Apoptosis of human neutrophils induced by protein phosphatase 1/2A inhibition is caspase‐independent and serine protease‐dependent

Protein phosphatase (PP) activity is associated with the regulation of apoptosis in neutrophils. However, the underlying regulatory mechanism(s) in apoptosis remain unclear. The type of cell death induced by okadaic acid (OA), the inhibitor of PP1 and PP2A, is characterized by apoptotic morphological changes of the cells and annexin V‐positive staining without DNA fragmentation. The apoptotic effects of OA and calyculin A on neutrophils were observed at concentrations ranging from 50 to 200 nM, or 10 to 50 nM, respectively. Cyclosporine A (a PP2B specific inhibitor), however, did not exhibit any pro‐apoptotic effects. OA and calyculin A, but not cyclosporine A, exhibited significant effects on protein levels and on the electrophoretic mobility of Mcl‐1. zVAD‐fmk, a pancaspase inhibitor, failed to inhibit the effect of OA on the caspase‐3 activity, procaspase‐3 processing, and the apoptotic rate of neutrophils. However, 4‐(2‐aminoethyl) benzenesulfonylfluoride (AEBSF), a general serine protease inhibitor, significantly abrogated the OA‐induced mobility shift in procaspase‐3, caspase‐3 activation, and the apoptotic morphological changes in neutrophils. Moreover, OA enhanced the serine protease activity of the neutrophils. The addition of the proteinase‐3 protein increased the rate of neutrophil apoptosis, which was also blocked by AEBSF but not by zVAD‐fmk. These results suggest that OA induces procaspase‐3 processing but that OA‐induced apoptosis is caspase‐independent and serine protease‐dependent. J. Cell. Physiol. 212: 450–462, 2007. © 2007 Wiley‐Liss, Inc.

[1]  C. Haslett,et al.  NF-κB Activation Is a Critical Regulator of Human Granulocyte Apoptosis in Vitro* , 1999, The Journal of Biological Chemistry.

[2]  M. Davis,et al.  Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells , 2002, Apoptosis.

[3]  M. Thelen,et al.  Caspase‐mediated proteolysis during apoptosis: insights from apoptotic neutrophils , 1998, FEBS letters.

[4]  T. Andersson,et al.  Protein Phosphatase 2A Regulates Apoptosis in Neutrophils by Dephosphorylating Both p38 MAPK and Its Substrate Caspase 3* , 2005, Journal of Biological Chemistry.

[5]  V. Dixit,et al.  Death receptors: signaling and modulation. , 1998, Science.

[6]  J. Lord,et al.  Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase , 2000, Apoptosis.

[7]  W. Earnshaw,et al.  Phosphorylated forms of activated caspases are present in cytosol from HL-60 cells during etoposide-induced apoptosis. , 1998, Blood.

[8]  C. Owen,et al.  Bioactive Proteinase 3 on the Cell Surface of Human Neutrophils: Quantification, Catalytic Activity, and Susceptibility to Inhibition1 , 2000, The Journal of Immunology.

[9]  J. Nick,et al.  Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. , 1999, The Journal of clinical investigation.

[10]  M. Lanotte,et al.  The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells. , 1991, Experimental cell research.

[11]  S. Akira,et al.  Proteolytic Processing of Stat6 Signaling in Mast Cells as a Negative Regulatory Mechanism , 2002, The Journal of experimental medicine.

[12]  J. Weinberg,et al.  Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation. , 2000, The Biochemical journal.

[13]  M. Lavin,et al.  Calyculin A, a potent inhibitor of phosphatases-1 and -2A, prevents apoptosis. , 1993, Biochemical and biophysical research communications.

[14]  A. Malkinson,et al.  Calpain and calpastatin regulate neutrophil apoptosis , 1999, Journal of cellular physiology.

[15]  A. Eastman,et al.  The Involvement of Protein Phosphatases in the Activation of ICE/CED-3 Protease, Intracellular Acidification, DNA Digestion, and Apoptosis* , 1996, The Journal of Biological Chemistry.

[16]  E. Moon,et al.  PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A. , 2003, Blood.

[17]  A. Yamauchi,et al.  Thymocyte Apoptosis Induced by Phosphorylation of Histones is Associated with the Change in Chromatin Structure to Allow Easy Accessibility of DNase , 2002, IUBMB life.

[18]  R. Moots,et al.  Sodium Salicylate Promotes Neutrophil Apoptosis by Stimulating Caspase-Dependent Turnover of Mcl-11 , 2006, The Journal of Immunology.

[19]  P. Anderson,et al.  Kinase cascades regulating entry into apoptosis , 1997, Microbiology and molecular biology reviews : MMBR.

[20]  S. Edwards,et al.  Regulation of neutrophil apoptosis via death receptors , 2003, Cellular and Molecular Life Sciences CMLS.

[21]  U. Specks,et al.  Interaction of proteinase 3 with CD11b/CD18 (β2integrin) on the cell membrane of human neutrophils , 2003, Journal of leukocyte biology.

[22]  A. Takai,et al.  Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. , 1988, The Biochemical journal.

[23]  T. Kuijpers,et al.  Tumor necrosis factor alpha induces a caspase-independent death pathway in human neutrophils. , 2003, Blood.

[24]  J. Kwak,et al.  Modulation of constitutive and delayed apoptosis by brefeldin A in human neutrophils. , 2003, International immunopharmacology.

[25]  J. Klein,et al.  Granulocyte-Macrophage Colony-Stimulating Factor Delays Neutrophil Constitutive Apoptosis Through Phosphoinositide 3-Kinase and Extracellular Signal-Regulated Kinase Pathways1 , 2000, The Journal of Immunology.

[26]  P. Cohen,et al.  Specificity and mechanism of action of some commonly used protein kinase inhibitors. , 2000, The Biochemical journal.

[27]  Guido Kroemer,et al.  Caspase-independent cell death , 2005, Nature Medicine.

[28]  T. Andersson,et al.  p38 Mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase activities have opposite effects on human neutrophil apoptosis , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[29]  H. Nakayama,et al.  A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. , 2001, Molecular cell.

[30]  H. Xiang,et al.  Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis. , 2001, Experimental cell research.

[31]  A. Eastman,et al.  Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways. , 2004, Biochemical and biophysical research communications.

[32]  C. Borner,et al.  Apoptosis without caspases: an inefficient molecular guillotine? , 1999, Cell Death and Differentiation.

[33]  P. Cohen,et al.  Specificity and mechanism of action of some commonly used protein kinase inhibitors , 2000 .

[34]  B. Gjertsen,et al.  Protein phosphorylation in apoptosis. , 1995, Biochimica et biophysica acta.

[35]  N. Thornberry,et al.  Caspases: killer proteases. , 1997, Trends in biochemical sciences.

[36]  Young-Chae Chang,et al.  Anti‐apoptotic mechanism and reduced expression of phospholipase D in spontaneous and Fas‐stimulated apoptosis of human neutrophils , 2004, European journal of immunology.

[37]  David M. Conrad,et al.  Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cells is negatively regulated by PP2A-associated p38 mitogen-activated protein kinase. , 2007, Cellular signalling.

[38]  Dennis Davidson,et al.  Okadaic acid induces sustained activation of NFκB and degradation of the nuclear IκBα in human neutrophils , 2003 .

[39]  A. Newton,et al.  Regulation of protein kinase C. , 1997, Current opinion in cell biology.

[40]  Fredrik Melander,et al.  p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils , 2004, The Journal of experimental medicine.

[41]  P. Clarke,et al.  Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK , 2003, Nature Cell Biology.

[42]  R. Craig,et al.  Myeloid Cell Leukemia 1 Is Phosphorylated through Two Distinct Pathways, One Associated with Extracellular Signal-regulated Kinase Activation and the Other with G2/M Accumulation or Protein Phosphatase 1/2A Inhibition* , 2000, The Journal of Biological Chemistry.

[43]  J. Lieberman,et al.  Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. , 1999, Immunity.

[44]  J. Savill.  Apoptosis in resolution of inflammation , 1997, Journal of leukocyte biology.

[45]  C. Haslett,et al.  Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro. , 1993, The Journal of clinical investigation.

[46]  P. Vogt,et al.  Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Lavin,et al.  Inhibition of apoptosis in human tumour cells by okadaic acid , 1992, Journal of cellular physiology.

[48]  V. Witko-Sarsat,et al.  Apoptosis‐induced proteinase 3 membrane expression is independent from degranulation , 2004, Journal of leukocyte biology.

[49]  M. Macey,et al.  Proteinase-activated receptor-2: expression by human neutrophils. , 1997, Journal of cell science.

[50]  J. Kwak,et al.  Anti‐apoptotic role of phospholipase D in spontaneous and delayed apoptosis of human neutrophils , 2002, FEBS letters.

[51]  V. Witko-Sarsat,et al.  Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils12 , 2005, The Journal of Immunology.

[52]  R. Falk,et al.  Novel effects of neutrophil-derived proteinase 3 and elastase on the vascular endothelium involve in vivo cleavage of NF-kappaB and proapoptotic changes in JNK, ERK, and p38 MAPK signaling pathways. , 2002, Journal of the American Society of Nephrology : JASN.

[53]  E. Chilvers,et al.  z-VAD-fmk augmentation of TNF alpha-stimulated neutrophil apoptosis is compound specific and does not involve the generation of reactive oxygen species. , 2005, Blood.

[54]  Jerry A Nick,et al.  A Role for Protein Phosphatase-2A in p38 Mitogen-activated Protein Kinase-mediated Regulation of the c-Jun NH2-terminal Kinase Pathway in Human Neutrophils* , 2002, The Journal of Biological Chemistry.

[55]  A. Spector,et al.  Okadaic acid-induced lens epithelial cell apoptosis requires inhibition of phosphatase-1 and is associated with induction of gene expression including p53 and bax. , 1998, European journal of biochemistry.

[56]  Ruedi Aebersold,et al.  Molecular characterization of mitochondrial apoptosis-inducing factor , 1999, Nature.

[57]  J. Nadel,et al.  Proteinase 3, a potent secretagogue in airways, is present in cystic fibrosis sputum. , 1999, American journal of respiratory cell and molecular biology.

[58]  H. Schulman,et al.  Ca2+/Calmodulin-dependent Protein Kinase II Is Required for Microcystin-induced Apoptosis* , 2002, The Journal of Biological Chemistry.

[59]  J. Feeney,et al.  Unequivocal assignments of NH proton magnetic resonance bands in oxytocin using 2H‐ and 15N‐substituted molecules , 1974, FEBS letters.

[60]  J. Savill,et al.  Constitutive Apoptosis in Human Neutrophils Requires Synergy between Calpains and the Proteasome Downstream of Caspases* , 1998, The Journal of Biological Chemistry.

[61]  A. Khwaja,et al.  Caspase-mediated proteolysis and activation of protein kinase Cdelta plays a central role in neutrophil apoptosis. , 1999, Blood.

[62]  R. Moots,et al.  Granulocyte Macrophage Colony-stimulating Factor Signaling and Proteasome Inhibition Delay Neutrophil Apoptosis by Increasing the Stability of Mcl-1* , 2004, Journal of Biological Chemistry.

[63]  C. Haslett,et al.  Pharmacological manipulation of granulocyte apoptosis: potential therapeutic targets. , 1999, Trends in pharmacological sciences.

[64]  T. Kolb,et al.  Biochemical and Morphological Events During Okadaic Acid-Induced Apoptosis of Tsc2-Null ERC-18 Cell Line , 2002, Toxicologic pathology.

[65]  A. Cavalli,et al.  Calpain-1 Regulates Bax and Subsequent Smac-dependent Caspase-3 Activation in Neutrophil Apoptosis* , 2004, Journal of Biological Chemistry.

[66]  M. Lanotte,et al.  Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line. , 1994, Journal of cell science.

[67]  B. Zhivotovsky,et al.  Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors , 1999, Cell Death and Differentiation.

[68]  S. Ansorge,et al.  More than destructive: neutrophil‐derived serine proteases in cytokine bioactivity control , 2001, Journal of leukocyte biology.