1
Geldanamycin Induces Transactivation of Hsp70a in Human Non-Small
Cell Lung Cancer H460 Cells through Calcium Sensitive and Non-Sensitive
Pathway
Nai-Lin Cheng, Jun-Hung Cho, Yiu-Kay Lai§
From the Department of Life Science, National Tsing Hua University, Hsinchu,
Taiwan 30013, Republic of China
Running Title:
GA-induced hsp70a expression through calcium sensitive and
non-sensitive pathway
Please send correspondence to:
Yiu-Kay Lai
Department of Life Science
National Tsing Hua University
Hsinchu, Taiwan 30013
Republic of China
Fax:
886-3-5715934
E-mail: yklai@life.nthu.edu.tw
URL: http://life.nthu.edu.tw/
2
ABSTRACT
The benzoquinoid ansamycin geldanamycin (GA) interferes with many
cell-signaling pathways and is currently being evaluated as an anticancer agent.
Recent studies have shown that GA induces the expression of heat shock proteins
(HSPs), including the HSP70s. The family of 70-kDa heat-shock proteins (HSP70s)
is evolutionarily highly conserved and is assumed that serve an important protective
function from a variety of adverse envirnmental conditions.
In human cells, two
stress inducible hsp70 genes, hsp70a and hsp70b, have often been studied.
By using
metabolic labeling followed by gel electrophoresis, Western and Northern blotting
techniques, we found that hsp70a is much more inducible than hsp70b in GA-treated
human non-small cell lung cancer H460 cells.
The induced expression of HSP70A
by GA is dependent on both concentration and the duration. And both actinomycin
D and cycloheximide block HSP70A induction. This shows that the effect of GA on
HSP70A induction is in transcription level.
On the other hand, GA causes
intracellular calcium ([Ca2+]i) mobilization, we further analyzed the effect of [Ca2+]i
on HSP70A induction in the above processes by exploiting a set of drugs that affect
the [Ca2+]i mobilization, including EGTA, BAPTA-AM, A23187, and thapsigargin
(TG).
It was found that the induced synthesis of HSP70A in GA-treated H460 cells
could be attenuated by these drugs, but failed to completely block the HSP70A
production and hsp70a mRNA accumulation. Taken together, our results lead us to
conclude that GA transactivated hsp70a in H460 cells through calcium-dependent and
-independent pathway.
3
INTRODUCTION
Geldanamycin (GA)1, a benzoquinoid ansamycin, is an natural-fermentation
product isolated from Streptomyces hygroscopicus (1, 2).
It was originally thought
to be a direct tyrosine-kinase inhibitor. However, subsequent studies revealed that
GA bind to 90-kDa heat-shock protein (HSP90) which are abundant molecular
chaperones in animal cells (3-8). HSP90 is constitutively expressed and serve as a
chaperone for a growing list of cell signaling proteins, including many tyrosine and
serine/threonine kinases, involved in proliferation and/or survival. (9-19). Among
others, HSP90 substrates also include steroid receptors (20, 21), and some
transcription factors such as hypoxia-inducible factor (22) and heat shock factor (HSF)
(23). Since many substrates of the HSP90 play crucial roles in cell proliferation
and/or survival, inhibition of this chaperone’s function by binding with GA would
result in client protein destabilization and lead cell death.
Therefore, this agent may
be of clinical benefit and has been deemed as a potential anti-cancer drug (24). On
the other hand, treatment with GA at sublethal doses has been shown to regulate a
number of specific genes. For instance, GA simultaneously suppresses c-MYC and
enhances the expression of pRB gene in HL-60 cells (25), up-regulates the
gadd153/CHOP transcription factor in CHO and COS-1 cells (26).
It also has been
reported that GA can induce the expression of many stress proteins, e.g., HSP70 and
HSP90 in rat cardiomyocytes (27); HSP70, HSP90, GRP78 and GRP94 in CHO and
COS-1 cells (26); HSP28 and HSP70 in K562 erythroleukemic cells (28), as well as
HSP70 in HT22 mouse hippocampal cells (29).
4
Heat-shock proteins (HSPs) with molecular masses ranging from 28 to 174 kDa are
highly conserved evolutionarily that can be found in bacteria, yeast, Drosophila, and
other organisms, including humans (30-32) and have been shown to enhance cell
survival from damage by noxious stimuli (33, 34). These proteins are classified into
two major groups, HSPs and GRPs; further subcategorized by their apparent
molecular mass. Among them, the members of 70-kDa stress protein family are the
most extensively studied (35-37).
In humans, this family encompasses at least 11
genes which encode a group of highly related proteins, including the cytosol/nuclear
resided HSC70 (the cognate/constitutive form, HSP72/73) and HSP70 (the inducible
form, HSP70/72); the mitochondria resided GRP75 and the endoplasmic resided
GRP78.
These genes corresponding to the above proteins are hsc70 on 11q23.3 for
HSC70, hsp70a and hsp70-hom on 6q23.1 for HSP70A and HSP70-HOM, hsp70b on
1q for HSP70B, grp78 on 9q34 for GRP78, and grp75 on 21 for GRP75 (38).
It is
interesting to note that the widely reported “inducible form” HSP70 may be coded by
at least two genes, hsp70a and hsp70b.
Unfortunately, the specific isoform(s) of
HSP70 induced by GA has not been elucidated.
Transactivations of the hsp genes under stress are mainly controlled by the binding
of the heat shock transcription factor (HSF) to its corresponding transcription heat
shock elements (HSEs) found in the promoter regions (39).
But the molecular
mechanisms responsible for signal transduction that leads to HSF activation and
phosphorylation have not been completely elucidated.
Increased Ca2+ mobilization
has also been recently shown to be an early event in the signal transdution pathway
mediating the activation of hsp70 gene expression by prostaglandin A2 (40), heat
5
shock (41-44) and ischemia/reperfusion (45).
It has been suggested that a
calcium-dependent metabolic process is involved in the generation these stress signal,
and calcium has also been shown to activate HSF DNA-binding ability (41, 46) and to
be essential for multistep activation of the heat shock factor in permeabilized murine
cells (47) and for induction of heat shock protein synthesis in rat hepatoma cells (48).
In addition specifically to inducing hsp70a of the hsp70 family, GA also cause a
pulse of [Ca2+]i mobilization change immediately.
[Ca2+]i alter metabolic systems.
It is known that change in
Herein, we examined the effects of several kinds of
drugs that affect the [Ca2+]i mobilization, including EGTA, BAPTA-AM, A23187,
and thapsigargin (TG), on GA-induced HSP70A expression in human non-small cell
lung cancer H460 cells as a source of insight into [Ca2+]i contributing to this process.
In this report, we characterized the the effect of GA treatment on hsp70a expression
and the relationship between GA-induced HSP70A and [Ca2+]i mobilization in H460
cells. We found that there are Ca2+-dependent and -independent processes involved
in the GA-induced HSP70A expression.
6
MATERIALS AND METHODS
Materials─Geldanamycin was purchased from Sigma (St. Louis, MO), dissolved
in dimethylsulfoxide at a concentration of 1 mM, and stored in the dark at –20 C.
All cultureware was purchased from Corning (Corning, NY) and culture medium
components were purchased from Gibco (Grand Island, NY).
[35S]methionine
(specific activity >800 Ci/mmole) and [-32P]dCTP (3,000 Ci/mmole) were purchased
from Amersham (Buckinghamshire, England).
Mouse monoclonal antibodies
against HSP70 (HSP72) were purchased from Stressgen (Victoria, BC, Canada).
Monoclonal antibodies against actin and AP-conjugated goat antibodies against
mouse IgG were purchased from Promega (Madison-Wisconsin, USA). Peroxidase
conjugate goat antibodies against mouse IgG were purchased from Sigma (Missouri,
USA). For visualization of the immunoblots, the AP detection system was purchased
from Bio-Rad (Richmond, CA) and chemiluminescence reagent was purchased from
PerkinElmer (Boston, USA).
DNAFax (Taipei, Taiwan).
(Richmond, CA).
Synthetic oligonucleotides were ordered from
Chemicals for electrophoresis were from Bio-Rad
BAPTA-AM (cell permeant) and Indo-1-acetoxymethylester
(Indo-1, AM) were purchased from Molecular Probes (Eugene, Oregon USA).
Thapsigargin and A23187 were purchased from Calbiochem (San Diego, CA). Other
chemicals were purchased from Sigma or Merk (Darmstadt, Germany).
Cells and Drug Treatment─The human non-small cell lung cancer (NSCLC) H460
cells from ATCC were maintained in RPMI-1640 minimum essential medium plus
7
10% fetal bovine serum supplemented with 100 units/ml penicillin G and 100 g/ml
streptomycin in a 37 C incubator under 5% CO2 and 95% air.
Prior to each
experiment, stock cells were plated in culture flasks or six-well plates at a density of
46 x 104 cells per cm2. Exponentially growing cells at 80-90% confluence were
used.
For the studies concerning the effects of the inhibitors, the cells were
respectively preincubated with the inhibitors for 15 min followed by treatment with
GA in the presence of the inhibitors.
Metabolic Labeling and Gel Electrophoresis─De novo protein synthesis was
revealed by [35S]methionine labeling at a concentration of 20 Ci/ml. At the end of
various treatments, the cells were labeled for 1 h before harvested. The cells were
washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, pH 7.4), and lysed in sample buffer (49). The cell lysates were resolved by
sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
After
electrophoresis, the gels were fixed, dried, and processed for autoradiography as
described previously (50).
Protein bands of interest were quantified by
densitometric scanning (Molecular Dynamics, Sunnyvale, CA).
Immunoblot Analysis—After electrophoresis, the proteins were electro-transferred
onto nitrocellulose membranes in TBE buffer (50 mM Tris, 50 mM boric acid, 1 mM
EDTA, pH 7.0) by using a semidry transfer apparatus according to the manufacturer’s
protocols (OWL Scientific, Woburn, MA). After being blocked with 5% nonfat milk
in TTBS (0.5% Tween-20, 20 mM Tris-HCl, pH 7.4, 0.5 M NaCl) for 2 h, the
membranes were incubated with a 1:2,000 dilution of mouse monoclonal Anti-HSP70.
8
Alternatively, monoclonal antibodies against actin were used as the primary antibody
at 1:2,000 dilutions, and then the membranes were washed three times with TTBS and
incubated with 1:5,000 dilutions of AP-conjugated or peroxidase conjugate
anti-mouse IgG antibodies for 4 h in a 4 C cold room. The AP detection system or
chemiluminescence reagents detected the immunocomplexes and bands of interest in
the immunoblots were quantified by densitometric scanning.
Intracellular Calcium Measurements─The cytoplasmic calcium was determined
according to the methods of Nuccitelli (51, 52).
The cell used for calcium tracing
were culture on coverslip in a 35 mm culture dish.
Before the experiments, the cells
were washed with cockroach saline (140 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM
MgCl2, buffered with 10 mM HEPES and balanced to pH 7.2 with NaOH) one times
and loaded with probe for calcium by placing in darkness, for 45 min at 25 oC, with 2
Mm indo-1-acetoxymethylester in normal cockroach saline containing 0.1%pluronic
acid, and 1% Fetal Bovine Serum. After loading, excess of the probe, was removed
from the cells by washing twice with normal cockroach saline. The cells were then
maintained in darkness at 25 oC, until calcium concentration was measured.
Changes in intracellular calcium concentration were monitored by single-cell
dual-wavelength microfluorymetry (PhoCal Pro, Life Science Resources, UK). The
indo-1 loaded cells were illuminated at an excitation wavelength of 340 nm.
The
fluorescent intensities at emission wavelength of 405 nm (Ca2+-binding form) and 490
nm (Ca2+-free form) were measured simultaneously by two photomultipliers, and
integrated in 100 ms intervals.
The concerntration of intracellular calcium was
estimated from the ratio R of the two emitted fluorescence according to the following
9
formula (53).
[Ca2+] = Kd [(R – Rmin)/(Rmax – R)] (Sf2/Sb2)
Where Kd; 250 nM, and (Sf2/Sb2); 2.01 after Grynkiewicz et al. (53), but Rmin and
Rmax, were determined in situ, according to Kao (54). Briefly, the Rmin is the limiting
value of the ratio R when the entire indicator is in the Ca2+-free form and the Rmax is
the limiting value of R when the indicator is saturated with calcium.
RNA Extraction and Northern Blotting─Total RNA was isolated from H460 cells
according to the method of Chomczynski and Sacchi (55) with minor modifications as
previously described (56). The same amount of RNA was fractionated on 1 %
agarose gels.
After electrophoresis, the gels were incubated in 0.05 N NaOH for 30
min and washed once with DEPC water (0.1%DEPC) and then were incubated with
20  standard saline citrate (SSC) buffer (1 SSC = 0.15 M NaCl, 0.015 M sodium
citrate, pH 7.0).
The RNA samples were then blotted onto nylon membranes
(Hybond-N, Amersham) in 20  SSC buffer for at least 12 h. Subsequently, the
membranes were dried and the RNA samples were fixed onto the membranes by using
an ultraviolet cross-linker (Stratagene, La Jolla, CA). The specific oligonucleotide
probes were from the cloning of the PCR products that hsp70a primer sets was
according to Wang (57) and hsp70b primer sets was purchased from Stressgen (Cat.#:
STM-507) and labeled with [-32P]dCTP by Rediprime DNA Labeling System
(Amersham).
Following prehybridization, hybridization and three times high
stringency washing step (0.1 SSC, 0.1% SDS, at 65 oC for 15 min), the membranes
were then dried and processed for autoradiography. Bands on autoradiograms were
quantified by densitometric scanning using rRNAs as the internal controls.
10
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RESULTS
Effects of GA on HSP70 Synthesis in H460 cells─To investigate the effect of GA
on HSP70 synthesis, H460 cells were treated with different concentration of GA and
the De novo protein synthesis was monitored by metabolic labeling with
[35S]methionine (Fig. 1). Western blotting verified the identity of HSP70 (Fig. 4B).
Samples containing an equal amount of radioactivity were processed for SDS-PAGE
and autoradiography, and synthesis of HSP70 was determined by densitometric
analysis as described.
The autoradiograms as shown in Fig. 1A indicate that GA did
not inhibit the general translational process in H460 cells and the synthesis rate of
several stress proteins including HSP90, HSC70 and HSP70 was enhanced. The
increase depended on the treatment concentration of GA and exposure to above 0.5
M GA produced maximal induction of HSP70 (Fig. 1).
Further, the induction
kinetic of HSP70 was analyzed. As shown in Fig. 2, the amount of HSP70 produced
by the cells was also dependent on the duration of GA treatment.
Under 0.5 M GA
treatment, HSP70 synthesis in H460 cells was enhanced and reached a maximum for
6 h treatment and the induction kinetic was different with heat shock 45 oC for 15 min
which reached a maximum for the recovery period at 37 oC 5 h (Fig. 2), suggesting
that the cellular mechanisms involved in HSP70 synthesis may be different between
GA treatment and heat shock. Taken together, the results indicated that GA induced
HSP70 production, which was dependent on both concentration and the duration of
GA treatment.
12
Effect of GA on HSP70A transcription and translation─We performed a series of
experiments to determine whether the promotion of HSP70 induction by GA
treatment occurred at the transcriptional or the translational level and to unequivocally
distinguish the inducible HSP70 genes that may differentially induced by GA in H460
cells. Northern blotting analyses were employed and the specific probes for hsp70a
and hsp70b mRNAs were generated as described under “Materials and Methods”.
At different times (0 – 24 h) after 0.5 M GA treatment, aliquots of cells were
collected from each culture, and total RNA was extracted to distinguish the levels of
hsp70a and hsp70b mRNAs. Although the mRNA levels of both hsp70a and hsp70b
were found to be enhanced in GA-treated H460 cells, the data indicated that hsp70a
was much more responsive compared to hsp70b (Fig. 3). Upon GA treatment, the
relative amount of hsp70a mRNA increased as the treatment time extended; reached a
maximum after 6 h, and then returned to baseline within 24 h.
This mRNA
accumulation time course correlated with the time course for protein synthesis.
In
another experiment, cells were treated with actinomycin D (a transcription inhibitor) 5
μg/ml or cycloheximide (a protein synthesis inhibitor) 25 μg/ml for 15 min before
GA treatment.
Fig. 4A shows that pretreatment with actinomycin D completely
prevented GA-induced HSP70 synthesis, and pretreatment with cycloheximide also
completely blocked the increase in HSP70 induced by GA (Fig. 4B).
This shows
that the elevated levels of HSP70 after GA treatment are result of new synthesis.
The requirement for transcription and the mRNA acummulation indicate that hsp70a
is transactivated in the presence of GA and its gene product (HSP70A), but not
HSP70B, is the major HSP70 induced in GA-treated H460 cells.
13
Effects of Ca2+ on the GA-induced HSP70─To elucidate the possible involvement
of [Ca2+]i mobilization in the induction of HSP70A under GA treatment, [Ca2+]i
mobilization in GA-treated H460 cells was monitored microspectrophotometrically
as described under “Materials and Methods”. H460 cells treated with 0.5 M GA in
normal cockroach saline at 25 oC produced a pulse of [Ca2+]i mobilization change
immediately, and then returned to slight high level, nearing basal level (Fig. 5).
In a
parallel experiment, analyses by using a set of drugs that affect the [Ca2+]i
mobilization, including EGTA (an external Ca2+ chelator), BAPTA-AM (a
intracellular Ca2+ chelator), A23187 (Ca2+ ionophore), and thapsigargin (TG; ER
Ca2+-ATPase inhibitor) were then performed to determine if the expression of hsp70a
could be attribute to different in Ca2+ mobilization after GA treatment.
As shown in
Fig. 6, cells were respectively pretreated with EGTA 1 mM, BAPTA-AM 15 M,
A23187 4 M, or TG 0.1 M for 15 min prior to exposure to GA, all of them could
attenuated different degrees of GA-induced HSP70A production in H460 cells and
treatment alone with each one of these drugs without GA treatment could not alter the
basal level of HSP70. Above these shows that GA course Ca2+ influx and Ca2+
mobilization influenced GA-induced HSP70A production. Besides, the result also
suggest that a change in [Ca2+]i without GA treatment does not promote HSP70
induction.
As expected, BAPTA-AM pretreatment inhibits GA-induced HSP70A
synthesis in time course analysis (Fig. 7). Similar results we also can find in the
level of hsp70a mRNA accumulation (Fig. 8) correlated with the protein production to
indicate that effect of Ca2+ mobilization should be an early event in the signal
transduction pathway mediating the activation of hsp70a gene expression by GA
treatment.
To evaluate the reaction time of GA to occur the signal that mediate the
14
activation of hsp70a gene expression, cells were incubated with various periods of GA
and then the culture medium was replaced with new one without GA treatment until 6
h. The result shows that HSP70 synthesis reached the maximal rate at the time when
GA incubation only 15 min (Fig. 9).
Besides, GA only need to be treated 1 min to
induce approximately 56% HSP70 synthesis that compare with the maximum
induction rates. Again, it emphasizes the important of the start period reaction by
GA. On the other hand, in order to determine whether the effect of Ca 2+ could be
affecting HSP70A translation activity, H460 cells were treated with or without
BAPTA-AM for 15 min after 4h GA treatment, and then incubated with actinomycin
D 1 h; finally pulse labeled with [35S]methionine for another 1 h to measure the rates
of protein synthesis at that time. The incorporation of [35S]methionine into HSP70
was not different between BAPTA-AM-treated and untreated cells (Fig. 10). These
suggesting that Ca2+ mobilization may only affect in hsp70a gene transcriptional level,
not translation.
15
DISCUSSION
This study demonstrates that the GA-induced the expression of a concentration- and
duration-dependent HSP70 in H460 cells is mainly through the transactivation of the
hsp70a, that the process is mediated by intracellular calcium mobilization. Hsp70a
and hsp70b are the only two genes coded for the “inducible forms” of the HSP70s
reported thus far; their gene products are respectively designated as HSP70A and
HSP70B (58, 59). Unfortunately, all studies concerning the regulation of these two
genes are always performed separately.
By different research groups, it has been
shown that both hsp70a and hsp70b are transactivated by heat shock (60, 61).
Additionally, hsp70a has been shown to be induced by postaglandins (62), serine
protease inhibitor (63), hemin (64), as well as heavy metal ions (65); while hsp70b
has been shown to be induced by non-steroidal anti-inflammatory drugs (NSAIDs)
(66).
Therefore, hsp70a and hsp70b, like most members of other stress gene
families, can be differentially regulated, apparently depends on the stress conditions
and/or cell types.
By using specific oligonucleotide probes, we have for the first
time successfully studied the regulation of these two genes simultaneously and our
result clearly indicated that hsp70a, but not hsp70b, is significantly transactivated by
GA in H460 cells.
It is also worth to note that another gene in the same family,
hsc70 that coded for HSC70 (the constitutive form/cognate of HSP70), was only
slightly induced by GA in H460 cells.
The cytoprotective effect of heat shock proteins has been related to their role in the
folding, assembly, and intracellular translocation of newly synthesized proteins;
16
refolding of partially denatured proteins; and ATP-dependent catalysis of protein
assembly/disassembly reactions (67). The regulation of heat shock gene expression
has attracted a great deal of interest because of its importance to the survival cells
under stressful condition.
The understanding of the mechanism by which
mammalian cells detect physiological stress at the molecular level and transduce the
stress signal to the transcriptional apparatus is an important goal in view of the
possibility of pharmacological manipulation of the stress response. The finding that
the mechanism of GA-induced HSP70 could represent a new way in the
comprehension of heat shock gene regulation.
It has been suggested that a
calcium-dependent metabolic process is involved in the generation of the heat shock
signal, and calcium has been shown to activate HSF DNA-binding ability (41, 46) and
to be essential for multistep activation of the heat shock factor, including HSF
phosphorylation (47, 68).
Increased Ca2+ mobilization has also been recently shown
to be an early event in the signal transduction pathway mediating the activation of
hsp70 gene expression by prostaglandin A2 (40), heat shock (41-44) and
ischemia/reperfusion (45).
In this report, we show that it cause a pulse of [Ca2+]i
mobilization change within 10 seconds after 0.5 M GA treatment in H460 cells, and
GA only need to be treated 1 min to induce approximately 56% HSP70 synthesis that
compare with the maximum induction rates. Besides, depletion of extracellular Ca2+
by treatment with the calcium chelator EGTA (1mM) prevented some effect of
GA-induced HSP70 synthesis.
These suggest that Ca2+ influx into H460 cells after
GA treatment course a signal transduction to affect HSP70 expression. Moreover,
pretreatment with BAPTA-AM, A23187, TG, respectively, after GA treatment, could
inhibit different degree of HSP70A expression in protein synthesis and mRNA
17
accumulation,
confirming
previous
observations
that
suggest
that
a
calcium-dependent metabolic process is involved in the regulation of the GA-induced
signal to affect HSP70 synthesis, as well as in heat shock condition that has been
reported (40, 46-48, 69).
And they seem only to effect on transcriptional level, not
translation.
Actually, it has been reported that A23187 and BAPTA-AM could affect HSF
phosphorylation and DNA-binding activity, respectively (46, 70). Besides, A23187
and TG elicit a transcriptional response though a rapid and transient increase of
intracellular Ca2+, with the implication of calmodulin kinase in the phosphorylation of
transcription factors (71, 72). They activation of c-fos transcription was shown to be
dependent on a cAMP response element adjacent to the TATA element in PC12 cells
(73).
The mechanism of this induction involves the phosphorylation of the
transcription factor cAMP response element-binding protein by a calmodulin kinase
(73). On the other hand, a different mechanism for induction of GRP genes by
A23187 and TG have been shown, which involves endoplasmic reticulum Ca2+
discharge and a novel pathway in which a Ca2+ signal is transduced through redundant
elements containing CCAAT box-like motifs flanked by GC-rich regions, without the
involvement of cAMP response element-like elements (74). A different well known
effect of A23187 is its ability to cause the release of eicosanoids, which are converted
to cyclooxygenase or lipoxygenase products and may consequently stimulate the
adenylate cyclase, giving rise to a mixed Ca2+/cAMP signal (75, 76).
The fact that
arachidonic acid (77) and cyclopentenone prostaglandins (78, 62) are known to induce
HSF activation, while cyclooxygenase and lipoxygenase inhibitors can modulate the
18
heat shock response in human cells (79-85), suggests the interesting possibility that an
eicosanoid-mediated signal could be involved in A23187 inhibitory activity. There
may be many different regulation pathways by Ca2+ mobilization to directly or
indirectly involved in the enhanced expression of HSPs in human cells. And these
different effect of Ca2+ mobilization on between HSF DNA-binding ability and
activity have not understood very clearly yet.
HSF may serve as a substrate for a
number of protein kinases in different regulation systems.
Although, in our
preliminary studies, pretreatment with BAPTA-AM, A23187, TG, respectively, after
GA treatment, all could inhibit different degrees of HSP70A expression, they may
through different regulation pathway to reach the same result in mRNA accumulation
and protein synthesis.
It is still investigated deeply.
But these all indicate the effect
of Ca2+ mobilization play an important role in GA-induced HSP70A expression.
In summary, GA induced a concentration- dependent HSP70 that was related to the
duration of GA treatment is mainly through the transactivation of the hsp70a. The
increase resulted from a GA-induced stimulation of transcription through calcium
sensitive and non-sensitive regulation pathway.
Acknowledgment─We thank Dr. Margaret Dah-Tsyr Chang, Whei-meih Chang,
and Chen-Yi Su for their assistance.
19
FOOTNOTES
1
The abbreviations used are:
GA, geldanamycin; HSP90, 90-kDa heat-shock protein;
HSF, heat shock factor; HSP70, 70-kDa heat-shock protein; HSPs, heat shock
proteins; GRPs, glucose-regulated proteins; HSE, heat shock element; AP, alkaline
phosphatase; NSCLC 460 cells, human non-small cell lung cancer H460 cells; SDS,
sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis
20
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FIGURE LEGENDS
Fig. 1. Effect of GA concentration on HSP70 synthesis in H460 cells.
A, H460
cells were pulse labeled with [35S]methionine for 1 h after treated with GA at
different concentrations as indicated for 5 h to measure the rates of protein
synthesis.
Equal amounts of cell lysates were processed for SDS-PAGE
and the de novo synthesized proteins were visualized by autoradiography.
The HSP90, HSC70, HSP70, and actin are indicated.
B, bands of HSP70
and actin in the autoradiograms as shown in A were quantified by
densitometric scanning, and the relative synthesis rate of HSP70 was
presented as sum of the pixel values of each band divided by that of actin in
the same lane (internal control).
Fig. 2. Time course effect of GA on HSP70 synthesis compare with heat shock
in H460 cells.
H460 cells were treated with 0.5 M GA for various
durations or heated (45 oC, 15 min) at different times during the recovery
period at 37 oC as indicated. The [35S]methionine incorporated synthesis of
HSP70 was quantitated densitometrically and the relative synthesis rate was
determined as described.
Fig. 3. Time course of hsp70a and hsp70b mRNAs accumulation in GA-treated
H460 cells.
A, H460 cells were treated with 0.5 M GA for various
durations as indicated.
After treatment, total RNA was extracted and
analyzed to distinguish the expression of hsp70 mRNAs by Northern blotting
28
with
32
P-labeled hsp70a and hsp70b probes as indicated. The specific of
these probes were tested in the left side.
B, the relative levels of hsp70
mRNAs accumulation were presented as the sum of the pixel values, using
18S rRNAs as the internal controls.
Fig. 4. Inhibition of HSP70 by actinomycin D and cycloheximide.
A, H460
cells were treated with nothing (lanes 1) or 0.5 M GA only (lanes 2) as
control; cells were treated with actinomycin D (5 μg/ml; lanes 3) or
cycloheximide (25μg/ml; lanes 4) 15 min before 0.5 M GA treatment.
B,
Western blotting verified the identity of HSP70.
Fig. 5. Effect of GA on changes in cytosolic calcium concentration in H460 cells.
Cells grown on coverslips were loaded with Indo-1-AM for 45 min at 25 oC
and then stimulated with 0.5 M GA at time 0.
calcium
concentration
microphotospectrometry.
were
determined
Changes in cytosolic
by
fluorescence
The horizontal bars indicate period of GA
superfusion.
Fig. 6. Effect of Ca2+ mobilization on HSP70 production in GA-treated H460
cells.
H460 cells were respectively pretreated with BAPTA-AM 15 M,
EGTA 1 mM, CaCl2 5 mM, A23187 4 M, or thapsigargin (TG) 0.1 M for
15 min prior to exposure to GA 0.5 M.
A, cells were pulse labeled with
[35S]methionine for 1 h after treatments to measure the rates of protein
29
synthesis.
B, Western blotting verified the identity of HSP70 to evaluate
this protein’s accumulation.
C, shown is the quantitative evaluation by
PhosphorImager analysis of the samples in B.
Fig. 7. BAPTA-AM pretreatment inhibits GA-induced HSP70 synthesis in time
course analysis.
A, H460 cells were pretreated with BAPTA-AM 15 M
for 15 min before 0.5 M GA treatment for various durations as indicated.
The [35S]methionine incorporated synthesis of HSP70 was visualized by
autoradiography.
B,
shown
is
the
quantitative
evaluation
by
PhosphorImager analysis of the samples in A.
Fig. 8. Effect of Ca2+ mobilization on hsp70a mRNA accumulation in
GA-treated H460 cells.
H460 cells were respectively pretreated with
BAPTA-AM 15 M, A23187 4 M, or thapsigargin (TG) 0.1 M for 15 min
prior to exposure to GA 0.5 M for various durations as indicated.
After
treatment, total RNA was extracted soon and processed for Northern blot
analysis using
32
P-labeled hsp70a probes as indicated and rRNAs as the
internal controls.
Fig. 9. The reaction time of GA to occur the signal that mediate the activation of
hsp70a gene expression.
H460 cells were incubated with various periods
of GA as indicated in the horizontal bars and then the culture medium was
replaced with new one without GA treatment until 6 h. The [35S]methionine
incorporated synthesis of HSP70 were quantified by densitometric scanning,
30
and the relative synthesis rate was presented, using actin as internal control.
Fig.10. Determine whether the effect of Ca2+ could be affecting HSP70A
translation activity.
H460 cells were treated with or without BAPTA-AM
after 4h GA (0.5 M) then 1 h actinomycin D (5 μg/ml) treatment, and then
pulse labeled with [35S]methionine for another 1 h to measure the rates of
protein synthesis at that time.
indicated.
The HSP90, HSC70, HSP70, and actin are