Critical Review Regulation of Apoptosis by Heat Shock Proteins Donna Kennedy1 €ger2 Richard Ja Dick D Mosser3 Afshin Samali1* 1

Department of Biochemistry, Apoptosis Research Centre, Biosciences Research Building, Corrib Village, NUI Galway, Dangan, Galway, Ireland 2 Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany 3 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

Abstract Thermotolerance, the acquired resistance of cells to stress, is a well-established phenomenon. Studies of the key mediators of this response, the heat shock proteins (HSPs), have led to the discovery of the important roles played by these proteins in the regulation of apoptotic cell death. Apoptosis is critical for normal tissue homeostasis and is involved in diverse processes including development and immune clearance. Apoptosis is tightly regulated by both proapoptotic and antiapoptotic factors, and dysregulation of apoptosis plays a significant role

in the pathophysiology of many diseases. In the recent years, HSPs have been identified as key determinants of cell survival, which can modulate apoptosis by directly interacting with components of the apoptotic machinery. Therefore, manipulation of the HSPs could represent a viable strategy for the treatment of diseases. Here, we review the current knowledge with regard to the mechanisms of HSP-mediated regulation of apoC 2014 IUBMB Life, 00(00):000–000, 2014 ptosis. V

Keywords: cell death; apoptosis; intrinsic pathway; heat shock proteins; caspases; heat shock response

Introduction Heat shock proteins (HSPs) are an evolutionarily conserved superfamily (1). As their name suggests, HSPs were originally discovered to be upregulated after exposure of cells to elevated temperatures; however, they are also induced in response to a variety of other stress stimuli (2), and the heat shock response is now recognized as being one of the most ancient and evolutionarily conserved cytoprotective mechanisms found in nature (3). Subjecting cells to a bout of mild thermal stress will confer protection against a subsequent and more severe insult (4). This is also true for pathological stressors; for example, a brief period of ischemia can mediate protection against a subsequent long-term ischemic insult (5). Protection from cell death C 2014 International Union of Biochemistry and Molecular Biology V

Volume 00, Number 00, Month 2014, Pages 00–00 *Address correspondence to: Afshin Samali, Apoptosis Research Centre, Biosciences Research Building, Corrib Village, NUI Galway, Dangan, Galway, Ireland. Tel: 1353-9149-2440. Fax: 1353-9149-4596. E-mail: [email protected] Received 4 April 2014; Accepted 1 May 2014 DOI 10.1002/iub.1274 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)

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afforded by thermal preconditioning is mediated by HSPs as it can be abrogated by inhibitors of HSPs such as triptolide (6). However, although a recent study revealed that many of these inhibitors are not as specific as initially thought (7), more specific methods, such as knockdown of HSPs, have shown their critical role in protection from cell death (8). Interestingly, cells exposed to a particular stress stimulus also develop cross-tolerance to a different stress stimulus (9). For example, thermal preconditioning can protect cells against the toxicity of anticancer drugs (10) and the neurotoxin N-methyl-4-phenylpyridine (11). The phenomenon of cross-tolerance supports the notion that HSP induction serves as a general cytoprotective mechanism in cells.

The HSP Superfamily HSPs function as “molecular chaperones,” proteins that guard against “illicit or promiscuous interactions” between other proteins. These chaperones protect the proteome from the dangers of misfolding and aggregation by facilitating protein folding, trafficking, complex assembly, and ubiquitination, as well as proteasomal degradation (12). This protection is achieved in a number of ways including through de novo protein folding, refolding of misfolded proteins, and oligomer

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assembly. It now appears that cytoprotection is not simply a matter of chaperoning damaged proteins but also involves direct interference with apoptotic pathways and the maintenance of cytoskeletal integrity under conditions of stress facilitated by the chaperoning action of HSPs (13). HSPs are divided into families based on molecular weight: HSPA (HSP70), HSPH (HSP110), HSPC (HSP90), DNAJ (HSP40), HSPB (small HSPs) (14) and chaperonin families HSPD/E (HSP60/HSP10) (15). The human genome encodes 13 HSPA family members including constitutively expressed and stress-inducible members (see Table 1 for details). They all display a high degree of sequence and domain homology. Additional characteristics include (i) a conserved ATPase domain at the N-terminus; (ii) a flexible C-terminal peptide substrate binding domain (SBD); and (iii) a G/P-rich C-terminal region containing an EEVDmotif that enables the proteins to bind co-chaperones and other HSPs. Cooperation with cofactors regulates the many functions of HSPA proteins (16). The HSPB family consists of 11 members, HSPB1–11. Some are ubiquitously expressed, whereas others display tissuerestricted patterns of expression (see Table 1 for details). Furthermore, some members of stress-inducible HSPBs are highly diverse in sequence, size, client protein specificity, and function (17). Post-translational modifications of HSPBs adds an additional layer of regulation, which is particularly important in stress conditions (18). The dynamic organization of HSPB oligomers appears to be a crucial feature governing HSPB activity. It has been demonstrated that in response to different stress stimuli, HSPB1 alters its oligomerization status, which may enable differential client–protein interactions (19). HSPD1 and HSPE1 are classed as chaperonins. HSPD1 and HSPE1 form a complex in which HSPE1 regulates the substrate binding and ATPase activity of HSPD1. A majority (60– 80%) of HSPD1 and HSPE1 proteins are located in the mitochondria where they are involved in folding of a subset of mitochondrial proteins (20). Roles for these proteins in the cytoplasm are also emerging. The HSPC family is composed of five members designated as HSPC1–5. Until recently, the literature has not differentiated between the various family members. However, HSPC1 and HSPC3 turned out to have non-overlapping functions (21). Specifically, HSPC1 and HSPC3 show similar interaction profiles with co-chaperones but differ in their substrate interactome. The HSPC family functions as part of a multichaperone complex via association with a variety of co-chaperones. Similar to the HSPA family, HSPC proteins have the ability to hydrolyze ATP and to bind and modify the conformations of client proteins (15,21). The human genome encodes four HSPH family members (HSPH1–4). The HSPH family is important for preventing protein aggregation. HSPH proteins are thought to primarily act as nucleotide exchange factors for the HSPA family; however, they have also been shown to be able to refold misfolded luciferase in the absence of HSPA proteins (15).

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Molecular Basis of Apoptosis Apoptosis is a caspase-dependent form of cell death. In the adult organism, apoptosis is responsible for the removal of damaged, aged, or superfluous cells in a manner that avoids unwanted activation of the immune system (22). Dysregulation of apoptosis is associated with a number of pathological processes; resistance to apoptosis can cause autoimmune disease and cancer. Excessive apoptosis is linked with inflammatory diseases and neurodegenerative diseases (23). The stereotypical pattern of cell demolition during apoptosis is mediated by a class of intracellular proteases, the caspases. In mammals, caspases (cysteine-dependent aspartatespecific proteases) are a family of 15 proteases. Seven family members (the “apoptotic” caspase-2, -3, -6, -7, -8, -9, and -10) are involved in apoptosis, whereas the remaining members play roles in inflammation and other processes. Within the wide range of cellular targets, apoptotic caspases are key substrates that mediate the demolition of the cell (24). Caspases are present in cells as inactive dimeric proenzymes. Activation involves two consecutive cleavage events, between the prodomains and the small and large subunits, respectively, which generates the active heterotetrameric caspase. Their prodomains and activation mechanisms classify the apoptotic caspases into two subgroups: initiator and executioner caspases. Executioner caspases (caspase-3, -6, and -7) have small prodomains, and their cleavage and activation are mediated by other caspases. Therefore, cleavage of executioner caspases sets in motion an irreversible chain reaction of further caspase cleavage and activation. This cascade is initiated by initiator caspases (caspase-2, -8, -9, and -10) that are characterized by large prodomains that contain homotypic protein interaction motifs facilitating protein–protein interactions, such as caspase activation and recruitment domains (CARD) in caspase-9 and -2 and death effector domains (DED) in caspase-8 and caspase-10. CARD and DED domains mediate recruitment of capases to the so-called activation complexes whose assembly controls the activation of the recruited initiator caspases. There are two major pathways of initiator caspase activation. The extrinsic pathway is initiated at the cell surface by death receptors that are members of the tumor necrosis factor (TNF) receptor gene family. Most cellular stresses, however, trigger the intrinsic pathway that is initiated inside cells by mitochondrial release of proapoptotic factors.

The “Extrinsic” Pathway The extrinsic pathway can be triggered by ligands of members of the death receptor family, including TNFR, Fas/CD95, TRAIL-R1, and TRAIL-R2 (25). The ligation of Fas or TRAIL receptors by their specific ligands triggers their trimerization and activation, which is followed by recruitment of the adaptor protein Fas-Associated protein with Death Domain (FADD) at the cytoplasmic side and assembly of the so-called deathinducing signaling complex (DISC) that recruits procaspases8/-10, mediating their oligomerization and autoactivation. The

Heat Shock Proteins and Apoptosis

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3

HSP70-1; HSP72

HSP70-B; HSP70-2

HSP70T; hum70t; HSP70-1L; HSP70-HOM

HSP70-2; HSP70-3

BIP, GRP78, MIF2

HSP70B

Possible pseudogene of HSPA1B

LAP1; HSC54; HSC70; HSC71; HSP71; HSP73; NIP71; HSPA10

GRP75; HSPA9B; MOT; MOT2; PBP74; mot-2

HSPA1A

HSPA1B

HSPA1L

HSPA2

HSPA5

HSPA6

HSPA7

HSPA8

HSPA9

5q31.1

11q24.1

1q23

9q33.3

14q24.1

6p21.3

6p21.3

6p21.3

Location

3313

3312

3310

3309

3306

3305

3304

3303

ID

Mitochondria

Nucleus

Cytoplasm

Nucleus

Cytoplasm

Endoplasmic reticulum

Nucleus

Cytoplasm

Nucleus

Cytoplasm

Lysosomes

Nucleus

Cytoplasm

Lysosomes

Nucleus

Cytoplasm

Cellular location

Ubiquitous

Ubiquitous

Ubiquitous

Ubiquitous

Testis

Testis

Ubiquitous

Ubiquitous

Expression

Yes

No

Yes

Yes

?

No

Yes

Yes

Poor prognosis in cancer

Polymorphisms linked with MS

Resistance to chemotherapeutics

Malignancy and metastasis

Poor prognosis in cancer

Associated with poor prognosis and metastasis in cancer

Polymorphisms associated with AD

Associated with poor prognosis and metastasis in cancer

Reduced in AD; protective in PD

Stress inducible Disease relevance

Overview of the HSPA and HSPB families expression profile, cellular localization, stress inducibility and disease association

Alternative names

HSPA family

Name

TABLE 1

4

Alternative names

(Continued)

CMT2F, HMN2B, HS.76067, HSP27, HSP28, Hsp25, SRP27

MKBP; HSP27; Hs.78846; LOH11CR1K

HMN2C; DHMN2C; HSPL27

CRYAA, CRYA1, CTRCT9

CRYAB, CMD1II, CRYA2, CTPP2

HSPB1

HSPB2

HSPB3

HSPB4

HSPB5

HSPB family

11q22.3-q23.1

21q22.3

5q11.2

11q22-q23

7q11.23

21q11

HSPA13

STCH

20p13

10q26.12

Location

HSPA12b C20orf60; dJ1009E24.2

HSPA12A FLJ13874; KIAA0417

Name

TABLE 1

Cellular location

1410

1409

8988

3316

3315

6782

Yes

Nucleus

Cytoplasm

Nucleus

Cytoplasm

Nucleus

Cytoplasm

Mitochondria

Ubiquitous

Lens

Cardiac, skeletal muscle

Yes

No

No

Protective in ischemia/reperfusion

Metastasis

Cancer

Cataracts

Myopathy, neuropathy

Cataracts

Neuropathy

dHMN

Cancer

Myopathy

Williams syndrome

Protective in ischemia/reperfusion

Metastasis

Cancer

CMT, dHMN

Cancer

Nucleus

No

Yes

?

Ischemia

Cardiac, skeletal muscle

Ubiquitous

Ubiquitous

Protective in ischemia/reperfusion

Required for angiogenesis

Reduced in schizophrenia

Resistance to chemotherapeutics

Stress inducible Disease relevance

Yes Ubiquitous (enriched in endothelial cells)

Ubiquitous

Expression

Cytoplasm

Cytosol

Nucleus Perikaryon

Cytoplasm

Microsomes

116835 Cytoplasm

259217 Cytoplasm

ID

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HSP20

cvHSP

H11; HMN2; CMT2L; DHMN2; E2IG1; HMN2A; HSP22

CT51

ODF1; RT7; ODF2; ODFP; SODF; CT133; ODF27; ODFPG; HSPB10; ODFPGA; ODFPGB

PP25; IFT25; C1orf41; HSPCO34

HSPB6

HSPB7

HSPB8

HSPB9

HSPB10

HSPB11a

4956

8q22.3

51668

94086

26353

12q24.23

17q21.2

27129

1p32

Cellular location

Nucleus

Cytoplasm

Sperm tail

Nucleus

Cytoplasm

Nucleus

Cytoplasm

Mitochondria

Nucleus

Cytoplasm

Nucleus

126393 Cytoplasm

ID

1p36.23-p34.3

19q13.12

Location

Placenta

Testis

Testis

Ubiquitous

Cardiac, skeletal muscle

Ubiquitous

Expression

Cardiomyopathy

Ischemia

Neuropathy

Yes

?

?

Cancer

Cancer

Cancer

Cell type CMT and dHMN specific

?

Yes

Stress inducible Disease relevance

Abbreviations: AD, Alzheimer’s disease; PD, Parkinson’s disease; MS, multiple sclerosis; CMT, Charcot Marie Tooth; dHMN, distal hereditary muscular neuropathy. a Controversial HSPB member as it does not have a classical a-crystallin domain.

Alternative names

(Continued)

Name

TABLE 1

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activated initiator caspases can directly cleave executioner caspases, thus initiating the caspase cascade, or they cleave the BH3-only protein BID that subsequently translocates to mitochondria and triggers the intrinsic pathway in parallel (described below). The Fas receptor can also bind an alternative adaptor protein, Daxx, leading to activation of the JNK pathway. Activation of the TNF receptor allows recruitment of the adaptor proteins (26), TNFR-Associated Death Domain and FADD. FADD can recruit and activate procaspase-8 in a manner similar to the Fas signaling pathway. Moreover, the TNFR DISC can prevent the activation of the prosurvival transcription factor NF-jB, thus promoting TNF-induced apoptosis (26). Apoptosis triggered by death receptors can be impaired or modulated by variants of the catalytically inactive caspase-8 homolog, cFLIP that can be recruited to the DISC. Important additional regulators of DISC signaling are the inhibitor of apoptosis proteins (IAPs), which mediate the ubiquitinylation of interacting proteins at the DISC and can also impair caspase activity (26).

The “Intrinsic” Pathway Activation of the intrinsic pathway leads to mitochondrial outer membrane permeabilization (MOMP) and release of apoptogenic factors from the mitochondria, such as cytochrome c, second mitochondrial-derived activator of caspases (SMAC), and Omi stress-regulated endoprotease/high-temperature requirement protein A2 (HTRA2) (27). This is a crucial event for driving caspase activation and subsequent apoptosis. Cytochrome c binds to the apoptotic protease-activating factor 1 (Apaf-1), causing it to assemble into a large protein complex termed the apoptosome that recruits and activates procaspase-9 resulting in activation of downstream executioner caspases including caspases-3, -7, and -6. SMAC and HTRA2 block X-linked inhibitor of apoptosis, a direct caspase inhibitor of the IAP family, and thus allow for unrestrained caspase activity (28). MOMP is controlled by a family of proteins called the BCL2 family. In mammals, there are 15 core BCL-2 proteins with varying degrees of structural similarity in short amino acid motifs called BH (BCL-2 homology) domains designated as BH1–4. The proapoptotic family members include the multidomain members BAX and BAK (containing BH1, 2, and 3 as well as a carboxy terminal stretch of hydrophobic amino acids serving for membrane insertion) and the BH3-only proteins BAD, BIM, BIK, BID, PUMA, BMF, HRK, and NOXA, whose sequence homology to the other family members is restricted to the BH3 domain. Antiapoptotic BCL-2 family members, BCL2, BCL-XL, MCL-1, BCL-w, BCL-2A1, and BCL-B, typically have all four BH domains and a carboxy terminal membrane insertion domain. The dynamic interplay between these proteins regulates MOMP. Proapoptotic multidomain family members BAX and BAK act on the outer mitochondrial membrane and mediate MOMP via oligomerization once they become activated by

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physical interaction with BH3-only proteins. Antiapoptotic BCL-2 members can interact with distinct BH3-only proteins, and this interaction impairs their antiapoptotic function (29). Thus, the BH3-only proteins trigger MOMP, and they are activated at the transcriptional or post-translational level by apoptotic signaling pathways and transduce apoptotic stimuli to the mitochondria.

Regulation of Apoptosis by HSPs Induction of HSPs has been shown to increase resistance to cell death induced by a number of conditions. Whether or not increased HSP expression proves beneficial or detrimental depends on the disease in question. Some forms of cancer are associated with increased expression of HSPs, which enhances tumorgenicity and resistance to cell death. On the other hand, increased expression of HSPs associated with enhanced survival of postmitotic cells such as cardiomyocytes and neurons is considered beneficial.

HSPs and Resistance to Apoptosis It is well established that the acquisition of thermotolerance or the overexpression of specific HSPs can attenuate apoptotic cell death (10,30). In particular, HSPA1 and HSPB1 have consistently proven to be inhibitors of apoptosis in multiple cell types. A number of studies have provided insight into the mechanisms involved and have shown that individual HSPs can interact with the apoptotic pathway at several levels (31), and these are discussed below.

HSPA Regulation of the Intrinsic Pathway Overexpression of HSPA1 protects cells against a range of stimuli that engage the intrinsic pathway such as hyperthermia (32), etoposide (33), staurosporine (34), and endoplasmic reticulum stress (35). HSPA1 is overexpressed in many cancers where it is associated with poor prognosis (36) and increased metastasis, and silencing of HSPA1 has been shown to promote apoptosis in human cancer cells (37). Conversely, overexpression of HSPA1 is protective in neurodegenerative disease models (38). HSPA1 was shown to attenuate the intrinsic pathway at three levels, upstream of, at the level of and downstream of MOMP (see Figure 1). It has been shown that HSPA1 can act upstream of the mitochondria by inhibiting BAX activation. In nonapoptotic cells, BAX is present primarily in the cytosol. Apoptotic stimuli cause a conformational change in BAX resulting in its translocation to the mitochondria, where it mediates MOMP. In heat-induced apoptosis models, HSPA1 can inhibit BAX activation but is unable to prevent cell death when a constitutively membrane-targeted mutant BAX protein is overexpressed. HSPA1 did not directly associate with BAX, indicating that it acts upstream of BAX activation (39). Consistent with this interpretation, HSPA1 overexpression prevented the heat-induced downregulation of the antiapoptotic BCL-2 family protein MCl-1, which is an important regulator of BAX oligomerization (40). Other studies have reported that the

Heat Shock Proteins and Apoptosis

HSPA1/Dna-J co-chaperone pair directly interacts with BAX preventing its translocation to the mitochondria and in this way prevents nitric oxide-induced apoptosis in macrophages (14). Similarly, HSPA1 overexpression in HL-60 cells inhibits Ara-C- and etoposide-induced BAX conformational change and translocation to the mitochondria via a direct interaction (41). Potentially, HSPA1 might bind to a protein(s) present in a complex with BAX that is present in some cells but not in others. HSPA1 was also shown to interact with the antiapoptotic BCL2 family member BCL2L12 preventing its proteasomal degradation (42). Several reports demonstrate that HSPA1 can interfere with cytochrome c release. HSPA1 overexpression in macrophages prevents cytochrome c release from mitochondria, thereby preventing nitric oxide-induced apoptosis (43). In HSPA1-transfected Jurkat cells, early apoptotic events such as mitochondrial depolarization and cytochrome c release are inhibited following H2O2 treatment (44). HSPA1 can reduce cytochrome c release in a manner that is dependent on the chaperoning function of HSPA1 in PEER cells subjected to heat shock (45). However, in U937 cells, overexpression of HSPA1 failed to prevent heat-induced cytochrome c release but was sufficient to reduce caspase activation suggesting cell typespecific effects (46). Downstream of the mitochondria, HSPA1 can interact directly with components of the mitochondrial pathway and potentially impede apoptosis. Evidence for a role downstream of cytochrome c release has been suggested by the finding that HSPA1 interacts with the proforms of caspases-3 and -7, but not their activated forms, indicating that HSPA1 might act by preventing procaspase processing (47). HSPA1 has been reported to inhibit cytochrome c-mediated caspase activation in vitro (8), although another study did not observe an inhibitory role (32). In vitro studies have also suggested a role for HSPA1 in the prevention of apoptosome assembly and procaspase-9 recruitment to the apoptosome (48). However, caution has been noted in the interpretation of these in vitro results (49). An inhibitory role for HSPA1 even later in the apoptotic pathway was reported downstream of caspase-3-like proteases, where it inhibits events such as phospholipase A2 activation and changes in nuclear morphology (50).

Other Apoptosis-Regulating Mechanisms of HSPA1 There are few reports implicating HSPA1 in inhibiting the extrinsic apoptotic pathway. In human leukemia cells, HSPA1 was shown to interact directly with TRAIL receptors, inhibiting DISC formation and apoptosis (51). In other studies using colon cancer cell lines, however, HSPA1 protected against TRAIL treatment only in cell lines where the intrinsic pathway was coactivated by TRAIL treatment. Activation of the intrinsic pathway following activation of the extrinsic pathway can occur in some cell types, referred to as type 2 cells. Similar observations were made for HSPA1-mediated protection from Fas receptor-triggered apoptosis (52), suggesting that HSPA1

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mediates cytoprotection against death receptor-mediated apoptosis often by impairing the intrinsic pathway. There are several additional mechanisms, apart from the interaction with components of the extrinsic or intrinsic apoptotic pathways, by which HSPA1 can inhibit apoptosis. HSPA1 can modulate expression/activity of several prodeath stress kinases, including apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38. HSPA1 was shown to directly bind ASK1, preventing its homo-oligomerization, thereby protecting cells from oxidative stress and death through inhibition of ASK1mediated cytochrome c release (53). HSPA1 can also inhibit stress-induced JNK activation thereby preventing JNK pathway signaling to apoptosis (32). JNK regulates the activities of both proapoptotic and antiapoptotic members of the BCL-2 family controlling BAX activation, and the JNK inhibitor SP600125 prevents the heat-induced depletion of MCL-1 as effectively as HSPA1 overexpression (40). In addition, the presence or absence of co-chaperones such as BAG-1, BAG-3, or CHIP, which have potent antiapoptotic properties, may be a decisive factor under specific stress stimuli. For example, BAG-1/HSPA1 interaction favors proteasomal degradation of certain client proteins, whereas BAG-3/ HSPA1 can protect clients such as IKKc from proteasomal degradation thereby promoting the prosurvival NF-jB pathway (54).

HSPB Regulation of the Intrinsic Pathway HSPB1 is the best characterized member of the HSPB family and has diverse functions, including inhibition of apoptosis, reducing proteotoxic stress, and regulation of cytoskeleton dynamics. Similar to HSPA1, studies revealed that HSPB1 can impair the intrinsic pathway upstream of, at the level of, or downstream of MOMP (see Figure 1). Several studies demonstrated the regulation of stress kinases such as AKT and JNK by HSPB1. These kinases modulate the intrinsic pathway upstream of MOMP by phosphorylating several BCL-2 family members. HSPB1 activates AKT by promoting PI3-kinase activity, an upstream activator or AKT (55). HSPB1, AKT, p38 MAPK, and MK2 were reported to coexist in a signaling complex that phosphorylates AKT on Ser-473. HSPB1 regulates AKT activation and promotes cell survival by scaffolding MK2 to the AKT signal complex (56). HSPB1 was reported to inhibit conformational BAX activation, oligomerization, and translocation to the mitochondria following metabolic stress (55). These experiments revealed no direct interaction between HSPB1 and BAX, but suggested that HSPB1 prevented BAX activation via PI3-kinase-mediated activation of AKT. AKT-mediated phosphorylation of BAX has been demonstrated to suppress its translocation to mitochondria (57). HSPB1-mediated AKT activation has also been shown to cause inactivation of BH3-only protein BAD by phosphorylation, precluding its interaction with antiapoptotic BCL2 family members and promoting cell survival (55). JNK can phosphorylate BCL-2 and BCL-XL thereby impairing their antiapoptotic potential. Furthermore, JNK can

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mediate phosphorylation of BH3-only proteins BIM and BMF preventing their sequestration by dynein and myosin motor complexes (58). Furthermore, JNK can enhance translocation of BAX to the mitochondria (59). Similar to HSPA1, HSPB1 has been shown to reduce JNK activity and thereby promote cell survival in stressed cells. Stetler et al. (60) pinpointed the mechanism by which HSPB1 overexpression protected against ischemic brain injury to regulation of the ASK1-JNK pathway via a physical interaction with ASK1. HSPB1 was found to impair the intrinsic pathway at the level of MOMP by delaying cytochrome c release in staurosporine-treated murine fibroblasts (61), and there are several reports showing that HSPB1 blocks apoptosis downstream of MOMP. One study showed that a proportion of cytosolic cytochrome c interacts with HSPB1 thereby inhibiting activation of caspases and reducing the efficacy of apoptosome formation (62). A direct protein–protein interaction between HSPB1 and caspase-3 was demonstrated both in vivo and with purified proteins in vitro. Interaction of HSPB1 with the prodomain of caspase-3 inhibits the second proteolytic cleavage step necessary for full caspase-3 activation (63).

Other Apoptosis-Regulating Mechanisms of HSPB1 HSPs, in particular HSPB1, play an important role in the regulation of cytoskeleton. HSPB1 behaves as an F-actin cap-binding protein and has been shown to inhibit actin polymerization in a phosphorylation-dependent manner as nonphosphorylatable HSPB1 shows reduced capacity to resist F-actin fragmentation induced by H2O2 and menadione (61). HSPB1-mediated regulation of the cytoskeleton indirectly reduces activation of the mitochondrial pathway. By binding to F-actin, HSPB1 prevents cytoskeletal disruption, intracellular redistribution of BID, cytochrome c release, and caspase-3 activation (61). Phosphorylated HSPB1 was also found to be required for maintenance of cell adhesion, thus suppressing apoptosis in renal epithelial cells (64). Reactive oxygen species (ROS) can lead to lethal oxidative damages when the antioxidant capacity of the cell is overwhelmed. HSPB1 can protect against ROS generated through TNFa stimulation or ROS-inducing agents H2O2 and menadione (65). HSPB1 has been shown to bind ubiquitin and stimulate the proteasome. Expression of HSPB1 leads to enhanced degradation of the cell cycle regulator, p27KIP1, while having no effect on other cell cycle proteins such as cyclins (66). In addition, by promoting the degradation of IjBa, HSPB1 allows NF-jB to translocate to the nucleus promoting transcription of prosurvival genes (66). In contrast, a negative regulation of NF-jB by HSPB1 has also been reported as HSPB1 was found to associate with the IKK complex, which is also involved in phosphorylation and ubiquitination of IjBa. In this case, however, HSPB1 downregulated IKK signaling and thereby NF-jB activation (67). This response was specific for TNFa as the HSPB1 interaction with IKK did not change in response to interleukin1 treatment. Thus, similar to HSPA1, although being generally

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antiapoptotic, HSPB1’s effects may be stimulus and/or cell type dependent. In addition, the phosphorylation status of HSPB1 is an important modulator of its function as revealed in the latter study where the phosphorylation of HSPB1 was important for its interaction with the IKK complex. HSPB1 can protect the eukaryotic initiation factor eIF2E from ubiquitination and subsequent proteasomal degradation (68). Therefore, HSPB1 appears to exert substrate-specific effects on proteasomal degradation and stability of client proteins.

Role of HSPD1 and HSPE1 in Apoptosis The role of HSPD1 and HSPE1 in apoptosis is complex with reports demonstrating both positive and negative regulation of the intrinsic pathway (69–71). Studies have shown that HSPD/ HSPE1 can promote caspase-3 activation. In Jurkat T cells, HSPD1 and HSPE1 form a multiprotein complex with mitochondrial procaspase-3 (69,72). These data are consistent with the chaperone function of HSPs. In contrast, studies in cardiomyocytes show that HSPD1 and HSPE1 are cytoprotective. The use of adenoviral vectors to overexpress both HSPD1 and HSPE1 as well as the individual overexpression of either HSPD1 or HSPE1 demonstrated that these HSPs protect rat neonatal cardiomyocytes and H9c2 cells against simulated ischemia, reoxygenation, and glucose-mediated apoptosis (70,71). In general, these studies show that the combined overexpression of HSPD1 and HSPE1 is cytoprotective and is associated with decreased cytochrome c release, caspase activity, and DNA fragmentation (71). The cytoprotective effects of HSPD1 were confirmed using antisense oligonucleotides to knockdown the expression of this HSP in rat cardiomyocytes (73). The cytoprotective effects of HSPD1 are linked with regulation of BAX. Therefore, perhaps in a manner similar to HSPA1, HSPD1 can sequester BAX in the cytosol keeping it in an inactive conformation (74). Overexpression of HSPD1 and HSPE1 leads to an increase in the antiapoptotic BCL-2 and BCL-XL via post-transcriptional mechanisms (74).

Other HSPs and Apoptosis Other HSPs such as HSPB5 (aB-crystallin), HSPC (HSP90), and HSPH1 (HSP105) have also been implicated in apoptosis regulation. HSPB5 has been shown to protect cells from apoptosis induced by staurosporine, TNFa, and Fas (65). There is evidence that HSPB5 negatively regulates apoptosis by preventing the maturation of active caspase-3 (75). Furthermore, HSPB5 can inhibit procaspase-3 processing in cytosolic extracts of Jurkat T cells incubated with either caspase-8 or cytochrome c/dATP, demonstrating that HSPB5 can inhibit both the “intrinsic” and the “extrinsic” apoptotic pathways. It has also been reported that the overexpression of BCL-2 in rabbit lens epithelial cells sensitizes these cells to oxidative stress-induced apoptosis through the downregulation of HSPB5 (75). Decreasing BCL-2 levels through antisense RNA restored HSPB5 levels and increased the resistance of the cells to apoptosis. Mao et al. (76) also showed that HSPB5 is capable of binding directly to the proapoptotic BCL-2-family members, BAX and

Heat Shock Proteins and Apoptosis

FIG 1

Proposed mechanisms of heat shock protein intervention in apoptotic pathways. Both HSPA1 and HSPB1 blocked MOMP upstream of mitochondria by inhibiting JNK and JNK activation, respectively. In addition, both HSPs were reported to directly interfere with Bax activation and translocation, and HSPA1 was shown to prevent stress-induced MCL-1 degradation. Furthermore, HSPB1 activated AKT leading to inactivation of BH3-only proteins. HSPA1 and HSPB1 were found to delay cytochrome c release, whereas HSPB1 bound cytochrome c and thus prevented apoptosome formation. Interference with apoptosome activity has been demonstrated for both HSPA1 and HSPB1. Finally, both HSPs inhibited the full activation of executioner procaspases. HSPA1 impaired death receptor signaling at the DISC, whereas HSPB1 augmented NF-jB activation by promoting the degradation of IjB. Note that for simplicity, different death receptor signaling platforms have been unified into one. Activating or inhibitory interactions are depicted using arrows or flat lines, respectively.

BCL-XS, blocking the translocation of these proteins to the mitochondria. HSP90 is reported to have both proapoptotic and antiapoptotic effects depending on the stimulus. For example, in U937 monoblastoid cells, HSP90 has been shown to promote apoptosis induced by TNFa and cycloheximide while protecting against UVB-induced cell death (77). Silencing of HSP90 in PC-12 cells exposed to 6-OHDA resulted in the inhibition of BAX activation and caspase-3 cleavage with concomitant upregulation of the antiapoptotic

Kennedy et al.

protein BCL-2. This was suggested to be due to increased activation of HSF1 and a compensatory increase in HSPA1 (78). On the other hand, HSPC1 was shown to inhibit staurosporineinduced apoptosis in L929 and 293T cells (79). HSP90 (HSPC3) can bind to Apaf-1 thereby impeding apoptosome formation (79). HSP90 is able to form a complex with the antiapoptotic RIP-1 kinase, promoting its stability, leading to increased cellular survival (80). Similar to HSPB1, HSP90 (HSPC1) also regulates AKT survival signaling by preventing its inactivation. The inhibition of HSP90 binding to AKT results in the loss of

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AKT activity and increased sensitivity to apoptosis (81). In addition, HSP90 associates with and stabilizes the IAP family protein survivin. Inhibition of HSP90 chaperone function promotes degradation of survivin leading to mitochondrialdependent apoptosis (82). In a similar fashion to HSP90, the effects of HSPH1 (HSP105a) can be either proapoptotic or antiapoptotic. The protective role of HSPH1 was shown in PC12 cells subjected to various stresses, including heat shock, hydrogen peroxide, and etoposide (83). However, a transient increase in HSPH1 expression was seen during mouse embryogenesis and, based on experiments using mouse embryonic F9 cells, was proposed to be associated with increased PARP cleavage, caspase-3 activation, and cytochrome c release (84).

Concluding Remarks HSPs play a pivotal role in regulating apoptosis. HSPA1 and HSPB1 are well-established regulators of the intrinsic pathway by interacting with BCL-2 proteins or by modulating the activity of kinases that modulate the function of BCL-2 proteins. A similar way of promoting cell survival has been demonstrated for HSPC1. Furthermore, HSPB1 and HSPC1 have been suggested to delay cytochrome c release in a direct fashion at the level of mitochondria. Both HSPA1 and HSPC1 have been shown to interfere with apoptosome formation, and all three HSP families can interact with caspases, inhibiting their full activation. Additional roles for HSPs in modulating DISC signaling and NF-jB activation are emerging. Given the potential contribution of alterations in HSPs expression to human disease, understanding the regulation of cell death by HSPs is a prerequisite for novel approaches for the treatment of conditions such as cancer and heart disease. In time, it is hoped that regulation of HSPs expression, either by pharmacological means or by gene therapy, will allow us to manipulate apoptotic pathways to promote or prevent cell death as required.

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