Activation of caspase-8 and Erk-1/2 in Domes Regulates Cell Death Induced by Conﬂuence in MDCK Cells YUNG-HENG CHANG, 1 HSI-HUI LIN, 1 YANG-KAO WANG, 1 WEN-TAI CHIU, 2 HSIAO-WEN SU, 2 AND MING-JER TANG 1,2 * 1 Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan 2 Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan Under normal culture conditions, cells adhere to culture dish, spread out, proliferate, and ﬁnally cover all areas and reach conﬂuence. During the conﬂuent stage, cell proliferation ceases and differentiation is enhanced. Meanwhile, cell death also appears as the monolayer conﬂuence proceeds. To delineate the mechanism of cell death induced by the conﬂuent process, we employed Madin-Darby canine kidney (MDCK) cells. When approaching conﬂuence, MDCK cells exhibited increase the levels of caspase-2 and enhanced the activity of caspase-8. Using various caspase inhibitors to block apoptosis, we found that only z-VAD-fmk and z-IETD-fmk can inhibit conﬂuent cell death, indicating that conﬂuent cell death is mediated by activation of caspase-8. Overexpression of Bcl-2 inhibited conﬂuent cell death, suggesting the involvement of mitochondria-dependent pathway in conﬂuent cell death. Interestingly, the activity of phospho-Erk (p-Erk) was initially decreased before conﬂuence, but markedly increased after conﬂuence. Immunoﬂuorescence staining studies showed that p- Erk was expressed exclusively on dome-forming cells that underwent apoptosis. Treatment of conﬂuent MDCK cells with PD98059 and UO126, the inhibitors of MEK, enhanced apoptosis as well as activity of caspase-8. These data indicate that elevation of p-Erk activity during conﬂuence may serve to suppress conﬂuent cell death. Taken together, activation of caspase-8 contributes to and results in conﬂuent cell death, whereas elevated p-Erk activity serves to prevent conﬂuent cell death by regulating activation of caspase-8. J. Cell. Physiol. 211: 174–182, 2007. ß 2007 Wiley-Liss, Inc. Cellular conﬂuence is a natural process and phenomenon used to describe the end point of cell proliferation. When cells reach conﬂuence, cell differentiation is enhanced and spontaneous cell loss can be found in the post-conﬂuent stage (Hosick, 1976). Although this observation was advanced in early 1970s, the underlying mechanism of conﬂuence-induced cell death remains unclear. To delineate what intracellular signals mediate cell death induced by conﬂuence, we employed Madin-Darby canine kidney (MDCK) cells. MDCK cells were derived from the distal tubule/collecting duct of normal dog kidney (Gaush et al., 1966). When the cells are cultured to the conﬂuent stage, they spontaneously form domes (Lever, 1979). Domes are ﬂuid-ﬁlled multi-cellular structures regulated by ﬂuid and ion transport activity and cell–cell interaction (Rindler and Saier, 1981; Kennedy and Lever, 1984). Our previous study showed that MDCK cells gradually underwent apoptosis after conﬂuence (Lin et al., 1997, 1999). However, the cellular mechanism of conﬂuence-induced apoptosis and the relationship between apoptosis and dome formation remain to be elucidated. Caspases are highly associated with apoptosis and are thought to play as ‘‘killers’’ in apoptosis. Caspase is a family of cysteine proteases and at least 14 members of the family have been described (Thornberry and Lazebnik, 1998). Caspases are sorted into two subfamilies by functions: pro-inﬂammatory caspase and pro-apoptotic caspase (Wang and Lenardo, 2000). The pro-inﬂammatory caspases are highly associated with cytokine processing (Wang et al., 1994), whereas the apoptotic caspases are involved in induction of cell death (Thornberry and Lazebnik, 1998). There are two apoptotic pathways that require caspase activation: the death receptor pathway and the mitochondrial pathway (Green, 2000; Hengartner, 2000). In the death receptor pathway, death receptors transmit death signals by activation of caspases. When death signal (death ligand) binds to death receptor, they recruit adaptor proteins to form a complex. These adaptor proteins usually contain a death domain, which serves to recruit initial caspases like caspase-8 and caspase-2. Upon the recruitment of procaspases, they can be auto-processed by cleaving each other to become active caspases and thereby transmit the death signals through activating downstream effector caspases (Kumar et al., 1994; Boldin et al., 1996; Muzio et al., 1996, 1998). In the mitochondrial pathway, cytochrome c is released from mitochondria by VDAC, which is regulated by Bcl-2 family (Kluck et al., 1997; Shimizu et al., 1999). Apaf-1/cytochrome c complex triggers downstream caspase activation that results in activation of procaspase-9. Active caspase-9 can subsequently cleave and activate downstream caspases to amplify death signals (Liu et al., 1996; Kroemer et al., 1998; Desagher and Martinou, 2000). In our previous studies, we showed that overexpression of Bcl-2 prevented conﬂuence-induced cell death (Lin et al., 1997, 1999). In this study, we examined the role of mitochondria pathways in conﬂuent cell death. Mitogen-activated protein kinases (MAPK) are a family of serine-threonine protein kinases which function may be related Contract grant sponsor: National Health Research Institute; Contract grant number: NHRI-EX91-9031SL. Contract grant sponsor: Ministry of Education; Contract grant number: 91-FA09-1-4. *Correspondence to: Ming-Jer Tang, Department of Physiology, College of Medicine, National Cheng Kung University, No.1, Ta-Hsueh Road, Tainan 701, Taiwan. E-mail: mjtang1@mail.ncku.edu.tw Received 20 July 2006; Accepted 19 September 2006 DOI: 10.1002/jcp.20926 ß 2007 WILEY-LISS, INC. ORIGINAL ARTICLE 174