J Appl Physiol 92: 1725–1742, 2002;
10.1152/japplphysiol.01143.2001.
highlighted topics
Molecular Biology of Thermoregulation
Invited Review: Effects of heat and cold stress
on mammalian gene expression
LARRY A. SONNA,1 JUN FUJITA,2 STEPHEN L. GAFFIN,1 AND CRAIG M. LILLY3
Thermal and Mountain Medicine Division, United States Army Research Institute of
Environmental Medicine, Natick 01760; 3Division of Pulmonary and Critical Care Medicine,
Brigham and Women’s Hospital/Harvard Medical School, Boston, Massachusetts 02115;
and 2Department of Clinical Molecular Biology, Faculty of Medicine,
Kyoto University, Sakyo-ku, Kyoto 6068507, Japan
1
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Sonna, Larry A., Jun Fujita, Stephen L. Gaffin, and Craig M.
Lilly. Invited Review: Effects of heat and cold stress on mammalian gene
expression. J Appl Physiol 92: 1725–1742, 2002; 10.1152/japplphysiol.
01143.2001.—This review examines the effects of thermal stress on gene
expression, with special emphasis on changes in the expression of genes
other than heat shock proteins (HSPs). There are ⬃50 genes not traditionally considered to be HSPs that have been shown, by conventional
techniques, to change expression as a result of heat stress, and there are
⬍20 genes (including HSPs) that have been shown to be affected by cold.
These numbers will likely become much larger as gene chip array and
proteomic technologies are applied to the study of the cell stress response. Several mechanisms have been identified by which gene expression may be altered by heat and cold stress. The similarities and differences between the cellular responses to heat and cold may yield key
insights into how cells, and by extension tissues and organisms, survive
and adapt to stress.
heat shock proteins; heat shock; cold shock; cell stress response
a complex program of gene
expression and biochemical adaptive responses (34,
64). These cell stress responses are of great interest
both to basic biology and to medicine. Biologically, the
ability to survive and adapt to thermal stress appears
to be a fundamental requirement of cellular life, as cell
stress responses are ubiquitous among both eukaryotes and prokaryotes, and key heat shock proteins
(HSPs) involved in these responses are highly conserved across evolutionary lines (64, 87). Even in euthermic species, in which core temperature is tightly
regulated, considerable variations in core temperature
can occur during severe environmental stress, exercise,
and fever. In medicine, evidence is mounting that the
THERMAL STRESSES TRIGGER
Address for reprint requests and other correspondence: L. A.
Sonna, Thermal and Mountain Medicine Division, US Army Research Institute of Environmental Medicine, 42 Kansas St., Natick,
MA 01760 (E-mail: [email protected]).
http://www.jap.org
ability to survive and adapt to severe systemic physiological stress is critically dependent on the ability of
cells to mount an appropriate compensatory stress
response. In addition, the observation that induction of
a cell stress response by one type of stressor (such as
heat) often leads to cross-protection to other stressors
has raised the interesting possibility that the pathways involved in the cell stress response might present
useful targets for therapeutic manipulation. For example, in a number of animal models of ischemia-reperfusion, the extent of organ damage produced is mitigated by prior induction of a cell stress response (50).
Conversely, cancer cells that express HSPs at a high
level are often relatively resistant to cytotoxic therapies, and a therapeutic intervention aimed at selectively diminishing the ability of malignant cells to
mount a stress response could, in principle, enhance
the effectiveness of chemo- and radiotherapy (50).
However, there are important differences in the bio1725
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INVITED REVIEW
HEAT STRESS
lar to the response to heat) display enhanced resistance to a subsequent endotoxin challenge, they will
undergo apoptosis if endotoxin exposure precedes exposure to either arsenite or heat (7). The mechanism by
which this occurs has not been fully elucidated (21),
but this experiment elegantly illustrates how seemingly subtle differences in the cellular responses to
different stressors can produce dramatic differences in
physiological outcome.
Heat-induced changes in gene expression occur both
during hyperthermia as well as after return to normothermia. In some experimental systems, hyperthermia
inhibits transcription of all but a few genes (mostly
HSPs). However, important additional changes in gene
expression can be detected for hours after return to
normothermic temperatures; to date, ⬃50 genes not
traditionally considered HSPs have been found to undergo changes in expression during or after heat stress
(discussed below). Many of these genes will likely prove
to be important mediators and effectors of the cell
stress response.
Heat Shock Factors and Heat Shock Proteins
Cell Stress Response
The effects of heat stress on cellular function have
been reviewed in detail elsewhere (57, 64). Briefly, they
include 1) inhibition of DNA synthesis, transcription,
RNA processing, and translation; 2) inhibition of progression through the cell cycle; 3) denaturation and
misaggregation of proteins; 4) increased degradation of
proteins through both proteasomal and lysosomal
pathways; 5) disruption of cytoskeletal components;
6) alterations in metabolism that lead to a net reduction in cellular ATP; and 7) changes in membrane
permeability that lead to an increase in intracellular
Na⫹, H⫹, and Ca2⫹.
In mammalian cells, nonlethal heat shock produces
changes in gene expression and in the activity of expressed proteins, resulting in what is referred to as a
cell stress response (50, 64). This response characteristically includes an increase in thermotolerance (i.e.,
the ability to survive subsequent, more severe heat
stresses) that is temporally associated with increased
expression of HSPs. A cell stress response can be induced by other stressors [including exposure to toxins
such as arsenite, bacterial lipopolysaccharide (LPS),
and so forth], and the response initiated by one stressor
often leads to cross-tolerance to others (87). The degree
to which changes induced by different stressors overlap
with each other has not been fully characterized, but
some of the most important ones (such as increased
expression of HSP70) are widely shared. At increasingly severe heat exposures, heat shock leads to activation of the apoptotic program and, in the extreme, to
cellular necrosis (16) . Importantly, the fate of cells
exposed sequentially to different stressors appears to
depend critically on the sequence of exposure (21). For
example, although porcine endothelial cells stressed
with arsenite (which produces a stress response simiJ Appl Physiol • VOL
Heat shock factors. Heat shock factors (HSFs) have
been the subject of recent detailed reviews (73, 88).
HSFs are transcription factors that regulate HSP expression through interaction with a specific DNA sequence in the promoter [the heat shock element
(HSE)]. The HSE is a stretch of DNA located in the
promoter region of susceptible genes containing multiple sequential copies (adjacent and inverse) of the
consensus pentanucleotide sequence 5⬘-nGAAn-3⬘ (73)
and has been found in both HSPs and in a number of
other genes. Three HSFs have been identified in mammalian systems: HSF-1, HSF-2, and HSF-4 (73). A
fourth HSF, HSF-3, is present in avian species but not
in humans. HSF-1 is involved in the acute response to
heat shock; the others are involved in a number of
different regulatory and developmental processes and,
until recently, were not generally thought to play roles
in the cellular response to heat (73, 88). Very recent
evidence indicates, however, that heat shock can produce reversible inactivation of HSF-2 (69).
Before heat-induced activation, HSF-1 exists as a
monomer localized to the cytoplasm. The initial stimulus for activation of HSF-1 appears to be the exposure
of hydrophobic domains of denatured proteins, such as
occurs during heat stress. Because much of the cytoplasmic HSF-1 coprecipitates with HSP70 and HSP90
in unstressed cells and because HSPs preferentially
bind to denatured proteins, it has been postulated that
HSF-1 in unstressed cells is bound to HSPs and that
activation of HSF-1 may occur as a result of competitive release of this transcription factor from HSPs
when the concentration of denatured cytoplasmic proteins increases as a result of heat shock (73). After
activation by thermal stress, HSF-1 is found primarily
in the nucleus in trimeric form, concentrated (in human cell lines) in granules (92). It is this activated,
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chemical pathways activated by different stressors; as
a result, the fate of cells (survival and adaptation vs.
apoptosis) depends critically on the sequence in which
these stressors are applied (7, 21). Thus a high degree
of understanding of the pathways that are shared by
various stressors and of those that are unique to particular stressors is critical to our ability to manipulate
cell stress responses for therapeutic purposes.
It is widely accepted that changes in gene expression
are an integral part of the cellular response to thermal
stress. Although the HSPs are perhaps the best-studied examples of genes whose expression is affected by
heat shock, it has become apparent in recent years that
thermal stress also leads to induction of a substantial
number of genes not traditionally considered to be
HSPs. Some of these genes are affected by a wide
variety of different stressors and probably represent a
nonspecific cellular response to stress, whereas others
may eventually be found to be specific to certain types
of stress. In this article, we review the effects of thermal stress on gene expression.
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INVITED REVIEW
J Appl Physiol • VOL
Studies of mice carrying a homozygous hsf-1(⫺/⫺) null
mutation (hsf-1 knockouts) illustrate the importance of
this transcription system to survival and stress adaptation (71, 114). Cells derived from these animals
lacked any detectable HSF-1 protein and had an impaired ability to increase expression of a number of
HSPs after either an in vitro or an in vivo heat shock,
although baseline expression was relatively preserved
(71, 114). The hsf-1(⫺/⫺) knockout mice had a higher
prenatal mortality and lower birth weight and exhibited postnatal growth retardation compared with wildtype [hsf-1(⫹/⫹)] animals (114). Cultured embryonic fibroblasts obtained from hsf-1(⫺/⫺) mice displayed an
impaired ability to acquire thermotolerance after an in
vitro sublethal heat shock (43°C for 30 min) (71), and
the survival of hsf-1 knockout mice after a single intraperitoneal endotoxin challenge was significantly diminished compared with wild-type and heterozygous
[hsf-1(⫹/⫺)] animals (114). Interestingly, the hsf-1(⫺/⫺)
mice also displayed an exaggerated increase in plasma
levels of the tumor necrosis factor-␣ (TNF-␣) after
endotoxin challenge compared with heterozygous animals but no difference in levels of the anti-inflammatory cytokine IL-10 (114). These experiments demonstrate that HSF-1 plays a central physiological role in
survival after severe stress, both in vitro and in vivo.
Heat shock proteins. HSPs were originally identified
as proteins whose expression was markedly increased
by heat shock (64). Several HSPs are expressed even in
unstressed cells and play important functions in normal cell physiology. Although the intensity and duration of the heat stimulus needed to induce HSP expression vary considerably from tissue to tissue, a typical in
vitro exposure involves heating mammalian cells to
42–45°C for 20–60 min and then reverting them to
normothermic temperatures (37°C). Induction of HSP
expression typically starts within minutes after the
initiation of thermal stress, with peak expression occurring up to several hours later. Importantly, several
experiments have found that, during the period of
hyperthermia and shortly thereafter, HSPs become the
predominant proteins synthesized by cells (64). Interestingly, most HSP genes lack introns (64), which may
facilitate their rapid expression and which may also
help explain how they can be expressed in the presence
of stressors (such as heat) that can interfere with RNA
splicing.
The HSPs are traditionally classified by molecular
weight (Table 1). As proteins, HSPs possess three principal biochemical activities. The first activity is chaperonin activity (36, 87). HSPs with this function help
prevent misaggregation of denatured proteins and assist the refolding of denatured proteins back into native conformations. Even in unstressed cells, some of
the chaperonin HSPs play a role in the folding of
nascent polypeptides into native conformations during
protein synthesis. Additionally, their ability to stabilize proteins in specific conformations is used by a
variety of normal cellular regulatory processes, such as
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trimeric form of HSF-1 that binds to the HSE and is
involved in increased HSP gene transcription during
heat stress (92).
Binding of HSF-1 to an HSE consensus site does not
invariably induce transcription. For example, in LPSstimulated human monocytes, HSF-1 acts as a repressor of interleukin (IL)-1␤ transcription (9). Furthermore, the net effect of HSF-1 binding to DNA
(induction or repression of transcription) may be affected by its state of phosphorylation. Although monomeric HSF-1 is constitutively phosphorylated, trimeric
HSF-1 can bind DNA but is transcriptionally inert in
some systems unless it undergoes additional (hyper-)
phosphorylation, which in turn can be induced by heat
shock (15). In other systems, hyperphosphorylation of
HSF-1 leads to a decrease in transcriptional activity at
normothermic temperatures (37°C) (13). HSF-1 can be
hyperphosphorylated at serine residues by mitogenactivated protein (MAP) kinases of the extracellularregulated kinase-1 family (12), by protein kinases C-␣
and C-␨ (13), and by glycogen synthase kinase 3-␣ (12,
13), all of which inhibit its transcriptional activity.
HSF-1 can also be hyperphosphorylated by c-Jun NH2terminal kinase (JNK), which activates its transcriptional activity under some conditions (86) and inhibits
its transcriptional activity in others (18). These findings provide important potential mechanisms whereby
key cellular signal transduction pathways may influence HSF-1-mediated transcription.
Recent evidence has also indicated that heat stress
induces tagging of HSF-1 with SUMO-1, a ubiquitinlike protein that is used by the cell to mark proteins for
transport into different cellular compartments and to
alter their activities (48). Importantly, in these experiments, HSF-1 in vitro was incapable of binding DNA
unless it had first acquired a SUMO-1 tag at lysine
298.
HSF-1 may also have effects on transcription that do
not require direct interaction with a gene’s promoter.
In Chinese hamster ovary fibroblasts, heat shock inhibits serum-induced c-fos expression (11). The mechanism appears to involve inhibition of Ras-induced
activation of the c-fos promoter. Interestingly, cotransfection experiments found that Ras-mediated activation of the c-fos promoter can also be inhibited with a
mutant form of HSF-1 that is incapable of binding
DNA (11). A similar effect was observed on the urokinase promoter, another gene regulated by the Ras
signal transduction pathway. These results show that
HSF-1 can antagonize Ras-mediated transcriptional
activation of c-fos by a mechanism that does not require binding of HSF-1 to DNA.
In addition to positive regulation of the HSE through
HSF-1, evidence also exists for negative regulation of
this promoter element in mice by means of a constitutively expressed protein known as the HSE binding
factor (HSE-BF) (55, 66). Thus, in addition to a phosphorylation state, the ability of HSF-1 to activate transcription may also be modulated by regulatory processes that affect the binding of HSE-BF to the HSE.
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Table 1. Human HSP genes
Gene Symbol
Gene Name
Chromosome
Location
Small HSPs
HSPE1
HSPB1
HSPB2
HSPB3
HSPB7
HSPB9
CRYAB
HMOX1
DNAJA1
DNAJA2
DNAJA3
DNAJA4
DNAJB1
DNAJB2
DNAJB4
DNAJB5
DNAJB6
DNAJB9
DNAJB11
DNAJB12
DNAJC3
DNAJC4
DNAJC6
DNAJC7
DNAJC8
SERPINH1
SERPINH2
FKBP5
HSPD1
HSPA1A
HSPA1B
HSPA1L
HSPA2
HSPA3
HSPA5
HSPA6
HSPA7
HSPA8
HSPA9A
HSPA9B
HSPCA
HSPCB
TRAP1
HSP105A;
HSP105B
APG-1
HSPA4; APG-2
UBB
UBC
UBD
UBF-fl
HSPABP1; ST13
Heat shock 10-kDa protein 1 (chaperonin 10)
Heat shock 27-kDa protein 1
Heat shock 27-kDa protein 2
Heat shock 27-kDa protein 3
Heat shock 27-kDa protein 7 (cardiovascular)
Small heat shock protein B9
Crystallin, alpha B
Heme oxygenase-1
DnaJ (Hsp40) homolog, subfamily A, member 1
DnaJ (Hsp40) homolog, subfamily A, member 2
DnaJ (Hsp40) homolog, subfamily A, member 3
DnaJ (Hsp40) homolog, subfamily A, member 4
DnaJ (Hsp40) homolog, subfamily B, member 1
DnaJ (Hsp40) homolog, subfamily B, member 2
DnaJ (Hsp40) homolog, subfamily B, member 4
DnaJ (Hsp40) homolog, subfamily B, member 5
DnaJ (Hsp40) homolog, subfamily B, member 6
DnaJ (Hsp40) homolog, subfamily B, member 9
DnaJ (Hsp40) homolog, subfamily B, member 11
DnaJ (Hsp40) homolog, subfamily B, member 12
DnaJ (Hsp40) homolog, subfamily C, member 3
DnaJ (Hsp40) homolog, subfamily C, member 4
DnaJ (Hsp40) homolog, subfamily C, member 6
DnaJ (Hsp40) homolog, subfamily C, member 7
DnaJ (Hsp40) homolog, subfamily C, member 8
Serine (or cysteine) proteinase inhibitor, clade H, member 1
Serine (or cysteine) proteinase inhibitor, clade H, member 2
FK506-binding protein 5 (HSP56)
Heat shock 60-kDa protein 1 (chaperonin)
Heat shock 70-kDa protein 1A
Heat shock 70-kDa protein 1B
Heat shock 70-kDa protein-like 1
Heat shock 70-kDa protein 2
Heat shock 70-kDa protein 3
Heat shock 70-kDa protein 5 (glucose-regulated protein, 78-kDa; grp78; BiP)
Heat shock 70-kDa protein 6 (HSP70B⬘)
Heat shock 70-kDa protein 7 (HSP70B)
Heat shock 70-kDa protein 8 (HSC70)
Heat shock 70-kDa protein 9A (mortalin-1)
Heat shock 70-kDa protein 9B (mortalin-2)
Heat shock 90-kDa protein 1, alpha
Heat shock 90-kDa protein 1, beta
Heat shock protein 75
Heat shock 105-kDa protein
2q33.1
7q11.23
11q22-q23
5q11.2
1p36.23-p34.3
17q21.2
11q22.3-q23.1
22q13.1
9p13-p12
16q11.1-q11.2
16p13.3
15q24.1
19p13.2
2q32-q34
1p22.3
9p12
11q24.3
7q31
3q28
10q22.1
13q32
11q13
1pter-q31.3
17q11.2
1p35.3
11
11q13.5
6p21.3-21.2
12q13.2
6p21.3
6p21.3
6p21.3
14q24.1
21
9q33-q34.1
1cen-qter
1q23.1
11q23.3-q25
Unknown
5q31.1
1q21.2-q22
6p12
16p13.3
13q12.3
Heat shock 110-kDa protein; osmotic stress protein 94
Heat shock 70-kDa protein 4 (heat shock protein apg-2)
Ubiquitin B
Ubiquitin C
Di-ubiquitin
Ubiquitin UBF-fl
Suppression of tumorigenicity 13 (colon carcinoma); HSP70-interacting
protein
Torsin family 1, member B (torsin B)
Tumor rejection antigen 1 (gp96)
4q28
5q31.1-q31.2
17p12-p11.2
12q24.3
6p21.3
19q13.43
22q13.2
HSP32
HSP40
HSP47
HSP56
HSP60
HSP70
HSP90
HSP110
Ubiquitins
Other
TOR1B
TRA1
cell cycle control, steroid and vitamin D receptor processing, and antigen presentation by cells with immune function. The prototypical chaperonin HSPs are
the members of the HSP40, HSP60, HSP70, and
HSP90 families of proteins. The second activity is regulation of cellular redox state, of which the best example is HSP32, better known as heme oxygenase-1
(HO-1) (84). This enzyme catalyzes the breakdown of
heme to biliverdin, carbon monoxide, and free iron
J Appl Physiol • VOL
9q34
12q24.2-q24.3
(which is rapidly incorporated into ferritin). Biliverdin
is subsequently converted to bilirubin, a potent antioxidant with cytoprotective effects. The release of free
iron by HO-1 also leads to increased expression of
ferritin, which is thought to exert its cytoprotective
effect in part by sequestering prooxidant free iron (84).
The third principal biochemical activity of HSPs is
regulation of protein turnover (87). An example is
ubiquitin, which is expressed in unstressed cells, up-
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Family
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regulated by heat shock, and that serves as a molecular
tag to mark proteins for degradation by proteasomes.
Other important biochemical activities of HSPs have
recently come to light. For example, HO-1 likely plays
a role in signal transduction in neural tissue and vascular smooth muscle cells by means of the generation of
carbon monoxide as part of heme degradation (84). The
released carbon monoxide interacts with guanylate cyclase and results in increased cGMP production, which
in turn has a vasodilatory effect on vascular smooth
muscle. This provides a potential mechanism whereby
stressed tissues may be able to modulate local blood
flow (84). Another interesting recent report has found
that exogenously added HSP70 can trigger CD14 receptor-mediated release of TNF-␣, IL-1␤, and IL-6 by
human monocytes (4). This suggests that HSP70 can
function as a proinflammatory cytokine when released
from injured cells or when secreted by activated immune cells (4).
Changes in transcriptional systems other than
HSF-1. Several transcriptional systems other than
HSF-1 are affected by heat shock. At least three mechanisms have been identified through which heat shock
specifically affects these systems: 1) changes in the
level of expression of transcription factors themselves
(through a variety of mechanisms, discussed below), 2)
changes in activity of transcription factors that are
already expressed (for example, by phosphorylation),
and 3) changes in cellular location of transcription
factors (such as translocation to the nucleus or sequestration in the cytoplasm).
Table 2 lists genes (including transcription factors)
whose expression is reported to be affected by heat.
We excluded genes whose change in expression has
been identified only by genomic or proteomic techniques. Transcriptional systems that show heat-induced changes in expression include 1) the AP-1 system, in which expression of the mRNA (2, 8, 24) and
protein (24) of the two key components ( fos and jun)
have been found to be increased by heat shock; 2) c-myc
(2, 6, 8, 108), which is downregulated by heat stress
through enhanced degradation of cytoplasmic mRNA
(108); 3) egr-1, whose heat-induced increased expression in mouse NIH/3T3 fibroblasts is thought to be
mediated, in analogy with arsenite-induced cell stress,
by p38- and JNK-mediated phosphorylation of transcription factor elk-1 (63); and 4) C/EBP-␣ and
C/EBP-␤, whose changes in expression and DNA binding activity as a result of heat shock appear to involve
both a change in mRNA expression and a shift in the
relative expression of different protein isoforms (120),
perhaps by alternative splicing.
Heat shock affects DNA binding by transcription
factors other than HSF-l, such as p53 in human glioblastoma A-172 cell lines [in which increased p53 DNA
binding precedes the heat-induced increase in cellular
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Changes in Expression of Other Genes as a Result
of Heat Stress
p53 mRNA and protein (79)] and Oct-1 and CREB in
rat thymocytes [in which DNA binding is decreased
(99)]. It is unknown whether the decrease in Oct-1 and
CREB binding after heat shock involves a change in
expression, a change in the binding activity of previously expressed protein, or both.
Another example of heat shock-induced changes in
transcription factor activity occurs in the AP-1 system.
Heat shock-induced phosphorylation of c-jun by JNK
has been demonstrated in mouse 3T3 cells (1), and this
was accompanied by an increase in AP-1-specific binding to DNA (1). Interestingly, immunodepletion of the
multifunctional DNA repair enzyme and redox sensor
Ref-1 (redox factor-1, also known as APE-1) prevented
heat shock-induced binding of AP-1 to its consensus
sequence (24). This activity was restored by reintroduction of reduced but not oxidized Ref-1 (24), suggesting
that cellular redox state, as sensed by Ref-1, may
strongly affect AP-1-mediated gene expression after
heat stress in some systems.
Heat shock can affect transcription by affecting the
cellular distribution of transcription factors other than
HSF-1. This has been shown to activate transcription
in some experimental systems. For example, in a colon
cancer cell line, heat shock produced translocation of
Y-box transcription factor 1 from the cytoplasm to the
nucleus, leading to increased expression of multidrug
resistance transporters MDR-1 and MRP-1 (103).
Conversely, heat shock can also inhibit transcription
by preventing transcription factor translocation to the
nucleus. The best-studied example of this is the heat
stress-mediated inhibition of cytokine-induced, or LPSinduced, nuclear factor (NF)-␬B translocation to the
nucleus. This inhibition is thought to be mediated by
effects on I␬B␣, a protein that traps NF-␬B in the
cytoplasm. Specifically, heat shock inhibits the activation of I␬B kinase (IKK) activity that normally occurs
in response to proinflammatory stimuli (17, 121), leading to decreases in phosphorylation (17, 121) and subsequent ubiquination (95) of I␬B␣. The net effect is an
inhibition of the degradation of I␬B␣ that normally
occurs after exposure to proinflammatory stimuli (17,
93, 95, 112, 121), which prevents NF-␬B translocation
to the nucleus by maintaining it in an inactive, bound
state in the cytoplasm. This mechanism functions in
the presence of inhibitors of protein synthesis (17) and
does not require increased expression of IKK (121).
Additionally, heat stress has been shown to increase
I␬B␣ mRNA expression in some experimental systems
(89, 113), which may help prevent the pool of available
I␬B␣ protein from diminishing after a proinflammatory stimulus (89), and it has been suggested that
HSPs may also stabilize the cytoplasmic I␬B␣/NF-␬B
complex (111), thus contributing to the inhibitory effect. These mechanistic redundancies, coupled with the
observation that delivery of a heat shock after exposure
to proinflammatory stimuli such as LPS (which activate NF-␬B) triggers apoptosis (7, 21), suggest that
inhibition of NF-␬B activity in the setting of thermal
stress is highly important to cell survival.
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Table 2. Genes other than HSPs whose expression is affected by heat stress [table excludes genes
whose change in expression has only been observed by genomic or proteomic technologies
(i.e., not yet confirmed by other techniques)]
Functional Class
Acute phase
reactant
Gene
Albumin
Cell adhesion
Cell cycle
Cell activation
marker
Species/Tissue
Exposure Model
Up
Mouse liver
Intact animal
Up
Mouse liver
Intact animal
Up
Mouse liver
Intact animal
Recovery
period
Down
Cell culture
Up
Human leukemia
cells HL60
Mouse liver
ICAM-1
Down
Rat pancreas
Intact animal
p53
Up
Cell culture
p21 (WAF-1)
Up
ED-1
Down
Human glioblastoma
A-172 cells; human
colorectal carcinoma
cells RKO.C: human
fibroblasts
Human glioblastoma
A-172 cells; human
colorectal carcinoma
cells RKO.C; human
fibroblasts
Rat brain
macrophages/
microglia
Human embryonal
carcinoma MCR-G3
cells
Human embryonal
carcinoma MCR-G3
Cells
Human embryonal
carcinoma MCR-G3
Cells
Rat liver
Recovery
period
Recovery
period
Recovery
period
Recovery
period
Cell
mcl-1
differentiation
Up
Cell
Cytokeratin 8
differentiation
marker
Hcg
Up
Up
Coagulation
␤-Fibrinogen
Down
Cytokine
Interferon-␥
and -␣
Interleukin-1␤
Variable
Down
Interleukin-6
Up
Interleukin-8
Down
Osteopontin
Down
RANTES
Down
Intact animal
Mouse peritoneal
macrophages;
Human astrocytes
U-373; Human
monocytes THP-1
Human intestinal
epithelial cells
(Caco-2); mouse
jejunal mucosa
Human airway
epithelial cells
(BEAS-2B) and
alveolar
pneumocytes (A549)
Human peripheral
blood mononuclear
cells
Human alveolar
pneumocytes (A549)
J Appl Physiol • VOL
Recovery
period
Recovery
period
Proposed
Mechanism/Comments
Ref.
HSF-1 binding to HSE 120
in the promotor
Increased expression
120
of specific isoforms
of transcription
factors C/EBP␣ and
C/EBP␤
Same as for Orm1
120
6
HSF-1 binding to HSE 120
in the promoter
I␬B␣ sequestration of
33
NF-␬B
78, 79, 82
Cell culture
Recovery
period
May involve p53
activation
35, 78, 79,
82
Intact animal
Recovery
period
I␬B␣ sequestration of
NF-␬B
46
Cell culture
Recovery
period
104
Cell culture
Recovery
period
68
Cell culture
Recovery
period
68
Intact animal
Recovery
period
Cell culture
Both
Cell culture
(Caco-2
cells);
intact
animal
Cell culture
Recovery
period
Recovery
period
Cell culture
Recovery
period
Cell culture
Recovery
period
92 • APRIL 2002 •
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Transcriptional effect, 38
mediator unknown
Reviewed in detail
45
elsewhere
HSF-1 binding to HSE 9, 101, 105
in the promoter
85, 106
I␬B␣ sequestration of
NF-␬B
121
102
I␬B␣ sequestration of
NF␬B
5
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Apoptosis
inhibitor
Blood protein
C-reactive
protein (CRP)
Orm1
(orsomucoid
1, acid
glycoprotein
1, AGP-1)
Orm 2
(orsomucoid
2, AGP-2)
bcl-2
Change
Reported
Timing
of Change
1731
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Table 2.—Continued
Functional Class
Cytoskeleton
Exposure Model
Down
Mouse macrophage Raw Cell culture;
264.7; mouse
tissue bath
peritoneal
(mouse
macrophages; human
liver); intact
mononuclear cells;
animal (rat
human astrocytes
pancreas)
U-373; mouse Kupffer
cells; mouse liver
slices; rat pancreas;
human airway
epithelial cells
(BEAS-2B); human
alveolar pneumocytes
(A549)
Both
Glial fibrillary
acidic protein
Basic fibroblast
growth factor
Up
Rat brain
Intact animal
Up
Human breast
carcinoma MCF-7/
ADR
Mouse SCC VII tumors
Cell culture
During heat
exposure
During heat
exposure
Metallothionein
Membrane
transport
MDR1 (ATPbinding
cassette
transporter)
MRP1 (ATPbinding
cassette
transporter)
Protease
Protease HTRA2
Protease inhibitor Amyloid
precursor
proteins
Receptor
Species/Tissue
TNF-␣
Vascular
endothelial
growth factor
(VEGF)
Transforming
growth factor
␤ (TGF-␤)
Heavy metal
binding
Change
M1 muscarinic
receptor
Up
Intact animal
Recovery
period
Up
Rat heart, cultured rat
cardiac fibroblasts,
rat model of
pancreatitis
Cell culture,
intact
animal
Recovery
period
Down
Rat neonatal
cardiomyocytes
Rat liver; human HeLa
cells
Cell culture
Recovery
period
Both
Down
Termination of TNF-␣
transcription,
increased degradation
of TNF-␣ mRNA,
I␬B␣ sequestration of
NF-␬B. The TNF-␣
promoter does not
contain a HSE. In the
mouse model, in vivo
heat exposure led to
transient increases in
plasma TNF-␣,
suggesting
dissociation between
gene expression and
secretion.
Activation of AP-1
Human clone carcinoma Cell culture
cell lines HCT15 and
HCT116
Recovery
period
Up
Human colon carcinoma Cell culture
cell lines HCT15 and
HCT116
Recovery
period
YB-1 translocation to
the nucleus
Up
Human neuroblastoma
SHSY5Y cells
Human fetal astrocytes
CC2565
Cell culture
During heat
exposure
Recovery
period
Reporter assays in rat
glial cells CCL-107
and mouse
neuroblastoma cells
CCL-147
Cell culture
Up
J Appl Physiol • VOL
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Recovery
period
www.jap.org
28, 29, 33,
51, 72,
100, 101,
105, 121
30
Unclear whether
52
hyperthermia or ischemia accounts for the
observed upregulation
In hearts from rats
32, 107
exposed to heat stress,
the response is
biphasic (initial
decrease followed by
subsequent increase)
and localizes to the
myocytes
32
Up
Cell culture
Ref.
97
In HeLa cells, mRNA
levels had returned to
baseline at the first
recovery time point
examined (3 hours
after heat shock)
YB-1 translocation to
the nucleus
Variable
Intact animal;
cell culture
Proposed
Mechanism/Comments
2, 38
103
103
41
Affects splicing of the
gene rather than
expression, as there
was an increase in
APP-751, no change
in APP-695 and a
decrease in APP-770
(splice variants of the
same gene)
HSF-1 binding to HSE
in the promoter
98
56
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Growth factor
Gene
Reported
Timing
of Change
1732
INVITED REVIEW
Table 2.—Continued
Functional Class
Gene
NMDA receptor
subunits
NMDAR1,
NMDAR2A,
and
NMDAR2B
Bradykinin
receptor B1
Redox control
Change
Exposure Model
Proposed
Mechanism/Comments
Ref.
Down
Rat hippocampus
Intact animal
During heat
exposure
Up
Rat aorta and heart; rat
aorta smooth muscle
cells
Human T lymphocytes
Intact animal;
cell culture
Recovery
period
Activation of p38 and
p42/p44 kinases
58, 59
Cell culture
Recovery
period
74
Cell culture
Both
Activation of
transcription factor
Elf-1
HSF-1 binding to HSE
in the promoter
Intact animal;
cell culture
Recovery
period
In intact animals,
MnSOD enzymatic
activity showed a
biphasic increase; the
early phase (seen
immediately after
heating) was not
accompanied by
increased expression
of MnSOD protein
117, 118
Cell culture
Recovery
period
Recovery
period
T-cell receptor
␨-chain
Down
Cu, Zn
superoxide
dismutase
(SOD-1)
MnSOD
Up
Up
Ribosomal RNA
Ribosomal RNA
Down
Signal
transduction
Annexin I
(lipocortin)
cNOS
Up
Up
DUSP1 (CL-100)
Up
DUSP5
Human leukemia cells
HL60; reporter assay
in human hepatoma
HepG2 cells
Rat myocardium and
rat cardiomyocytes
Mouse lung epithelium
MLE-15 cells
Mouse lymphosarcoma
P-1798 cells
Human lung A549 and
HeLa cells
Rat hippocampus
Cell culture
Cell culture
Intact animal
6, 122
110
Decreased expression of
the p72 subunit of
transcription factor
E1BF/Ku
HSF-1 binding to HSE
in the promoter
37
90
61, 96
Up
Human skin fibroblasts
EK4
Human skin fibroblasts
Cell culture
iNOS
Up
Rat brain
Intact animal
iNOS
Down
Cell culture;
tissue bath
(pancreatic
islets); intact
animal (rat
brain)
macrophages/
microglia)
Rad (Ras
associated
with diabetes)
Ran (Ras-related
nuclear
protein)
C/EBP-␣
(CCAATenhancer
binding
protein ␣)
Up
Rat pulmonary artery
smooth muscle cells;
rat hepatocytes;
human hepatocytes
(AKN-1); mouse lung
epithelium (MLE-15);
rat and human
pancreatic islets; rat
astrocytes; rat brain
macrophages/
microglia
Human PBMCs
Cell culture
Recovery
period
102
Down
Human leukemia cells
HL60
Cell culture
Recovery
period
6
Down
Mouse liver
Intact animal
Recovery
period
J Appl Physiol • VOL
Cell culture
Recovery
period
During heat
exposure
Recovery
period
Recovery
period
During heat
exposure
Recovery
period
61
92 • APRIL 2002 •
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54
49
96
I␬B␣ sequestration of
NF-␬-B
Heat stress affects both
the level of mRNA
expression and the
relative expression of
different protein
isoforms of this gene
22, 23, 31,
46, 93,
109, 110,
112
120
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Down
Transcription
factor
Species/Tissue
Reported
Timing
of Change
1733
INVITED REVIEW
Table 2.—Continued
Functional Class
Translation
Unknown
Change
Species/Tissue
Exposure Model
Proposed
Mechanism/Comments
Ref.
C/EPB-␤
(CCAATenhancer
binding protein
␤)
Up
Mouse liver
Intact animal
Recovery
period
c-fos
Up
Cell culture
Both
c-fos
Down
Human HeLa cells;
mouse NIH/3T3 cells;
human Hyon (pre-B)
cells; pre-T cells (DND41) and thymocytes
Chinese hamster ovary
K-1 fibroblasts and
reporter assay in same
Cell culture
During heat
exposure
c-jun
Up
Cell culture
Both
c-myc
Down
Human HeLa cells;
mouse NIH3T3 cells;
human Hyon (pre-B)
cells; pre-T cells (DND41), B cells (Daudi), T
cells (PEER);
thymocytes
Human leukemia cells
HL60; HeLa cells;
human B cell lines
BJAB and BJAB-6A;
human Hyon (pre-B)
cells; pre-T cells (DND41), B cells (Daudi), T
cells (PEER);
thymocytes
Mouse lymphosarcoma
P-1798 cells
Cell culture
Both
Enhanced cytoplasmic
degradation of c-myc
mRNA
2, 6, 8, 108
Cell culture
During heat
exposure
Leads to decreased RNA
polymerase I-directed
transcription
37
Mouse NIH/3T3 cells
Cell culture
Human lung carcinoma
A549 cells; human
bronchial epithelial
BEAS-2B cells; rat
brain macrophages/
microglia; mouse
jejunal mucosa
Human pancreatic
carcinoma cells FG2
and immortalized
human keratinocytes
HaCaT
Human leukemia cells
HL60
During heat
exposure
Recovery
period
Cell culture;
intact animal
(rat brain
macrophages/
microglia,
mouse jejunal
mucosa)
Cell culture
Recovery
period
p38- and JNK-mediated
63
phosphorylation of elk-1
HAF-1 binding to HSE in 46, 89, 111,
the promoter
113
Cell culture
Recovery
period
6
Mouse liver, spleen,
kidney, testis; human
HeLa cells, mouse
NIH/3T3 and rabbit
skin fibroblast RAB-9
cells
Intact animal;
Cell culture
Recovery
period
62, 67
E1-BF (enhancer- Down
1 binding
factor), subunit
p72
egr-1 (early growth Up
response-1)
I␬B␣
Up
Integrin beta-4
binding protein
(ITGB4BP;
eIF6; p27-BBP)
Down
Hypothetical
Up
protein TI-227H
(GenBank
#D50525)
Untranslated RNA B1 and B2 short
Up
interspersed
elements (SINE)
RNase; (Alu
repeats)
HSF-1 binding to HSE in
the promoter; heat
stress also affects the
relative expression of
different protein
isoforms of this gene
Post-transcriptional
mechanism; the c-fos
promoter does not have
a HSE
120
HSF-1 mediated
inhibition of the Rasmediated increase in
fos, by a mechanism
that does not involve
HSF-1 binding to the
fos promoter
11
2, 8, 24
8, 24
I␬B␣ sequestration of
NF␬B
26
Down, heat-induced decreases in expression or increases in expression (in response to other stimuli) that are prevented by heat. ICAM,
intracellular adhesion molecule; IL, interleukin; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; TGF-␤, transforming
growth factor-␤; MnSOD, Mn superoxide dismutase; DUSP, dual specificity phosphatase; iNOS, inducible nitric oxide synthetase; cNOS,
constitutive nitric oxide synthetase.
J Appl Physiol • VOL
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Transcription
factor inhibitor
Gene
Reported
Timing
of Change
1734
INVITED REVIEW
J Appl Physiol • VOL
The effect of heat shock on expression of non-HSP
genes appears to be tissue specific to some degree. For
example, heat stress increases expression of manganese superoxide dismutase (MnSOD) in rat cardiac
myocytes in vitro and in vivo (117, 118). By contrast, in
murine type II alveolar pneumocytes, heat shock
causes no induction of MnSOD expression and inhibits
cytokine-stimulated MnSOD expression (110). The
mechanisms responsible for these different effects remain to be determined; however, the existence of diverse responses among different cell types suggests the
possibility of tissue-specific mechanisms that modulate
the cellular response to heat stress. For example, given
the high oxygen consumption capacity of myocardium
relative to other tissues, heat shock might stimulate
MnSOD expression in this tissue through an effect on
the redox state that does not come into play in tissues
with lower oxidative capacity. Additionally, because
myocardial MnSOD enzymatic activity does not correspond one-to-one with MnSOD protein levels as measured by ELISA (118), the activity of this molecule may
be regulated by mechanisms other than, or in addition
to, expression. This could be one explanation of an
experiment in which rats exercised in the heat showed
a decrease rather than an increase in myocardial
MnSOD enzymatic activity compared with rats exercised under conditions that did not cause a rise in core
temperature (44). It is also possible that physiological
changes induced by exercise in the heat led to repression of this otherwise heat-inducible gene. Thus, for
MnSOD (and probably for many other genes), the net
in vitro effect of heat shock is susceptible to modulation
by a host of in vivo physiological variables. Accounting
for such differences is critically important when attempting to extrapolate in vitro data to higher-order
systems and vice versa.
The number of reported genes involved in the cell
stress response to heat is rapidly increasing, as more is
learned about the cellular processes affected by heat
shock and those that interact with HSPs. Additionally,
gene chip arrays, which allow researchers the ability to
investigate the expression of thousands of sequences
simultaneously, are likely to substantially increase the
number of genes known to be involved in the cellular
response to heat shock. Several gene chip array experiments have been performed that specifically examined
the role of heat shock on gene expression. An experiment reported in 1996 (94) used an array containing
1,000 genes to examine changes in gene expression in
human T cells and demonstrated the feasibility of
using this technology to identify new candidate heatresponsive genes. Another study involved exposing cultured human retinal pigment epithelial cells to sublethal heat shock (55°C for 3 s) (25). In a third, mice were
immersed in 43°C water for 20 min and changes in
gene expression in testes were examined (91). The
most recent experiment involved exposing peripheral
blood mononuclear cells obtained from adult male volunteers to a conventional in vitro heat shock (43°C for
20 min) and examining changes in gene expression in
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Changes in the expression and activation of transcription factors other than HSF-1 as a result of heat
stress are likely to be important to the physiology of
the cellular stress response. For example, myc (downregulated by heat, Table 2) is involved in a wide range
of biological functions (27) including apoptosis, cell
growth, differentiation, and division. When human B
lymphoid cells were prevented from downregulating
myc after heat shock (by transfecting cells with a myc
gene fused to an HSP70 promoter), there was a marked
decrease in cell viability (defined as an ability to restart proliferation) after heat shock (108). This suggests that downregulation of myc is important to recovery from heat shock. Other changes in transcription
factors that are likely of physiological importance include increased expression and activation of p53 by
heat shock, which leads to increased expression of cell
cycle regulator p21/WAF-1 (79) and to cell cycle arrest
(78); activation of AP-1, which affects the expression of
many cellular genes involved in stress responses (24);
and increased expression of transcription factor egr-1,
which also affects cell stress responses and is involved
in cell proliferation and differentiation (63).
Changes in expression of other, non-HSP genes. Approximately 50 genes not traditionally considered to be
HSPs have been found to undergo changes in expression in response to heat stress (Table 2). This table
includes both genes whose expression changes during
hyperthermia and genes whose expression changes after return to normothermia. We excluded genes whose
changes in expression as a result of heat stress have
been identified only by gene chip array or proteomic
technologies (i.e., not yet confirmed by other techniques).
Several heat-responsive genes encode molecules that
modulate the MAP kinase pathways, which play a role
in the cellular response to a variety of different environmental stressors. Of particular physiological interest is the increase in the MAP kinase phosphatases,
DUSP-1 (54) and DUSP-5 (49), both of which dephosphorylate components of the MAP kinase pathways.
MAP kinases are known to be activated at the onset of
heat stress. In principle, subsequent expression of the
DUSP phosphatases might allow the MAP kinase signal transduction pathway to be “reset,” thus rendering
the cell responsive to subsequent stressors after an
initial thermal stress (49). This interesting hypothesis,
along with its implications for the physiology of cells
with acquired thermotolerance, warrants further experimental exploration.
Another important effect of heat shock is the arrest
of the cell cycle, which is mediated by both changes in
gene expression and changes in the activity of previously expressed proteins. Genes affecting cell cycle
that have been shown to be affected by heat shock
include p53 and p21 (Table 2). Induction of p53 after
heat shock appears to be critical to this process in some
cell lines, as no cell cycle arrest was noted in cells that
were homozygous for a defective p53 gene (78).
1735
INVITED REVIEW
cells that showed a typical heat shock response (a
twofold or greater increase in HSP70 protein expression at 3 or 4 h) (102). Together, these experiments
reveal that the gene expression response to heat shock
is far broader than previously realized and involves
every major functional category involved in the cellular
response to heat stress. However, these findings require confirmation by other techniques as well as rigorous experimental evaluation to establish their physiological importance.
limited number of genes have been found to be induced
during the period of exposure to moderate hypothermia
(25–33°C). In addition, with the possible exception of
apoptosis-specific protein (40), no genes have been
found to be upregulated during exposure to temperatures below 5°C (34). However, as occurs after a heat
shock, many of the genes that are induced by cold
exposure (including a number of HSPs) do not increase
during the period of thermal stress itself but during the
cell stress response that ensues after rewarming (34).
COLD STRESS
Cold-Induced Changes in Gene Expression
and Cold Shock Proteins
Cellular Responses to Cold
J Appl Physiol • VOL
Mechanisms of cold-induced changes in gene expression. Five mechanisms have been identified by which
cold produces changes in mammalian gene expression
during the period of hypothermic exposure itself. The
first (as mentioned) is generalized cold-induced inhibition of transcription and translation. A second mechanism involves inhibition of RNA degradation, which is
used by bacteria to increase cold shock protein expression (116) and by hepatoblastoma cell lines to increase
expression of ATPase 6⫹8 subunits (81). A third mechanism involves increased transcription, mediated by a
cold response element in the promoter region of coldinducible RNA-binding protein (CIRP), a cold shock
protein (J. Fujita, unpublished observations). A fourth
mechanism is alternative splicing of pre-mRNA, which
occurs in neurofibromatosis type 1 mRNA during exposure to temperatures of 20°C-32°C (3). A fifth mechanism involves an enhanced efficiency of translation at
lower temperatures that is mediated by specialized
regions within the mRNA 5⬘ leader sequence (internal
ribosome entry sites, or IRESs) of RBM3, another cold
shock protein (10).
Three mechanisms have been postulated by which
changes in gene expression might occur after return to
normothermia following a hypothermic exposure. In
the first, it is proposed that severe cold exposure activates signals for a cell stress response (such as protein
denaturation or MAP kinase phosphorylation) but also
interferes sufficiently with processes such as transcription and translation as to preclude stress protein expression until rewarming occurs. This could explain
the HSF-1-mediated induction of HSP70 and HSP90
expression by human fibroblasts and HeLa cells after
cold shock at 4°C (65). In these experiments, coldinduced trimerization of HSF-1, binding of HSF-1 to
the HRE, and increases in HSP expression occurred
not during the period of hypothermia but rather after
rewarming to 37°C. This mechanism can also explain
cold-induced IL-8 expression by human bronchial epithelial cells (39). These cells exhibited increased tyrosine phosphorylation of p38 MAP kinase during exposure to 1°C but did not exhibit p38-dependent
increases in IL-8 mRNA expression and protein secretion until rewarmed to 37°C. A second hypothesis is
that rewarming after exposure to cold leads to gener-
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The responses of mammalian cells to cold exposure
have been reviewed in detail elsewhere (34). In principle, cold should reduce rates of enzymatic reactions,
diffusion, and membrane transport (whereas heat
would tend to accelerate these processes). Interestingly, many of the net cellular physiological effects of
cold exposure are similar to those seen in heat-stressed
cells. These include 1) an increase in the denaturation
and misaggregation of proteins; 2) a slowing of progression through the cell cycle, with phase G1 typically
being the most sensitive; 3) an inhibition of transcription and translation, leading to a generalized reduction
in protein synthesis; 4) a disruption of cellular cytoskeletal elements; and 5) changes in membrane permeability leading to increases in cytosolic Na⫹ and H⫹
(albeit with a decrease in intracellular K⫹). Cold stress
can also produce alterations in the properties of the
lipid bilayer, some of which (such as phase transitions)
are simply due to the reduction in temperature,
whereas others (such as changes in the fatty acid
composition of the membrane) likely reflect a cellular
physiological response to cold stress.
Depending on the intensity of the exposure, cold
stress can trigger a cell stress response, activate the
apoptotic program, or lead to necrosis. Evidence of a
cold-induced cellular stress response has included reports of induction of HSPs on rewarming (47, 60, 65),
phosphorylation of p38 MAP kinase during hypothermia (39), and translocation of ␤-crystallin from the
nucleus to the cytoplasm (14). Cold-induced apoptosis
occurs and appears to be sensitive to both the minimum temperature achieved and the duration of the
exposure. For example, Burkitt lymphoma cells underwent apoptosis on return to 37°C after only a 20- to
30-min exposure at 1°C but required a 4-h exposure to
achieve the same effect at 25°C (42). Importantly, the
proapoptotic effects of cold shock depend not only on
the exposure but also on the cell line, intracellular
Ca2⫹ levels, cell cycle phase, and cytoskeletal stability.
Necrosis occurs at the most severe cold exposures,
through mechanisms such as the formation of ice crystals, leading to disruption of membranes and subcellular organelles.
Table 3 lists genes whose expression has been found
to change as a result of cold exposure. To date, a
1736
INVITED REVIEW
Table 3. Genes whose expression is affected by cold stress. [table excludes genes whose change in expression
has only been observed by genomic or proteomic technologies (i.e., not yet confirmed by other techniques)]
Functional
Class
Apoptosis
Gene
Change
Cell adhesion
Apoptosis specific
protein (ASP)
E selectin
Down
Cell cycle
p53
Up
WAF1/p21
Up
Cytokines
IL-8
Heat shock
proteins
Exposure Model
Cell culture
Both
Human bronchial epithelial
cells NCl-H292
Cell culture
After
rewarming
Up
Rat neonatal cardiomyocytes
Cell culture
HSP70 (HSP72)
Up
Tissue bath
(keratinocytes,
testis), cell culture
(others)
HSC70 (HSP73;
HSPA8)
HSP90
Up
Human keratinocytes,
squamous cell carcinoma
SCC12F cells, IMR-90
fibroblasts and HeLa
cells; mouse testis, TAMA
26 Sertoli cells, NIH3T3
fibroblasts; rat neonatal
cardiomyocytes
Human keratinocytes
After
rewarming
After
rewarming
Up
Human keratinocytes, IMR90 fibroblasts and HeLa
cells
HSP105 (HSP110)
Up
Mouse testis, TAMA 26
Sertoli cells, NIH3T3
fibroblasts
Tissue bath
(keratinocytes),
cell culture
(others)
Tissue bath (testis),
cell culture
(others)
APG-1
Up
Metallothionein
Up
Mouse testis, TAMA26
Sertoli cells, NIH3T3
fibroblasts
Rat keratinocytes HT-1213
Tissue bath (testis),
cell culture
(others)
Cell culture
ATPase subunit 6⫹8
(mitochondrial
gene)
CIRP
Up
Human Hep G2 hepatocytes
Cell culture
Up
Cell culture
RBM3
Up
Signal
transduction
NF-1 variant
Up
Unknown
KIAA0058
Up
Mouse BALB/3T3
fibroblasts, BMA1 bone
marrow stromal cells,
TAMA26 Sertoli cells;
human K562 leukemia
cells, Hep G2 hepatocytes,
HeLa, NC65 renal cell
carcinoma and T24
bladder carcinoma; rat
PC12 pheochromocytoma
Human K562 leukemia
cells, HeLa, Hep G2
hepatocytes, T24 bladder
carcinoma, and NC 65
renal cell carcinoma cells;
mouse BALB/3T3
fibroblasts, BMA-1 bone
marrow stromal cells, and
TAMA26 Sertoli cells;
reporter constructs
transfected into human,
rat and mouse cell lines
Human peripheral blood
lymphocytes; human
fibroblasts (primary
culture); human
osteoblastoma line U2OS
Human endothelial cells
ECV304
RNA binding
Human Burkitt’s lymphoma
cells (MUTU-BL)
Human umbilical vein
endothelial cells (HUVEC)
Human AG01522
fibroblasts, glioblastoma
A-172 cells
Human AG01522
fibroblasts; human
glioblastoma A-172 cells
Cell culture
Up
HSP25
Cell culture
Tissue bath
After
rewarming
After
rewarming
After
rewarming
After
rewarming
After
rewarming
During cold
exposure
Refs.
40
43
Inhibits cell cycle
progression
70, 80
Induced in cells
carrying wildtype but not
mutant p53 genes
Cold-induced
phosphorylation
of p38
mRNA induced but
not protein
Activation of HSF-1
on rewarming
70, 80
39
60
47, 53, 60, 65
47
Activation of HSF-1
upon rewarming
47, 65
Dual control: HSF-1
binding to HSE
in the promoter
plus a resetting
of the set point of
a cellular
temperature
sensor
Same as for
HSP105
53
53
83
Inhibition of RNA
degradation
81
During cold
exposure
Putative cold
response element
in the promoter*
75–77, 115
Cell culture
During cold
exposure
Enhanced efficiency
of translation
through IRESs in
the 5⬘ leader
sequence
10, 19, 20
Cell culture
During cold
exposure
Alternative splicing,
leading to mRNA
with an
additional exon
3
Cell culture
During cold
exposure
* Unpublished observation.
J Appl Physiol • VOL
Proposed
Mechanism/
Comments
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34*
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Cell culture
After
rewarming
During cold
exposure
Both
Heavy metal
binding
Membrane
transport
Up
Species/Tissue
Time of
Change
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INVITED REVIEW
J Appl Physiol • VOL
tion of a reporter construct containing the target CIRPbinding region was significantly enhanced in cells
overexpressing CIRP. Furthermore, RNA constructs
containing the CIRP-binding region displayed enhanced resistance to degradation by RNases in the
presence of CIRP, compared with constructs lacking
this 3⬘-UTR. Together, these results suggest that CIRP
enhances translation of its target RNA species, at least
in part through stabilization of the mRNA. Importantly, cells transfected with an antisense CIRP vector
had decreased levels of CIRP protein and a diminished
ability to survive ultraviolet irradiation, illustrating
the physiological importance of this molecule to the
stress response (119).
CIRP may have functional roles other than as a
stress protein, such as a role in cell cycle control, as a
suppressor of mitosis, and as a molecule involved in
maintenance of differentiated states (34). CIRP may
also play a role in the cold-induced cell cycle arrest,
because cells that overexpressed CIRP exhibited reduced growth rates at 37°C and a prolonged G1 phase
(77). Conversely, inhibition of CIRP induction at 32°C
(by addition of antisense CIRP mRNA to cultured
BALB/3T3 cells) attenuated the slowing of cellular
growth that occurred at this temperature in normal
cells (77).
Another well-characterized cold shock protein is
RBM3. This molecule is structurally similar to CIRP,
and expression of its mRNA is increased by cooling to
32°C (20). However, unlike CIRP, RBM3 does not appear to be involved in the cold-induced growth suppression (19). The tissue distribution of RBM3 appears to
be more limited than that of CIRP, as RBM3 was not
detected (by Northern blot analysis) in either heart or
thyroid, both of which expressed CIRP (20). Interestingly, the RBM3 mRNA 5⬘ leader sequence contains a
number of specialized sequences that allow initiation
of translation independently of the methylated G nucleotide 5⬘-cap that is typically used by cells to tag an
mRNA molecule for initiation of protein synthesis.
These IRESs appear to facilitate translation at 33°C
(10), a temperature that inhibits rates of protein synthesis. Other genes known to contain IRESs that enhance translation of reporter constructs at 33°C are
c-myc and sequences from poliovirus (10).
A third candidate cold shock protein is KIAA0058
(unpublished observations and Ref. 34), also referred to
as DAZ-associated protein 2. Although the sequence of
this protein is known and it has been detected in cDNA
libraries in a wide variety of tissues, its physiological
function and its role in the cold shock response both
remain to be characterized.
The number of identified cold shock genes is likely to
increase as new technologies are introduced to answer
these questions. For example, through the use of cDNA
subtraction and gene chip array methods, several
genes have recently been identified whose expression is
increased at 32°C (cold-inducible cDNA clones) (Fujita,
unpublished observations).
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ation of free radicals and other toxic metabolites that
are capable of inducing a stress response. The finding
that HSF-1 activation following cold stress only occurs
on rewarming can also be explained by this hypothesis
(65). A third hypothesis, however, is required to explain the peculiar induction pattern of APG-1 (a HSP)
and HSP105 in mouse somatic cells (Sertoli cells and
NIH/3T3 fibroblasts). In these cells, induction of APG-1
and HSP105 did not occur after a conventional heat
shock (by raising the temperature from 37 to 42°C);
rather, it was required that cells first be incubated at
32°C and then heated to 39°C (53). Temperature shifts
both from 32 to 42°C and from 32 to 39°C led to
increases in the binding of HSF-1 to HSEs in the
APG-1 promoter. However, whereas both temperature
shifts induced HSP70 expression, only the latter (32–
39°C) led to an increase in APG-1 expression. A possible (although untested) explanation for these findings
would be that the APG-1 and HSP105 promoters are
under dual control of both HSF-1 and a hypothetical
repressor that becomes inactive at low temperatures
and whose rate of reactivation is substantially faster at
42°C than at 39°C.
Cold shock proteins. Cold shock proteins can be defined as proteins that are induced during the period of
exposure to moderate hypothermia (typically, 25–
33°C). Under this definition, the best-characterized
cold shock protein to date is CIRP, a 172-amino acid (in
mouse) protein containing an RNA-binding domain
(77). A CIRP gene has been detected in mouse, rat, and
human cells, and its sequence is highly conserved in
these species (76, 77, 115). The mRNA encoding this
protein is expressed constitutively in most tissues of
adult mice at relatively low levels. However, it is
strongly induced by cold; in a cell culture model (BALB/
3T3 mouse fibroblasts), CIRP was induced within 3 h
after the ambient temperature was reduced to 32°C,
with maximal expression detected between 6 and 24 h
of exposure (77). CIRP expression can also be increased
by a variety of stressors other than cold, such as ultraviolet irradiation and hypoxia [reviewed by Fujita
(34)], although it has not been found to be induced by
heat stress (76, 77).
CIRP shares structural similarity with a number of
other known RNA-binding proteins; accordingly, it has
been speculated that one of its physiological functions
is to protect and restore native RNA conformations
during stress (i.e., to serve as a chaperonin for RNA).
Evidence for this role has recently been reported in
human colorectal carcinoma (RKO) cells, where CIRP
(also known as hnRNP A18) was found by immunofluorescence to translocate from the nucleus to the cytoplasm after ultraviolet irradiation and was noted to
bind specifically to a limited number of RNA species,
including several stress-inducible molecules (119).
Binding was specific to the 3⬘ untranslated region
(UTR) of susceptible RNA, which is known to be involved both in the efficiency of translation and in RNA
stability. Cotransfection assays showed that transla-
1738
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Changes in Expression of Other Genes as a Result
of Cold Stress
As summarized in Table 3, several genes with wellestablished physiological functions have been reported
to be induced by cold stress. Two genes likely to play
important roles in cell physiology during cold exposure
are cell cycle proteins p53 and p21, which are induced
by exposure to temperatures that produce cell cycle
arrest (70, 80). Importantly, p53 appears to play an
integral role in the cold-induced cell cycle arrest, as
cells lacking a wild-type p53 gene escaped this arrest
both in human fibroblasts (70) and in glioblastoma cell
lines (80).
CONCLUSIONS
J Appl Physiol • VOL
The views, opinions, and findings contained in this publication are
those of the authors and should not be construed as an official United
States Department of the Army position, policy, or decision, unless so
designated by other documentation. Approved for public release;
distribution unlimited.
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