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

Abstract

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. 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. Proteinase 3 (PR3) 1The abbreviations used are: PR3, proteinase 3; FCS, fetal calf serum; MALDI-TOF, matrix-assisted laser desorption ionization time-offlight; MD, molecular dynamics; PBS, phosphate-buffered saline; PMA, phorbol myristate acetate; siRNA, small interfering RNA; aa, amino acids; RT, reverse transcription. is a neutrophil-derived proteinase originally described in azurophilic granules along with its homologues elastase, cathepsin G, and azurocidin (1Baggiolini M. Bretz U. Dewald B. Feigenson M.E. Agents Actions. 1978; 8: 3-10Crossref PubMed Scopus (144) Google Scholar, 2Campanelli D. Melchior M. Fu Y. Nakata M. Shuman H. Nathan C. Gabay J.E. J. Exp. Med. 1990; 172: 1709-1715Crossref PubMed Scopus (141) Google Scholar, 3Zimmer M. Medcalf R.L. Fink T.M. Mattmann C. Lichter P. Jenne D.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8215-8219Crossref PubMed Scopus (134) Google Scholar). Despite the strong homology between PR3 and elastase, PR3 appears to display some functional and structural features of its own (4Witko-Sarsat V. Rieu P. Descamps-Latscha B. Lesavre P. Halbwachs-Mecarelli L. Lab. Invest. 2000; 80: 617-653Crossref PubMed Scopus (877) Google Scholar, 5van der Geld Y.M. Limburg P.C. Kallenberg C.G. J. Leukocyte Biol. 2001; 69: 177-190PubMed Google Scholar). Interestingly, PR3 was cloned in HL-60 myeloid cells and was called myeloblastin, because its mRNA was down-regulated during myeloid differentiation (6Bories D. Raynal M.C. Solomon D.H. Darzynkiewicz Z. Cayre Y.E. Cell. 1989; 59: 959-968Abstract Full Text PDF PubMed Scopus (259) Google Scholar). 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 (7Dengler R. Munstermann U. al-Batran S. Hausner I. Faderl S. Nerl C. Emmerich B. Br. J. Haematol. 1995; 89: 250-257Crossref PubMed Scopus (97) Google Scholar, 8Molldrem J.J. Clave E. Jiang Y.Z. Mavroudis D. Raptis A. Hensel N. Agarwala V. Barrett A.J. Blood. 1997; 90: 2529-2534Crossref PubMed Google Scholar). Further studies have shown that PR3 is a granulocyte-macrophage colony-stimulating factor-responsive gene, which can confer serum independence to precursor cells (9Lutz 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: 1601-1606Crossref PubMed Scopus (41) Google Scholar). Although several molecular targets have been postulated, such as the transcription factor Sp1 (10Rao J. Zhang F. Donnelly R.J. Spector N.L. Studzinski G.P. J. Cell. Physiol. 1998; 175: 121-128Crossref PubMed Scopus (48) Google Scholar) or the 28-kDa heat shock protein (11Spector N.L. Hardy L. Ryan C. Miller Jr., W.H. Humes J.L. Nadler L.M. Luedke E. J. Biol. Chem. 1995; 270: 1003-1006Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), 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 (12Witko-Sarsat V. Canteloup S. Durant S. Desdouets C. Chabernaud R. Lemarchand P. Descamps-Latscha B. J. Biol. Chem. 2002; 277: 47338-47347Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). p21 belongs to the family of cyclin-dependent kinase inhibitors (13Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3215) Google Scholar). It inhibits cyclin-dependent kinase-1, -2, -4, and -6 (14Gu Y. Turck C.W. Morgan D.O. Nature. PubMed Scopus Google Scholar), and its by a of R. J.M. D. B. Cell. Full Text PDF PubMed Scopus Google Scholar) cell cycle the the cell to the cell cycle and N. Cell. Full Text PDF PubMed Scopus Google Scholar). In several hematopoietic cell U937, the p21 is a primary to early of the differentiation B. A. C. M.E. 9: Google Scholar). myeloid of precursor cells is with a in p21 expression J. B. A. Blood. 1998; PubMed Google Scholar). The concomitant expression of both PR3 and p21 during the early phase of myeloid cell differentiation the that to a of differentiation. In the we a molecular analysis of the cleavage site on p21 by PR3 in to p21A45R, a PR3-resistant the of the of the p21 cleavage by PR3 in myeloid cells The of U937 which expressed PR3 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), to the of an of PR3 in and the of the of PR3 or the of the expression of PR3-resistant p21, to the of PR3 in monocytic differentiation. evidence that of PR3 in the cell U937 p21 cleavage and that of PR3 activity either by small interfering or by expression of the the early steps of monocytic differentiation. PR3 was the expression between and (12Witko-Sarsat V. Canteloup S. Durant S. Desdouets C. Chabernaud R. Lemarchand P. Descamps-Latscha B. J. Biol. Chem. 2002; 277: 47338-47347Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The the p21 with an was a S. D. B. Nature. PubMed Scopus Google Scholar). The was by p21 the The was to express the PR3-resistant p21 by the the was the to the were by not p21 was Escherichia with the expression a the S. D. B. Nature. PubMed Scopus Google Scholar). p21 protein was were by and in a The was a and the p21 was by a to The presence of p21 was by and the were and in p21A45R, the PR3-resistant p21, was as a recombinant in E. the of the presence of the on recombinant p21, the Ala45 is in we to the of wild type the recombinant PR3-resistant p21 called in to the wild type PR3 was granules of as described V. Halbwachs-Mecarelli L. A. P. I. Canteloup S. Descamps-Latscha B. J. Biol. PubMed Scopus Google Scholar). of was used to the of p21 and PR3 and to not in vitro recombinant p21 and PR3 the were in of was to the and an of the was on and by or by spectrometry to the p21 cleavage analysis was with a with with a laser B. der M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were a with an of a a and a of the recombinant p21 and with an of a a and a of the p21 the laser of the recombinant p21, treated with PR3 or PBS, were with an of and on a as by the The was and with an of a of in a of of of this were on the and to was with the of a of were treated with PR3 or as in the to and of were with of a of in a of on the and to was with the of the of the and The expressed are and to the of the on the or on the cleavage of p21 by PR3, by cleavage of the p21 with the The p21 was treated with PR3 a of during of were with of a of in a of on the and in the to was with the of the of the and cleavage of the p21 with the in the the cleavage of the p21 with the in in that the the cleavage of the p21 with the in in the cleavage of the p21 with the in in the cleavage of the p21 with the in in that the the of the of PR3 was the M. R. J. Biol. PubMed Scopus Google the of the were no of the proteinase 3 with a we on the elastase, its used the of the between elastase and and the structural between elastase and PR3 to the in The of was S. M. J. Chem. Scopus Google Scholar) and the D. M. R.L. S. J. H. S. D. L. C. S. B. B. M. R. J. M. J. D. M. J. Chem. B. 1998; PubMed Scopus Google Scholar). were with L. R. M. R. A. N. J. A. J. Scopus Google Scholar) and in the to control the and to control the J. Scopus Google Scholar, J. Scopus Google Scholar) was used to the between and were a of the PubMed Scopus Google Scholar, U. L. H. J. Chem. 1995; Scopus Google Scholar). The of were a M.E. Scopus Google Scholar, S. J. Chem. Scopus Google Scholar). The were to and then and of the along the were the in to the an analysis of the of the of the were the the and with steps of and steps of U937 cells were in with and then transfected the as by the cells were in of cell in the presence of of or were and cells were on the on their to and cloned by differentiation of U937 was by analysis was an with a some U937 cells were treated with the serine proteinase inhibitor, or with the proteasome inhibitor PR3 expression was by analysis the as the primary by a a as described S. M. Y. Canteloup S. P. Lesavre P. V. J. Biol. PubMed Scopus Google Scholar). The expression of was by with and compared with control PR3 of a of in the presence of in were to the of This was to U937 cells 3 in of of in a with was in to a in were during the in the presence of PR3 target were as and The control was used to the of the of PR3 The control was used to by the of a and not a analysis of the U937 cells were a in with with or and to in The was in serum the cells were with The cells were with during and then with were treated with with a and and were by cells were and the of cells the and of the cell cycle were by M. C. V. S. V. J. PubMed Scopus Google Scholar). U937 were a proteinase inhibitors was the were by the as or were the primary The was with and the was a of PR3 and p21 mRNA were by was U937 cells 3 reverse transcription was on of leukemia reverse The mRNA of either PR3 or p21 or which was used as control was by a with the PR3, and J. Moog-Lutz C. A. Canteloup S. A. V. Cayre Y.E. PubMed Scopus Google p21, and and B. J. C. 2002; PubMed Scopus Google Scholar). were as PR3 and p21, and and the of was PR3 p21 and to that of the was in a of The were by in The was used to the as described B. J. C. 2002; PubMed Scopus Google Scholar). analysis was the were by analysis of were significant when the was are expressed as of the by PR3 on p21 by Mass demonstrated that neutrophil-derived PR3 recombinant p21 expressed in E. (12Witko-Sarsat V. Canteloup S. Durant S. Desdouets C. Chabernaud R. Lemarchand P. Descamps-Latscha B. J. Biol. Chem. 2002; 277: 47338-47347Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). shown in p21 cleavage was on PR3 a a the the p21 had a molecular of This was to a which we observed a PR3 of The cleavage site was identified by Mass analysis was on recombinant p21 with or In the of PR3, recombinant p21 with a molecular of In the presence of PR3, cleavage and were were more a PR3 of analysis suggested that the cleavage Ala45 in In to the cleavage a p21 on the was by spectrometry with or the presence of The p21 a molecular of was and generated a of to the of the the Ala45 the not we that the was or by In to and cleavage we a were to the presence of the and a of the This p21 was Ala45 in the presence of the and the were and of with PR3 cleavage a cleavage or was this which can a and we also a with a serine in of the The was the not demonstrated that the cleavage either the or the p21 was on In to the on p21, which p21 resistant to PR3 we the of an a amino the to the Interestingly, the the cleavage of p21 by PR3 the is a small the amino in both and and were not The of the in the and in the was mutated p21 on the or the were in the the p21 was In when the was in the the cleavage was was used to the of the of the amino in the of the p21 when the or was in the the the cleavage It can that PR3 can p21 on Ala45 with amino of in and in the has been in PR3 we used molecular to the of the the of of the between p21 and PR3 by the the cleavage site of with PR3, we of a between PR3 and a the of a we also the on a the p21 we a on the The 3 amino and the site studies the of the B. S. E. M. M. L. F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and not of the M. R. J. Biol. PubMed Scopus Google Scholar). The in PR3 is The during the were to and a of the and and of and respectively. between amino of the and the This is in and are between and In and have been as in L. Chem. 2002; PubMed Scopus Google Scholar). The of is in the to in a The between the of the serine and the of the is and In the of with the of and Our of the between and PR3 the structural of a that is a the type of a p21A45R, this PR3, a and the between the and the between the and the with the between the site and the along that the not a which is the of the of its the of with PR3, structural of the Interestingly, the is not between PR3 and and of the in Moreover, the that the and of and which are an and L. Chem. 2002; PubMed Scopus Google Scholar) not with the of has with The between PR3 and of are of with were not to between the and a the of the and are of with PR3, as was the with and Our that with PR3 to a not has of the but the is the of p21 to PR3 by the of Ala45 to Ala45 as the the cleavage of p21 by PR3, a recombinant mutated was generated by site-directed mutagenesis in E. of the mutagenesis by E. were with mutated was expressed and wild type p21 and were E. and by of the p21 and were by in to the p21 and to a in the cleavage analysis of the cleavage of by PR3 that was resistant wild type p21 was by PR3 in a It can that the cleavage site of PR3 on p21 was the The mutated was resistant to in vitro PR3 cleavage and used in a model to the of the specific cleavage of p21 by PR3 in myeloid cells. of PR3 PMA-induced and p21 in U937 U937 cells were transfected with either the control or PRCRSV/PR3 and were with In that overexpressed PR3, analysis a significant in PR3 expression as compared with control U937, which expressed PR3 was used to monocytic differentiation in both control and The of cells as a of monocytic was by and shown in the monocytic differentiation was in as compared with control with and and and Interestingly, the expression of was with cell and of the cells not In to the expression of p21 in and in control U937 by the protein was in cell and then to p21 expression was in both control and in U937/PR3, and no clear was observed In p21 expression was significantly as early as and was a the the PMA-induced p21 expression not observed in a or Interestingly, when were with the serine proteinase inhibitor, the PMA-induced in p21 expression was that p21 was by PR3 in analysis of the protein on the was used as an control to that the was in of the and In analysis of p21 mRNA that a strong of p21 mRNA in both control U937 and This of data that the decreased of p21 expression in U937/PR3, was the of its proteolytic cleavage by of PR3 either by PR3 or by of PR3-resistant p21 PMA-induced in U937 the of PR3 in U937 we used the which in the specific PR3 but is not specific a protein target of of PR3 mRNA one of but not a with an is shown in In p21 mRNA and mRNA were Interestingly, analysis that the of p21 protein was in U937 cells treated with PR3 as compared with U937 cells treated with siRNA, that specific of PR3 by in an in p21 protein in the of p21 mRNA the of PR3 on monocytic U937 cells were with either PR3 or with of PR3 on cell of either PR3 or cells was observed in the of In was a in cell and of a of monocytic in PR3 U937, This was not observed in U937 with In to that the specific cleavage of p21 by PR3 was to the differentiation we the of the expression of either wild type p21 or the PR3-resistant p21, p21A45R, in U937. U937 cells were transfected with PRCRSV/p21 or with and were with analysis that p21 mRNA expression was both in U937/p21 and which express recombinant p21 and p21, as compared with which express p21 Interestingly, was no in the expression of p21 mRNA between U937/p21 and in the of mRNA was observed between control and analysis of the cell cycle in U937/p21 and U937/p21A45R no in or in the S phase as compared with the control the p21 proteolytic regulation the of both PR3 and proteasome cell cycle analysis was in the presence of PS-341, a selective proteasome shown in we a decrease in the of cells in S phase in U937/p21A45R treated with Interestingly, a significant decrease in the S phase and a concomitant blockade in the G0-G1 phase were in as compared with U937/p21 or control U937 data suggested that both PR3 and the proteasome in the proteolytic regulation of p21 expression in myeloid cells. The of the cleavage of p21 by PR3 was of p21 or protein was by of analysis a p21 expression was in both U937/p21 and as compared with control This was in with the expression in p21 mRNA observed in both U937/p21 and U937/p21A45R as compared with control described a in p21 expression were more pronounced of p21 by was in by and was in control U937. a p21 expression was more in both U937/p21 and U937/p21A45R as compared with the analysis of the PR3 mRNA in U937 demonstrated that PR3 mRNA is in U937 but is significantly a and a observed of PR3 mRNA in U937/p21 and Conversely, the p21 mRNA was in control shown in in U937/p21 and in The expression of mRNA was not and was used as an control This of data demonstrated that the of the p21 cleavage by PR3 in the early steps of PMA-induced monocytic differentiation. Interestingly, a the in p21 expression observed in U937/p21A45R was concomitant with morphological features of early differentiation U937/p21A45R the morphological that during such as cell and a morphological were more pronounced in U937/p21A45R as compared with no of differentiation was observed in a was no in the of U937/p21A45R and which both in was a clear between U937 U937/p21 and and the which a In PR3 with with expression of PR3-resistant p21, demonstrated that monocytic differentiation is when the p21 cleavage by PR3 is This is the that the of PR3 cleavage of Our data that in myeloid p21 cleavage prevents differentiation. a differentiation such as PMA, are that to cell cycle and the strong of the p21 and the of the PR3 The is an in p21 protein because of transcription and decreased cleavage. can that the cleavage of p21 by PR3 is of the mechanisms that monocytic differentiation. is an that p21 cleavage by PR3 cell cycle which is a differentiation. the of the p21 cleavage by PR3 the of cell cycle on the differentiation by the when cells not cycle not p21 and PR3 data demonstrated that the primary site of the cleavage of p21 by PR3 the Ala45 Our data not with a cleavage site on p21 by PR3, which was on a in which is a in this was no spectrometry and no p21 with mutant studies to this site R.J. J.E. M. L. Full Text Full Text PDF PubMed Scopus Google Scholar). The PR3 an in and a in and 1992; PubMed Scopus Google Scholar). The cleavage of the p21 was when Ala45 was by the the of PR3 a small amino in analysis of the in vitro cleavage by PR3 on the mutant expressed in E. that not by PR3 as compared with the recombinant wild type to the and are small The cleavage site on Ala45 has an in and in the Moreover, analysis has shown that the p21 cleavage was in the presence of in and respectively. Interestingly, molecular provided evidence that the between Ala45 and the with the cleavage by to the M. R. J. Biol. PubMed Scopus Google Scholar), PR3 can a small The also have can either or can a and can small and can a The is because of the and which are has been shown that the a a B. S. E. L. F. J. Biol. PubMed Scopus Google Scholar). 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