Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock.. When ansamycin-treated cells are h
Trang 11 Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, CA, USA
2 Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine Los Angeles, CA, USA
Exposure of cells to stress inactivates proteins and can
result in cell death, unless protective responses occur
[1,2] Virtually all living organisms from bacteria to
humans counteract the detrimental effects of cell stress
by inducing a highly conserved response, termed the
heat shock response or stress response [3] The
response was initially described and detailed in heat
shocked Drosophila cells [4,5], among others
Subse-quent studies have shown that a wide array of stressful
circumstances, including oxidative stress, abnormal
protein accumulation, and endoplasmic reticulum
per-turbations, induce a coordinated molecular response
with two distinct facets: first, many metabolic
proces-ses, including mRNA processing and translation, are severely inhibited, presumably to minimize the accu-mulation of deleterious protein forms Second, there is
a massive, rapid synthesis of a small group of proteins termed the heat shock proteins (Hsps), or stress pro-teins, that requires unique transcriptional induction and mRNA processing, and preferential translation [3]
A key feature of the heat shock response is its sup-pression following restoration of normal environmental conditions [3,6] This entails the restoration of normal metabolic functions, such as protein synthesis, termin-ation of Hsp synthesis, and reduction in Hsp abun-dance The latter features appear to be required
Keywords
geldanamycin; heat shock; herbimycin;
Hsp90; Hsp function
Correspondence
R F Duncan, School of Medicine,
Department of Molecular Microbiology
and Immunology, University of Southern
California School of Pharmacy Los Angeles,
CA, USA
Fax: +1 323 442 1681
Tel: + 323 442 1449
E-mail: rduncan@usc.edu
(Received 6 July 2005, accepted 17 August
2005)
doi:10.1111/j.1742-4658.2005.04921.x
The induction of the heat shock response as well as its termination is auto-regulated by heat shock protein activities In this study we have investi-gated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation The elevated levels of Hsps 22–28 (the small Hsps) and Hsp70 are not able to promote thermo-tolerance when Hsp90 activity is repressed by ansamycins; rather a suppres-sion of thermotolerance is observed These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration
of normal translation, and likely the establishment of thermotolerance Impaired function of either component is sufficient to alter the heat shock response
Abbreviations
2DGE, two-dimensional IEF ⁄ SDS ⁄ PAGE; eIF, eukaryotic initiation factor; EMSA, electrophoretic mobility shift assay; HSF, heat shock transcription factor; Hsp, heat shock protein; PI3K, phosphatidylinositol 3-kinase; SM, Schneider’s Drosophila growth medium.
Trang 2because some Hsps are detrimental to normal growth
and hence are sequestered or degraded during recovery
[3,7] Molecular programs that regulate the termination
of the heat shock response thus significantly influence
cellular and organismal health
The accumulation of a threshold level of Hsp70
sig-nals the cessation of Hsp mRNA transcription and the
restoration of the normal rate of protein synthesis
This has been termed ‘autoregulation’ of the stress
response, and is proposed to reflect the ability of Hsps
to inhibit the heat shock transcription factor (HSF)
[6,8,9] Hsp70-dependent autoregulation of the stress
response has been amply documented in cells from
yeast to Drosophila to humans [8,10,11]
In this study we have investigated whether and how
Hsp90 influences the duration or characteristics of the
stress response, about which little is known to date
Hsp90 has been directly implicated in the regulation of
HSF, partnering with Hsp70 to control its activity [12]
Through application of benzoquinone ansamycin
anti-biotics that compete with ATP for the Hsp90 nucleotide
binding site and inhibit Hsp90 activity [13,14], the
role(s) of Hsp90 in the heat shock response can be
straightforwardly investigated We find that in
Droso-philacells latent HSF is activated by Hsp90 inhibition
leading to a ‘heat shock response’ in the absence of
stress, as reported previously for other cell types When
ansamycin-treated cells are heat shocked, the restoration
of a normal metabolic function, protein synthesis, is
sig-nificantly repressed, even though supra-physiological
levels of Hsp70 accumulate due to the failure of these
cells to terminate the heat shock response In the absence
of Hsp90 function cells cannot establish a
thermo-tolerant state, even with the accentuated abundance of
the other classes of Hsps It has been well-established
that Hsp70 collaborates with Hsp90 to carry out many
of its chaperone functions [15,16], and these results
indi-cate that such collaboration is required for efficient cessation of the heat shock response
Results
Benzoquinone ansamycins induce a heat shock response in the absence of heat shock
We hypothesize that Hsp90 function regulates facets of the heat shock response, as has been documented for Hsp70 [3,6,8] Ansamycin Hsp90 inhibitors were used
to determine whether two key elements of the heat shock response – (a) induction and suppression of heat shock protein synthesis, and (b) normal protein synthe-sis recovery – were altered when Hsp90 function was abrogated Initially, the effects of ansamycins on non-heat protein synthesis rate and type were examined to exclude the possibility that effects observed during recovery from heat shock resulted from a general inhi-bition of the translational machinery First, there was
no overall inhibition or activation of translation rate
as assessed by trichloroacetic acid (TCA) precipitation
of pulse-labeled protein over 6 h of treatment (data not shown) and IEF⁄ SDS ⁄ PAGE analysis (Fig 1), thus excluding the possibility that a deficit in protein synthesis recovery from heat shock could be due to a direct effect of the inhibitors on the translational machinery Second, there is a striking synthetic induc-tion of a limited number of proteins (Fig 1) Benzo-quinone antibiotics induce the expression of heat shock proteins at the transcriptional level in mamma-lian cells, presumably because Hsp90 sequesters HSF
in an inactive state [17–19] Our results show the same induction of Hsps occurs in Drosophila, based on the precise comigration of the ansamycin-induced proteins with heat-induced proteins Similar results were obtained with both ansamycins: geldanamycin
Fig 1 Ansamycins induce Hsp synthesis in the absence of heat shock in Drosophila cells Drosophila S2 cells were incubated with
1 lgÆmL)1 geldanamycin or 1 l M herbimycin A, or left untreated Cells were pulse-labeled for 15 min at intervals with 20 lCiÆmL)1 [ 35 S]methionine Protein samples were prepared as described for 2DGE (see Experimental procedures) and analyzed by IEF ⁄ SDS ⁄ PAGE Equal amounts of protein (equal cell numbers) were loaded onto each first dimension gel (based on Bradford assays, and confirmed by Co-omassie Brilliant Blue staining of the gels after electrophoresis) Labeled proteins were detected by autoradiography The basic region of the gel is to the right, the acidic end to the left Cells labeled after 6 h incubation without (A) or with (B) geldanamycin (C, D) Cells labeled after
2 h incubation with or without herbimycin A Only sectors containing Hsp70 ⁄ 68 (left sectors in C, D) and Hsp22 ⁄ 23 (right sectors in C, D) are shown (E) EMSA analysis of ansmycin-treated and heat shocked cells Whole cell lysates were prepared from cultures treated without
or with herbimycin A, as well as a positive control heat-shocked culture Each lysate was incubated with a 32 P-labeled DNA probe containing the HS element, analyzed by PAGE and autoradiography [41]; only the region corresponding to the migration of protein-bound, retarded mobility probe is shown (F) Northern analysis RNA was extracted from cultures treated without or with herbimycin A for 3 h, displayed by formaldehyde ⁄ agarose gel electrophoresis, probed with a 32 P-labeled DNA probe for Hsp70, and visualized by autoradiography Only the region corresponding to the migration of Hsp70 mRNA is shown (G) Western EMSA analysis of HSF phosphorylation Extracts were pre-pared from cells treated with geldanamycin (upper) or herbimycin A (lower) were analyzed by SDS ⁄ PAGE and immunoblotting with anti-HSF sera; retarded bands have been correlated with HSF phosphorylation and activation [22] An extract from cells heat shocked for 30 min at
36 C is shown at the lower right, for reference (P)HSF, phosphorylated HSF isoforms nsb, nonspecific band.
Trang 3(Fig 1A,B) and herbimycin A (Fig 1C,D) The
activa-tion of Hsp transcripactiva-tion by ansamycins at normal
temperature is not subject to autoregulation as little to
no difference in Hsp synthetic rate was observed
between 2 and 6 h of treatment; e.g panel B shows
abundant Hsp synthesis after 6 h of treatment based
on pulse-label analysis Notably, as will be detailed
subsequently, suppression of Hsp synthesis is observed
when ansamycin-treated cells are heat shocked, then
recovered The activation of HSF was confirmed by DNA-binding gel shift assay (Fig 1E) and northern blot analysis using Hsp70 probes (Fig 1F) Curiously, HSF phosphorylation, which is frequently used as a surrogate marker for HSF activation, was not detected following ansamycin treatment and robust Hsp tran-scription (Fig 1G), though phosphorylated isoforms can be readily detected following heat shock (Fig 1G, lower right lane)
B
F
G E D
Trang 4We were also interested to determine if evidence for
Hsp90-mediated regulation of expression of other
genes, either at a transcriptional or translational level,
could be discerned that might implicate other
path-ways that could influence the heat shock response
However, careful comparative examination of the 2D
IEF⁄ SDS ⁄ PAGE spot patterns of several untreated
and treated samples failed to uncover any additional
reproducibly altered spots (e.g Fig 1A,B)
Benzoquinone ansamycins delay heat shock
recovery and prolong Hsp synthesis
To determine whether Hsp90 function affects
characteristics of the heat shock response,
geldanamycin-treated Drosophila S2 cells were heat
shocked for 30 min at 36C, then restored to their
nor-mal growth temperature Samples were pulse-labeled
for 15 min with [35S]methionine⁄ cysteine at intervals
during heat shock and recovery In keeping with the
observation that geldanamycin did not inhibit protein
synthesis per se, no difference in protein synthesis rate
during heat shock or early in the recovery phase was
observed (Fig 2A)
However, geldanamycin treatment causes a modest
inhibition of protein synthesis rate during later
recov-ery that is more evident at 3–4 h (Fig 2A; at recovrecov-ery
times of 2 h and thereafter differences between
untreated and ansamycin-treated cells were
signifi-cantly negative at the P¼ 0.05 level based on a paired
t-test) As will be described, this overall measurement
significantly underestimates the delay in recovery,
because the synthesis of heat shock proteins dominates
the overall measurement Recovery rates for specific
nonheat shock mRNA translation requires more
pre-cise analysis, as will be presented below Herbimycin
treatment during heat shock and recovery produced
results indistinguishable from those obtained with
gel-danamycin (data not shown) To begin to address how
the translation of specific mRNAs was affected,
pulse-labeled samples were analyzed by one-dimensional
SDS⁄ PAGE This provides an informative overview of
translational changes, but is insufficient to accurately
quantify individual protein synthetic changes Several
features stand out First, the duration of heat shock
protein synthesis is extended (Fig 3); this is most
evi-dent in the Hsp70 band, though detectable for all
clas-ses of Hsp synthesis Second, there is a delay in the
restoration of normal translation This is most evident
in the 1D analysis in lanes wherein significant
restor-ation is just beginning, e.g in the 2 h recovery samples
in Fig 3B These two events are likely coupled and
both result from inadequate Hsp90 activity
To precisely characterize and quantify the influence
of ansamycin treatments on normal translation recov-ery and Hsp synthesis, cells were pulse-labeled as des-cribed and proteins analyzed by two-dimensional IEF⁄ SDS ⁄ PAGE, autoradiography, and densitometry (Fig 4) From a qualitative perspective a significant impairment in normal translation restoration is evident
in the reduced intensity of virtually all nonheat protein spots To provide quantitative data a random sample of
A
B
Fig 2 Ansamycin treatment suppresses the recovery of normal pro-tein synthesis following heat shock Drosophila S2 cells were pre-treated with ansamycin for 10 min, then heat shocked in a water bath equilibrated to 36 C for 30 min, then returned to normal growth temperature for the duration of the experiment Aliquots were pulse-labeled for 15 min with 20 lCiÆmL)1[35S]methionine at intervals (A) Protein samples prepared as described (see Experimental proce-dures) were quantified for incorporation by TCA precipitation on
GF ⁄ C filters and liquid scintillation counting Results represent the average and standard deviation of 3–5 replicates with values from geldanamycin- and herbimycin A-treated samples combined (B) Spe-cific protein synthesis was quantified by 2D IEF ⁄ SDS ⁄ PAGE, auto-radiography, and densitometry of an array of 14 nonheat shock protein spots (black bars; see Fig 4 for identification) from samples pulse-labeled 3 h into recovery.
Trang 5spots (identified in Fig 4A) was measured by
densito-metry; the extent to which recovery of these spots was
retarded is reported in Fig 2B For spots 1–9, the range
of inhibition is 70–95%, with an average of 81%
(black bars) Recovery of a minor class of spots was less
affected by ansamycin treatment Selected members of
this group, labeled a-e, were inhibited only 40–60%
(right five gradient-filled bars) In summary, ansamycin
treatment significantly delays restoration of cell
func-tion following heat shock, implicating Hsp90 activity in
this process
Heat shock proteins 90, 70, 22 and 23 were similarly quantified The rate of synthesis of these proteins achieves a higher maximum and remains activated longer in the ansamycin-treated cells (Fig 4C–I; gel sectors are shown in panels C–E, and densitometric quantitation in panels F–I) For example, Hsp70 synthe-sis rapidly achieves its maximum synthetic rate in untreated cells at 1–2 h, and then rapidly declines, whereas in ansamycin-treated cells its synthetic rate at
2 h is approximately threefold elevated relative to untreated cells, and its synthesis at 3 h remains high,
Fig 3 Ansamycin effects on Hsp synthesis and normal protein synthesis recovery analyzed by SDS ⁄ PAGE Cells were treated and labeled
as described in Fig 2, legend Extracts were prepared using hSDS buffer (see Experimental procedures) and analyzed by SDS ⁄ PAGE and autoradiography Equal amounts of protein (equal cell numbers) were loaded into each lane (based on Bradford assays, and confirmed by Coomassie Brilliant Blue staining of the gel after electrophoresis) (A) Cells pretreated with (right) or without (left) 1 lgÆmL)1geldanamycin (B) Cells pretreated with (right) or without (left) 1 l M herbimycin A The location of bands corresponding to heat shock proteins Hsp83, Hsp70, Hsp26 ⁄ 28, and Hsp22 ⁄ 23, as well as actin, are indicted to the right The extent of induction of Hsp90 during recovery varies; the herbimycin panels (without, with treatment) represent an example of extensive induction, which is not due to the herbimycin treatment itself (as it is equally observed in the untreated portion of the panel) This analysis has been repeated in part or completely three times; a repre-sentative result is shown.
Fig 4 Ansamycin effects on Hsp synthesis and normal protein synthesis recovery analyzed by 2-dimensional IEF ⁄ SDS ⁄ PAGE Cells were treated and labeled as described in Fig 2, legend Protein samples were prepared as described for 2DGE (see Experimental procedures) and analyzed by IEF ⁄ SDS ⁄ PAGE Equal amounts of protein (equal cell numbers) were loaded onto each first dimension gel (based on Bradford assays, and confirmed by Coomassie Brilliant Blue staining of the gels after electrophoresis), exposed to film and labeled proteins detected
by autoradiography For quantitation, films were scanned with a densitometer, and the intensity of spots determined using the Labworks software (UV Products) The basic region of the gel is to the right, the acidic end to the left (A, B) Cells treated without or with herbimycin
A beginning 10 min prior to heat shock ⁄ recovery; the 3 h recovery samples are shown The location of spots corresponding to heat shock proteins Hsp90, Hsp70, Hsp26 ⁄ 28, and Hsp22 ⁄ 23, and nonheat shock protein actin, are indicated with arrows These presentations are overexposed relative to the ones used for densitometric quantification (panels F–I, and Fig 2B) to emphasize nonheat shock protein synthe-sis Correspondingly, Hsp synthetic differences are significantly underrepresented due to film saturation 14 nonheat shock protein spots used for monitoring nonheat shock protein recovery are indicated in panel A with numbers, letters above the spots (with the exception of
‘9’, which is written to the right of the spot); see text for details (C–E) Sectors from 2D gels analyzing protein labeled at the indicated times (F–I) Densitometric quantitation of Hsp synthesis Black bars, without treatment; gradient-filled bars, ansamycin-treated.
Trang 6G F
Trang 7similar to the rate in untreated cells 1–2 h earlier (and
fourfold higher than the rate in untreated cells at
3 h) For the other Hsps the onset of repression during
the recovery interval is delayed relative to untreated
cells, such that the maximum rate is synthesis occurs
1–2 h later These elevated synthetic rates result in
increased Hsp accumulation, as described in the next
section
The prolonged rate of Hsp protein synthesis is
paral-leled by a prolonged activation of the heat shock
tran-scription factor HSF, as assessed by HSF
phosphorylation (Fig 5) Slower migrating HSF
iso-forms are induced by heat shock and increase to 1 h
of recovery, then rapidly decline such that they are no
longer detectable by 3 h recovery (Fig 5A) On the
other hand, phosphorylated HSF isoforms peak later
(at 2 h into recovery) in ansamycin-treated cells,
and remain in their activated form after they have
disappeared from the untreated cells These results
confirm that ansamycins cause a prolongation of the
heat shock response, which can be considered a deficit
in autoregulation as Hsp70 levels are high
Notably, the transcription and translation of Hsp70
mRNA is largely suppressed by 5 h recovery in
ansa-mycin-treated cells exposed to heat shock⁄ recovery,
whereas in cells exposed to ansamycin treatment at
normal temperature its gene expression continues
un-abated (Fig 1) This suggests that high levels of Hsp70
are sufficient to suppress HSF activity even in the
absence of Hsp90, albeit less efficiently than in its
pres-ence Similarly, restoration of normal metabolic
func-tion (e.g protein synthesis) occurs in the absence of
Hsp90 function, but with delayed kinetics
The abundance of Hsps ultimately determines their functional significance The influence of ansamycins on Hsp abundance during heat shock recovery was deter-mined by Coomassie staining 2DGE-resolved proteins, and densitometry Throughout the first hours of recov-ery no significant differences in abundance of any Hsp was observed (data not shown), consistent with the sim-ilar rates of synthesis Protein abundance at times cor-responding to the maximum Hsp accumulation (4–5 of recovery) showed modest to significantly elevated levels
of Hsps (Fig 6) For Hsp70, the major Drosophila heat shock protein and the one that primarily determines heat shock sensitivity and resistance, ansamycin treat-ment increased its levels by approximately threefold (see also Fig 7, inset, for confirmation by western blot analysis) Hsp22 was approximately twofold increased, whereas Hsp23 and Hsp90 were 20–40% increased These results suggest that ansamycins might enhance thermotolerance, with the caveat that Hsp90 is non-functional and might mitigate benefits provided by higher levels of the other Hsp classes This issue is addressed in the next section
Benzoquinone ansamycins decrease thermotoler-ance
To determine whether the increased Hsp abundance caused by ansamycin treatment had functional conse-quences on cells’ ability to tolerate stress, restoration of cell proliferation following a moderately severe chal-lenge heat shock (30 min at 38C) was determined, with
or without ansamycin pretreatment As noted above the contrasting influences of various Hsp classes could
Fig 5 Ansamycin prolongs the activity of HSF as assessed by phosphorylation Cells were treated as described in Fig 2, legend Extracts were prepared using hSDS buffer (see Experimental procedures) and analyzed by SDS ⁄ PAGE and immunoblotting with antisera to Drosophila HSF Equal amounts of protein (equal cell numbers) were loaded into each lane (based on Bradford assays, and confirmed by Amido schwartz staining
of the gel after electrophoretic transfer) Cells without (A) or with (B) pretreatment with 1 l M herbimycin A Locations of the major HSF band as well as the more slowly migrating phosphorylated isoforms [(P)HSF] are indicated to the right of panel B Three to four differentiated bands are detected on the original film; nsb, nonspecific band.
Trang 8produce accentuated thermotolerance, reduced
thermo-tolerance if Hsp90 function is crucial, or no
distinguishable effect if the influences of distinct Hsp
classes counterbalance each other
Without ansamycin treatment or a
tolerance-indu-cing heat shock (control conditions) cell numbers
progressively increase over several days (Fig 7, open
triangles; top curve) Addition of ansamycin to the
cul-ture medium at reseeding had no effect, by itself, on
cell growth (open diamond, day 4) Hence its
suppres-sion of cell proliferation, described below, cannot be
ascribed to a basal toxic effect The heat shock⁄ recovery
protocol described in previous experiments produces
no distinguishable effect on subsequent cell growth at
normal growth temperature (solid diamonds) The
more severe challenge heat shock modestly delays cell
growth (open squares) In cells that have undergone
a tolerance-inducing heat shock⁄ recovery cycle, as
detailed in the previous figures, the severe heat shock appears less detrimental as under these conditions cells increase in number indistinguishably from wholly untreated control cells (open circles) In summary, proliferation under the four cited regimens was indistinguishable
If cells are pretreated with ansamycin before the severe challenge heat shock then cell proliferation is significantly retarded (solid squares) Under these conditions modestly elevated levels of Hsps are syn-thesized, but the activity of Hsp90, and perhaps Hsp70 indirectly, is repressed The elevated levels of Hsps provide no discernable stress-resistance benefits
If cells are both ansamycin-treated and provided a priming heat shock-recovery cycle, leading to sub-stantially higher levels of all Hsps (e.g Fig 7, inset), then the growth retardation is less than with ansa-mycin treatment alone (compare solid circles (both
A
C
B
Fig 6 Accumulation of Hsps in ansamycin-treated and untreated cells following heat shock Cells were treated and labeled as described in Fig 2, legend Protein samples were prepared as described for 2DGE (see Experimental procedures) and analyzed by IEF⁄ SDS ⁄ PAGE Equal amounts of protein (equal cell numbers) were loaded onto each first dimension gel (based on Bradford assays, and confirmed by Coomassie Brilliant Blue staining of the gels after electrophoresis) Only a sector of the entire gel is shown in the panels (A) Sectors containing Hsp90 and Hsp70 from samples 4 h and 5 h of recovery from heat shock The locations of Hsp68 and Grp78 (also referred to as Hsc72 in Droso-phila) are indicated as well (B) Sectors containing Hsp22 and Hsp23, as in A (C) Densitometric quantitation of Hsp abundance The maxi-mum amount for each Hsp protein was set to 100% Black bars, without treatment; gradient-filled bars, ansamycin-treated.
Trang 9treatments) to solid squares), but remains
distinguish-ably reduced relative to cells heat shocked⁄ recovered
in the absence of ansamycin (compare open to solid
circles) Similar results were obtained when the
ansa-mycins were removed after 12 h exposure Thus,
even though heat shock protein production is
accen-tuated by ansamycin treatment the abundant Hsps
rather correlate with reduced thermotolerance when
Hsp90 is rendered nonfunctional, implicating Hsp90
in restoration of cell proliferation following heat
shock Because ansamyin did not suppress cell
prolif-eration in nonheat shocked cells, the results suggest
that Hsp90 function is specifically needed to restore
function to critical, heat-impaired targets Results by
others have suggested that ansamycin-induced Hsp
expression can provide thermotolerance to
mamma-lian cells (e.g [18]) The discrepant results may
that the primary thermotolerance-inducing Hsp dif-fers between species⁄ cell type [20]
Discussion
Molecular chaperones have distinct, specific roles in cel-lular physiology as well as coordinated functions in pro-tein folding and refolding In Drosophila, Hsp70 is the principal stress-induced chaperone and its activity has been shown to determine many features of the heat shock response [3] In particular experimental manipula-tions that restrict Hsp70 accumulation or activity pro-long the heat shock response [3,6,8] In this study we have addressed the requirement for Hsp90 function dur-ing the early inductive events of the heat shock response,
in its cessation via autoregulation by Hsps, and for the activity of Hsps in providing thermotolerance The results suggest that Hsp90 activity contributes to all aspects of the heat shock response, perhaps as part of a chaperone protein refolding system in coordination with Hsp70 [15,16,21]
Inhibition of Hsp90 by ansamycins does not influ-ence normal protein synthesis, either on a global or protein-specific scale As reported by others studying mammalian cells [17–19], ansamycin treatment acti-vates HSF leading to the production of Hsps in Drosophila Intriguingly, this appears to occur in the absence of detectable HSF phosphorylation, contrary
to other chemically induced activations of HSF such
as are caused by proteasome inhibitors [22] Suppres-sion of Hsp90 function prolongs the activity of HSF and the synthesis of Hsp mRNAs following heat shock, paralleling the effect of blocking Hsp70 func-tion Ultimately, HSF is inactivated suggesting that Hsp70 is sufficient to autoregulate HSF transcription even in the absence of Hsp90 function As a conse-quence of ansamycin pretreatment, the rate of Hsp synthesis is maintained for a longer duration and excess amounts of each of the major classes of Hsps accumulate However, the enhanced Hsp complement does not enhance restoration of cell proliferation fol-lowing a moderately severe heat shock, but rather sub-stantially reduces it This suggests that Hsp90 function
is critical for thermotolerance and heat shock recovery This impairment is not a general consequence of Hsp90 inhibition, as retarded cell growth was only observed following heat shock, and not in cells main-tained at their normal growth temperature (the repor-ted cytotoxic effects of ansamycin treatment on tumor cells (e.g [23,24]) were not observed in Drosophila S2 cells) Thus, the effects are specific to the refolding of
Fig 7 Ansamycin-treated cells overexpressing Hsps are sensitized
to cell growth inhibition by heat shock Drosophila S2 cells were
untreated or treated with ansamycin (Ansa Rx) and ⁄ or heat
shocked in a water bath equilibrated to 36 C for 30 min followed
by recovery at normal growth temperature for 5 h (PreHS) and ⁄ or
heat shocked in a water bath equilibrated to 38 C for 30 min
(chal-lenge, or Ch) Following the challenge heat shock (or in its absence
for nonshocked cells) cells were placed in a 23 C incubator for up
to 4 days Cell samples were removed at intervals for
determin-ation of cell number by counting in a hemocytometer The symbol
designations are shown to right of the 4 d points Experiments
were performed using either geldanamycin (1 lgÆmL)1) or
herbimy-cin A (1 l M ); cell counts are combined The day-by-day analysis for
each condition was repeated three to eight times, and the average
is shown Deviation for all points overlaps, and so is not presented
for clarity For the 4 d points the deviations were: No challenge
(n), ± 161; no challenge, PreHS (m), ± 284; PreHs (s), ± 100;
chal-lenge only (h), ± 152; PreHs, Herb Rx (d), ± 191; no PreHs, Herb
Rx (n), ± 152 Cells treated with ansamycin only [e, shown only for
4 d (no PreHS, no challenge)] increased cell numbers
indistinguish-ably from the control cells (‘no challenge: n’) Inset: protein
sam-ples removed after 5 h recovery from the PreHS, or after an
equivalent incubation, without or with ansamycin treatment (– ⁄ –),
no treatment; (A ⁄ –), ansamycin, no PreHS; (– ⁄ H), PreHS, no
ansa-mycin; (A ⁄ H), ansamycin present prior to PreHS and for the 5 h of
recovery.
Trang 10heat-denatured proteins, or some other detrimental
consequence of heat shock
Inhibition of Hsp90 function by ansamycins also
sig-nificantly delays the recovery of normal, nonheat
shock mRNA translation following heat shock Global
translation rates are modestly reduced during recovery
(Fig 2), but nonheat shock mRNA translation rates as
determined by 2DGE analysis of specific protein spots
reveals that the translation of most mRNAs is
inhib-ited by 80% (Fig 2) during intermediate stages of
recovery ( 1–3 h) The quantitative discrepancy arises
because heat shock mRNAs are super-induced by
ansamycin treatment and constitute the majority of
overall translation, masking the extensive inhibition of
most nonheat shock mRNAs’ translation A subset
of nonheat shock mRNAs recovers more rapidly
These translation recovery patterns likely are linked to
molecular events that regulate heat shock-mediated
translation repression and subsequent restoration of
the affected factors to their active state As the
molecu-lar mechanism for translation inhibition in Drosophila
is not currently known, formulating a more precise
hypothesis becomes speculative For example, if
inhibi-tion involves diminished eIF4F activity, as substantial
evidence suggests [25–28], then as eIF4F activity is
progressively restored during recovery mRNAs with
the least dependence on eIF4F (e.g those with
rela-tively unstructured 5¢ untranslated regions) would be
predicted to recover activity earlier In the event where
eIF4F activity restoration is delayed, as may occur
when chaperone activity is inhibited by
ansa-mycin treatment, mRNAs with very little requirement
for eIF4F, such as the heat shock mRNAs and the
subset of nonheat shock mRNAs with diminished
eIF4F requirements, will be preferentially translated
Hsp90 functions as a homodimer to fold several key
classes of proteins, including protein kinases and
tran-scription factors [15,16,21] In these roles it collaborates
with Hsp70-family proteins, which bind to the newly
synthesized proteins and transfer them to the Hsp90
folding machine via the intermediates Hop (for
tran-scription factors) or cdc37 (for protein kinases) for final
folding and stabilization of the activation-primed
con-formation Numerous specific protein substrates of this
coordinated folding system have been identified, but
with specific regards to protein synthetic events during
the heat shock response the eIF2a protein kinase and
Akt are noteworthy eIF2a phosphorylation has been
implicated by many in the repression of protein
synthe-sis during heat shock [25,29–31], and thermotolerance
has been shown to correlate with accelerated
restor-ation of eIF2a to its active state [31] Hsp70 and Hsp90
association with the heat shock-activated eIF2a kinase
regulates its activity [32,33] Hence, impairment via Hsp90 and the ansamycins provides an avenue through which the heat shock response could be influenced In addition, the PI3K⁄ Akt kinase axis regulates protein synthesis rate and type in several circumstances, inclu-ding growth factor stimulation and recovery from stress [34,35] Suppressing its activity via ansamycins [36] may delay recovery from heat shock paralleling the reduc-tion in recovery from other forms of stress observed by directly inhibiting targets of Akt such as Tsc2 and mTOR with U0126 and rapamycin, respectively [35] A final potential target of Hsp90 is eIF4F itself, that has been observed to enter into heavy-sedimenting particu-late granules in heat-shocked mammalian cells [28,37]
If Hsp90 is involved in resolubilizing eIF4F from par-ticulate storage granules, and if this is critical for recov-ery of normal translation, then Hsp90 inhibition by ansamycin would be predicted to delay restoration fav-oring the translation of Hsp mRNAs and nonheat shock mRNAs with relatively low 5¢ untranslated region secondary structure
The influence of Hsp90 on heat shock⁄ recovery is only moderate, suggesting that the other heat shock pro-teins that accumulate in supra-physiological amounts in ansamycin-treated cells can compensate for Hsp90 though less efficiently This mirrors results in other cir-cumstances documenting partial redundancy in protein folding systems, based in part on the ability of an over-expressed chaperone class to substitute for a deficit in a wholly different class [21] The contribution of Hsp90 function to the heat shock response is clearly documen-ted in this study, though in keeping with previous char-acterizations of the Drosophila heat shock response Hsp70 predominates Considering that both heat and ansamycin treatments are used as cancer treatments [38], these results suggest that their coapplication could provide cooperative, more efficacious cell killing
Experimental procedures
Chemicals Chemicals were purchased from Sigma Chemical Company (St Louis, MO, USA) unless otherwise indicated
Drug treatment, heat shock, [35S]methionine labeling, and protein extraction and analysis Schneider S2 cells were cultured at 22–24C in Schneider’s Drosophila Medium (SM; Invitrogen, Inc., Carlsbad, CA, USA) containing 10% (v⁄ v) fetal bovine serum, 20 mm
l-glutamine, 100 unitsÆmL)1penicillin, 0.1 mgÆmL)1 strepto-mycin, 0.25 mgÆmL)1 amphotericin B (Invitrogen) At the