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持续性细胞皱缩在人上皮细胞凋亡中的必要性

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Acta Physiologica Sinica , August 25, 2007, 59 (4): 512-516

https://www.wendangku.net/doc/438092464.html,

Review

Received 2007-04-19 Accepted 2007-07-03

This work was supported by Grants-in Aid for Scientific Research from MEXT and JSPS. *Corresponding author. Tel: +81-564-557731; Fax: +81-564-557735; E-mail: okada@nips.ac.jp

Prerequisite role of persistent cell shrinkage in apoptosis of human epithelial cells

SHIMIZU Takahiro, MAENO Emi, OKADA Yasunobu *

Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan

Abstract: Persistent cell volume reduction is a major hallmark of apoptosis. Recent studies have demonstrated that cell volume reduction is not a passive, secondary event of the apoptotic cell death process. Whole-cell shrinkage, termed apoptotic volume decrease (A VD), takes place soon after stimulation with apoptogen and precedes caspase activation, DNA and cell fragmentation in a variety of cell types including human epithelial cells. The A VD induction is the result of KCl efflux attained by activation of K + and Cl - channels.Inhibition of A VD induction leads to rescue of the cells from apoptosis. Since the A VD process is coupled to dysfunction of the regulatory volume increase (RVI), apoptotic cells undergo persistent cell shrinkage in human epithelial HeLa cells. When the RVI mechanism was impaired, hypertonic stress itself induced not only persistent cell shrinkage but also apoptotic cell death in HeLa cells.Even under normotonic apoptogen-free conditions, exposure of HeLa cells to Na +- or Cl --deficient solution alone can bring about persistent cell shrinkage and thereafter apoptotic cell death. Thus, it is concluded that persistent cell shrinkage, which comprises A VD induction and RVI dysfunction, is a prerequisite to apoptosis induction in human epithelial cells.

Key words: apoptosis; apoptotic volume decrease; persistent cell shrinkage; cell volume regulation; human epithelial cells

持续性细胞皱缩在人上皮细胞凋亡过程中的必要性

SHIMIZU Takahiro ，MAENO Emi ，OKADA Yasunobu *

国立生理学研究所细胞生理学部，冈崎 444-8585，日本

摘要：持续性细胞皱缩是凋亡发生的一个主要标志。近期研究发现细胞皱缩在细胞凋亡过程中并不是一个被动的次要事件。在各种细胞中，包括人上皮细胞，凋亡因子(apoptogen)刺激后马上发生全细胞皱缩，又称为凋亡性容积减小(apoptotic volume decrease, AVD)，继而发生caspase 激活、DNA 片段化、细胞破裂死亡。K +和Cl -通道的激活导致KCl 外流，诱导AVD 发生。抑制AVD 发生可以抑制细胞凋亡。AVD 与调节性容积增加(regulatory volume increase, RVI)异常相伴发生时，人上皮性HeLa 细胞发生持续性细胞皱缩。RVI 功能受损时，高渗本身就能诱导HeLa 细胞持续性细胞皱缩，继而凋亡。即使在正常渗透压、无凋亡因子刺激的情况下，将HeLa 细胞置于缺乏Na + 或Cl - 的溶液也会导致细胞持续性皱缩，继而凋亡。因此，AVD 诱导和RVI 异常所导致的持续性细胞皱缩是人上皮细胞发生凋亡的首要条件。关键词：凋亡；凋亡性容积减小；持续性细胞皱缩；细胞容积调节；人上皮细胞中图分类号：Q 255；Q 256

1 Introduction

Apoptosis, also called programmed cell death, is essential to maintaining somatic cell turnover and tissue homeostasis.

This type of cell death is morphologically and biochemi-cally characterized by cell shrinkage, caspase activation,cytochrome c release, nuclear condensation, DNA laddering, and cell fragmentation into apoptotic bodies. In

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the last ten years, scientific interest in apoptotic cell shrin-kage has upsurged [1-3], not only because it is a common phenomenon in apoptotic cell death [4] but also because block-ing of cell shrinkage was found to rescue cells from apoptosis [2,5]. Previously, it was believed that apoptotic cell shrinkage is observed only after biochemical changes such as caspase activation, proteolytic events and DNA cleavage [6].However, it is now noted that apoptotic cell shrinkage must be divided into two phases: the early-phase whole-cell vo-lume decrease, termed apoptotic volume decrease (A VD)[2,5]and the late-phase cell fragmentation into apoptotic bodies.Our recent studies demonstrated that the early-phase A VD is independent of caspase activation whereas the late-phase cell fragmentation is dependent on caspase activation [2,7].A VD takes place within 0.5-1 h after stimulation either with a mitochondrion-mediated apoptosis inducer (stauros-porine) or with a death receptor-mediated apoptogen (TNF α,Fas ligand)[2,5,7]. The A VD event precedes caspase activation,cytochrome c release, DNA fragmentation and apoptotic body formation [2,7]. These facts indicate that A VD is one of the requisite or causative processes of apoptosis, but not a secondary feature or the result of executive apoptotic events.

2 A VD induction and RVI dysfunction

It is well known that animal cell volume is elaborately con-trolled and the cell volume regulation is underlying a variety of cell functions [8]. Osmotically swollen cells regulate their volume mainly by inducing KCl efflux which drives out-flow of osmotically obliged water, whereas shrunken cells restore their volume mainly by inducing NaCl uptake and water inflow. These mechanisms are called regulatory volume decrease (RVD) and regulatory volume increase (R VI), respectively, and are accompanied by activation of

a number of ion channels and transporters existing on the plasma membrane [9,10].

Hazama and Okada, through collaboration with Ishizaki,accidentally found that the RVD process is facilitated in human epithelial HeLa and lymphoid U937 cells undergoing A VD [2]. Thus, it was hypothesized that A VD shares a simi-lar induction mechanism with R VD which is known to be accomplished by parallel operation of Ca 2+-activated K +channels [11,12] and swelling-activated, volume-sensitive out-wardly rectifying (VSOR) Cl - channels [11,13]. In fact, Shimizu and Okada recently demonstrated that even though HeLa cells are not undergoing swelling but rather shrinking, the VSOR Cl - channel is activated by staurosporine, TNF α or Fas ligand [14]. So far, apoptotic stimuli have been shown to activate a variety of K + channels [15] including voltage-gated K + channels [16,17], Ca 2+-activated K + channels [17-19], two-pore domain K + channels [20], ROMK [21], HERG [22] and Kv2.1[23].It is now finally established that the A VD induction is brought about by activation of K + and Cl - channels which results in KCl efflux and exit of osmotically obliged water (Fig.1).

When the A VD induction was prevented by blocking K +or Cl - channels, apoptotic biochemical events and cell death were found to be abolished in human epithelial HeLa, lym-phoid U937, rat pheochromocytoma PC12, and mouse neuroblastoma×rat glioma hybrid NG108-15 cells stimu-lated with staurosporine or TNF α[2] as well as mouse cardiomyocytes stimulated with staurosporine [24,25]. Also,we showed that preventing the A VD induction by anion channel blockers inhibited apoptosis in rat cardiomyocytes stimulated with staurosporine [26], mouse cardiomyocytes subjected to ischemia-reperfusion [27], human adenocarci-noma KB cells challenged with an anti-cancer drug cisplatin [28],and mouse hippocampal CA1 neurons subjected to tran-sient forebrain ischemia [29]. Thus, it appears that the A VD

Fig. 1. Schematic illustration of roles of persistent cell shrinkage in apoptosis of cells stimulated with a mitochondrion-mediated apoptogen (STS) or a death receptor-mediated apoptogen (FasL or TNF α). Activation of volume-regulatory K + channels and Cl - channels (VSOR) is involved in the A

VD induction, and inhibition of volume-regulatory cation channels (HICC) and transporters (NHE and AE) may be involved

in the RVI dysfunction in apoptotic cells (see text for details).

Acta Physiologica Sinica, August 25, 2007, 59 (4): 512-516 514

event is an essential process of apoptosis in a large variety

of non-excitable and excitable cells, including epithelial cells.

In apoptotic cells, persistent whole-cell shrinkage takes

place. However, most types of normal cells possess the

ability to regulate their volume even after osmotic shrinkage,

a process called R VI which involves not only the operation

of Na+/H+ exchanger (NHE) and anion exchanger (AE)

but also activation of hypertonicity-induced cation channel

(HICC) in HeLa cells[30]. In apoptotic cells, thus, the RVI

process must be overridden by the A VD process or the

RVI mechanism must be impaired[1]. Our recent study[31]

clearly demonstrated that the latter is the case in HeLa cells

stimulated with a Fas ligand, TNFα or staurosporine.

Therefore, it appears that persistent cell shrinkage com-

prises A VD induction and R VD dysfunction (Fig.1).

3 Persistent cell shrinkage as a prerequisite to

apoptosis

It has been reported that hypertonicity-induced cell shrink-

age often induces apoptotic cell death. Bortner and

Cidlowski[1], for the first time, showed that hypertonicity-

induced cell shrinkage leads to apoptotic death in lymphoid

cells that lack the R VI ability but not in several other R VI-

exhibiting cell types including HeLa cells. We have con-

firmed their findings, as presented in Fig.2. When human

lymphoid U937 cells that lack the R VI ability (Fig.2A, filled

triangles) were exposed to hypertonic solution prepared

by adding either 300 mmol/L mannitol or 150 mmol/L NaCl,

activation of caspase-3 was induced (Fig.2B, filled

symbols). However, hypertonic stimulation failed to in-duce capase-3 activation in human epithelial HeLa cells (Fig. 2B, open symbols) that can exhibit the R VI response (Fig. 2A, open triangles).

In addition, we have recently shown that hypertonic stress induced apoptosis even in HeLa cells when the R VI was inhibited by combined application of blockers for NHE and AE[31] or by application of HICC blocker[32]. Furthermore, we found that hypertonicity-induced apoptosis in NHE1-deficient PS120 fibroblasts which lack the RVI response, but hypertonicity-induced apoptosis was completely pre-vented when R VI was restored by transfection with NHE1[31]. Thus, it is evident that hypertonicity-induced cell shrin-kage causes apoptosis, if persisted due to dysfunction of RVI. However, there remains a possibility that hypertonic stress may have activated some intracellular signals that are essentially related to apoptosis induction.

To test whether persistent shrinkage per se is a causative factor for apoptosis induction under normotonic conditions,we have examined effects of deprivation of extracellular Cl-, because Na+-K+-2Cl- cotransporters (NKCC) and K+-Cl- cotransporter (KCC) are known to be involved in cell volume maintenance in many cell types[33,34]. When an elec-trochemical driving force for facilitation of KCC-mediated Cl- efflux and for inhibition of NKCC-mediated Cl- influx was imposed by reducing the extracellular Cl- concentra-tion (Fig.3), normotonic cell shrinkage was actually ob-served in HeLa cells[35]. Under low Cl- conditions, cell shrinkage should persist because of inhibition of RVI due to inability to accomplish volume-regulatory Cl- influx via AE and background Cl- channels (Fig.3). Also reduction of cell viability coupled to caspase-3 activation was found to be induced after exposure to low Cl- solution for over 2 h[35]. Next, we examined effects of deprivation of extracellular Na+, because this maneuver is supposed to induce not only inhibition of forward-mode operation of NKCC and Na+/ Ca2+ exchanger (NCX) but also induction of reverse-mode operation of NKCC and NCX (Fig.3). Also, Na+ deprivation Fig. 2. Hypertonicity-induced apoptosis in RVI-lacking U937 cells but not in RVI-exhibiting HeLa cells. Each symbol stands for the means±SEM (vertical bar) of 5 observations. A: Changes in mean cell volume upon hypertonic stress (300 → 600 mosM) produced by adding 300 mmol/L mannitol in HeLa cells (open triangles) and U937 cells (filled triangles). B: Changes in caspase-3 activity upon hypertonic stress (300 →600 mosM) produced by adding 300 mmol/L mannitol

(triangles) or 150 mmol/L NaCl (circles) in HeLa cells (open) and

U937 cells (filled symbols).

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SHIMIZU Takahiro et al : Persistent Cell Shrinkage in Apoptosis

may inhibit the R VI mechanism by inhibiting Na + influx via HICC [30] and via NHE (Fig.3). In fact, extracellular Na +deprivation brought about persistent shrinkage and apoptosis in HeLa cells [36]. When Na + deprivation-induced cell shrin-kage was prevented by a blocker of NKCC or NCX, HeLa cells were found to be rescued from apoptosis under Na +-deficient conditions [36].

4 Concluding remark

Considering all the data provided, it is concluded that per-sistent cell shrinkage per se provides a sufficient condi-tions for apoptosis induction in human epithelial cells. Fur-ther studies are required to elucidate how apoptosis indu-cers inhibit RVI and how persistent cell shrinkage per se triggers apoptosis cell death.

* * *

ACKNOWLEDGEMENTS: The authors thank K.Shigemoto, C. Kondo, M. Yamasawa and M. Ohara for technical assistance, and T. Okayasu for secretarial assistance.

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Yasunobu Okada, M.D., Ph.D.

Yasunobu Okada is the Director-General, National Institute for Physiological Sciences (NIPS); the Vice President, Na-tional Institutes of Natural Sciences (NINS); and the Professor, School of Life Science, Graduate University for Ad-vanced Studies (SOKENDAI); the President of The Physiological Society of Japan (PSJ); the President of The Federa-tion of Asian and Oceanian Physiological Societies (FAOPS); as well as the Vice President and the Secretary General of the Organizing Committee of The 36th International Congress of Physiological Sciences (IUPS2009). Born in 1943, graduated at Kyoto University Faculty of Medicine in 1970, obtained a medical doctor license in 1970, and studied Physiology at Department Physiology, Kyoto University Faculty of Medicine as Research Fellow (~1974), Research Associate (~1981) and Assistant Professor (~1992). Awarded a scholarship from Japan Government to study Biophysics at Montreal University (1976-1977) and received a Ph.D. in 1981 at Kyoto University Faculty of Medicine. Moved in 1992 to National Institute for Physiological Sciences in Okazaki as Professor, and took the present position in April, 2007.