1: Life Sci. 2005 Oct 14;77(22):2783-93. 

Arsenic trioxide induces Hsp70 expression via reactive oxygen species and JNK
pathway in MDA231 cells.

Kim YH, Park EJ, Han ST, Park JW, Kwon TK.

Department of Immunology, School of Medicine, Keimyung University, 194
DongSan-Dong Jung-Gu, Taegu, 700-712, South Korea.

In the present study, we determined the molecular pathways that induce the heat
shock proteins (Hsps) after treatment of cells with arsenic trioxide.
Administration of arsenic trioxide to MDA231 cells leads to induce Hsp70, which
is accompanied by generation of reactive oxygen species (ROS) and activation of
c-Jun N-terminal kinase (JNK). We showed that arsenic trioxide-induced Hsp70
expression was caused by activation of ROS and prevented by the antioxidant
N-Acetyl-Cysteine (NAC). SP600125 and dominant-negative SEK suppressed Hsp70
promoter-driven reporter gene expression, suggesting that JNK would be
preferentially associated with the protective heat shock response against
arsenic trioxide stress. In addition, SP600125, a specific JNK inhibitor,
significantly reduced the amount of phosphorylated HSF1 upon administration of
arsenic trioxide. It is likely that Hsp70 expression against arsenic trioxide
exposure protects cells from oxidative injury and apoptotic cell death by means
of JNK activity.

PMID: 15978632 [PubMed - in process]


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2: Circulation. 2003 Dec 16;108(24):3024-30. Epub 2003 Nov 17. 

Heat shock transcription factor 1 protects cardiomyocytes from
ischemia/reperfusion injury.

Zou Y, Zhu W, Sakamoto M, Qin Y, Akazawa H, Toko H, Mizukami M, Takeda N,
Minamino T, Takano H, Nagai T, Nakai A, Komuro I.

Department of Cardiovascular Science and Medicine, Chiba University, Graduate
School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
komuro-tky@umin.ac.jp

BACKGROUND: Because cardiomyocyte death causes heart failure, it is important to
find the molecules that protect cardiomyocytes from death. The death trap is a
useful method to identify cell-protective genes. METHODS AND RESULTS: In this
study, we isolated the heat shock transcription factor 1 (HSF1) as a protective
molecule by the death trap method. Cell death induced by hydrogen peroxide was
prevented by overexpression of HSF1 in COS7 cells. Thermal preconditioning at 42
degrees C for 60 minutes activated HSF1, which played a critical role in
survival of cardiomyocytes from oxidative stress. In the heart of transgenic
mice overexpressing a constitutively active form of HSF1, ischemia followed by
reperfusion-induced ST-segment elevation in ECG was recovered faster, infarct
size was smaller, and cardiomyocyte death was less than wild-type mice. Protein
kinase B/Akt was more strongly activated, whereas Jun N-terminal kinase and
caspase 3 were less activated in transgenic hearts than wild-type ones.
CONCLUSIONS: These results suggest that HSF1 protects cardiomyocytes from death
at least in part through activation of Akt and inactivation of Jun N-terminal
kinase and caspase 3.

PMID: 14623809 [PubMed - indexed for MEDLINE]


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3: J Cell Biochem. 2001;82(2):326-38. 

JNK phosphorylates the HSF1 transcriptional activation domain: role of JNK in
the regulation of the heat shock response.

Park J, Liu AY.

Department of Cell Biology and Neuroscience, Rutgers, The State University of
New Jersey, Piscataway 08854-8082, USA. JPARK@molbio.princeton.edu

The role of c-Jun NH2-terminal kinase (JNK) signaling cascade in the
stress-inducible phosphorylation of heat shock factor 1 (HSF1) was investigated
using known agonists and antagonists of JNK. We showed that treatment of HeLa
cells with MG132, a proteasome inhibitor and known INK activator, caused the
transcriptional activation domain of HSF1 to be targeted and phosphorylated by
JNK2 in vivo. Dose-response and time course studies of the effects of heat shock
and anisomycin treatment showed a close correlation of the activation of JNK and
hyperphosphorylation of HSF1. SB203580 inhibited INK at the 100 microM
concentration and significantly reduced the amount of hyperphosphorylated HSF1
upon heat shock or anisomycin treatment. SB203580 and dominant-negative JNK
suppress hsp70 promoter-driven reporter gene expression selectively at 45
degrees C but not at 42 degrees C heat stress, suggesting that JNK would be
preferentially associated with the protective heat shock response against severe
heat stress. The possibility that JNK-mediated phosphorylation of HSF1 may
selectively stabilize the HSF1 protein and confers protection to cells under
conditions of severe stress is discussed.

PMID: 11527157 [PubMed - indexed for MEDLINE]


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4: J Hepatol. 2001 Jul;35(1):53-61. 

A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses
apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol.

Ikeyama S, Kusumoto K, Miyake H, Rokutan K, Tashiro S.

First Department of Surgery, School of Medicine, The University of Tokushima,
Japan.

BACKGROUND/AIMS: A stress-inducible heat shock protein 70 (HSP70) is one of the
best-known endogenous factors protecting cell injury under various pathological
conditions. The aim of this study was to examine anti-apoptotic actions of a
non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to
hydrogen peroxide (H2O2) or ethanol. METHODS: Primary cultures of rat
hepatocytes were treated with different concentrations of GGA and exposed to 0.5
mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the
activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and
accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA
fragmentation. RESULTS: Pretreatment with 1 microM GGA for 2 h enhanced nuclear
translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA
expression, and its accumulation, when the cells were exposed to H202 or
ethanol. In association with this accelerated response, GGA suppressed the
insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase
3-like proteases, leading to significant inhibition of apoptosis. CONCLUSIONS:
GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for
enhanced HSP70 induction. Our results suggest that GGA may have a potential
benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.

PMID: 11495042 [PubMed - indexed for MEDLINE]


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5: Mol Endocrinol. 2001 Aug;15(8):1396-410. 

Inhibition of heat shock transcription factor by GR.

Wadekar SA, Li D, Periyasamy S, Sanchez ER.

Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614, USA.

The GR is a hormone-activated transcription factor that acts to regulate
specific gene expression. In the absence of hormone, the GR and other steroid
receptors have been shown to form complexes with several mammalian heat shock
proteins. As heat shock proteins are produced by cells as an adaptive response
to stress, speculation has existed that communication between the heat shock and
glucocorticoid hormone signal pathways must exist. Only recently has evidence to
support this hypothesis been reported. In almost all cases, the evidence has
been of an ability of heat shock to cause a potentiation of the glucocorticoid
hormone response. In this proposal, evidence is now presented that heat shock
signaling can, in turn, be regulated by glucocorticoids. In mouse L929 cells
stably expressing a chloramphenicol acetyltransferase reporter controlled by the
human heat shock protein70 promoter and containing known binding sites for heat
shock transcription factor 1 treatment with glucocorticoid agonist
(dexamethasone) results in a dose-dependent decrease of stress-induced
chloramphenicol acetyltransferase gene expression. In these cells, inhibition of
heat shock protein70 promoter activity by dexamethasone was completely blocked
by GR antagonist (RU486). Similar treatment of L929 cells stably expressing a
chloramphenicol acetyltransferase reporter under the control of the
constitutively active SV40 promoter showed no such inhibition by dexamethasone.
More importantly, dexamethasone was also found to inhibit heat shock-induced
expression of the major heat shock proteins-heat shock proteins70, 90, and 110.
Thus, the inhibitory effect of dexamethasone appears to apply to most, if not
all, heat shock transcription factor 1-regulated genes. Although dexamethasone
did not prevent the DNA-binding function of heat shock-activated heat shock
transcription factor 1, it did inhibit a constitutively active mutant of human
heat shock transcription factor 1 under nonstress conditions, suggesting that
dexamethasone repression of heat shock transcription factor 1 was primarily
through an inhibition of heat shock transcription factor 1 transcription
enhancement activity. To more accurately characterize the stage of GR signaling
responsible for inhibition of heat shock transcription factor 1, a series of
Chinese hamster ovary cells containing either no GR, wild-type mouse GR, or
single-point mutations of GR were employed. Dexamethasone inhibition of heat
shock-induced heat shock transcription factor 1 activity was observed in the
presence of wild-type GR, but not in Chinese hamster ovary cells lacking GR,
suggesting that signaling cascades other than GR were not involved in this
effect of dexamethasone. Consistent with this conclusion was the observation
that dexamethasone had no effect on activity of the MAPKs (ERK1, ERK2, or c-jun
N-terminal kinase), which are known to negatively regulate heat shock
transcription factor 1. Dexamethasone inhibition of heat shock transcription
factor 1 was not seen in Chinese hamster ovary cells expressing GR defective for
DNA-binding function. Moreover, dissociation of GR/Hsp90/Hsp70 complexes was
observed in response to hormone for both the wild-type and DNA binding-defective
forms of GR, demonstrating that release of Hsp90 or Hsp70 (both of which are
known to keep heat shock transcription factor 1 in its inactive state) could be
ruled out as a potential mechanism. Thus, it appears that GR-mediated
transactivation or transrepression is required for the inhibitory effect of
dexamethasone on heat shock transcription factor 1 activity. Taken as a whole,
these results provide evidence for a novel mechanism of cross-talk in which
signaling by the GR can attenuate the heat shock response in cells through an
inhibition of the transcription enhancement activity of HSF1.

PMID: 11463862 [PubMed - indexed for MEDLINE]


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6: J Cell Physiol. 2000 Dec;185(3):348-57. 

Pervanadate induces the hyperphosphorylation but not the activation of human
heat shock factor 1.

Park J, Liu AY.

Graduate Program in Cell and Developmental Biology, Rutgers State University of
New Jersey, Piscataway, New Jersey, USA.

In this study, we evaluated the effects of pervanadate, a tyrosine phosphatase
inhibitor, on the regulation and function of heat-shock factor 1 (HSF1) in HeLa
cells. We showed that 50-100 microM pervanadate induced the hyperphosphorylation
of the latent HSF1, as demonstrated by a retarded mobility of the HSF1 protein
in SDS-polyacrylamide gel electrophoresis and as supported by the reversal of
this mobility shift upon treatment of the cell extract with acid phosphatase.
Pervanadate by itself had no effect on the monomeric stoichiometry and
DNA-binding activity of HSF1. Upon heat shock, the pervanadate-induced
hyperphosphorylated HSF1 formed DNA-binding trimers and translocated into the
nuclear compartment. At high concentration (approximately 500 microM),
pervanadate also induced the tyrosine phosphorylation of many cellular proteins
and blunted the heat-induced transcription of hsp 70. N-acetyl cysteine
inhibited these effects of pervanadate, suggesting a redox-based mechanism for
its activity. Analysis of the activation of mitogen-activated protein kinases
(MAPKs) using antibodies specific for the phospho-form (activated) of the
kinases in Western blot showed that pervanadate activated extracellular
signal-regulated kinase (ERK1/2), c-Jun-N-terminal kinase 1/2 (JNK1/2), and p-38
kinase. Pharmacological inhibitors of the ERK1/2 kinase pathway or the p38
kinase had little or no effect on the pervanadate-induced hyperphosphorylation
of HSF1. Our results show that hyperphosphorylation of hHSF1 can occur prior to
and independent of other events involved in the activation of hHSF1. The
possibility that activation of the MAPK signaling cascade, notably JNK, may
contribute to the hyperphosphorylation of human HSF1 (hHSF1) is discussed.
Copyright 2000 Wiley-Liss, Inc.

PMID: 11056005 [PubMed - indexed for MEDLINE]


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7: J Biol Chem. 2001 Jan 19;276(3):1889-95. Epub 2000 Oct 24. 

Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase
and heat shock factor in response to heat shock.

Han SI, Oh SY, Woo SH, Kim KH, Kim JH, Kim HD, Kang HS.

Department of Molecular Biology, College of Natural Sciences, Pusan National
University, Pusan 609-735, Korea.

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat
shock protein (HSP) expression through activation of the heat shock factor
(HSF), but its signal pathway is not clearly understood. Since a small GTPase
Rac1 has been suggested to participate in the cellular response to stresses, we
examined whether Rac1 is involved in the heat shock response. Here we show that
moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and
membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant
negative mutant of Rac1, significantly inhibited JNK activation by heat shock.
Since Rac1V12 was able to activate JNK, it is suggested that heat shock may
activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in
response to heat shock was observed. However, inhibitory effects of Rac1N17 on
heat shock-induced JNK and HSF activation were reduced as the heat shock
temperature increased. Rac1N17 also inhibited HSF activation by
l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)),
suggesting that Rac1 may be linked to HSF activation by denaturation of
polypeptides in response to various proteotoxic stresses. However, Rac1N17 did
not prevent phosphorylation of HSF1 in response to these proteotoxic stresses.
Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF.
Therefore, Rac1 activation may be necessary, but not sufficient, for heat
shock-inducible HSF activation and HSP expression, or otherwise a signal
pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In
sum, we suggest that Rac1 may play a critical role(s) in several aspects of the
heat shock response.

PMID: 11050083 [PubMed - indexed for MEDLINE]


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8: Circ Res. 2000 Jun 9;86(11):1122-8. 

Comment in:
    Circ Res. 2000 Jun 9;86(11):1101-3.

Mechanical stress-induced heat shock protein 70 expression in vascular smooth
muscle cells is regulated by Rac and Ras small G proteins but not
mitogen-activated protein kinases.

Xu Q, Schett G, Li C, Hu Y, Wick G.

Institute for Biomedical Aging Research, Austrian Academy of Sciences,
Innsbruck, Austria. qingbo.xu@oeaw.ac.at

-Previous studies have documented that acute elevation in blood pressure results
in heat shock protein (hsp) 70-mRNA expression followed by hsp70-protein
production in rat aortas. In this article, we provide evidence that mechanical
forces evoke rapid activation of heat shock transcription factor (HSF) and hsp70
accumulation. In our study, Western blot analysis demonstrated that
hsp70-protein induction peaked between 6 and 12 hours after treatment with
cyclic stain stress (60 cycles/minute, up to 30% elongation). Elevated protein
levels were preceded by hsp70-mRNA transcription, which was associated with HSF1
phosphorylation and activation stimulated by mechanical forces, suggesting that
the response was regulated at the transcriptional level. Conditioned medium from
cyclic strain-stressed vascular smooth muscle cells (VSMCs) did not result in
HSF-DNA-binding activation. Furthermore, mitogen-activated protein kinases
(MAPKs), including extracellular signal-regulated kinases, c-Jun NH(2)-terminal
protein kinases or stress-activated protein kinases, and p38 MAPKs, were also
highly activated in response to cyclic strain stress. Inhibition of
extracellular signal-regulated kinase and p38-MAPK activation by their specific
inhibitors (PD 98059 and SB 202190) did not influence HSF1 activation.
Interestingly, VSMC lines stably expressing dominant-negative rac (rac N17)
abolished hsp-protein production and HSF1 activation induced by cyclic strain
stress, whereas a significant reduction of hsp70 expression was seen in ras
N17-transfected VSMC lines. Thus, our findings demonstrate that cyclic strain
stress-induced hsp70 expression is mediated by HSF1 activation and regulated by
rac and ras GTP-binding proteins. Induction of hsp70 could be important in
maintaining VSMC homeostasis during vascular remodeling in response to
hemodynamic stimulation.

PMID: 10850962 [PubMed - indexed for MEDLINE]


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9: Cancer Res. 1999 Aug 15;59(16):4012-7. 

Ku autoantigen affects the susceptibility to anticancer drugs.

Kim SH, Kim D, Han JS, Jeong CS, Chung BS, Kang CD, Li GC.

Department of Biochemistry, College of Medicine, Pusan National University,
South Korea.

The Ku70/80 autoantigens (Ku) are the DNA-binding components of a DNA-dependent
protein kinase (PK) involved in DNA double strand breaks repairing a V(D)J
recombination. Because apoptosis is associated with DNA fragmentation and,
consequently, creation of double strand breaks, and a variety of DNA-damaging
drugs kill tumor cells by apoptosis, we tested the impact of Ku deficiency on
the sensitivity of anticancer drugs. Ku-null mutant cell lines Ku70-/- and
Ku80-/- were highly sensitive to anticancer drugs, compared with their wild-type
cells. Ku-deficient cells were more sensitive to bleomycin-induced DNA
fragmentation and exhibited a higher level of c-jun NH2-kinase/stress-activated
PK activity than wild-type cells, whereas R7080-6 cells overexpressing both
human Ku70 and Ku80 were resistant to bleomycin-induced apoptosis and exhibited
a lower level of c-jun NH2-kinase/stress-activated PK activity. The Ku-protein
level and Ku DNA binding activity were decreased after treatment with bleomycin,
adriamycin, or vincristine, and the decreases were blocked by the treatment of
z-DEVD-fmk, a specific inhibitor of caspase-3, suggesting that loss of Ku DNA
binding is, in part, due to a caspase-mediated decrease in Ku protein levels. By
contrast, HSF1 DNA-binding activity was increased by the treatment of these
anticancer drugs and, subsequently, mitochondrial heat shock protein HSP75 was
specifically induced. Our data suggest that Ku can affect the susceptibility to
anticancer drug-induced apoptosis.

PMID: 10463600 [PubMed - indexed for MEDLINE]


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10: Biochem Biophys Res Commun. 1999 Aug 27;262(2):516-22. 

Involvement of c-Jun NH(2)-terminal kinase pathway in differential regulation of
heat shock proteins by anticancer drugs.

Kim SH, Kim D, Jung GS, Um JH, Chung BS, Kang CD.

College of Medicine, Pusan National University, Pusan, 602-739, Korea.

In the present study, we examined the modulation of heat shock factor 1 (HSF1)
activity and expression of heat shock proteins (HSPs) after exposure to
anticancer drugs. Anticancer drugs induced HSF1 DNA-binding activity, and this
was followed by an increase of mitochondrial HSP75 and HSP60 levels and
concurrent decrease of cytoplasmic HSP70 levels. Unlike heat shock-induced full
phosphorylation, HSF1 was partially phosphorylated after exposure to
vincristine, and this result was tightly correlated with the kinetics of
JNK/SAPK activation, and up-regulation of mitochondrial HSP75 level and
concurrent down-regulation of HSP70. Furthermore, the dominant-negative mutant
of SEK1 blocked the phosphorylation of HSF1 and up-regulation of mitochondrial
HSP75 in response to vincristine or vinblastine. These data suggest that
anticancer drugs regulate the HSF1 transcriptional activity differently from
heat shock, and JNK/SAPK pathway appears to be involved in anticancer
drug-induced HSF1 phosphorylation and consequently differential regulation of
mitochondrial HSP75 and HSP60 and cytoplasmic HSP70. Copyright 1999 Academic
Press.

PMID: 10462506 [PubMed - indexed for MEDLINE]


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11: Exp Cell Res. 1995 Nov;221(1):103-10. 

Activation of multiple transcription factors and fos and jun gene family
expression in cells exposed to a single electric pulse.

Pazmany T, Murphy SP, Gollnick SO, Brooks SP, Tomasi TB.

Department of Molecular Medicine, Roswell Park Cancer Institute, Buffalo, New
York 14263, USA.

We report that exposure of cells to a single electric pulse (250-1250 V/cm)
results in the rapid and persistent activation of the DNA binding activities of
a number of transcription factors, including AP-1, SP1, AP-2, and NF-kappa B,
and the transient expression of select members of the fos and jun gene families.
Induction of gene expression occurs primarily at the level of transcription,
although c-jun expression also appears to be regulated posttranscriptionally.
Interestingly, maximal induction of gene expression is detected at electrical
field strengths that do not result in pore formation in the plasma membrane and
that do not significantly affect cell viability. Exposure of cells to electric
pulses does not result in the activation of HSF1 DNA binding activity, or the
induction of hsp70 or p53 protein synthesis, indicating that the induction of
fos and jun gene expression is not coincident with protein or DNA damage. The
results of these studies suggest that electrical pulses may represent a novel
mechanism for inducing the activities of multiple transcription factors and the
expression of select members of the fos and jun gene families.

PMID: 7589234 [PubMed - indexed for MEDLINE]


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