The exact mechanism of HSF1 hyperphosphorylation is cur-rently unknown, and the regulation of the mamma-lian heat shock response appears to be more complex Keywords local anesthetics; mo
Trang 1as a signal to initiate the heat shock protein response
Ga´bor Balogh1, Ibolya Horva´th1, Eniko˜ Nagy1, Zso´fia Hoyk2, Sa´ndor Benko˜3, Olivier Bensaude4 and La´szlo´ Vı´gh1
1 Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
2 Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
3 Outpatient Medical Centre, Municipality of Szeged, Hungary
4 De´partement de Ge´ne´tique Mole´culaire, Ecole Normale Supe´rieure, Paris, France
Cellular stress response is a universal mechanism of
extraordinary pathophysiological and pharmacological
significance [1] Dysregulation of the stress protein
expression is known to play a determining role in the
pathology of different human diseases and aging [2]
Identification of the primary sensors that perceive
var-ious stress stimuli and of the transducers that carry,
amplify and integrate the signals culminating in the
expression of a particular heat shock protein (HSP) is
therefore of key importance [3,4]
HSP expression in mammalian cells is primarily regulated at the level of transcription and, although not exclusively, is mainly mediated by heat shock fac-tors (HSF), especially HSF1 [5] The conversion of HSFs to their active, DNA-binding form involves oligomerization to a trimeric state and reversible hyperphosphorylation at multiple sites [6] The exact mechanism of HSF1 hyperphosphorylation is cur-rently unknown, and the regulation of the mamma-lian heat shock response appears to be more complex
Keywords
local anesthetics; molecular chaperones;
membrane fluidity; membrane
microdomains; stress proteins
Correspondence
L Vı´gh, Institute of Biochemistry, Biological
Research Centre, Hungarian Academy of
Sciences, Szeged, POB 521, H-6701,
Hungary
Tel ⁄ Fax: +36 62 432048
E-mail: vigh@brc.hu
(Received 18 July 2005, revised 27
September 2005, accepted 3 October 2005)
doi:10.1111/j.1742-4658.2005.04999.x
The concentrations of two structurally distinct membrane fluidizers, the local anesthetic benzyl alcohol (BA) and heptanol (HE), were used at con-centrations so that their addition to K562 cells caused identical increases in the level of plasma membrane fluidity as tested by 1,6-diphenyl-1,3,5-hexa-triene (DPH) anisotropy The level of membrane fluidization induced by the chemical agents on isolated membranes at such concentrations corres-ponded to the membrane fluidity increase seen during a thermal shift up to
42C The formation of isofluid membrane states in response to the administration of BA or HE resulted in almost identical downshifts in the temperature thresholds of the heat shock response, accompanied by increa-ses in the expression of genes for stress proteins such as heat shock protein (HSP)-70 at the physiological temperature Similarly to thermal stress, the exposure of the cells to these membrane fluidizers elicited nearly identical increases of cytosolic Ca2+ concentration in both Ca2+-containing and
Ca2+-free media and also closely similar extents of increase in mitochond-rial hyperpolarization We obtained no evidence that the activation of heat shock protein expression by membrane fluidizers is induced by a protein-unfolding signal We suggest, that the increase of fluidity in specific mem-brane domains, together with subsequent alterations in key cellular events are converted into signal(s) leading to activation of heat shock genes
Abbreviations
BA, benzyl alcohol; DPH, 1,6-diphenyl-1,3,5-hexatriene; ERK, extracellular signal-regulated kinase; HE, heptanol; HSF, heat shock factor; HSP, heat shock protein; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; DW m , mitochondrial membrane potential.
Trang 2than previously thought [7] The existence of
interac-tions between stress-activated signaling pathways and
HSPs is well established [8] The overall interplay of
different stress-sensitive signaling pathways ultimately
determines the magnitude of the transcriptional
activ-ity of HSF1 [2,8,9]
Hitherto, most of the published studies have focused
predominantly on the cellular responses to severe heat
stress, which causes the unfolding of pre-existing
pro-teins and the misfolding of nascent polypeptides [6] It
is suggested therefore that the denaturation of a
pro-portion of the cellular proteins during severe heat
serves as the primary heat-sensing machinery which
triggers the up-regulation of the HSP gene expression
Because mild heat stress is not coupled with the
exten-ded unfolding of cellular proteins, it may be assumed
that it is sensed by a different mechanism [10] A
num-ber of data support the notion that, indeed, instead of
proteotoxicity, a change in the fluidity of membranes
may be the first event that signals a change in
tempera-ture and may, thus, be regarded as a thermosensor
under such conditions [3,4,11–13] By affecting the
membrane microdomain structure and mobility,
fever-range hyperthermia may result in the activation of
membrane proteins, e.g multiple growth factor
recep-tors [10] Following such a typical scenario, the
activa-tion of growth factor receptors may in turn activate
the Ras⁄ Rac1 pathway, which has been shown to play
a critical role in HSF1 activation and HSP
up-regula-tion [14]
We have reported that specific alterations in the
membrane physical state for prokaryotes and yeasts,
can act as an additional stress sensor [11–13] We
assumed that membrane-controlled signaling events
might exist temporarily if the adjustment of the
mem-brane hyperstructure is completed subsequent to stress
[3,4] Here, we furnish the first evidence that
chemic-ally induced membrane perturbations of K562
ery-throleukemic cells, analogously with heat-induced
plasma membrane fluidization, are indeed capable of
activating HSP formation even at the growth
tempera-ture, without causing measurable protein denaturation
We also demonstrate that, just as in response to heat
treatment, there are immediate increases in
intracellu-lar free Ca2+ level and mitochondrial membrane
potential, DYm, following the administration of
mem-brane fluidizers Hence, it is highly conceivable that
changes in the fluidity of the plasma membrane, which
is affected considerably by environmental stress, are
well suited for cells to sense stress In a wider sense,
even subtle alterations or defects of the lipid phase of
membranes (known to be present during aging or
under pathophysiological conditions) should influence
membrane-initiated signaling processes, leading to a dysregulated stress response
Results
Selection of the critical concentrations of membrane perturbers equipotent in fluidization with temperature upshifts
We proposed that the lipid phase of membranes plays a central role in the cellular responses that occur during acute heat stress and pathological states [3,4,11–13] A direct correlation between the membrane fluidization of the lipid region and the HSP response, however, has not been unambiguously established for mammalian cells By intercalating between membrane lipids the two structurally unrelated membrane fluidizers that we selected benzyl alcohol (BA) and heptanol (HE), we induced a disordering effect by weakening the van der Vaals interactions between the lipid acyl chains [13] As
in the case of heat stress, the initial fluidity increases induced by these membrane perturbants in vivo are fol-lowed by a rapid relaxation period (G Balogh et al., unpublished results) Thus, for a correct assessment and comparison of the levels of the thermally and chemically induced primary changes in the membrane physical orders, we used isolated membranes As shown
by Fig 1A, the plasma membrane fraction of K562 cells was labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) and the steady-state fluorescence anisotropy [11–13] was monitored as a function of temperature Simultaneously, the fluidity changes were recorded at the different concentrations of the two alcohols (Fig 1B,C) In this way it was possible to determine the critical concentrations of each of the two fluidizers
at which their addition to membrane preparations caused increases in the level of membrane fluidity iden-tical to that found after a temperature change to 42 C
As highlighted by the arrows in Fig 1A–C, plasma membrane hyperfluidization resulting from heat treat-ment at 42C (i.e a reduction of the steady-state DPH anisotropy value by 0.015 units) can be attained by the administration of 30 mm BA or 4.5 mm HE The critical concentrations of the membrane perturbers proved to be essentially equipotent in causing mem-brane hyperfluidization in vivo (Fig 2) The decrease in the lipid order was followed in the membrane interior
of the K562 cells by monitoring the DPH anisotropy change The fluidizing effects of the alcohols in the gly-cerol and upper acyl regions were also determined
by means of the charged, not membrane permeable derivative of DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH)
Trang 3Membrane fluidizers lower the set-point
temperature of HSP-70 synthesis
K562 cells were treated at different temperatures in the
presence or absence of different concentrations of BA
or HE for 60 min Following a 3-h recovery period at
37C, the cells were then labeled with a 14C amino
acid mixture for an additional 60 min to follow the level of the de novo synthesized HSP-70 Co-treatment
of the cells with BA or HE during heat stress resulted
in a dose and temperature-dependent synthesis of HSP-70 (Fig 3) Obviously, gradual rising of the tem-perature shifted the peak heat stress response towards the lower alcohol concentration range, indicating a
Fig 2 Membrane fluidity measurements in vivo K562 cells were labeled with 0.2 l M DPH (¤) or TMA-DPH (h) for 40 or 5 min, respect-ively, and then further incubated with different concentrations of BA or HE The fluorescence steady-state anisotropy was measured and the differences from the controls were calculated The arrows indicate the concentrations of the alcohols at which similar levels of HSP-70 syn-thesis were detected at 37 C Mean ± SD, n ¼ 6.
Fig 3 HSP-70 induction in K562 cells treated with BA or HE and subjected to heat stress Cells were treated with various concentrations of
BA or HE for 1 h at different temperatures After a 3 h recovery period, the cells were labeled for 1 h with14C protein hydrolysate and, after SDS ⁄ PAGE, prepared for fluorography The HSP-70 lane of the fluorograph is presented The arrows indicate the most effective concentra-tions of the alcohols at 37 C.
Fig 1 Heat stress- or membrane fluidizer-induced changes in isolated plasma membrane fluidity, tested with DPH Isolated plasma mem-branes were labeled with DPH and (A) the effects of heat or (B) different concentrations of BA or HE on the steady-state fluorescence anisotropy were measured The arrows indicate the concentrations of the alcohols that exert a fluidizing effect equivalent to that caused by exposure to 42 C Mean ± SD, n ¼ 4.
Trang 4cooperative triggering mechanism in the induction of
HSP-70 synthesis The maximum responses at 37C
were obtained by the administration of 30 mm BA or
4.5 mm HE, these critical concentrations of the
fluidiz-ers being exactly those that caused identical levels of
in vitro and in vivo plasma membrane fluidization
(Figs 1 and 2) In other words, elevation of the plasma
membrane fluidity as a consequence either of heat
exposure or of chemical membrane perturbations is
equally followed by the activation of HSP formation
The higher doses of BA or HE in synergy with heat
stress caused a complete inhibition of protein
synthe-sis Thus, at 42C the highest tolerable concentrations
of BA and HE were 10 and 2 mm, respectively
Effects of heat and membrane fluidizers on the
cellular morphology and the cytosolic free Ca2+
level
Heat stress is known to produce distinct morphological
changes in mammalian cells [15] Using electron
micro-scopy, a moderate level of membrane blebbing was
also detected in the present study when K562 cells
were heat shocked at 42C or incubated with 30 mm
BA or 4.5 mm HE for 1 h However, no major
altera-tions in cell ultrastructure were observed following
these treatments (data not shown)
The intracellular calcium [Ca2+]i concentration,
which is tightly regulated, is known to be a key
signa-ling element of the heat shock response in mammalian
cells Whereas the synthesis of HSP-70 has been
dem-onstrated to be promoted by an increase in [Ca2+]i,
the overexpression of HSP-70 attenuates increases in
[Ca2+]i [16,17] It was earlier documented that
mem-brane fluidizer anesthetics may displace Ca2+ from
internal and external binding sites and alter the
func-tioning of different Ca2+regulatory systems [18,19]
Therefore, we monitored any dose-dependent
increa-ses in cytosolic [Ca2+]i following treatment with the
membrane fluidizer alcohols and to compare the
find-ings with the [Ca2+]iincrease resulting from heat shock
By continuous monitoring of Fura-2 fluorescence when
the cells were treated with these alcohols at
concentra-tions equipotent in membrane fluidization and in the
induction of HSP-70, it was found that BA and HE
enhanced the level of [Ca2+]i in a closely similar and
strictly dose-dependent fashion (Fig 4A) [Ca2+]i rose
to its plateau level within 30 s (from 185 nm to 290
nm and 305 nm) To compare the effects of heat with
these alcohols on the free cytosolic Ca2+levels, the cells
were heated at 42C for 5 min The averaged [Ca2+]i
value obtained is displayed by the bar in Fig 4A
Obvi-ously, the heat stress at 42C caused a similar elevation
of [Ca2+]i (from 185 nm to 296.5 ± 16.5 nm) to that produced by the corresponding alcohol doses at which equal HSP-70 synthesis was documented
In order to estimate the contribution of intracellular
Ca2+-mobilizing compound, cells were suspended in a buffer without Ca2+, but containing the Ca2+chelator EGTA Whereas the absolute values dropped to about one-third, the pattern of [Ca2+]iobtained by treatment with heat stress and the membrane fluidizer alcohols was not affected by the depletion of external Ca2+ (Fig 4B)
The effects of heat stress and membrane fluidizers on DWm
Together with several other stimuli, via the activation
of phospholipase A2or by other mechanisms, an intra-cellular free Ca2+ overload is known to elicit struc-tural and functional changes in the mitochondria These include swelling, the disruption of electron transport, and the opening of mitochondrial membrane
Fig 4 Intracellular free Ca 2+ concentration increase induced by heat or membrane fluidizers [Ca 2+ ]i was measured at 37 C by using fura-2 ⁄ AM (A) Time course of [Ca 2+
] i rise induced in 1.2 m M CaCl2-containing buffer by BA or HE or treatment at 42 C (B) [Ca 2+ ]iconcentrations in Ca 2+ -free buffer containing EGTA, meas-ured in samples treated with alcohol or heat for 5 min Mean ± SD,
*P < 0.05 compared with control, n ¼ 4.
Trang 5permeability transition pores [20] Recent studies
pro-vided evidence that the change in DYmduring cellular
insults exhibits a biphasic profile and is not associated
exclusively with apoptosis Instead, acting as one of
the major checkpoints of cell death pathway selection,
mitochondrial hyperpolarization may represent an early and reversible switch in cellular signaling [21,22]
In line with the above reasoning, we addressed the question of whether the strikingly similar changes in [Ca2+]i seen following membrane hyperfluidization induced either by mild heat or by equipotent mem-brane fluidizers are paralleled by similar tendencies
in changes in DYm A two-dimensional display of 5,59,6,69-tetrachloro-1,19,3,39-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) red fluorescence vs green fluorescence illustrates the changes in DYm that occur following membrane manipulations (Fig 5A) A higher intensity of red fluorescence is supposed to indicate
a higher DYm (hyperpolarization) Cells treated with carbonyl cyanide p-chlorophenylhydrazone (CCCP) served as methodological control for mitochondrial depolarization Figure 5B depicts histograms in which
DYm(detected via the J-aggregate fluorescence) is plot-ted against the number of cells As for heat stress at
42C and BA at 30 mm, two treatments at which equal extent of membrane hyperfluidization are cou-pled with identical degrees induction of HSP-70 syn-thesis, we observed a noteworthy uniform increase in
DYm The quantification of DYm in arbitrary units in response to gradually increasing heat and increasing concentrations of the membrane fluidizers is displayed
on Fig 6 Both heat treatment and membrane hyper-fluidization with these alcohols led to the closely sim-ilar extent of mitochondrial hyperpolarization
The chemical membrane fluidizers do not exert a measurable effect on protein denaturation Firefly luciferase can be inactivated by heat shock when it is expressed in mammalian cells The loss of enzymatic activity correlates with the loss of its
Fig 5 Flow cytometric analysis of mitochondrial membrane
poten-tial of K562 cells after heat treatment, or incubation with BA or
CCCP Cells were left untreated or treated with BA, heat or CCCP
for 1 h as indicated Cells were then stained with JC-1 and assayed
by flow cytometry (A) Dot plots of JC-1 red fluorescence vs green
fluorescence (B) corresponding histograms, in which the
J-aggre-gate fluorescence is plotted against the number of cells.
Fig 6 Quantification of the DWm changes caused by gradually increasing heat stress or increasing concentrations of membrane fluidizers Cell were treated with BA, HE or subjected to heat stress for 1 h as indicated The samples were analyzed as in Fig 5 The mean fluorescence intensity of J-aggregates was used to determine the DW m Mean ± S.D, *P < 0.05 compared with con-trol, n ¼ 4.
Trang 6bility and can be taken as direct evidence of protein
denaturation This method served as a sensitive tool
with which to test the proteotoxicity of HSP-inducing
compounds [23] In the present study, we used HeLa
cells expressing cytoplasmic firefly luciferase The
pres-ence of either 30 mm BA or 4.5 mm HE did not exert
a significant effect on luciferase activity when the cells
were tested at their growth temperature In contrast,
loss of enzyme activity was detected in cells exposed to
42C (Fig 7) The same tendency was observed in an
in vitro protein denaturation assay, using lysates of
K562 cells (data not shown)
Discussion
Whereas the importance of HSPs in the pathogenesis of
many diseases is well established together with their
potential therapeutic value, our knowledge of the stress
sensing and signaling that lead eventually to an altered
HSP expression is still very limited [1] The early finding
that most of the stressors and agents with the ability to
induce HSPs appeared to be proteotoxic gave rise to the
suggestion that protein denaturation may be the sole
initiating signal for the activation of HSP genes [24]
In the course of the present study, we treated K562
cells with BA or HE at concentrations that induce a
heat shock response at the normal growth temperature,
as highlighted by monitoring of the synthesis of the
major HSP, HSP-70 The critical concentrations of
each of the two fluidizers were selected so that their
addition to the cells caused identical increases in the
plasma membrane fluidity level, corresponding to the
fall in membrane microviscosity induced by heat stress-ing at 42C We have demonstrated that, irrespective
of the origin of the membrane perturbations, the formation of isofluid membrane states is accompanied
by an essentially identical heat shock response in K562 cells Heat shock at 42 C or the administration of
30 mm BA or 4.5 mm HE, structurally distant com-pounds, proved equally effective in the up-regulation
of HSP-70 formation
At the cellular level, Ca2+ is derived from external and internal sources We assume that the mechanism
by which heat stress and these alcohols alter the Ca2+ homeostasis in the present study basically results from their action on Na+⁄ Ca2+ exchangers and subsequent
Ca2+ mobilization from different intracellular Ca2+ pools [17] Lipid rearrangement induced changes in membrane permeability, and the activity of mechano-sensitive ion channels during stress may also promote
Ca2+ influx into the cytosol [18] In parallel with the induction of HSP synthesis, heat stress and the admin-istration of these membrane fluidizers elicited nearly identical elevations of the cytosolic Ca2+ concentra-tion, in both Ca2+-containing and Ca2+-free media It
is suggested that the increase in intracellular free Ca2+ level that occurs during the cellular responses to heat shock, serum or growth factors is due to the release of the Ca2+-regulatory compound inositol 1,4,5-triphos-phate and coupled to the activation of phospho-inositide-specific phospholipase C (PLC) [25] The costimulation of phospholipases such as PLC and PLA2 by heat shock and the resultant release of lipid mediators could also enhance the subsequent mem-brane association and activation of protein kinase C (PKC), found to drive the phosphorylation of HSFs [18,23] In separate studies, an intracellular Ca2+ level elevation was shown to stimulate HSF1 translocation into the nucleus, resulting in HSP-70 expression [26], and proved to be essential for the multistep activation
of HSFs [27] Similar to our findings, an immediate change in intracellular free Ca2+ level and an in vivo change in membrane lipid order following treatment with the calcium ionophore ionomycin have been repor-ted, in parallel with the activation of stress-activated protein kinase, an enhanced HSF (heat shock element) interaction and the increased synthesis of HSP-70 [28]
Ca2+ can be released from internal Ca2+ stores, through channels in the endoplasmic reticulum Spatio-temporal studies are in progress in our laboratory to elu-cidate the role and contribution of intracellular Ca2+ reservoirs (i.e endoplasmic reticulum and mitochon-dria) to the cytosolic rise of this ion observed upon heat shock and administration of different membrane fluidizers
Fig 7 In vivo protein denaturation assay The effects of heat or BA
or HE treatment on protein denaturation were monitored by
meas-urement of the activity of cytosolic luciferase expressed in HeLa
cells Cells were treated with 30 m M BA (¤), 4.5 m M HE (n) or
submitted to heat-shock at 42 C (n) At different time points cells
were lysed and analyzed for luciferase activity Enzyme activity of
control cells was taken as 100% Mean ± SD, n ¼ 3.
Trang 7Both heat treatment and membrane
hyperfluidiza-tion with the simultaneous induchyperfluidiza-tion of the synthesis
of HSPs were parallel by closely similar extent of
mitochondrial hyperpolarization While representing
early and reversible steps in apoptosis [21,22], the
documented change in DWm which peaked at the dose
(or concentration) of the stressors that elicited the
maximum HSP response may be assumed with high
probability to serve as a key event in the stress
signa-ling of K562 cells Mitochondrial hyperpolarization
can develop in several ways, including the Ca2+
-over-load activated dephosphorylation of cytochrome c
oxidase, and is a likely cause of subsequent reactive
oxygen species production [21] The composition of
reactive oxygen intermediates and their
compartmen-talization during activation of the stress response by
heat or membrane perturbants await further studies
As an indication of their delicate and hitherto
unex-plored interrelationship, disruption of HSF1, while
resulting in a reduced HSP expression also increased
DWm in renal cells [29] On the other hand, the
over-production of HSP-70 by heat shock prevented the
H2O2-induced decline in mitochondrial permeability
transition and the swelling of the mitochondria [29]
Previous studies on the regulation of the heat shock
response in different prokaryotic model organisms
revealed that the threshold temperature of activation
of the major heat shock genes is significantly lowered
by BA treatment [12,13] Whereas BA stress activated
the entire set of heat shock genes when the solubility
of the most aggregation-prone protein homoserine
trans-succinylase was tested, it failed to cause in vivo
protein denaturation in Escherichia coli cells [13] The
overexpression of a desaturase gene in Saccharomyces
cerevisiae, or the addition of exogenous fatty acids,
can change the unsaturated⁄ saturated fatty acid ratio
and exert a significant effect on the expression of heat
shock genes [11] The HSP co-inducer bimoclomol and
its derivatives, just like other chaperone inducers and
coinducers, appear to be nonproteotoxic [20,30–32] It
has been suggested that bimoclomol and related
com-pounds selectively interact with acidic membrane
lipids, modifying those membrane domains where the
thermally or chemically induced perturbation of the
lipid phase is sensed and transduced into a cellular
sig-nal, leading to the enhanced activation of heat shock
genes [20] In the present study, we tested the possible
effects of BA and HE on protein stability at
non-heat-shock temperatures via the heat-induced inactivation
of heterologously expressed cytoplasmic firefly
lucif-erase in HeLa cells Neither of the fluidizers exerted
measurable effect on protein denaturation Taken
together, the above findings lend further support to
the view that, besides the formation of denatured pro-teins, alterations in the lipid phase of cell membranes, alone or together with consequent elevation of the intracellular cytosolic Ca2+level and DYm, may parti-cipate in the sensing and transduction of environmen-tal stress into a cellular signal
It has been demonstrated that shear stress-induced fluidity changes in endothelial cells are sufficient to initi-ate signal transduction [33], i.e changes in lipid dynam-ics in the plasma membrane can serve as a link between mechanical force and chemical signaling In fact, BA has been shown to mimic the effect of step-shear stress
by increasing ERK and JNK activities In contrast, the experimental reduction of the membrane fluidity by cho-lesterol administration resulted in the opposite effect Cell activation by shear stress is hypothesized to occur via the lipid modification of integral and peripheral membrane proteins, or signaling complexes organized in cholesterol-rich microdomains (rafts, focal adhesions, caveoli, etc., see [34]) The phospholipid bilayer is able
to mediate the shear stress-induced activation of mem-brane-bound G proteins, even in the absence of G-pro-tein receptors, similarly by changing the composition and physical properties of the lipid phase [35]
The mechanisms highlighted above conceivably also operate in the present case The heat-induced activation
of kinases such as Akt has been shown to increase HSF1 activity Enhanced Ras maturation by heat stress was associated with a heightened activation of extra-cellular signal-regulated kinase (ERK), a key mediator
of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation [36] Given the importance of the plasma membrane in link-ing growth factor receptor activation to the signallink-ing cascade, it is likely that any alteration in surface mem-brane fluidity could greatly influence ERK activation
In fact, ERK activation in aged hepatocytes is reduced
in response to either proliferative stimuli or stressful treatments [37] The level of membrane-associated PKC
is also reduced in elderly, hypertensive subjects [38] It
is proposed that this effect is strictly controlled by age-related alterations in fluidity and the polymorphic phase state of the membranes [38] Thus, strategies aimed at altering the physical state of the membranes can be used to enhance stress responsiveness in aged cells or in disease conditions such as diabetes, where reduced HSP levels are causally linked to stiffer, less fluid membranes as a result of glycation, oxidative stress or an insulin deficiency [39]
Finally, heat and other types of stress are associated not only with changes in the tension, fluidity, permeab-ility or surface charges of membranes, and in lipid and protein rearrangements, but are also coupled with the
Trang 8formation of lipid peroxides and lipid adducts [40] It
may be noted that 4-hydroxynonenal a highly reactive
end-product of lipid peroxidation, is an inducer of
HSPs and has been suggested to play an important
role in the initial phase of stress-mediated signaling in
K562 cells [41]
In conclusion, our results strongly indicate that the
membranes of mammalian cells play a critical role in
thermal sensing as well as signaling The exact
mech-anism of the perception of membrane stress imposed
on K562 cells by BA and HE, coupled with the
activa-tion of HSP expression, awaits further studies We
propose that, rather than the overall changes in the
physical state of membranes, the appearance of specific
microdomains [34] with an abnormal hyperfluid state,
locally formed nonbilayer structures [38] or changes in
the compositions of particular lipid molecular species
involved directly in lipid–protein interactions [3,4], are
potentially equally able to furnish a stimuli for the
activation of heat shock genes [42] Identification, by
single molecule microscopy [43], of the critical local
membrane microdomains that may act as primary
thermosensors during heat stress is in progress in our
laboratory
Experimental procedures
Cell culture
K562 cells were cultured in RPMI-1640 medium
supple-mented with 10% fetal calf serum and 2 mm glutamine in
a humidified 5% CO2, 95% air atmosphere at 37C and
routinely subcultured three times a week
Membrane fluidity measurements
The plasma membrane fraction of K562 cells was isolated
according to Maeda et al [44] Isolated plasma membranes
were labeled in 10 mm Tris, 10 mm NaCl (pH 7.5) with
0.2 lm DPH at a molar ratio of 1 : 200
probe–phospho-lipid for 10 min, and steady-state fluorescence anisotropy
was measured as in [45] When the temperature dependence
of fluidity was followed, the temperature was gradually
(0.4CÆmin)1) increased and the anisotropy data were
col-lected every 30 s
DPH-labeled membranes were incubated with different
concentrations of BA or HE for 5 min at 37C, and DPH
anisotropy was measured at 37C
For in vivo fluidity measurements, K562 cells were
labe-led with 0.2 lm DPH or TMA-DPH, for 40 min or 5 min,
respectively, and incubated further with BA (0–50 mm) or
HE (0–6 mm) for an additional 5 min Steady-state
fluores-cence anisotropy was determined as in [45]
In vivo protein labeling Cells (1 mL of 106
ÆmL)1) were treated with different con-centrations of BA or HE for 1 h at various temperatures,
as indicated in Fig 3 The cells were then washed and fur-ther incubated in complete medium for 3 h at 37C The medium was next replaced with 1 mL buffer A (1.2 mm CaCl2, 2.7 mm KCl, 1.5 mm KH2PO4, 0.5 mm MgCl2,
136 mm NaCl, 6.5 mm Na2HPO4, 5 mm d-glucose) contain-ing 10 lL 14C protein hydrolysate (Amersham CFB25, radioactive concentration 50 lCiÆmL)1) and the cells were incubated for 1 h at 37C Following this, the cells were harvested and resuspended in sodium dodecyl sulfate sam-ple buffer Proteins were separated on 8% SDS⁄ PAGE and prepared for fluorography
Measurement of intracellular free Ca2+level K562 cells were washed in buffer A and loaded with 5 mm Fura-2⁄ AM at 37 C for 45 min They were then washed with buffer A and placed in the measuring cell at D510¼ 0.25
at 37C and treated with BA or HE or subjected to 42 C The fluorescence signal was measured with a PTI spectrofluo-rometer (Photon Technology International, Inc., South Brunswick, NJ, USA) with emission at 510 nm and dual exci-tation at 340 and 380 nm (slit width 5 nm) The autofluores-cence from the cells not loaded with the dye was subtracted from the Fura-2 signal The rate of leakage this fluorescent dye at 37C and the method of determining [Ca2+]iare des-cribed in [46] When the contribution of the intracellular
Ca2+mobilization was tested, the cells were resuspended in buffer A without Ca2+, but containing 10 mm EGTA
Measurement of DWm
DWmwas analyzed as in [47], by using the fluorescent lipo-philic cation, JC-1 K562 cells (0.5· 106) were incubated with JC-1 (5 lgÆmL)1) during the last 15 min of any treat-ment in the dark and were immediately analyzed with a FACScan flow cytometer (Becton-Dickinson) equipped with
a 488 nm argon laser Dead cells were excluded by forward and side scatter gating JC-1 aggregates were detectable in the FL2 (585 ± 21 nm), and JC-1 monomers were detect-able in the FL1 (530 ± 15 nm) channel Data on 104 cells per sample were acquired and analyzed with Cell Quest software The mean fluorescence intensity of J-aggregates was used to determine the DWm
Estimation of the level of in vivo protein denaturation in response to heat stress and membrane fluidizing alcohols
The effects of heat or BA or HE treatment on protein denaturation were monitored via measurement of the
Trang 9activity of luciferase expressed in HeLa cells as in [20] The
cells were incubated at 37C with 30 mm BA or 4.5 mm
HE or at 42C for 30 min Immediately after treatment,
the cells were cooled to 4C and lysed Luciferase activity
was measured as described in [48]
Statistical analysis
All data are expressed as mean ± SD Student’s paired
t-test (a¼ 0.05) with the Bonferroni adjustment was used
to compare groups
Acknowledgements
This work was supported by grants from the
Hungar-ian National Scientific Research Foundation (OTKA:
TS 044836, T 038334) and Agency for Research Fund
Management and Research Exploitation (RET
OMFB00067⁄ 2005 and Bio-00120 ⁄ 2003 KPI)
References
1 Pockley GA (2001) Heat shock proteins in health and
disease: therapeutic targets or therapeutic agents? Expert
Rev Mol Med 2001, 1–21
2 Calderwood KS (2005) Regulatory interfaces between
the stress protein response and other gene expression
programs in the cell Methods 35, 139–148
3 Vigh L, Maresca B & Harwood J (1998) Does the
mem-brane physical state control the expression of heat shock
and other genes? Trends Biochem Sci 23, 369–373
4 Vigh L & Maresca B (2002) Dual role of membranes in
heat stress: as thermosensors they modulate the
expres-sion of stress genes and, by interacting with stress
proteins, re-organize their own lipid order and
functional-ity In Cell and Molecular Responses to Stress (Storey, KB
& Storey, JM, eds), pp 173–188 Elsevier, Amsterdam
5 Holmberg IC, Tran EFS, Eriksson JE & Sistonen L
(2002) Multisite phosphorylation provides sophysticated
regulation of transcription factors Trends Biochem Sci
27, 619–627
6 Sarge PK, Murphy SP & Morimoto RI (1993)
Activa-tion of heat shock gene transcripActiva-tion by heat shock
factor 1 involves oligomerization, acquisition of
DNA-binding activity, and nuclear localization and can
occur in the absence of stress Mol Cell Biol 13,
1392–1407
7 Trinklein ND, Murray JI, Hartman SJ, Botstein D &
Myers RM (2004) The role of heat shock transcription
factor1 in the genome-wide regulation of the
mamma-lian heat shock response Mol Biol Cell 15, 1254–1261
8 Dorion S & Landry J (2002) Activation of the
mitogen-activated protein kinase pathways by heat shock Cell
Stress Chaperones 7, 200–208
9 Gabai VL & Sherman MY (2002) Interplay between molecular chaperones and signaling pathways in survi-val of heat shock J Appl Physiol 92, 1743–1748
10 Park HG, Han SI, Oh SY & Kang HS (2005) Cellular responses to mild heat stress Cell Mol Life Sci 62, 10–23
11 Carratu L, Franceschelli S, Pardini C, Kobayashi GS, Horvath I, Vigh L & Maresca B (1997) Membrane lipid perturbation sets the temperature of heat shock response
in yeast Proc Natl Acad Sci USA 93, 3870–3875
12 Horvath I, Glatz A, Varvasovszki V, Torok Z, Pali T, Balogh G, Kovacs E, Nadasdi L, Benko S, Joo F et al (1998) Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a ‘fluidity gene’ Proc Natl Acad Sci USA 95, 3513–3518
13 Shigapova N, Torok Z, Balogh G, Goloubinoff P, Vigh
L & Horvath I (2005) Membrane fludization triggers membrane remodeling which affects the thermotolerance
in Escherichia coli Biochem Biophys Res Comm 328, 1216–1223
14 Han SI, Oh SY, Woo SH, Kim KH, Kim JH, Kim HD
& Kang HS (2001) Implication of small GTPase Rac1
in the activation of c-junN-terminal kinase and heat shock factor in response of heat shock J Biol Chem
276, 1889–1895
15 Welch JW & Suhan PJ (1985) Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments
in rat fibroblasts after heat-shock treatment J Cell Biol
101, 1198–1211
16 Kiang JG, Carr FE, Burns MR & McLain DE (1994) HSP-72 synthesis is promoted by increase in [Ca2+]ior activation of G proteins but not pHior cAMP Am J Physiol Cell Physiol 267, C104–C114
17 Kiang GK, Ding XZ & McClain DE (1998) Overexpres-sion of HSP-70 attenuates increases in [Ca2+]iand pro-tects human epidermoid A-431 cells after chemical hypoxia Toxicol Appl Pharmacol 149, 185–194
18 Kultz D (2005) Molecular and evolutionary basis of the cellular stress response Annu Rev Physiol 67, 225–257
19 Grant RL & Acosta D Jr (1994) A digitized fluores-cence imaging study on the effects of local anasthetics
on cytosolic calcium and mitochondrial membrane potential in cultured rabbit corneal epithelial cells Toxicol Appl Pharmacol 129, 23–35
20 Qian L, Song X, Ren H, Gong J & Cheng S (2004) Mito-chondrial mechanism of heat stress-induced injury in rat cardiomyocyte Cell Stress Chaperones 9, 281–293
21 Perl A, Gergely P, Nagy Gy Koncz A & Banki K (2004) Mito-chondrial hyperpolarization: a checkpoint of T-cell life, death and autoimmunity Trends Immunol 25, 360–367
22 Zaragoza A, Diez-Fernandez C, Alvarez AM, Andres D
& Cascales M (2001) Mitochondrial involvement in
Trang 10cocaine-treated rat hepatocytes: effect of N-acetylcysteine
and deferoxamine Br J Pharmacol 132, 1063–1070
23 Torok Z, Tsvetkova NM, Balogh G, Horvath I, Nagy
E, Penzes Z, Hargiati J, Bensaude O, Csermely P, Crow
JH et al (2003) Heat shock protein coinducers with no
effect on protein denaturation specifically modulate the
membrane lipid phase Proc Natl Acad Sci USA 100,
3131–3136
24 Hightower LE & White FP (1981) Cellular responses to
stress: comparison of a family of 71–73-kilodalton
pro-teins rapidly synthesized in rat tissue slices and
canava-nine-treated cells in culture J Cell Physiol 108, 261–275
25 Calderwood SK & Stevenson MA (1993) Inducers of
the heat shock response stimulate phospholipase C and
phospholipase A2activity in mammalian cells J Cell
Physiol 155, 248–256
26 Ding XZ, Smallridge RC, Galloway RY & Kiang JG
(1996) Increases in HSF1 translocation and synthesis in
human epidermoid A-431 cells: Role of protein kinase
C and [Ca2+]i J Invest Med 44, 144–153
27 Price BD & Calderwood SK (1991) Ca is essential for
multistep activation of the heat shock factor in
permea-bilized cells Mol Cell Biol 11, 3365–3368
28 Sreedhar AS & Arinivas UK (2002) Activation of stress
response by ionomycin in rat hepatoma cells J Cell
Biochem 86, 154–161
29 Yan L-Y, Rajasekaran NS, Sathyanarayanan S &
Benjamin IJ (2005) Mouse HSF1 disruption perturbs
redox state and increases mitochondrial oxidative
stress in kidney Antioxidants Redox Signaling 7,
465–471
30 Vigh L, Literati NP, Horvath I, Torok Zs Balogh G,
Glatz A, Kovacs E, Boros I, Ferdinandy P, Farkas B,
Jaszlits L et al (1997) Bimoclomol: a novel non-toxic,
hyroxylamine derivative with stress protein inducing
activity and wide cytoprotective effects Nature Med 3,
1150–1154
31 Yan D, Saito K, Ohmi Y, Fujie N & Ohtsuka K (2004)
Paeoniflorin, a novel heat shock protein-inducing
com-pound Cell Stress Chaperones 9, 378–389
32 Sachidhanandam SB, Lu J, Low KSY & Moochala SM
(2003) Herbimycin A attenuates apoptosis during heat
stress in rats Eur J Pharmacol 474, 121–128
33 Butler PJ, Tsou TC, Li JYS, Usami S & Chien S (2001)
Rate sensitivity of shear-induced changes in the lateral
diffusion of endothelial cell membrane lipids: a role for
membrane perturbation in shear-induce MAPK
activa-tion Am J Physiol 280, C962–C969
34 Vereb G, Szollosi J, Matko J, Farkas T, Vigh L,
Matyus L, Waldmann TA & Damjanovich S (2003)
Dynamic, yet structured: the cell membrane three
dec-ades after the Singer–Nicolson model Proc Natl Acad
Sci USA 100, 8053–8058
35 Gudi S, Nolan JP & Frangos JA (1998) Modulation of
GTPase activity of G proteins by fluid shear stress and
phospholipid composition Proc Natl Acad Sci USA 95, 2515–2519
36 Shack S, Gorospe M, Fawcett TW, Hudgins WR & Holbrook NJ (1999) Activation of the cholesterol path-way and Ras maturation in response to stress Oncogene
18, 6021–6028
37 Guyton GZ, Gorospe M, Wang X, Kokkonen YD, Liu GC, Roth GS & Holbrook NJ (1998) Age-related changes in activation of MAPK cascades by oxidative stress J Invest Dermatol 3, 23–27
38 Escriba PV, Sanchez-Dominguez JM, Alemany R, Perona JS & Ruiz-Gutierrez V (2003) Alterations
of lipids, G proteins, and PKC in cell membranes
of elderly hypertensives Hypertension 41, 176–182
39 Hooper PL & Hooper JJ (2005) Loss of defense against stress: diabetes and heat shock proteins Diabetes Tech-nol Ther 7, 204–208
40 Garbe TR & Yukawa H (2001) Common solvent toxicity: autooxidation of respiratory redox-cyclers enforced by membrane derangement Z Naturforsch 56c, 483–491
41 Cheng JZ, Sharma R, Yang Y, Singhal SS, Sharma A, Saini MK, Sing SV, Zimniak P, Awasthi S & Awasthi
YC (2001) Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress J Biol Chem 276, 41213–41223
42 Vigh L, Escriba P, Sonnleitner A, Sonnleitner M, Piotto
S, Maresca B, Horva´th I & Harwood LJ (2005) The sig-nificance of lipid composition for membrane activity: new concepts and ways of assessing function Prog Lipid Res 44, 303–344
43 Schutz GJ, Kada G, Pastushenko VP & Schindler H (2000) Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy EMBO J 19, 892–901
44 Maeda T, Balakrishnan K & Mehdi SQ (1983) A simple and rapid method for the preparation of plasma mem-branes Biochim Biophys Acta 731, 115–120
45 Torok ZS, Horvath I, Goloubinoff P, Kovacs E, Glatz A, Balogh G & Vigh L (1997) Evidence for a lipochaperonin: Association active protein-folding GroESL oligomers with lipids can stabilzie membranes under heat shock conditions Proc Natl Acad Sci USA 94, 2192–2197
46 Kiang JG (1991) Effect of intracellular pH on cytosolic free [Ca2+] in human epidermoid A-431 cells Eur J Pharmacol 207, 287–296
47 Khaled AR, Reynolds DA, Young HA, Thompson CB, Muegge K & Durum SK (2001) Interleukin-3 withdra-wal induces an early increase in mitochondrial mem-brane potential unrelated to the Bcl-2 family: roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase
J Biol Chem 276, 6453–6462
48 Nguyen VT & Bensaude O (1994) Increased thermal aggregation of proteins in ATP-depleted mammalian cells Eur J Biochem 220, 239–246