Vital functions for lethal caspases

REVIEW

Vital functions for lethal caspases

Sophie Launay1, Olivier Hermine2, Michaela Fontenay3, Guido Kroemer4, Eric Solary*,1

and Carmen Garrido*,1

1INSERM U-517, IFR100, Faculty of Medicine, 7 Boulevard Jeanne d’Arc, 21033 Dijon, France; 2Department of Adult Hematology,Hopital Necker-Enfants Malades, 149-161 rue de Sevres, 75743 Paris Cedex 15, USA; 3Department of Hematology, InstitutCochin, INSERM U-567, 27 rue du Faubourg Saint-Jacques, 75679 Paris, USA; 4CNRS-UMR8125, Institut Gustave Roussy, 39 rueCamille-Desmoulins, F-94805 Villejuif, France

Caspases are a family of cysteine proteases expressed asinactive zymogens in virtually all animal cells. Theseenzymes play a central role in most cell death pathwaysleading to apoptosis but growing evidences implicatecaspases also in nonapoptotic functions. Several of theseenzymes, activated in molecular platforms referred to asinflammasomes, play a role in innate immune response byprocessing some of the cytokines involved in inflammatoryresponse. Caspases are requested for terminal differentia-tion of specific cell types, whether this differentiationprocess leads to enucleation or not. These enzymes playalso a role in T and B lymphocyte proliferation and, insome circumstances, appear to be cytoprotective ratherthan cytotoxic. These pleiotropic functions implicatecaspases in the control of life and death but the fineregulation of their dual effect remains poorly understood.The nonapoptotic functions of caspases implicate thatcells can restrict the proteolytic activity of these enzymesto selected substrates. Deregulation of the pathways inwhich caspases exert these nonapoptotic functions issuspected to play a role in the pathophysiology of severalhuman diseases.Oncogene (2005) 24, 5137–5148. doi:10.1038/sj.onc.1208524

Keywords: caspase; differentiation; programmed celldeath; immune system; cell cycle

Introduction

A total of 12 cysteine proteases known as caspases havebeen identified in mammals: caspase-1 to -10, caspase-12and caspase-14. The protein initially named caspase-13was later found to represent a bovine homolog ofcaspase-4, and caspase-11 is most likely the murinehomolog of human caspase-4 and -5. These enzymes,which are expressed in virtually all animal cells, play anessential role in many forms of cell death by apoptosis(Nicholson and Thornberry, 1997). They are synthesizedas inactive proenzymes (procaspases) and can be

classified into two main groups according to the lengthof their N-terminal prodomain.

Procaspases with a short prodomain (procaspase-3,-6 and –7) exist in the cells as dimers that requireproteolysis at internal aspartate residues to generate twolarge and two small subunits. Active enzymesresult from heterodimerization of these subunits, thusinclude two active sites. These caspases are themain effectors of apoptotic cell death by cleavingcellular substrates, either a downstream procaspaseor other cellular proteins, on the carboxy-terminal sideof an aspartate residue. Other procaspases suchas procaspase-8, -9 and -10 are characterized by theirlong prodomain. They exist in living cells as monomersand require dimerization or oligomerization for activa-tion that can occur in the absence of any proteolyticcleavage (Salvesen and Abrams, 2004). These enzymesare usually the initiators of a caspase cascade althoughthe main functions of some of them do not relate toapoptosis.

Two main pathways of caspase activation leading toapoptotic cell death have been described. Schematically,the intrinsic pathway involves sentinel proteins of theBcl-2 family, also known as ‘BH3-only proteins’ that, inresponse to a cellular damage, migrate to the mitochon-dria to either antagonize antiapoptotic proteins of theBcl-2 family or activate multi-domain proapoptoticproteins Bax and Bak (Marsden and Strasser, 2003).In turn, the external membrane of the mitochondria ispermeabilized and soluble molecules are released fromthe mitochondrial intermembrane space into the cytosol.These molecules include cytochrome c that, in thepresence of ATP, triggers oligomerization of a platformprotein named Apaf-1 that recruits and activatescaspase-9. Other soluble molecules are simultaneouslyreleased from the mitochondria to antagonize theinhibitory functions of (inhibitor of apoptosis proteins)(IAPs) on caspases such as caspase-9, -3 and -7 (Acehanet al., 2002; Hill et al., 2004). A caspase cascade isactivated in the cytosol, leading to the limited proteo-lytic cleavage of intracellular, structural and regulatoryproteins, which leads to membrane blebbing, chromatincondensation and nuclear DNA fragmentation. Theextrinsic pathway starts at the level of plasma membraneby engagement of transmembrane death receptors such

*Correspondence: C Garrido;E-mail: [email protected] and E Solary

Oncogene (2005) 24, 5137–5148& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00

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as Fas/CD95, tumor necrosis factor receptor 1 (TNF-R1)and TNF-related apoptosis-inducing ligand (TRAIL)receptors. Multiprotein complexes are formed in whichcaspase-8 and -10 are recruited by the adaptor moleculeFas-associated death domain protein (FADD) andactivated. In turn, these enzymes either directly activatethe caspase cascade or connect the extrinsic to theintrinsic pathway through cleavage of the sentinel, BH3-only protein Bid (Figure 1).

Thus, in the setting of apoptosis, the roles of caspasesare: (i) to initiate (initiator caspase-8, -9) the caspasecascade and (ii) to execute (effector caspase-3, -6, -7) theapoptotic program through cleavage of plenty of vitalproteins. Their activation is under control of the balancebetween pro- and antiapoptotic proteins of the Bcl-2family, heat-shock proteins and IAPs (Fischer et al.,2003). Caspase-2 could play a role in both initiation andexecution phases of apoptotic cell death (Droin et al.,2001; Lassus et al., 2002). Target proteins cleaved bycaspases in cells undergoing apoptosis are eitherinactivated by the cleavage such as antiapoptoticproteins (Bcl-2 and Bcl-xL), actin, lamin, kinases (Akt,Raf-1 and MEK) and transcription factors (GATA-1and STAT1) or become functional to exert a discretefunction in propagation of cell death including kinases(MEKK1, PKC, PAK2, MST1), proapoptotic proteins(Bid, Bad) or signalling molecules for transcriptionfactors (IkBa) (Fischer et al., 2003). However, apoptosiscan occur in the absence of caspase activation (Garridoand Kroemer, 2004), whereas caspase activation doesnot systematically trigger cell death. Recent studies havepointed out the role of caspases in nonapoptoticpathways, including inflammatory response, immunecell proliferation, differentiation of various cell types

and others. This review will focus on these nonlethalfunctions of caspases, summarized in Table 1.

A role for caspases in innate immunity and inflammatoryresponse

A subfamily of caspases including human caspase-1, -4and -5 and mouse caspase-11 and -12, referred to as the‘caspase-1 subfamily’, has been identified in vertebrates.Some of these caspases could be involved in apoptoticpathways, for example, caspase-1 could play a role inneuronal cell death, but their main function is theregulation of inflammatory processes. Caspase-1 wasoriginally identified as interleukin-1b (IL-1b)-convertingenzyme, the enzyme responsible for the processing ofproIL-1b into active IL-1b in macrophages (Thornberryet al., 1992). The requirement for caspase-1 in IL-1bmaturation was confirmed by generation of micedeficient in caspase-1, which are resistant to the lethaleffect of endotoxins (Kuida et al., 1995; Li et al., 1995).Analysis of these mice indicated that caspase-1 wasrequired for migration of epidermal dendritic cells(Langerhans cells) from epidermis to draining lymphnodes in cutaneous immune response, which wasprobably a consequence of defective IL-1b activation(Antonopoulos et al., 2001). Caspase-1 activates anothermember of the IL-1 cytokine superfamily, IL-18, alsoknown as interferon-g-inducing factor, whereas thecleavage of proIL-18 by caspase-3 generates inactivefragments (Gracie et al., 2003). Thus, caspase-1 con-tributes to inflammation by triggering the proteolysisand maturation of prointerleukins.

DR-Ligand

DR

FADD

Caspase-8/-10

M

BH3only

AA

Caspase cascade (caspase-3 and others)

MPA

Apoptosome

(caspase-9)

Cell damage

Plasma membrane

DISC

Figure 1 Schematic representation of the main caspase-involving apoptotic pathways. Cellular damage usually activate the so-called‘intrinsic pathway’ that involves sentinel proteins of the Bcl-2 family (BH3-only proteins). These sentinels modify the equilibriumbetween antiapoptotic (AA) and multidomain proapoptotic (MPA) proteins of the Bcl-2 family, thus induce permeabilization of themitochondrial external membrane (M¼mitochondria). Cytochrome c is released in the cytosol, induces the formation of theapoptosome in which the initiator caspase-9 is activated and activates the caspase cascade. The extrinsic pathway involves the deathreceptors (DR) at the plasma membrane level. In response to their specific ligand (DR-Ligand), these trimeric receptors recruit theadapter molecule FADD and either procaspase-8 or �10 in the death-inducing signaling complex (DISC). These enzymes eitherdirectly (T lymphocytes) or indirectly through cleavage of the BH3-only protein Bid (hepatocytes) activate the caspase cascade

Nonlethal roles for caspasesS Launay et al

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Human caspase-4 and -5 could arise from the geneduplication of a caspase-11-like ancestral gene. Likemurine caspase-11, expression of caspase-5 is inducibleby lipopolysaccharides (LPS), which is not the case forcaspase-1 (Wang et al., 1998; Lin et al., 2000). Caspase-5is involved in IL-1b maturation by interacting withcaspase-1 in multiprotein complexes called inflamma-somes in which caspase-1 is activated (Martinon andTschopp, 2004). According to caspase-4, its transcrip-tion is induced by interferons but little is known on itsfunctional role (Ahn et al., 2002). An effector caspase,caspase-3, could process another prointerleukin in CD8-positive lymphocytes, namely prointerleukin-16, torelease a biologically active IL-16, suggesting redundan-cies in the functions of proinflammatory and proapop-totic enzymes of the caspase family (Zhang et al.,1998b).

Inflammatory caspases belong to the group ofinitiator caspases that exist in the cells as inactivemonomers and require dimerization or oligomerization

to assume an active conformation, with or withoutproteolytic cleavage. Dimerization or oligomerizationoccur in specialized multimeric platforms that recruitmultiple procaspase molecules into close proximity toprovoke their activation. Several platforms in whichcaspase-1 could be activated have now been describedand named inflammasomes. A first one involves aninteraction between the adaptor protein Ipaf andcaspase-1 (Damiano et al., 2001; Geddes et al., 2001;Poyet et al., 2001). Caspase-1 is also activated intoNALP inflammasomes, which are multiproteincomplexes involving proteins of the NALP family.NALP1, the first identified activating platform in thisgroup, recruits caspase-5 and, through the adaptorprotein ASC, caspase-1 (Martinon et al., 2002). Twoother activating platforms of the NALP family,named NALP2 and NALP3, can recruit ASC andactivate caspase-1 in a multiprotein complex in which aprotein designated Cardinal is also recruited, whereascaspase-5 is not (Agostini et al., 2004). Caspase-1 can be

Table 1 Role of caspases in apoptosis and other cell processes

Roles in apoptosis Other roles

Conserved activation mechan-ism of initiator caspase

Caspase-1 � IL-1 production, inflammasome

� Differentiation of skeletal muscle� Cell migration

Initiator or executor caspase Caspase-2 � Differentiation of erythroblasts, osteoblasts and macrophages� DNA repair

Executor caspase Caspase-3 � Differentiation of erythroblasts, keratinocytes, macrophages, lens epithelial cells, sperm,skeletal muscle, osteoblasts and placental trophoblasts

� Negative cell cycle control in B cells� IL-16 production� Platelet formation� Brain development

� Conserved activation me-chanism of initiator caspase

Caspase-5 � IL-1 production, inflammasome

Executor caspase Caspase-6 � Differentiation of lens epithelial cells� Positive cell cycle control in B cells

� Executor caspase Caspase-7 � Differentiation of erythroblasts

Initiator caspase of the deathreceptor pathway

Caspase-8 � T cells proliferation and activation

� Positive cell cycle control in B cells� Differentiation of placental trophoblasts, osteoblasts, erythroblasts, monocytes� Internalization of death receptors

Initiator caspase of the mito-chondrial pathway

Caspase-9 � In most cell differentiation processes in which caspase-3 has been involved

� Differentiation of epithelial cells

� Initiator caspase of the deathreceptor pathway

Caspase-10 � Not determined

� Initiator caspase of the deathreceptor pathway

Caspase-11 � IL-1 production

Initiator caspase in endoplas-mic reticulum stress

Caspase-12 � Attenuates the inflammation

� Innate immune response

� Not determined Caspase-14 � Differentiation of keratinocytes


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activated by either bacterial products or intracellularionic changes but only ATP and LPS were shownto trigger the assembly of inflammasomes throughpoorly understood pathways. Following LPS stimula-tion of monocytic THP-1 cells, activated caspase-1and caspase-5 are rapidly released along withmature IL-1b into the supernatant, which couldbe required for a macrophage to survive (Martinonet al., 2002). Several mechanisms control cas-pase-1 activity, sometimes in negative feedback loopsthat regulate the inflammatory response, and de-fective control of inflammasome activity can causedeleterious diseases. Point mutations in the NALP3gene were identified in some hereditary feversyndromes such as the Muckle–Wells syndrome,whereas mutations in the gene product termed Pyrin,that interacts with the adaptor ASC, are involved in thefamilial Mediterranean fever (Martinon and Tschopp,2004). These mutations could cause high states ofactivation of one or several inflammasomes, resultingin local or systemic inflammation. In patients withMuckle–Wells syndrome or chronic infantile cutaneousand articular syndrome, administration of an IL-1inhibitor dramatically attenuates the disease symptoms(Hawkins et al., 2004).

Caspase-1 was recently proposed to contribute toinflammation by activating a distinct pathway in B cells.This pathway involves an interaction of the caspase-recruitment domain (CARD) of procaspase-1 with theCARD-containing kinase receptor-induced proteinRIP2, which, in turn, recruits the IkB kinase (IKK)complex to activate nuclear factor of the k-enhancer B(NF-kB) and p38 mitogen-activated protein kinase(MAPK) (Lamkanfi et al., 2004). Taken together, thesedata demonstrate that some caspases play a critical rolein the regulation of multiple proinflammatory pathwaysby activating interleukins and intracellular signalingpathways.

Caspase-12 was initially reported to play a role inapoptosis induced by endoplasmic reticulum stress inrodent cells but is phylogenetically related to inflamma-tory caspases (Nakagawa et al., 2000). In humans, asingle nucleotide polymorphism at amino-acid position125 in caspase-12 results in the synthesis of either atruncated (TGA stop codon) or a full-length (CGAread-through codon) proenzyme. The full-length allelewas identified only in populations of African descent(about 20% of them) and appeared to be increased inAfrican American individuals with severe sepsis. Thisfull-length caspase-12 reduced the magnitude of endo-toxin-induced immune response, acting as a dominant-negative regulator of the IL-1 and NF-kB pathways(Saleh et al., 2004).

The link between caspases and innate immunityextends to Drosophila. A fly screen to identify mutantsdefective in innate immunity revealed that a loss-of-function mutation in the gene encoding the caspaseDredd blocked the expression of all genes that code forpeptides with antibacterial activity, thus rendering flieshighly susceptible when challenged with Gram-negativebacteria (Leulier et al., 2000).

A role for caspases in adaptive immunity by controlinglymphocyte proliferation

Defects in Fas-mediated apoptosis, due to heterozygousmutations in Fas, its ligand or caspase-10 were shown toaccount for the autoimmune lymphoproliferative syn-drome (ALPS), which is also known as the Canale–Smith syndrome (Rieux-Laucat et al., 2003). ALPS ischaracterized by the accumulation of a polyclonalpopulation of T cells called double-negative T cells thatfail to produce growth and survival factors such asinterleukin-2. This population appears to originate fromantigen-exposed T cells that downregulate the expres-sion of CD8 and fail to undergo apoptosis. Analysis ofpatients with atypical ALPS identified a homozygouscaspase-8 mutation leading to inactivation of theenzyme. Interestingly, this mutation induced defects inthe activation and proliferation of T lymphocytes, Blymphocytes and natural killer cells, for example,caspase-8-deficient T cells had a defect in the productionof IL-2 (Chun et al., 2002). This observation explainedprevious reports suggesting that caspase inhibitors couldinterfere with T-cell proliferation and that Fas-ligandcould stimulate the proliferation and IL-2 secretion inresponse to a suboptimal dose of anti-CD3 antibody(Alam et al., 1999; Kennedy et al., 1999; Boissonnaset al., 2002). As indicated above, mice in which casp-8gene has been disrupted demonstrate embryonic leth-ality due to impaired heart muscle development andhematopoietic progenitor deficiency (Varfolomeev et al.,1998). Postnatal survival in humans may be due to thecompensatory function of caspase-10, the closest para-log of caspase-8 that exists in humans but has no knownortholog in mice. Targeted caspase-8 mutation restrictedto the T-cell lineage confirmed the role of caspase-8 inT-cell activation and led to immunodeficiency, forexample, affected the ability of T cells to clearlymphocytic choriomeningitis virus (Salmena et al.,2003). The mechanism whereby caspase-8 deletionaffects activation-induced T-cell expansion remainsunclear, although a role in cell response to cytokinessuch as IL-2 has been suspected. Caspase activity duringT-cell proliferation was shown to result in the cleavageof the kinase Wee1 (Alam et al., 1999). This cleavageprevents phosphorylation of the cell cycle-regulatingkinase Cdc2, thereby promoting kinase activity of Cdc2and progression through the cell cycle (Castedo et al.,2002). Since conditional ablation of caspase-8 inbone-marrow cells was observed also to compromisemacrophage proliferation, caspase-8 appears to play anessential role in immune system homeostasis that doesnot depend on its proapoptotic functions (Kang et al.,2004).

The cytoplasmic adapter molecule FADD, whichrecruits caspase-8 in the death-inducing signaling com-plexes, was shown also to play a role in T-cell activationand proliferation. Transgenic mice expressing a domi-nant negative mutant of FADD demonstrate defectiveT-cell activation-induced proliferation through theT-cell receptor and CD28 and fail to mount anantigen-specific immune response when infected with


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the lymphocytic choriomeningitis virus (Newton et al.,1998; Walsh et al., 1998; Zhang et al., 1998a). Thus, T-cell survival under conditions of proliferation couldrequire the FADD-mediated activation of caspase-8. Inthe absence of a functional FADD-caspase-8 pathway,T cells may die during the progression of the cell cyclethrough a necrosis-like death mechanism. In thepresence of a functional FADD-caspase-8 pathway,these T cells may eventually die by apoptosis. Eitherway, T cells are prevented from proliferating uncon-trollably (Beisner et al., 2003; Salmena et al., 2003). Therole of c-FLIPL, the long isoform of c-FLIP thatresembles caspase-8 but lacks its enzymatic activity, inT-cell growth is more confusing as enforced expressionof this protein in the T-cell compartment either increases(Lens et al., 2002) or decreases (Tai et al., 2004) T-cellreceptor-triggered proliferation. Anyway, whereas in-flammatory caspases play a central role in innateimmunity, caspase-8, in association with FADD, iscrucial to adaptive immunity.

In B cells, caspases also regulate cell cycle progres-sion. Mice deficient in caspase-3 have an increasednumber of splenic B cells that show normal apoptosisbut enhanced proliferation, both in vivo and aftermitogenic stimulation in vitro (Woo et al., 2003).Caspase-3 was proposed to cleave the cyclin-dependentkinase inhibitor (CDKI) p21Cip1/Waf1, thus removing itsC-terminus that interacts with proliferating cell nuclearantigen (PCNA) to promote proliferation. Interestingly,the expression of p27Kip1, another CDKI that is apotential target for caspase-3 (Eymin et al., 1999b),remains unchanged in mitogen-activated B cells, whichenforces the idea that substrate specificity is essential fornonapoptotic functions of caspases. In contrast to thisnegative effect of caspase-3 on B-cell proliferation,caspase-6 was proposed to mediate entry of quiescentB cells into G1 phase of the cell cycle upon stimulation.One of the selective targets of caspase-6 in this processcould be the transcriptional suppressor special AT-richsequence-binding protein 1 (Olson et al., 2003). Thus,B-cell proliferation analysis points out a dual effect ofcaspases in the same process. Caspases may serve asadditional checkpoints in the control of cell cycle in Tand B cells by specific cleavage of negative or positiveregulators of the cell cycle machinery.

Caspases and differentiation with enucleation

The involvement of caspases in cell differentiation wasinitially suspected in those whose terminal differentia-tion is associated with enucleation. Erythroblasts,keratinocytes and lens epithelial cells lose their nucleusand other subcellular organelles during their terminaldifferentiation but continue to be metabolically active.

Erythropoiesis is promoted by the hormone erythro-poietin (Epo) and involves the sequential formation inthe bone marrow of proerythroblasts and basophilic,polychromatophilic and orthochromatic erythroblasts.These latter cells extrude their nucleus and enter the

circulation as mature red blood cells. The terminaldifferentiation of erythroid cells exhibits some simila-rities with apoptosis such as chromatin condensationand the degradation of nuclear components (Moriokaet al., 1998). The process of enucleation itself couldinvolve p53 in erythroid cells (Peller et al., 2003) andDNase IIa from central macrophages in the erythro-blastic island (Kawane et al., 2001; Krieser et al., 2002).A transient activation of several effector caspases isrequired for Epo-induced erythroid differentiation inboth humans and mice. However, this transient caspaseactivation may not account for enucleation as it occursat earlier steps of red blood cell formation (Zermatiet al., 2001; Kolbus et al., 2002; Carlile et al., 2004).Accorcdingly, the large spectrum caspase inhibitorN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone(z-VAD.fmk) arrests the in vitro maturation of erythroidprogenitors before nucleus and chromatin condensation(Zermati et al., 2001). In addition, ablation of caspase-3through the use of small interfering RNAs blockserythropoiesis at the transition between pro- andbasophilic erythroblasts (Carlile et al., 2004). Caspasesare probably activated through a mitochondria-depen-dent pathway similar to that identified in various formsof apoptosis (Zermati et al., 2001; Kolbus et al., 2002)and cleave proteins involved in nucleus integrity (laminB) and chromatin condensation (acinus) (Zermati et al.,2001). Interestingly, caspase activation in erythroid cellsundergoing differentiation is negatively regulated by theRaf-1 kinase, which prevents premature differentiationof actively proliferating precursors (Kolbus et al., 2002).

The transcription factor GATA-1 plays a major rolein the control of erythroid differentiation, for example,it is required for Epo-mediated upregulation of theantiapoptotic protein Bcl-xL (Gregory et al., 1999).GATA-1 is cleaved by caspases in erythroid cellsundergoing apoptosis under Epo deprivation or deathreceptor stimulation (De Maria et al., 1999), but remainsuncleaved in erythroid cells undergoing terminal differ-entiation (Zermati et al., 2001). The differential cleavageof GATA-1 by caspases in erythroid cells undergoingdifferentiation versus apoptosis indicates that the abilityof caspases to cleave their cellular protein targetsdepends on the cellular context.

Targeted disruption of the casp-8 gene results inembryonic lethality with congested accumulation oferythrocytes in mice (Varfolomeev et al., 1998). Thephenotype of mice in which the gene encoding theadaptor molecule FADD is disrupted resembles that ofcaspase-8�/� animals (Yeh et al., 1998; Zhang et al.,1998a). FADD and caspase-8 could play a role in thenegative regulation of erythropoiesis through a negativefeedback loop where mature erythroblasts, whichexpress Fas-ligand, might exert a cytotoxic effect onimmature erythroblasts expressing the death receptorFas within the erythroblastic island (De Maria et al.,1999). In Fas-stimulated immature erythroblasts, thetranscription factor GATA-1 is cleaved by activatedcaspases, which impairs erythroid differentiation orleads to apoptosis, depending on Epo concentration inthe environment. Thus, caspase-mediated cleavage of


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GATA-1 represents a negative control mechanism thatregulates the total red cell mass (De Maria et al., 1999).

Another transcription factor, the basic helix–loop–helix protein SCL/Tal-1, was reported to be cleaved bycaspases when activated in erythroid cells understimulation of death receptors or Epo deprivation.Caspase-mediated cleavage of SCL/Tal1 was proposedto amplify caspase activation, which leads to GATA-1cleavage in Epo-deprived or death receptor-stimulatedimmature erythroid cells (Zeuner et al., 2003).

Myelodysplastic syndromes are a heterogeneousgroup of disorders of hematopoietic stem cells char-acterized by a hypercellular bone marrow with mor-phological features of dysplasia and peripheral bloodcytopenias with anemia dominating the clinical picture.At early stages of development of the disease, a high rateof apoptosis in the bone marrow hematopoietic cells isresponsible for ineffective erythropoiesis (Raza et al.,1995; Tehranchi et al., 2003; Testa, 2004) and severalstudies indicate that the activation of the Fas-ligand/Faspathway plays an important role in this pathogenicevent (Gersuk et al., 1996; Bouscary et al., 1997;Claessens et al., 2002). Thus, exacerbation of thephysiologic mechanisms of Fas-mediated control oferythropoiesis, which functions even in the presence ofhigh concentrations of Epo, may account for earlyelimination of erythroid precursors and anemia in thesesyndromes. Accordingly, lentivirus-mediated transduc-tion of a dominant-negative FADD construct inhematopoietic progenitors from patients with earlystage myelodysplasia prevents caspase-8 activation inerythroid cells cultured in vitro, thus restoring erythro-poiesis (Claessens et al., 2005). In some of thesemyelodysplastic syndromes, growth factors such asgranulocyte colony-stimulating factor were reported toinhibit Fas-induced caspase activation by inhibiting themitochondrial pathway to cell death (Schmidt-Mendeet al., 2001; Tehranchi et al., 2003). However, how thisgranulocyte-specific growth factor interferes witherythropoiesis remains unexplained. Amplification ofphysiological caspase activation associated with ery-throid differentiation may also account for apoptosis ofmegaloblastic erythroblasts that appears upon folate orcobalamine deficiency, as well as erythroblasts frompatients with b-thalassemia, parvovirus B19 infectionand congenital aplastic anemia. Activation of caspasesthrough the death receptor pathway could account alsofor anemia observed in diseases such as multiplemyeloma and rheumatoid arthritis (Testa, 2004).

Another differentiation process with enucleation inwhich caspases play a role is lens fiber differentiation.Primary fibers develop from lens epithelial cells thatform the posterior, half of early lens vesicle. Theseprimary fibers constitute the core at the centre of thelens whereas secondary fibers develop from epithelialcells at the equatorial region of the lens (Goss, 1978). Aseach fiber forms, the nucleus and other organelles arelost, which is of fundamental importance to the functionof the mature lens since it leads to the formation of atransparent region at its center. Disturbances in thisprocess lead to congenital cataracts, due to disordered

fiber cell packing and scattering of light by theorganelles (Dahm et al., 1998). Transgenic mice over-expressing the antiapoptotic Bcl-2 protein in their lensesdemonstrate disturbances of lens development, includ-ing the persistence of intact or fragmented nuclei(Fromm and Overbeek, 1997). In an explant culture ofanterior epithelium from rat lens, in which epithelialcells differentiate into fibers under exposure to bFGF(Fibroblast Growth Factor) and insulin, the formationof lentoid bodies is associated with poly(ADP-ribose)polymerase (PARP) cleavage and nuclear DNA frag-mentation and is completely prevented by the caspaseinhibitor z-VAD-fmk and some more specific caspaseinhibitors (Ishizaki et al., 1998; Wride et al., 1999;Sanders and Parker, 2002). Interestingly, caspase-6activation was detected in lens from rat embryos duringthe period when organelles are eliminated, 2–3 daysbefore enucleation (Foley et al., 2004). However,caspase 6�/� mice do not demonstrate any lens altera-tion, which suggests that other caspases are involved inlens differentiation. Whether caspases are directlyresponsible for organelle loss and enucleation or playa role in earlier stages of the differentiation processremains to be clarified.

A third cell type in which terminal differentiation isassociated with enucleation and caspase activation is theepidermal keratinocyte. Caspases are activated duringhuman keratinocyte differentiation in organotypiccultures and this activation has been suggested to berequired for the normal loss of the nucleus (Weil et al.,1999). In cultured keratinocytes undergoing differentia-tion, caspase activation is associated with a gradualdecrease in mitochondrial membrane potential and aprogressive release of cytochrome c from some mito-chondria (Allombert-Blaise et al., 2003). Caspase-14,which shows a restrictive tissue expression mainlyconfined to the epidermal keratinocyte, was suggestedto be the main caspase involved in this differentiationprocess (Eckhart et al., 2000; Lippens et al., 2000; Chienet al., 2002; Rendl et al., 2002). Interestingly, a recentstudy demonstrated that casp-3 gene was a transcrip-tional target of Notch1, a transmembrane receptorinvolved in keratinocyte differentiation. Elevated cas-pase-3 expression and activity were shown to partiallyaccount for the commitment of embryonic keratinocytesto terminal differentiation, which explains the increasedthickness and the reduced expression of terminaldifferentiation markers observed in caspase-3�/� miceepidermis (Okuyama et al., 2004).

Although terminal differentiation of red blood cells,lens fibers and keratinocytes is characterized by en-ucleation, most studies in these cell types suggest thatcaspase activation is not directly involved in theenucleation process and is required at earlier steps ofthe differentiation pathways. In all these cells, activatedcaspase levels are not sufficient to trigger apoptosis andresult in the more selective targeting of substrates suchas lamin B in erythroblasts (Zermati et al., 2001) andprotein kinase Cd in keratinocytes (Okuyama et al.,2004). In addition, the caspase targets may vary,depending on the differentiation pathway, as PARP is


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cleaved during lens fiber differentiation while remaininguncleaved in erythroblasts and keratinocytes.

Caspases and differentiation without enucleation

Caspase activity has been shown to be required also forthe differentiation of specific nucleated cell types. One ofthe best-studied example is skeletal myoblast differen-tiation in which activation of caspases was initiallyrelated to the death of a fraction of cells that is usuallyobserved in in vitro models of myoblast cultures (Deeet al., 2002). Actually, caspase-3-null mice that surviveto early perinatal life are strikingly smaller relative totheir wild-type littermates, with a reduction in totalskeletal muscle mass. Primary myoblast cultures demon-strated that casp-3 gene deletion altered the in vitroformation of myotubes and the appearance of severaldifferentiation-specific proteins without modifying therate of apoptosis associated with the differentiationprocess (Fernando et al., 2002). Interestingly, expressionof active caspase-3 into a subconfluent population ofmyoblasts initiated the differentiation program withoutinducing cell death. The serine/threonine kinase MST1(for Mammalial Steril Twenty-like kinase) appears tomediate caspase-3 function in muscle differentiation as itis activated through caspase-3-mediated removal of itsC-terminal negative regulatory domain in muscle cellsundergoing differentiation and active MST1 rescuesmyogenesis in caspase-3�/� mice (Fernando et al., 2002).Raf signaling could negatively regulate caspase-3activity in skeletal myoblasts undergoing differentiation,as observed in erythropoiesis (DeChant et al., 2002).Based on the pharmacological manipulation of caspasesby permeant inhibitory peptides, caspase-1 has alsobe involved in rat myoblast fusion and this effectwas related to calpastatin degradation (Barnoy andKosower, 2003).

In Drosophila melanogaster, caspases are involved insperm differentiation and possibly in oogenesis. InDrosophila males, several apoptotic proteins play a rolein spermatid individualization, including multiple cas-pases, dFADD, ARK (the Drosophila homolog of Apaf-1) and one of the two cytochrome c genes, cyt-c-d(Arama et al., 2003; Huh et al., 2004b). An apoptosome-like complex could be assembled to activate caspasesand remove the bulk cytoplasm from spermatids(Arama et al., 2003). Interestingly, caspase activationduring spermatid differentiation could be the mainfunction of cytochrome-c-d in this animal. Organellesand in particular sperm nucleus must be protectedagainst the potentially lethal activity of caspasesactivated during the differentiation process. dBruce, aDrosophila IAP that is homolog to human Apollon andencodes a E2 ubiquitin-conjugating enzyme, may exertthis function in specific cell compartments (Arama et al.,2003).

In the Drosophila ovary, one germ cell in an eggchamber becomes an oocyte, whereas the 15 sistergermline cells develop into nurse cells that are linked to

the oocyte by cytoplasmic bridges. These so-called ‘ringcanals’ are used by nurse cells to transfer RNA andproteins to the oocyte. This event requires actin- andmyosin-based contraction of the nurse cells that subse-quently degenerate and die by apoptosis. Loss offunction of dcp-1 gene, which encodes a caspase similarin sequence to mammalian caspase-3 and -6, wasproposed to inhibit the transfer of cytoplasm fromnurse cells to developing ovocytes, which causes sterility.Thus, caspases could be involved in cytoskeletal andnuclear events in nurse cells (McCall and Steller, 1998).However, several phenotypes attributed to the loss ofdcp-1 are due to the disruption of CG3941 gene or thecombined disruption of both genes, as dcp1 is located inan intron of CG3941 gene and P element allelesassociated with the sterile phenotype also disruptCG3941 (Corrections and Clarifications, 2004).

Caspase activation has been involved in thrombo-poietin-induced megakaryocyte differentiation and pro-platelet formation. As observed in erythropoiesis, anagonist anti-Fas antibody inhibits megakaryocytopoi-esis in vitro. This effect is credited to the caspase-inducedcleavage of transcription factors GATA-1 and NF-E2,suggesting a Fas-mediated negative regulation of mega-karyocytopoiesis (De Maria et al., 1999). Thrombopoie-tin is the ligand of Mpl receptor whose engagement onmegakaryocytes stimulates proliferation and promotespolyploidization and maturation. Interestingly, theserine/threonine kinase MST1 was shown to be acti-vated by Mpl-ligand and to participate in the Mpl-ligand-induced signalling pathways that potentiatepolyploidization and megakaryocyte differentiation(Sun and Ravid, 1999). Whether caspase-mediatedcleavage of MST1, identified in apoptotic B lymphocyte(Graves et al., 1998) and involved in myoblast differ-entiation (Fernando et al., 2002), is required for MST1function in megakaryocytopoiesis has to be explored.

Another function of caspases in megakaryocytopoi-esis is proplatelet formation. Platelets are enucleatedcells formed by the fragmentation of the maturemegakaryocyte cytoplasm and arise from the develop-ment of thin, long cytoplasmic extensions calledproplatelets. A localized activation of caspase-3mediated by cytochrome c released from the mitochon-dria and prevented by Bcl-2 overexpression activelyparticipates in the formation of proplatelets (De Bottonet al., 2002). Moreover, caspases also play a role inplatelet activation. Treatment with z-VAD.fmk, a pan-caspase inhibitor, significantly decreases ADP-inducedplatelet aggregation (Cohen et al., 2004).

Differentiation-associated caspase activation is notalways associated with phenotypic changes reproducingthose observed in cells dying by apoptosis. For example,several caspases are activated in peripheral bloodmonocytes undergoing differentiation into macrophageswhen cultured in the presence of M-CSF, whereas nocaspase activation can be detected when these peripheralblood monocytes undergo differentiation into dendriticcells under exposure to GM-CSF and IL-4. Caspaseactivation in monocytes undergoing differentiation intomacrophages involves the mitochondrial release of


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cytochrome c and leads to the cleavage of specificproteins such as acinus, whereas other well-character-ized caspase targets such as PARP remain uncleaved.Overexpression of Bcl-2 and inhibition of caspases byz-VAD-fmk or the baculoviral inhibitory protein p35inhibit the differentiation process (Sordet et al., 2002).The role of caspases in the differentiation of monocytesinto macrophages was recently confirmed by analysis ofa mouse model in which the casp-8 gene could beconditionally disrupted by using the Cre/loxP recombi-nation system. This model revealed several nonapopto-tic functions of caspase-8, for example, casp-8 genedeletion in bone marrow cells prevented hematopoieticprogenitors from forming myeloid and B-lymphoidcolonies, both in vitro and in vivo. When casp-8 genedeletion was restricted to cells of the myelomonocyticlineage, the differentiation of monocytes into macro-phages was arrested, whereas their differentiation intodendritic cells or granulocytes remained unaffected(Kang et al., 2004). Of interest, homozygous deletionof bid gene involved in death receptor signal amplifica-tion, induces a fatal, clonal malignancy closely resem-bling chronic myelomonocytic leukemia (Zinkel et al.,2003). This latter observation suggests a connectionbetween Bid protein and caspase-8 activation in mono-cytes undergoing differentiation into macrophages.

Caspase activation has also been tightly linked todifferentiation of an osteoblastic cell line exposed to thebone morphogenetic protein BMP-4 (Mogi and Togari,2003), to terminal differentiation of a colon carcinomacell line exposed to butyrate (Cai et al., 2004) and topostnatal development of rat cerebellum (Oommanet al., 2004).

A role for caspases in preventing cell death

Based on the assumption that apoptotic death ofmammalian cells usually depends on caspase activation,pharmacological inhibition of these enzymes may bebeneficial in diseases in which excessive apoptosis playsa pathophysiologic role. Actually, inhibition of caspasescan paradoxically enhance cell death. This was clearlydemonstrated by studying death receptor-mediatedapoptosis, both in vitro and in vivo. Inhibition ofcaspases, either with z-VAD.fmk or by transfection ofthe poxvirus-derived caspase inhibitor CrmA thatselectively inhibits caspase-8, prevents or delays death-receptor-mediated apoptosis in most cell lines, yetsensitizes some of these cell lines to death-receptor-mediated necrosis (Vercammen et al., 1998; Luschenet al., 2000; Chan et al., 2003). These deleterious effectsof caspase inhibition have been confirmed in vivo byshowing that pretreatment with z-VAD.fmk sensitizesmice to the lethal effect of human or mouse recombinantTNFa, resulting in hyperacute hemodynamic collapse.This latter effect probably involves mitochondriallygenerated reactive oxygen species and an increase inperoxidation of lipids (Cauwels et al., 2003). Interest-ingly, caspase-mediated cleavage of target proteins suchas p27Kip1 (Eymin et al., 1999a) and Rb (Rincheval et al.,

1999) can generate fragments that demonstrate aprotective effect towards cell death.

The death receptor-mediated caspase-independentdeath pathway involves the death domain of the adapterprotein FADD and the serine/threonine kinase activityof the receptor-induced protein (RIP), whereas thetranscription factor NF-kB is not activated (Vercammenet al., 1998; Fiers et al., 1999; Holler et al., 2000;Vonarbourg et al., 2002). The connection betweenFADD/RIP and mitochondrial production of reactiveoxygen species remains poorly understood, although apivotal role for phospholipase A2 has been suggested(Cauwels et al., 2003). Whether a limited activation ofcaspase-8 in the DISC in the presence of FLIPL

(Micheau et al., 2002) or a protease-independentfunction of caspase-8 (Hu et al., 2000) are involved inthis process remains to be clarified.

By arresting apoptosis, caspase inhibitors couldpromote other forms of cell death, for example,caspase-8 inhibition was shown recently to promoteautophagic cell death, a process involving the genesATG7 and beclin 1 genes. Again, this molecular pathwayinvolves the serine-threonine kinase RIP together withthe Jun amino-terminal kinase (JNK) (Yu et al., 2004).The suppression of necrotic and autophagic death bycaspases in mammalian cells indicates that caspasesregulate both apoptotic and nonapoptotic cell deathpathways. Thus, caspase inhibition as a therapeutic goalcould have the untoward effect of exacerbating celldeath.

Another protective effect has been proposed recentlyfor caspase-2, an enzyme whose role in apoptosis is stillcontroversial. This protease was shown to be recruitedto a platform named PIDD (p53-induced death domain)to form a large multiprotein complex involving theadaptor protein RAIDD. Spontaneous activation ofcaspase-2 by overexpression of PIDD is not sufficient totrigger cell death, although it sensitizes to genotoxicdamage-induced cell death. It is suggested that caspase-2in this setting may have nonapoptotic effects such asactivation of the DNA repair machinery (Tinel andTschopp, 2004).

A protective effect of caspases has been described inhuman dendritic cells undergoing maturation underexposure to LPS, a pathway that could involve LPS-induced activation of extracellular receptor kinases(ERKs) and upregulation of c-FLIPL (Franchi et al.,2003). Lastly, a dual role for the initiator caspase Droncwas recently identified during wing development ofDrosophila. Dronc-mediated ectopic cell death in somecells is associated with the compensatory proliferation ofother cells, a mechanism required to maintain tissue sizeand homeostasis (Huh et al., 2004a).

A role for caspases in cell motility and migration

Caspases were suggested to be involved in the main-tenance of cytoskeleton integrity. This assertion wasbased on the ability of the poorly specific pancaspase


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inhibitor benzyloxycarbonyl-L-aspartyl-[(2,6-dichloro-benzoyl)oxy] methane (z-Asp-CH2-DCB) to inhibit cellspreading on collagen-coated plates without affectingviability nor proliferation. A caspase-3-like activity(cleavage of a z-Asp-Glu-Val-Asp (DEVD)-containingpeptide) was detected in adherent cells while remainingundetectable in cells in suspension. However, morespecific caspase inhibitors failed to inhibit cell spreadingin this model (Watanabe and Akaike, 1999).

More recently, analysis of CD95-resistant tumor celllines again suggested a role for caspases in cellmigration. A significant number of these CD95-resistanttumor cell lines respondes to stimulation of CD95 byincreased motility and invasiveness through Matrigel-coated membranes. Among the different pathways thataccount for these effects of CD95 engagement, onecould involve caspase-8, downstream or in parallel to atranscriptional activation induced by activated NF-kB.Based on this study, it was suggested that CD95-ligandexpressed at the surface of numerous tumor cell typescould have tumorigenic effects through CD95 stimula-tion of tumor cells with caspase-8 activation facilitatingtheir migration (Barnhart et al., 2004). How theseobservations are connected to the frequent inactivatingmutations in the proximal pathway of CD95-mediatedapoptosis affecting CD95, FADD and caspase-10remains to be explained (Shin et al., 2002).

Conclusions

Caspase activation does not mean cell death but howcaspases are implied in different cell processes withoutkilling the cells remains poorly known. The nonapopto-tic functions of caspases implicate the selective cleavageof specific target proteins to avoid the cell dismantle.Various mechanisms could account for this restrictedcleavage, including post-translational modifications ofcaspases and substrates, subcellular compartmentaliza-tion of the proteases, protection of potential targetproteins by scaffold molecules, activation of antiapop-totic factors and recruitment of antagonistic proteins atthe level of caspase activation platforms (Figure 2).

Post-translational modifications concern caspasesthemselves, for example, Akt-mediated phosphorylationof caspase-9 was proposed to decrease its activity(Cardone et al., 1998). Adapter proteins such as FADDcould also be modified, for example, a slower migratingform of FADD has been detected in T cells exposed tomitogenic stimulation, while not being observed in Tcells undergoing death receptor-mediated cell death(O’Reilly et al., 2004). Post-translational modificationscould also concern caspase targets, for example, thephosphorylation of serine residues adjacent to thecaspase-3 cleavage site of presenilin-2 could protect theprotein from cleavage (Walter et al., 1999). Whethersuch modifications of caspases or their targets accountfor the Raf-mediated regulation of erythroid andskeletal muscle differentiation remains to be explored(DeChant et al., 2002; Kolbus et al., 2002). A model in

which RasGAP functions as a sensor of caspase activityto determine whether or not a cell should survive hasbeen proposed. According to this model, a limitedactivation of caspases, for example, in response to alimited stress, leads to the partial cleavage of RasGAPthat protects cells from apoptosis, whereas a strongeractivation of caspases allows completion of RasGAPcleavage and the resulting RasGAP fragments turn intopotent proapoptotic molecules (Yang and Widmann,2001, 2002). Authors indicate that executioner caspasestherefore control the extent of their own activation by afeedback regulatory mechanism initiated by the partialcleavage of RasGAP that is crucial for cell survivalunder adverse conditions (Yang et al. 2004).

Although caspases are mainly localized in the cytosol,a fraction of them has been identified in cellularorganelles, for example, procaspase-3 was identified inthe mitochondria, in a complex with HSP60 and HSP10(Zhivotovsky et al., 1999), a substantial portion ofprocaspase-2 was localized to the nucleus and Golgicomplex (Mancini et al., 2000) while procaspase-9 wasidentified in the nucleus (Sordet et al., 2001). A localizedactivation of caspases could account for some non-apoptotic functions of these enzymes, for example,activated forms of caspase-3 detected in maturingmegakaryocytes before proplatelet formation demon-strate a punctuate cytoplasmic distribution, whereas adiffuse staining pattern is observed in these cells whenbecoming apoptotic (De Botton et al., 2002). Aprocaspase redistribution is associated with phorbolester-induced differentiation of U937 monocytic leuke-mic cells (Sordet et al., 2001), a model in which caspase

Caspase activation

Accessibility of cleavable substrates

Accumulation of cleavable substrates(inhibition of proteasome)

Selective processing of substrates

Death Life

Anti-apoptoticfactors

Scaffoldproteins

Subcellular compartmentalization

Post-translational Modification/ Phosphorylation

Figure 2 Regulation of cell progression, differentiation, activationand cytoprotection by caspases requires the restricted cleavage ofspecific target proteins, which may be governed either by post-translational modifications of caspases, adapter molecules involvedin their activation and their substrates, for example, by phosphor-ylation, or by specific subcellular compartmentalization of caspasesor by protection of potential target proteins by scaffold proteins orby the activation of antiapoptotic factors


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activation is required for the differentiation process(Sordet et al., 2002). Whether the differentiation-associated redistribution of the caspase inhibitor cIAP-1, which is present in the nucleus of undifferentiatedcells and is relocalized to the Golgi apparatus in cellsundergoing differentiation (Plenchette et al., 2004;Vischioni et al., 2004), is involved in the control ofcaspase activation in some differentiation pathwaysremains unknown. However, another IAP, Dbruce,protects differentiating fly spermatids from destructionby caspases activated during the differentiation process(Arama et al., 2003).

At the level of death receptors, the role of moleculeredistribution into plasma membrane rafts remains acontroversial issue but the recruitment of regulatorymolecules such as FLIPL at the level of the death-inducing signaling complex appears to modulate thelevel of caspase-8 activation and downstream signaliza-tion (Micheau et al., 2002; O’Reilly et al., 2004). Onecould speculate that, in tumor cells, deregulated expres-sion of regulatory molecules such as IAPs and FLIP, inaddition to inhibiting cell death and inducing chemore-sistance (Kim et al., 2001; Liu et al., 2002; Shin et al.,2002; Yang, 2002), may favor nonlethal pathways ofsignalization that contribute to enhanced tumorigenesis(Barnhart et al., 2004).

Analysis of nonlethal functions of caspases mayclarify the role of enzymes such as of the evolutionarilyconserved caspase-2 whose apoptotic functions appearto be limited to amplification of other caspase cascades(Droin et al., 2001). Recent data suggest that caspase-2could play a role in other cellular functions such asDNA repair (Tinel and Tschopp, 2004) and cellular lipidmetabolism (unpublished results). After more than adecade of research dedicated to the study of apoptoticfunctions of caspases, several nonapoptotic functions ofthese enzymes have now been identified. It is likely thatother biological functions of caspases are still to beuncovered. Ongoing studies may provide more informa-tions of caspase targets associated with a specific cellularfunction and decipher the regulatory mechanisms thatcontrol the multiple functions of these versatile enzymes.They may also shed new lights on the role of caspaseactivity deregulation in the pathophysiology of varioushuman diseases.

Acknowledgements

Our group is supported by la Ligue Nationale Contre leCancer (label to ES and GK) and SL is supported by a post-doctoral fellowship of la Ligue Nationale Contre le Cancer.The work has been partially supported by the Canceropole, Ilede France (to OH, MF and GK).

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