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Critical Review

Regulation of Apoptosis by Heat Shock Proteins Donna Kennedy1Richard Jager2

Dick D. Mosser3

Afshin Samali1*

1Department of Biochemistry, Apoptosis Research Centre, Biosciences

Research Building, Corrib Village, NUI Galway, Dangan, Galway, Ireland2Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied

Sciences, Rheinbach, Germany3Department 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 proc-

esses including development and immune clearance. Apopto-

sis 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, manipula-

tion of the HSPs could represent a viable strategy for the treat-

ment of diseases. Here, we review the current knowledge with

regard to the mechanisms of HSP-mediated regulation of apo-

ptosis. VC 2014 IUBMB Life, 66(5):327338, 2014

Keywords: cell death; apoptosis; intrinsic pathway; heat shock

proteins; caspases; heat shock response

IntroductionHeat 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 evolu-

tionarily 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 subse-

quent long-term ischemic insult (5). Protection from cell death

afforded by thermal preconditioning is mediated by HSPs as itcan 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 spe-

cific 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-phe-

nylpyridine (11). The phenomenon of cross-tolerance supports

the notion that HSP induction serves as a general cytoprotec-

tive mechanism in cells.

The HSP SuperfamilyHSPs 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 pro-

tein folding, refolding of misfolded proteins, and oligomer

VC 2014 International Union of Biochemistry and Molecular Biology

Volume 66, Number 5, May 2014, Pages 327338

*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 26 May 2014 in Wiley Online Library

(wileyonlinelibrary.com)

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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 mainte-

nance of cytoskeletal integrity under conditions of stress facili-

tated 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 mem-

bers (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 EEVD-

motif 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, HSPB111. Some

are ubiquitously expressed, whereas others display tissue-

restricted 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 clientprotein interactions (19).

HSPD1 and HSPE1 are classed as chaperonins. HSPD1 andHSPE1 form a complex in which HSPE1 regulates the sub-

strate binding and ATPase activity of HSPD1. A majority (60

80%) of HSPD1 and HSPE1 proteins are located in the mito-

chondria 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 HSPC15. Until recently, the literature has not differentiated

between the various family members. However, HSPC1 and

HSPC3 turned out to have non-overlapping functions (21). Spe-

cifically, 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 com-

plex 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 pro-

teins (15,21).

The human genome encodes four HSPH family members

(HSPH14). The HSPH family is important for preventing pro-

tein 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 lucif-

erase in the absence of HSPA proteins (15).

Molecular Basis of ApoptosisApoptosis 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 proc-esses; 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 apopto-

sis is mediated by a class of intracellular proteases, the cas-

pases. In mammals, caspases (cysteine-dependent aspartate-

specific 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 sub-

strates 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 prodo-

mains and the small and large subunits, respectively, which

generates the active heterotetrameric caspase. Their prodo-

mains and activation mechanisms classify the apoptotic cas-

pases 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 interactionmotifs facilitating proteinprotein 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 activa-

tion. 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 PathwayThe 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 death-

inducing signaling complex (DISC) that recruits procaspases-

8/-10, mediating their oligomerization and autoactivation. The

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Overviewofthe

HSPAandHSPBfamiliesexpressionpro

file,cellularlocalization,stressinducibilityanddiseaseassociation

Name

Alternativenames

Location

ID

C

ellularlocation

Expression

Stress

inducible

Diseaserelevance

HSPAfamily

HSPA1A

HSP70-1;HSP72

6p21.3

3303C

ytoplasm

Ubiquitous

Yes

ReducedinAD;protectiveinPD

N

ucleus

Associatedwithpoo

rprognosis

andmetastasisin

cancer

L

ysosomes

HSPA1B

HSP70-B;HSP70-2

6p21.3

3304C

ytoplasm

Ubiquitous

Yes

PolymorphismsassociatedwithAD

N

ucleus

Associatedwithpoo

rprognosis

andmetastasisin

cancer

L

ysosomes

HSPA1L

HSP70T;hum70t;

HSP70-1L;HSP70-HOM

6p21.3

3305C

ytoplasm

Testis

No

N

ucleus

HSPA2

HSP70-2;HSP70-3

14q24.1

3306C

ytoplasm

Testis

?

Poorprognosisincancer

N

ucleus

HSPA5

BIP,GRP78,MIF2

9q33.3

3309E

ndoplasmic

reticulum

Ubiquitous

Yes

Malignancyandmetastasis

Resistancetochemo

therapeutics

HSPA6

HSP70B

1q23

3310C

ytoplasm

Ubiquitous

Yes

PolymorphismslinkedwithMS

N

ucleus

HSPA7

Possiblepseudogen

eof

HSPA1B

HSPA8

LAP1;HSC54;HSC7

0;

HSC71;HSP71;HSP73;

NIP71;HSPA10

11q24.1

3312C

ytoplasm

Ubiquitous

No

N

ucleus

HSPA9

GRP75;HSPA9B;M

OT;

MOT2;PBP74;mo

t-2

5q31.1

3313M

itochondria

Ubiquitous

Yes

Poorprognosisincancer

TABLE

1

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(Continued)

Name

Alternativenames

Location

ID

C

ellularlocation

Expression

Stress

inducible

Diseaserelevance

Resistancetochemo

therapeutics

HSPA12A

FLJ13874;KIAA0417

10q26.12

259217C

ytoplasm

Ubiquitous

Yes

Reducedinschizoph

renia

Requiredforangiogenesis

HSPA12b

C20orf60;dJ1009E2

4.2

20p13

116835C

ytoplasm

Ubiquitous

(enrichedin

endothelialce

lls)

Yes

Protectiveinischem

ia/reperfusion

HSPA13

STCH

21q11

6782M

icrosomes

Ubiquitous

?

Cancer

HSPBfamily

HSPB1

CMT2F,HMN2B,HS

.76067,

HSP27,HSP28,Hsp25,

SRP27

7q11.23

3315C

ytoplasm

Ubiquitous

Yes

CMT,dHMN

N

ucleusPerikaryon

Cancer

Metastasis

Protectiveinischem

ia/reperfusion

Williamssyndrome

HSPB2

MKBP;HSP27;Hs.7

8846;

LOH11CR1K

11q22-q23

3316C

ytosol

Cardiac,skeletal

muscle

No

Myopathy

C

ytoplasm

Ischemia

N

ucleus

Cancer

M

itochondria

HSPB3

HMN2C;DHMN2C;

HSPL27

5q11.2

8988C

ytoplasm

Cardiac,skeletal

muscle

No

dHMN

N

ucleus

Neuropathy

HSPB4

CRYAA,CRYA1,CT

RCT9

21q22.3

1409C

ytoplasm

Lens

No

Cataracts

N

ucleus

HSPB5

CRYAB,CMD1II,CR

YA2,

CTPP2

11q22.3-q23.1

1410C

ytoplasm

Ubiquitous

Yes

Myopathy,neuropat

hy

N

ucleus

Cataracts

Cancer

Metastasis

Protectiveinischem

ia/reperfusion

TABLE

1

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(Continued)

Name

Alternativenames

Location

ID

C

ellularlocation

Expression

Stress

inducible

Diseaserelevance

HSPB6

HSP20

19q13.12

126393C

ytoplasm

Ubiquitous

Yes

Neuropathy

N

ucleus

Ischemia

HSPB7

cvHSP

1p36.23-p34.3

27129C

ytoplasm

Cardiac,

skeletal

muscle

?

Cardiomyopathy

N

ucleus

M

itochondria

HSPB8

H11;HMN2;CMT2L

;

DHMN2;E2IG1;H

MN2A;

HSP22

12q24.23

26353C

ytoplasm

Ubiquitous

Celltype

specific

CMTanddHMN

N

ucleus

Cancer

HSPB9

CT51

17q21.2

94086C

ytoplasm

Testis

?

Cancer

N

ucleus

HSPB10

ODF1;RT7;ODF2;O

DFP;

SODF;CT133;OD

F27;

ODFPG;HSPB10;

ODFPGA;ODFPGB

8q22.3

4956S

perm

tail

Testis

?

HSPB11a

PP25;IFT25;C1orf41;

HSPCO34

1p32

51668C

ytoplasm

Placenta

Yes

Cancer

N

ucleus

Abbreviations:AD,

Alzheimersdis

ease;PD,

Parkinsonsdisease;MS,

multiplesclerosis;CMT,

CharcotMarieTooth;dHMN,

distalhereditarymuscularneuropathy.

aControversialHSPBmemberasit

doesnothaveaclassicala-crystallindomain.

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 pro-tein, Daxx, leading to activation of the JNK pathway. Activa-

tion 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 simi-

lar to the Fas signaling pathway. Moreover, the TNFR DISC

can prevent the activation of the prosurvival transcription fac-

tor 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 apo-

ptosis proteins (IAPs), which mediate the ubiquitinylation of

interacting proteins at the DISC and can also impair caspaseactivity (26).

The Intrinsic PathwayActivation of the intrinsic pathway leads to mitochondrial

outer membrane permeabilization (MOMP) and release of

apoptogenic factors from the mitochondria, such as cyto-

chrome c, second mitochondrial-derived activator of caspases

(SMAC), and Omi stress-regulated endoprotease/high-tempera-

ture requirement protein A2 (HTRA2) (27). This is a crucial

event for driving caspase activation and subsequent apoptosis.

Cytochrome cbinds to the apoptotic protease-activating factor

1 (Apaf-1), causing it to assemble into a large protein complex

termed the apoptosome that recruits and activatesprocaspase-9 resulting in activation of downstream execu-

tioner 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 BCL-

2 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

BH14. The proapoptotic family members include the multido-

main 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, BCL-

2, BCL-XL, MCL-1, BCL-w, BCL-2A1, and BCL-B, typically have

all four BH domains and a carboxy terminal membrane inser-

tion 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

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 acti-

vated at the transcriptional or post-translational level by apo-

ptotic signaling pathways and transduce apoptotic stimuli tothe mitochondria.

Regulation of Apoptosis by HSPsInduction 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 sur-

vival of postmitotic cells such as cardiomyocytes and neurons

is considered beneficial.

HSPs and Resistance to ApoptosisIt 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 con-

sistently 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 PathwayOverexpression of HSPA1 protects cells against a range of

stimuli that engage the intrinsic pathway such as hyperther-mia (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 pro-

mote apoptosis in human cancer cells (37). Conversely, overex-

pression 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). Consist-

ent 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

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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 com-

plex with BAX that is present in some cells but not in others.

HSPA1 was also shown to interact with the antiapoptotic BCL-

2 family member BCL2L12 preventing its proteasomal degra-

dation (42).

Several reports demonstrate that HSPA1 can interfere

with cytochrome c release. HSPA1 overexpression in macro-

phages 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 thechaperoning 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 type-

specific 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 crelease 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 activationin vitro (8), although another study did not observe an inhibi-

tory 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 HSPA1There 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

mediates cytoprotection against death receptor-mediated apo-

ptosis often by impairing the intrinsic pathway.

There are several additional mechanisms, apart from the

interaction with components of the extrinsic or intrinsic apo-

ptotic pathways, by which HSPA1 can inhibit apoptosis. HSPA1

can modulate expression/activity of several prodeath stresskinases, including apoptosis signal-regulating kinase 1 (ASK1),

JNK, and p38. HSPA1 was shown to directly bind ASK1, pre-

venting its homo-oligomerization, thereby protecting cells from

oxidative stress and death through inhibition of ASK1-

mediated 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 antiapop-totic properties, may be a decisive factor under specific stress

stimuli. For example, BAG-1/HSPA1 interaction favors protea-

somal degradation of certain client proteins, whereas BAG-3/

HSPA1 can protect clients such as IKKcfrom proteasomal deg-

radation thereby promoting the prosurvival NF-jB pathway

(54).

HSPB Regulation of the Intrinsic PathwayHSPB1 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, ordownstream of MOMP (see Figure 1).

Several studies demonstrated the regulation of stress

kinases such as AKT and JNK by HSPB1. These kinases modu-

late the intrinsic pathway upstream of MOMP by phosphoryl-

ating 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 follow-

ing metabolic stress (55). These experiments revealed no

direct interaction between HSPB1 and BAX, but suggested that

HSPB1 prevented BAX activation via PI3-kinase-mediated acti-

vation of AKT. AKT-mediated phosphorylation of BAX has

been demonstrated to suppress its translocation to mitochon-

dria (57). HSPB1-mediated AKT activation has also been

shown to cause inactivation of BH3-only protein BAD by phos-

phorylation, precluding its interaction with antiapoptotic BCL-

2 family members and promoting cell survival (55).

JNK can phosphorylate BCL-2 and BCL-XL thereby impair-

ing 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 themechanism 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 down-

stream of MOMP. One study showed that a proportion of

cytosolic cytochrome c interacts with HSPB1 thereby inhibi-

ting activation of caspases and reducing the efficacy of apop-

tosome formation (62). A direct proteinprotein interaction

between HSPB1 and caspase-3 was demonstrated both in

vivo and with purified proteins in vitro. Interaction of HSPB1with the prodomain of caspase-3 inhibits the second proteo-

lytic cleavage step necessary for full caspase-3 activation

(63).

Other Apoptosis-Regulating Mechanisms of HSPB1HSPs, in particular HSPB1, play an important role in the regu-

lation of cytoskeleton. HSPB1 behaves as an F-actin cap-bind-

ing protein and has been shown to inhibit actin polymerization

in a phosphorylation-dependent manner as nonphosphorylat-

able HSPB1 shows reduced capacity to resist F-actin fragmen-

tation 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 pre-vents 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 degrada-

tion 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 prosur-

vival genes (66). In contrast, a negative regulation of NF-jB by

HSPB1 has also been reported as HSPB1 was found to associ-

ate with the IKK complex, which is also involved in phospho-

rylation and ubiquitination of IjBa. In this case, however,

HSPB1 downregulated IKK signaling and thereby NF-jB acti-

vation (67). This response was specific for TNFa as the HSPB1

interaction with IKK did not change in response to interleukin-

1 treatment. Thus, similar to HSPA1, although being generally

antiapoptotic, HSPB1s 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 sub-sequent proteasomal degradation (68). Therefore, HSPB1

appears to exert substrate-specific effects on proteasomal deg-

radation and stability of client proteins.

Role of HSPD1 and HSPE1 in ApoptosisThe role of HSPD1 and HSPE1 in apoptosis is complex with

reports demonstrating both positive and negative regulation of

the intrinsic pathway (6971). Studies have shown that HSPD/

HSPE1 can promote caspase-3 activation. In Jurkat T cells,

HSPD1 and HSPE1 form a multiprotein complex with mito-

chondrial 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 individ-

ual 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

crelease, caspase activity, and DNA fragmentation (71). The

cytoprotective effects of HSPD1 were confirmed using anti-

sense oligonucleotides to knockdown the expression of this

HSP in rat cardiomyocytes (73). The cytoprotective effects of

HSPD1 are linked with regulation of BAX. Therefore, perhapsin a manner similar to HSPA1, HSPD1 can sequester BAX in

the cytosol keeping it in an inactive conformation (74). Over-

expression of HSPD1 and HSPE1 leads to an increase in the

antiapoptotic BCL-2 and BCL-XL via post-transcriptional

mechanisms (74).

Other HSPs and ApoptosisOther HSPs such as HSPB5 (aB-crystallin), HSPC (HSP90), and

HSPH1 (HSP105) have also been implicated in apoptosis regu-

lation. HSPB5 has been shown to protect cells from apoptosis

induced by staurosporine, TNFa, and Fas (65). There is evi-

dence 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). Decreas-

ing 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

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BCL-XS, blocking the translocation of these proteins to the

mitochondria.

HSP90 is reported to have both proapoptotic and antiapop-

totic effects depending on the stimulus. For example, in U937

monoblastoid cells, HSP90 has been shown to promote apopto-

sis 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

protein BCL-2. This was suggested to be due to increased acti-

vation of HSF1 and a compensatory increase in HSPA1 (78).

On the other hand, HSPC1 was shown to inhibit staurosporine-

induced 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 cel-

lular survival (80). Similar to HSPB1, HSP90 (HSPC1) also reg-

ulates AKT survival signaling by preventing its inactivation.

The inhibition of HSP90 binding to AKT results in the loss of

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. Further-

more, HSPB1 activated AKT leading to inactivation of BH3-only proteins. HSPA1 and HSPB1 were found to delay cytochrome c

release, whereas HSPB1 bound cytochromec and thus prevented apoptosome formation. Interference with apoptosome activ-

ity has been demonstrated for both HSPA1 and HSPB1. Finally, both HSPs inhibited the full activation of executioner procas-

pases. 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. [Color figure can be viewed in the online issue,

which is available atwileyonlinelibrary.com.]

FIG 1

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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 pro-

motes degradation of survivin leading to mitochondrial-

dependent 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 acti-

vation, and cytochrome crelease (84).

Concluding RemarksHSPs play a pivotal role in regulating apoptosis. HSPA1 and

HSPB1 are well-established regulators of the intrinsic pathwayby interacting with BCL-2 proteins or by modulating the activ-

ity 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 sug-

gested 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 sig-

naling and NF-jB activation are emerging. Given the potential

contribution of alterations in HSPs expression to human dis-

ease, understanding the regulation of cell death by HSPs is a

prerequisite for novel approaches for the treatment of condi-

tions 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 apopto-

tic pathways to promote or prevent cell death as required.

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