Med. 1990; 172: 1709-1715Google Scholar). 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 (5Witko-Sarsat V. Cramer E.M. Hieblot C. Guichard J. Nusbaum P. Lopez S. Lesavre P. Halbwachs-Mecarelli L. Blood. 1999; 94: 2487-2496Google Scholar). 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 necrosis factor-α (TNF-α) stimulation (6Zhou Z. Richard C. Ménard H.A. J. Rheumatol. 2000; 27: 2406-2411Google Scholar). Moreover, we recently demonstrated that PR3 biosynthesis is induced in vivo in monocytes from children with cystic fibrosis only during episodes of acute pulmonary inflammation (7Just J. Moog-Lutz C. Houzel-Charavel A. Canteloup S. Grimfeld A. Witko-Sarsat V. Cayre Y.E. FEBS Lett. 1999; 457: 437-440Google Scholar). Third, aside from its pro-inflammatory activity in mature phagocytes, PR3 has been implicated in autoimmunity and in myeloid progenitor differentiation. PR3 has pathophysiological importance because it is the main target of autoantibodies in Wegener's granulomatosis, a systemic form of necrotizing vasculitis (8Jenne D.E. Tschopp J. Ludemann J. Utecht B. Gross W.L. Nature. 1990; 346: 520Google Scholar). In another area of research focusing on myelopoietic mechanisms, PR3 (also termed myeloblastin) was described as a myeloid-specific serine protease (9Bories D. Raynal M.C. Solomon D.H. Darzynkiewicz Z. Cayre Y.E. Cell. 1989; 59: 959-968Google Scholar). PR3 is expressed in human progenitor CD34+ cells at the mRNA and protein levels and appears to be up-regulated by the granulocyte colony-stimulating factor (G-CSF) (10Lutz P.G. Moog-Lutz C. Coumau-Gatbois E. Kobari L. Di Gioia Y. Cayre Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1601-1606Google Scholar). Therefore, we hypothesized that PR3 could act as a protein involved in cell proliferation control and/or apoptosis during myeloid differentiation or in mature neutrophils. In this study, we investigated how PR3 and HNE influence cell proliferation. To differentiate between PR3 and HNE, both expressed in granulocytic cells, stably transfected mast cell lines (the rat basophilic/mast cell line RBL and the human mastocytic cell line HMC1) were used to express either PR3 or HNE. The expression of active recombinant PR3 and HNE has been shown to be tissue-specific and can be achieved only in myeloid cells. Mast cell lines have proven to be valuable models for myeloid-specific proteins and to be lacking endogenous PR3 and HNE (11Specks U. Fass D.N. Fautsch M.P. Hummel A.M. Viss M.A. FEBS Lett. 1996; 390: 265-270Google Scholar,12Garwicz D. Lindmark A. Hellmark T. Gladh M. Jogi J. Gullberg U. J. Leukocyte Biol. 1997; 61: 113-123Google Scholar). In this respect, investigation of the specific interactions between cell cycle regulatory proteins and myeloid serine proteinases might give some novel mechanisms of cell proliferation and/or apoptosis. Our results provide the first evidence that PR3, but not HNE, promotes cell proliferation via direct proteolytic cleavage of the cyclin-dependent kinase inhibitor (CDKI) p21waf1/cip1, which is a potent inhibitor of G1/S transition. In mature neutrophils, p21 was cytoplasmic and was cleaved after TNF-α treatment, which induces PR3 re-expression. This was reversed by a serine proteinase inhibitor. The plasmid pCR/PR3 was a gift from Dr. Pierre G. Lutz (10Lutz P.G. Moog-Lutz C. Coumau-Gatbois E. Kobari L. Di Gioia Y. Cayre Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1601-1606Google Scholar). PR3 cDNA was then subcloned into the expression vector pcDNA3/zeo (Invitrogen) between the HindIII and NotI restriction sites. The plasmid pcDNA/PR3(S203A) was prepared to express the enzymatically inactive mutant of PR3 by mutating the serine residue of the catalytic triad (Ser203) to alanine as previously described (11Specks U. Fass D.N. Fautsch M.P. Hummel A.M. Viss M.A. FEBS Lett. 1996; 390: 265-270Google Scholar). Mutagenesis was performed using the QuikChange method according to the instructions of the manufacturer (Clontech). The plasmid pRc/CMV/elastase, containing the cDNA for HNE, was a gift from Dr. Urban Gullberg (Lund University, Lund, Sweden) (13Gullberg U. Lindmark A. Lindgren G. Persson M.A. Nilsson E. Olsson I. J. Biol. Chem. 1995; 270: 12912-12918Google Scholar). HNE cDNA was subcloned into pcDNA3 between the HindIII and NotI restriction sites. The plasmid pET/p21/His, containing the human p21 cDNA with an N-terminal His tag, was a gift from Dr. Bruce Stillman (14Waga S. Hannon J.G. Beach D. Stillman B. Nature. 1994; 369: 574-578Google Scholar). All cDNA sequences were confirmed by direct sequencing. The plasmid pcDNA/p21 was a gift from Dr. Bernard Ducommun (15Cayrol C. Ducommun B. Oncogene. 1998; 17: 2437-2444Google Scholar). The p21 cDNA was subcloned into pRC/RSV plasmid (Invitrogen) between the HindIII and NotI restriction sites. To construct the adenoviral vector, PR3 cDNA was subcloned into the shuttle vector pDK6 between the EcoRV and NotI restriction sites under the control of a cytomegalovirus promoter. To obtain a replication-deficient, recombinant adenoviral vector, pDK6/PR3 was cotransfected into 293 cells (CRL 1573, American Type Culture Collection) with plasmid pJM17, containing the adenovirus type 5 genome (Microbix Biosystems), to produce Ad/PR3. Ad/PR3 vectors were propagated, purified, and titered as previously described (16Danel C. Erzurum S.C. Prayssac P. Eissa T. Crystal R.G. Herve P. Baudet B. Mazmanian M. Lemarchand P. Hum. Gene Ther. 1998; 9: 1487-1496Google Scholar). The rat basophilic/mast cell line, RBL, was a gift from Dr. Michel Dy (CNRS UMR 8603, Paris). The human mastocytic cell line, HMC1, was a gift from Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN). Both cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected using the electroporation system (Eurogentec) as described (11Specks U. Fass D.N. Fautsch M.P. Hummel A.M. Viss M.A. FEBS Lett. 1996; 390: 265-270Google Scholar, 12Garwicz D. Lindmark A. Hellmark T. Gladh M. Jogi J. Gullberg U. J. Leukocyte Biol. 1997; 61: 113-123Google Scholar). Transfected cells were selected on the basis on their resistance to Zeocin (1 μg/ml). Stable transfectants were then cloned by limited dilution and selected for high protein expression. For the RBL/PR3/p21 double transfectants, control RBL cells were first transfected with pcDNA/PR3 and selected for 1 month with Zeocin to obtain RBL/PR3 cells. They were then transfected with pRC/RSV/p21 and selected on the basis of a Zeocin and neomycin (1 mg/ml) double resistance. The inhibitors lactacystin and benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Calbiochem) were added to the culture as indicated. Human neutrophils were isolated from EDTA-anticoagulated blood from healthy donors by centrifugation on Polymorphprep (Nycomed, Oslo, Norway), and contaminating erythrocytes were lysed as previously described (5Witko-Sarsat V. Cramer E.M. Hieblot C. Guichard J. Nusbaum P. Lopez S. Lesavre P. Halbwachs-Mecarelli L. Blood. 1999; 94: 2487-2496Google Scholar). Ten milliliter (107 cells) of thrice-washed neutrophils were cultured in complete RPMI 1640 medium at 37 °C in 5% CO2. The neutrophils were stimulated with human recombinant TNF-α (Sigma) at a final concentration of 10 ng/ml. Neutrophils were harvested after 4 and 15 h by centrifugation. Cell pellets were stored at −80 °C. RBL or HMC1 cells were plated in 96-well plates (5000 cells/well) in the absence or presence of 10% serum and allowed to seed for 24 h in culture. Cell DNA synthesis was measured using a colorimetric immunoassay based on bromodeoxyuridine (BrdUrd) incorporation during DNA synthesis. After a 3-h labeling period, incorporated BrdUrd was combined with peroxidase-conjugated anti-BrdUrd antibody and then quantified by measuring the absorbance at 340 nm according to the instructions of the manufacturer (Roche Molecular Biochemicals). RBL or HMC1 cells (100 × 106 cells/ml) were lysed in lysis buffer (0.5% deoxycholate, 0.1% SDS, 1% Nonidet P-40, 150 mm NaCl, and 50 mm Tris, pH 8) containing proteinase inhibitors (0.5 μm aprotinin (Sigma), 1 mm phenylmethylsulfonyl fluoride, 1 μmleupeptin (Roche Molecular Biochemicals), and 1 mmchymostatin (Sigma)). Protein concentration was determined using the BCA method (Pierce). Proteins were analyzed by the standard immunoblot procedure previously described (5Witko-Sarsat V. Cramer E.M. Hieblot C. Guichard J. Nusbaum P. Lopez S. Lesavre P. Halbwachs-Mecarelli L. Blood. 1999; 94: 2487-2496Google Scholar). The primary antibodies used were rabbit polyclonal anti-PR3 (9Bories D. Raynal M.C. Solomon D.H. Darzynkiewicz Z. Cayre Y.E. Cell. 1989; 59: 959-968Google Scholar); mouse monoclonal anti-cyclin A (17McIntyre M. Desdouets C. Senamaud-Beaufort C. Laurent-Winter C. Lamas E. Brechot C. Oncogene. 1999; 18: 4577-4585Google Scholar); rabbit polyclonal anti-CDK2 and rabbit polyclonal anti-p21 (sc-397) (Santa Cruz Biotechnology); and rabbit polyclonal anti-cyclin E, mouse monoclonal anti-p27, and mouse monoclonal anti-p16 (Pharmingen). The secondary antibody was conjugated to horseradish peroxidase, and the blot was developed using the ECL detection kit (Amersham Biosciences). For Western blot analysis of p21 in neutrophils, cells were suspended in hypotonic buffer (50 mm HEPES) containing proteinase inhibitors (1 μm aprotinin, 1 mmphenylmethylsulfonyl fluoride, 2 μm leupeptin, and 2 mm chymostatin) and sonicated. The cell lysate was centrifuged at 100,000 × g to obtain the cytosolic fraction. The protein samples were then analyzed by Western blotting as described above. RBL or HMC1 cells were lysed in phosphate-buffered saline (PBS) containing 1% Nonidet P-40 and centrifuged at 10,000 ×g for 10 min. Protein concentration was adjusted to 0.5 mg/ml. The PR3 or HNE enzymatic activity was evaluated by measuring the hydrolysis of the tripeptide thiobenzyl ester Boc-Ala-Pro-Nva-SBzl (Sigma) in the presence of 5,5′-dithiobis(2-nitrobenzoic acid) atA 405 nm as previously described (18Witko-Sarsat V. Halbwachs-Mecarelli L. Schuster A. Nusbaum P. Ueki I. Canteloup S. Lenoir G. Descamps-Latscha B. Nadel J.A. Am. J. Respir. Cell Mol. Biol. 1999; 20: 729-736Google Scholar). The procedure for determination of the histone H1 kinase activity was performed as previously described (17McIntyre M. Desdouets C. Senamaud-Beaufort C. Laurent-Winter C. Lamas E. Brechot C. Oncogene. 1999; 18: 4577-4585Google Scholar). Briefly, cells (HMC1, HMC1/PR3, HMC1/PR3(S203A), and HMC1/HNE) were lysed in lysis buffer and immunoprecipitated for 1 h using mouse monoclonal anti-cyclin A antibody. After adding protein A-Sepharose beads, samples were washed twice with cold lysis buffer and twice with kinase buffer (50 mm HEPES, pH 7.6, 15 mm MgCl2, and 1 mm dithiothreitol). For the last wash, 5 μm ATP was added. Reactions were initiated by adding substrate protein (histone H1) and 5 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham Biosciences) in a final volume of 30 μl. Samples were incubated for 30 min at 30 °C. Phosphorylated histone H1 was then electrophoresed on SDS-polyacrylamide gels, and the gels were fixed, dried, and exposed to radiographic films at −70 °C. For flow cytometry analysis of the DNA content, HMC1 cells were resuspended in 100 μl of PBS. The cells were fixed by addition of 900 μl of 100% ethanol in drop form and incubation for 1 h at 4 °C. The fixed cells were washed once with PBS and resuspended in PBS containing 10 μg/ml DNase-free RNase and 25 μg/ml propidium iodide. DNA fluorescence was measured using a Coulter EPICS Profile II flow cytometer equipped with an argon laser to give 488-nm light. Data from 104 cells were collected, and the percentages of cells in the G1, S, and G2/M phases of the cell cycle were determined by Multicycle software (Phoenix Flow Systems). Total RNA was extracted from HMC1 cells using an RNeasy mini-kit (QIAGEN Inc.), run on a formaldehyde-containing 1% agarose gel, and transferred onto Hybond-N nylon filters (Amersham Biosciences). The p21 probe was obtained by digesting the pET/p21/His plasmid, containing the human p21 cDNA, with BamHI and NcoI to obtain the full-length p21 cDNA. This p21 probe was labeled with [α-32P]dCTP (3000 Ci/mmol) using a random primer labeling kit (Amersham Biosciences). Filters were prehybridized for 2 h at 42 °C in 50% formamide, 5× SSC, 0.5% SDS, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 50 mm sodium pyrophosphate, pH 6.5, 1% glycine, and 500 μg/ml single-stranded DNA. Hybridization was conducted for 15 h at 42 °C in 50% formamide, 5× SSC, 0.5% SDS, 0.04% polyvinylpyrrolidone, 0.04% Ficoll, 20 mm sodium pyrophosphate, pH 6.5, 10% dextran sulfate, and 100 μg/ml single-stranded DNA. Filters were washed for 30 min with 2× SSC and 0.1% SDS at room temperature, followed by 60 min with 0.1× SSC and 0.1% SDS at 60 °C. The glyceraldehyde-3-phosphate dehydrogenase probe was a PCR product as previously described (10Lutz P.G. Moog-Lutz C. Coumau-Gatbois E. Kobari L. Di Gioia Y. Cayre Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1601-1606Google Scholar) and was used to control the amount of loaded RNA. HMC1 cells were seeded at 2 × 106 cells/4 ml of medium and infected with Ad/PR3 at various multiplicities of infection from 0 to 500 pfu/cell in the presence of 4 μl of LipofectAMINE (2 mg/ml; Invitrogen). At 72 h post-infection, HMC1 cells were washed with PBS and lysed in lysis buffer. Protein concentrations were measured, and PR3 and p21 protein were analyzed by Western blotting. Human recombinant p21 was purified from Escherichia coli cells transformed with the expression plasmid pET7/p21/His (14Waga S. Hannon J.G. Beach D. Stillman B. Nature. 1994; 369: 574-578Google Scholar). Briefly, growing bacteria were induced by isopropyl-β-d-thiogalactopyranoside for 24 h at 30 °C, centrifuged, and lysed. The supernatant was loaded onto an S-Sepharose column and eluted with an NaCl gradient. The p21-positive fractions were then loaded onto a nickel-nitrilotriacetic acid column, and the p21 was eluted by 150 mm imidazole. PR3 and HNE were purified from granules of human neutrophils as previously described (18Witko-Sarsat V. Halbwachs-Mecarelli L. Schuster A. Nusbaum P. Ueki I. Canteloup S. Lenoir G. Descamps-Latscha B. Nadel J.A. Am. J. Respir. Cell Mol. Biol. 1999; 20: 729-736Google Scholar). The purity of p21, PR3, and HNE was assessed by silver staining of SDS-polyacrylamide gels (data not shown). Purified recombinant p21 (2 μg) and the indicated amount of PR3 or HNE were incubated in PBS, pH 7.4, in a final volume of 100 μl and incubated at 37 °C for 3 h. After incubation, an aliquot of the reaction mixture (40 μl) was subjected to 15% SDS-PAGE and analyzed by Western blotting. Subcellular fractionation was performed as previously described (19Gullberg U. Lindmark A. Nilsson E. Persson M.A. Olsson I. J. Biol. Chem. 1994; 269: 25219-25225Google Scholar). Briefly, RBL cells resuspended at 108cells/ml in homogenization buffer (0.34 m sucrose, 10 mm HEPES, pH 7.3, and 0.3 mm EDTA) containing proteinase inhibitors (0.5 μm aprotinin, 1 mm Pefabloc® (Roche Molecular Biochemicals), 1 mm chymostatin, and 1 μm leupeptin) were homogenized by 50 strokes with a Thomas potter. The remaining unbroken cells and nuclei were pelleted by centrifugation at 500 × g for 10 min, and the supernatant was layered on 6 ml of 20% Percoll containing 15 mm HEPES, pH 7.3, and 0.25 mm sucrose on top of 1 ml of saturated sucrose. Centrifugation was performed at 32,000 × g for 60 min. Three subcellular fractions, including granules, cytosol, and plasma membranes, were then visually identified. Each band was aspirated, resuspended in PBS, and ultracentrifuged at 100,000 ×g for 2 h. The pellet obtained after the first centrifugation was treated with 0.3% Nonidet P-40 to lyse the residual unbroken cells and centrifuged at 1000 × g for 10 min. This fraction was solubilized with lysis buffer and centrifuged at 10,000 × g to obtain a nuclear fraction. Each fraction was solubilized in 1% Triton X-100 and adjusted to a 2 mg/ml protein concentration to measure β-hexosaminidase and PR3 enzymatic activities. β-Hexosaminidase activity, taken as a marker of the granular fraction, was measured using p-nitrophenylN-acetyl-β-d-glucosaminide as substrate as previously described (20Hultberg B. Lindsten J. Sjöblad S. Biochem. J. 1976; 155: 599-605Google Scholar). PR3 enzymatic activity was measured using the chromogenic substrate Boc-Ala-Pro-Nva-SBzl. An aliquot of each fraction was boiled in reduced Laemmli sample buffer and analyzed by Western blotting for the presence of PR3 and p21 as described above. p21 and PR3 subcellular localization was analyzed by immunofluorescence in control RBL and RBL/PR3 cells, respectively. Cells were cytocentrifuged, fixed in 1% paraformaldehyde, and made permeable with PBS and 1% Triton X-100. Free binding sites were saturated with 1% bovine serum albumin solution for 15 min. Cells were incubated with the primary antibody (rabbit polyclonal anti-p21 (Pharmingen), or mouse monoclonal anti-PR3 (CLB12.8) for 1 h at 37 °C, washed with PBS, and incubated with the secondary antibody (rhodamine-conjugated anti-rabbit or fluorescein isothiocyanate-conjugated anti-mouse, respectively). Slides were washed with PBS and mounted in Fluoroprep. Cells were examined with a Leica DMRB fluorescence microscope using Leica narrow-range filters (fluorescein isothiocyanate selective L4, rhodamine filter N2.1, blue/green/red). For colocalization experiments, p21 and PR3 double labeling was performed in RBL/PR3(S203A) cells. p21 labeling was performed first, followed by PR3 labeling. Confocal microscopy was performed using a Zeiss LSM 510 inverted laser scanning confocal microscope. Neutrophils were cytocentrifuged, fixed in 1% paraformaldehyde, and permeabilized with PBS and 1% Triton X-100. Immunostaining was performed according to the instructions of the manufacturer (Dako) using mouse monoclonal anti-p21 (Santa Cruz Biotechnology) as the primary antibody. Co-immunoprecipitation of PR3 using rabbit polyclonal anti-p21 antibody (sc-397) was performed using the μMACS system as indicated by the manufacturer (Miltenyi Biotec, Paris, France). Briefly, RBL/PR3(S203A) cells were lysed in 50 mm Tris, pH 8, and 1% Nonidet P-40 supplemented with 1 μm leupeptin and 1 mm chymostatin for 20 min on ice. The cell lysate (800 μl) was incubated with 2 μg of either rabbit polyclonal anti-p21 antibody (sc-397) or control IgG and 50 μl of protein A-Sepharose magnetic microbeads. After mixing, the lysate was incubated for 30 min on ice. For magnetic immunoprecipitation, the lysate was applied to a microcolumn placed in the field of a magnetic separator. The microcolumn was washed with 4 × 200 μl of lysis buffer and rinsed with 600 μl of 20 mm Tris, pH 7.5. For elution, 60 μl of 2× nonreducing Laemmli sample buffer were added. The samples (exclusion, lavage, and elution) were analyzed by 12.5% SDS-PAGE and blotted on polyvinylidene difluoride membrane. PR3 was detected using rabbit polyclonal anti-PR3 antibody (1:2000), followed by horseradish peroxidase-conjugated anti-rabbit IgG (1:5000). Co-immunoprecipitation of CDK2 and p21 was performed using the same technique: immunoprecipitation of CDK2 using rabbit polyclonal anti-CDK2 antibody and analysis of the immunoprecipitated material by Western blotting using anti-p21 antibody. The direct impact of PR3 on cell proliferation was assessed using mast cell lines transfected with PR3 cDNA. Following a 1-month selection period, RBL and HMC1 cells transfected with PR3 cDNA showed increased proliferation capacity compared with controls transfected solely with the plasmid pcDNA. BrdUrd incorporation was significantly increased in RBL/PR3 and HMC1/PR3 cells compared with RBL/pcDNA and HMC1/pcDNA cells, respectively (Fig.1 A). Moreover, this increase in BrdUrd incorporation was much more pronounced when cells were tested in the absence of serum (Fig. 1 A). The use of an inactive PR3 mutant allowed us to further define whether PR3 serine protease activity is required for the induction of cell proliferation. This mutant (PR3(S203A)) was obtained by mutation of serine 203, which belongs to the catalytic triad, to alanine (11Specks U. Fass D.N. Fautsch M.P. Hummel A.M. Viss M.A. FEBS Lett. 1996; 390: 265-270Google Scholar). This mutation abolished the effect on cell proliferation (Fig. 1 A). Interestingly, RBL and HMC1 cells transfected with HNE showed no increase in cell proliferation. Western blot analysis provided evidence that recombinant PR3 and PR3(S203A) are expressed at similar levels in both RBL and HMC1 cells (Fig. 1 B). In addition, the level of PR3 expression was five times less than that obtained in control human neutrophils, thus providing evidence that there is no PR3 overexpression in RBL/PR3 or HMC1/PR3 cells. Measurement of serine proteinase activity using the chromogenic substrate Boc-Ala-Pro-Nva-SBzl confirmed that no serine proteinase activity was present in cells transfected with the inactive PR3(S203A) mutant, whereas a high level of serine proteinase was measured in RBL/PR3 and HMC1/PR3 cells as well as in RBL/HNE and HMC1/HNE cells (Fig.1 C). From this set of experiments, we concluded that the increase in cell proliferation observed in PR3-transfected cells requires PR3 serine proteinase activity. Western blot analysis of cyclin A showed that it appeared as a doublet in control HMC1, HMC1/PR3, HMC1/PR3(S203A), and HMC1/HNE cells with a molecular mass of 60 and 55 kDa. No difference in cyclin A expression was observed in any of the transfectants (Fig.2 A). Likewise, Western blot analysis of cyclin E and CDK2 expression showed no striking difference in any of the transfectants. However, a significant increase in CDK2 activity in HMC1/PR3 cells compared with control HMC1, HMC1/PR3(S203A), and HMC1/HNE cells was observed, thereby corroborating the increase in DNA synthesis and the increase in cells in S phase (Fig. 2 B). Flow cytometry analysis of the cell cycle was performed in HMC1 cells maintained in the absence of serum for 24 h. The results of cell cycle analysis are consistent with the results of BrdUrd incorporation and CDK2 activity, showing a significant increase in DNA synthesis in PR3-transfected cells, with 63.0% of the cells in S phase compared with 50.6, 50.4, and 45.5% in control HMC1, HMC1/PR3(S203A), and HMC1/HNE cells, respectively (Fig. 2 C). However, the percentage of HMC1/PR3 cells in S phase did not increase in the same proportion as the increase in BrdUrd incorporation, thereby suggesting that the cells that proliferated were no longer in S phase. We next sought to elucidate the mechanisms through which PR3 stimulates cell proliferation and facilitates G1/S transition. The cell cycle is negatively regulated by a series of CDKIs that fall into two classes: the INK4 family (p15, p16, p18, and p19) and the p21 family (p21waf1, p27kip1, and p57kip2) (21Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Google Scholar). p21 and p27 share a similar domain involved in CDK and cyclin binding (22Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Google Scholar), and both have been shown to mediate growth arrest when overexpressed (23Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Google Scholar, 24Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 79: 59-66Google Scholar), to contribute to restriction point G1 arrest (25Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Google Scholar, 26Coats S. Flanagan W.M. Nourse J. Roberst J.M. Science. 1996; 272: 877-880Google Scholar), and to be regulated in myeloid differentiation (27Steinman R.A. Huang J. Yaroslavskiy B. Goff J.P. Ball E.D. Nguyen A. Blood. 1998; 91: 4531-4542Google Scholar). We first analyzed the status of CDKIs, including p16, p27, and p21. Western blotting using anti-p21 antibody clearly showed an absence of detectable p21 in PR3-transfected cells in both RBL and HMC1 cells, whereas p21 remained present at a constant level in all other transfectants, including control/pcDNA, PR3(S203A), and HNE (Fig. 3 A). We found no difference in either p27, another member of the KIP family, or p16, a member of the INK family. In addition, under stress conditions such as serum withdrawal, p21 was also absent in PR3-transfected cells (Fig.3 B). Northern blot analysis using the human p21 probe demonstrated the presence of p21 mRNA in PR3-transfected cells as well as in all HMC1 transfectants (Fig. 3 B), thus indicating that PR3 induction of proliferation proceeds via a post-transcript
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