Apoptosis of Human Neutrophils Induced by Protein Phosphatase 1/2A Inhibition Is Caspase- independent and Serine Protease-dependent HAE-YOUNG PARK, 1,2 MIN-GYU SONG, 1,2 JUN-SIK LEE, 1 JA-WOONG KIM, 1,2 JUN-O JIN, 1,2 JOO-IN PARK, 1,2 YOUNG-CHAE CHANG, 3 AND JONG-YOUNG KWAK 1,2 * 1 Department of Biochemistry, School of Medicine and Medical Research Center for Cancer Molecular Therapy, Dong-A University, Busan, Korea 2 BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan, Korea 3 Department of Pathology, School of Medicine, Catholic University of Daegu, Daegu, Korea 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. ß2007 Wiley-Liss, Inc. Neutrophils have a limited life span and proliferative capacity. The apoptosis of neutrophils has been reported to be an important factor in reducing inflammation as well as terminating the activation status (Savill, 1997). The rate of neutrophil apoptosis is dependent on the presence of pro- or anti-inflammatory stimuli in the surrounding milieu (Ward et al., 1999b). Neutrophils from the human express a family of cysteine proteases called caspases (e.g., caspase-3, -8, and -9) (Sanghavi et al., 1998). The caspases have been implicated in neutrophil apoptosis, but neutrophil apoptosis can also be triggered by a caspase-independent pathway (Maianski et al., 2003). Other proteases, such as calpain or proteasome, might also contribute to the spontaneous apoptosis of human neutrophils (Knepper-Nicolai et al., 1998; Squier et al., 1999). Moreover, it has been suggested that proteasomes act downstream of the caspases in neutrophil apoptotic programs (Knepper-Nicolai et al., 1998) and that the delay of neutrophil apoptosis due to proteasome inhibitors is mediated by the stabilization of Mcl-1, the anti-apoptotic Bcl-2 family of proteins (Derouet et al., 2004). Reversible protein phosphorylation, which is catalyzed by protein kinases and phosphoprotein phosphatases, plays an important role in a variety of cellular processes, including apoptosis (Gjertsen and Doskeland, 1995; Anderson, 1997). The serine/threonine protein phosphatases (PP) are categorized into four major groups: PP1, PP2A, PP2B, and PP2C. Okadaic acid (OA) is a potent inhibitor of PP1 and PP2A (Bialojan and Takai, 1988) but is more selective in its action for PP2A (Chatfield and Eastman 2004; Boudreau et al., 2007). The inhibition of PP by OA has been associated with the induction of apoptosis in several cell types (Boe et al., 1991; Gjertsen et al., 1994; Fladmark et al., 1999). In contrast, OA and another PP1/ PP2A inhibitor, calyculin A (CA), which has equivalent activity against PP1 and PP2A, has also been reported to inhibit apoptosis (Song et al., 1992; Song and Lavin, 1993). The inhibition of PP activity by OA might enhance protein phosphorylation through various kinases. Signaling pathways that promote neutrophil survival include extracellular signal-regulated kinase (ERK) activation in the granulocyte macrophage colony-stimulating factor (GM-CSF)-induced delay of apoptosis (Klein et al., 2000), signaling though phosphatidylinositol-3-kinase (PI-3-K) and downstream pathways that include phosphoinositide-dependent kinase-1 and protein kinase B (Webb et al., 2000), p38-mitogen- activated protein kinase (MAPK) signal survival by Hae-Young Park and Min-Gyu Song have equally contributed to this work. Contract grant sponsor: The Korea Science and Engineering Foundation (KOSEF); Contract grant number: R13-2002-044-02003-0. *Correspondence to: Jong-Young Kwak, Department of Biochemistry, School of Medicine and Medical Research Center for Cancer Molecular Therapy, Busan 602-714, Korea. E-mail: jykwak@dau.ac.kr Received 10 May 2006; Accepted 28 December 2006 DOI: 10.1002/jcp.21039 ORIGINAL ARTICLE 450 ß 2007 WILEY-LISS, INC.