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Originally published In Press as doi:10.1074/jbc.M410838200 on January 25, 2005

J. Biol. Chem., Vol. 280, Issue 13, 13148-13152, April 1, 2005
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Heat Shock Protein 90 Stabilization of ErbB2 Expression Is Disrupted by ATP Depletion in Myocytes*

Xuyang Peng{ddagger}, Xinxin Guo{ddagger}, Steven C. Borkan§, Ajit Bharti{ddagger}, Yukio Kuramochi{ddagger}, Stuart Calderwood¶, and Douglas B. Sawyer{ddagger}||

From the {ddagger}Whitaker Cardiovascular Institute and Center for Molecular Stress Response, §Renal Division, Boston University Medical Center, Boston, Massachusetts 02118 and the Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115

Received for publication, September 21, 2004 , and in revised form, January 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with ~10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of {beta}-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The physiological mechanisms by which cells dynamically shift between steady states of tissue function, for example from tissue growth to cell survival during periods of metabolic stress, are incompletely understood. The activity of large sets of proteins must be coordinately regulated during these shifts. Many of the proteins involved in cell growth are stabilized by the constitutively expressed chaperone heat shock protein (Hsp)1 90 (1, 2). Dissociation of Hsp90 from its client proteins induced by Hsp90 inhibitors leads to rapid degradation of proteins involved in cell growth and the activation of a stress response (3). Thus, pharmacologically induced disruption in Hsp90 chaperone function alone can cause a cell to "shift gears" to a stress-mode.

Hsp90 inhibitors such as geldanamycin (GA) bind with high affinity to a conserved pocket in the Hsp90 family of proteins and thereby prevent the ATP binding that is required for chaperone function (4). The relative affinity of Hsp90 for GA, ADP, and ATP were recently measured and exhibited dissociation constants of 0.2, 12, and 124 µM, respectively (5). The relatively high affinity of Hsp90 for GA is consistent with its established potency as an inhibitor of Hsp90 chaperone function. The ~10-fold higher affinity of Hsp90 for ADP versus ATP is intriguing. Under basal conditions, when the intracellular [ATP]/[ADP] ratio is between 2 and 10:1 (6), the higher affinity of Hsp90 for ADP suggests that a small drop in the ATP/ADP ratio will result in a relatively large drop in the proportion of Hsp90 occupied by ATP and, therefore, available for client protein stabilization.

These observations led us to hypothesize that Hsp90 functions as a physiologic ATP sensor, regulating the stability of growth and stress signaling proteins in response to a reduction in cellular energy charge. We tested this hypothesis by examining the role of Hsp90 in the stabilization of ErbB2 in cardiac myocytes. We have previously demonstrated that ErbB2 activation by neuregulin regulates growth and stress signaling in cardiac myocytes (7), and ErbB2 is a well known Hsp90 client protein (8, 9). We reasoned that the high basal energy requirements of contracting ventricular myocytes might make them more likely to reveal such an ATP-sensing activity of Hsp90. We show that Hsp90 inhibitors induce the rapid dissociation of Hsp90 from ErbB2 and the degradation of ErbB2 via a proteasome pathway that involves Hsp70 and the carboxyl terminus Hsc70-interacting protein (CHIP). We demonstrate that ATP depletion has a similar effect on Hsp90/ErbB2 association and ErbB2 stability, along with other Hsp90 client proteins, and resistance to growth factor-stimulated kinase signaling. The potential role of Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under stressed conditions is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—GA and MG132 were purchased from EMD Biosciences Inc. (San Diego, CA). 2-Deoxy-D-glucose and antimycin A were purchased from Sigma. GA and MG132 were used at 1 µM. Neuregulin-1{beta} (NRG-1{beta}) was used at 50 ng/ml. L-Norepinephrine (NE; Sigma) was used at 0.1 and 1 µM for {beta}-adrenergic receptor ({beta}-AR) stimulation (NE{beta}) after pretreatment with prazosin (100 nM; Sigma) for 30 min. Acrylamide, bisacrylamide, and all other chemicals were from Sigma. A silver staining kit was from Bio-Rad. Anti-ErbB2 (clone b10, antibody 9) for immunoprecipitation antibody was purchased from Neomarker. Anti-ErbB2 (Neu (C-18)), Raf-1 (C-12), and mouse Hsp70 antibodies were from Santa Cruz Biotechnology, Inc. Rat anti-Hsp90 monoclonal antibody (SPA-835) was from Stressgen Biotech Corp. The rabbit anti-CHIP polyclonal antibody was from Oncogene Science Inc. Rabbit Akt, phospho-Akt, phospho-ERK, phospho-JNK, HSF-1, and the phospho-AMPK-(Thr-172) polyclonal antibody were purchased from Cell Signaling Technology Corp. Mouse anti-actin was from Sigma.

Preparation of Cardiac Myocytes—Adult rat ventricular myocytes (ARVMs) were isolated from male Sprague-Dawley rats weighing 150–200 g as described previously (7). For most experiments, ARVMs were maintained in Dulbecco`s modified Eagle's medium supplemented with 7% fetal calf serum, 2 mM glutamine, 1 mM Hepes, and 100 units/ml 1% penicillin/streptomycin (Invitrogen) and 100 µM bromodeoxyuridine culture medium. ARVMs were treated on day 7 in culture at a time when cells are spontaneously beating (10). For experiments involving electrical stimulation, ARVMs were maintained in serum-free media for 16 h prior to electrical stimulation at 5 Hz using a 2-ms pulse duration and 6.6 V/cm stimuli with alternating polarity using a cell culture pacing system (Ionoptix) as described (11). These conditions result in >70% capture of myocytes.

Lysate Preparation, Immunoprecipitation, and Western Blot Analysis—Cells were washed twice with cold phosphate-buffered saline (pH 7.0) and lysed by scraping in radioimmune precipitation assay buffer (50 mM Tris-HCl (pH 7.4), 0.25% deoxycholic acid, 1% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, and 1 mM sodium orthovanadate) supplemented with proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin). Cell lysates were clarified by centrifugation at 14,000 rpm (4 °C) for 15 min. For immunoprecipitation, 1 mg of lysate protein was incubated with 2 µg of mouse monoclonal antibodies at 4 °C overnight, followed by the addition of protein G-agarose beads (Santa Cruz Biotechnology) and rotation at 4 °C for 2 h. The beads were washed four times with lysis buffer, resuspended in 1x SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 0.0005% bromphenol blue), and boiled for 5 min. Immunoprecipitated proteins (or cell lysates mixed with 5x SDS sample buffer) were separated by 4–12% SDS-PAGE. Protein was detected with an antibody using a chemiluminescence-based Western blotting kit (DuPont) according to the supplier`s instructions. Membranes were exposed for various times to Kodak X-Omat AR film. Films were scanned and analyzed by densitometry to obtain semi-quantitative analysis of protein expression.

Metabolic Labeling of Cultured Cells—Cells-were washed and incubated for 12 h in medium free of methionine, followed by the addition of 5 Ci/ml 35S-labeled methionine for an overnight incubation (pulse). Cells were then washed thoroughly, incubated in media containing methionine, and given GA treatment for different times (chase). This was followed by cell lysis, immunoprecipitation, electrophoresis, and autoradiography of dry gels.

Luminescence ATP Detection Assay—1.0 x104 ARVMs per well in a 96-well microplate were washed and incubated for 12 h in fetal calf serum-free medium after culture for 7 days. Cells were treated by 2-deoxy-D-glucose and antimycin-A as indicated. Cellular ATP was measured using ATPLiteTM (12), an ATP monitoring system based on firefly (Photinus pyralis) luciferase according to the manufacturer's instructions. A LumiCount microplate reader was used to measure the luminescence.

Statistical Analysis—All data are presented as mean ± S.D. from at least four experiments in each group at each time point (except any-where mentioned in the text). Comparisons were made by unpaired t test or by one-way analysis of variance as appropriate. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hsp90 Inhibition Induces ErbB2 Degradation via a Proteasome Pathway with Disruption of Hsp90/ErbB2—We first examined the chaperone function of Hsp90 in cardiac myocytes using the Hsp90 inhibitor GA. As in the other cell types (9), GA treatment (1 µM) led to a significant decrease of ErbB2 expression in myocytes (Fig. 1A). Treatment of cells with the proteasome inhibitor MG-132 (1 µM) prevented GA-induced degradation of ErbB2 (Fig. 1B). GA disruption of Hsp90-ErbB2 interaction in ARVM was evident within 1 h of treatment (Fig. 1C). Recently, the Hsp70/Hsp90 co-chaperone CHIP has been shown to function as a ubiquitin ligase toward several Hsp90 client proteins including ErbB2 (8, 9). Upon GA treatment of cardiac myocytes, there was association of CHIP and Hsp70 with ErbB2 (Fig. 1D, lane 2), which further increased after 12 h of exposure to GA, despite the reduced ErbB2 expression at this time point (Fig. 1D, lane 3). Thus, ErbB2 stability is regulated by Hsp90 in myocytes and degraded via a proteasome pathway upon the disruption of Hsp-90-ErbB2, which appears to involve Hsp70 and the ubiquitin ligase CHIP.



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  		FIG. 1.
Hsp90 inhibition induces ErbB2 degradation via a proteasome pathway with disruption of Hsp90/ErbB2. A, the effect of GA on ErbB2 stability was demonstrated in [35S]methionine-labeled ARVMs by immunoprecipitating (IP) the ErbB2 protein at the indicated time points after GA treatment. Immunoprecipitates were electrophoresed, and autoradiograms were quantified by densitometry. By 24 h the Hsp90 inhibitor GA (1 µM) caused degradation of ~70% ErbB2 (*, p < 0.002; n = 4). Con, control. B, GA caused a similar decrease in total ErbB2 in whole cell extracts as detected by Western blot (WB). ErbB2 degradation was partially prevented by the proteasome inhibitor MG-132 (50 µM) (*, p < 0.001; **, p < 0.05; n = 4). C, ARVM lysates before and after treatment with GA were subjected to immunoprecipitation with an ErbB2 antibody and then probed after electrophoresis with an Hsp90 antibody. Under "No Ab" conditions, virtually no nonspecific precipitation of Hsp90 was observed (data not shown). Specific Hsp90-ErbB2 interaction is observed in the precipitates with anti-ErbB2. This complex is dissociated in the presence of GA. The blot shown is representative of three separate experiments. D, ARVM lysates before (Con) or after treatment with GA were subjected to immunoprecipitation and Western blot analysis as indicated, demonstrating that GA induces an association of ErbB2 with the ubiquitin ligase CHIP (molecular mass 35 kDa) and Hsp70 (middle lane).

 
GA is a quinone that not only inhibits Hsp90-ATP interactions but is also known to undergo redox cycling, increasing the generation of reactive oxygen species (13). To exclude nonspecific oxidant effects of GA on Hsp90 client protein stability, other agents that alter redox state were investigated. Equimolar concentrations of the quinone menadione, a potent ROS generator, did not alter ErbB2 stability (data not shown). Similarly, there was no effect of the antioxidants Mn(III) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP) or N-acetyl cysteine on ErbB2 degradation induced by GA (data not shown). In addition, the non-quinone Hsp90 inhibitor radicicol reproduced the same effect on ErbB2 degradation as GA (data not shown). Thus, oxidative stress does not appear to play a role in the disruption of Hsp90/client protein interactions.

Hsp90 Inhibition Alters Expression of Other Hsp90 Client Proteins—GA treatment caused alterations in the expression and function of other Hsp90 client proteins such as Akt, HSF-1, and Hsp70 that mediate cell growth and survival. With GA treatment, Akt was degraded (Fig. 2A) and HSF-1 was activated. HSF-1 is maintained in an inactive state by Hsp90 and, upon dissociation from this complex, is subjected to hyperphosphorylation with the subsequent activation of transcription (14). Consistent with this literature, GA caused a shift in the molecular weight of HSF-1 within 15 min of treatment (1Fig. 2C), with the subsequent induction of Hsp70 expression after several hours (Fig. 2B). These observations demonstrate that Hsp90 exerts a generalized chaperone activity in ARVMs.



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  		FIG. 2.
Effects of Hsp90 inhibition on expression and activity of Hsp90 client proteins. ARVM lysates were proved for Akt (A), Hsp70 (B), or HSF-1 (C) before (Con, control) or after treatment with GA. C, lysates probed for HSF-1 after GA treatment at early time points showed a shift in its apparent molecular mass consistent with the hyperphosphorylation and activation of HSF-1. The Western blots (WB) are representative of three separate experiments.

 
Metabolic Inhibition Triggers Hsp90 Client Protein Degradation—Treatment of ARVM with either 2-deoxyglucose or antimycin A causes a significant decrease in myocyte ATP (Fig. 3, A and B). Metabolic inhibition resulted in degradation of Hsp90 client proteins (Fig. 3C), with ErbB2 exhibiting the greatest sensitivity of these client proteins. Within 1 h of 2-deoxy-D-glucose or antimycin A treatment, ErbB2 degradation was evident. The Hsp90 client proteins Raf-1 and Akt were also degraded in response to ATP depletion, although later than ErbB2. The effect of metabolic stress on ErbB2/Hsp interaction was examined by immunoprecipitation and Western blot analysis 30 min and 1 h after 2-deoxyglucose treatment or 15 min and 1 h after antimycin A treatment. Like GA, metabolic stress caused dissociation of Hsp90 from ErbB2 (Fig. 3D), with evidence of Hsp70/ErbB2 interaction by1hof metabolic inhibition (data not shown).



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  		FIG. 3.
Effects of metabolic inhibition on Hsp90 client protein stability. A and B, luciferase assay was used to measure the concentration of ATP in ARVMs during treatment with 2-deoxy-D-glucose (DOG) (A) or antimycin A (B), respectively. Both compounds caused a significant decrease in [ATP] (*, p < 0.01; #, p < 0.05). C, the effect of metabolic inhibition on Hsp90 client protein stability was examined by SDS-PAGE. The phosphorylation state of {alpha}-AMPK was examined as an indicator of metabolic stress. D, the effect of metabolic stress on ErbB2/Hsp90 interaction was examined by immunoprecipitation (IP) and Western blot (IB) analysis 15 min, 30 min, and 1 h after 2-deoxyglucose (DOG) or antimycin A treatments. The amount of immunoprecipitated ErbB2 was examined by reprobing with anti-ErbB2. The blots are representative of at least three separate experiments. Con, control.

 
With ATP depletion we did not see any increase in Hsp70 expression (Fig. 3C), and the expression of Hsp70 and Hsp90 was stable until 8 h, when there was evidence of Hsp90 degradation (Fig. 3C). In contrast to Hsp90 or Hsp70, actin expression was stable.

Effects of Metabolic Inhibition on ErbB2 Ligand-dependent Signaling—We examined the effect of antimycin A or 2-deoxy-D-glucose on the activation of an intracellular kinase signal by exogenous NRG-1{beta} and epidermal growth factor (EGF), ligands that activate and signal in part through ErbB2 (Fig. 4, A and B). Under control conditions, Akt and ERK are activated by NRG (50 ng/ml) and 10% fetal calf serum, with EGF causing only weak activation of ERK. After 1 h of antimycin A treatment, all ligand-dependent signaling was suppressed. 2-Deoxy-D-glucose treatment for 2 h suppressed ERK and Akt signaling by NRG and fetal calf serum but had no clear effect on EGF-dependent ERK activation. As kinase signaling is itself dependent on ATP, this result could be explained by either a disruption of Hsp90-ErbB2 interactions leading to ErbB2 degradation or simply to an insufficient supply of ATP to allow kinase activity. Therefore, we allowed cells to recover by washing away antimycin A and adding back glucose. After 1 h of antimycin A treatment followed by ATP recovery, there was no increase in ErbB2. Interestingly, recovery induced Akt phosphorylation but did not restore NRG-dependent signaling (Fig. 4C). Phosphorylation of ERK1/2 was completely suppressed by antimycin A treatment, and induced during ATP recovery, with no discernible effect of NRG in the presence of antimycin A (Fig. 4C). NRG activation of JNKs was also suppressed by antimycin A treatment, although JNK phosphorylation could be seen with ATP recovery alone, similar to ERK1/2 (Fig. 4C). The increased phosphorylation of AMPK during antimycin A treatment demonstrates that ATP levels are sufficient to support protein kinase activity in general. It is interesting that neuregulin alone appears to induce phosphorylation of {alpha}-AMPK, which has not previously been reported. Collectively, these data demonstrate that ATP depletion activates Hsp90 client protein degradation, which renders myocytes insensitive to growth factor-stimulated kinase signaling.



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  		FIG. 4.
Effects of metabolic inhibition on ErbB2 signaling. Phosphorylated Akt (pAkt), phosphorylated ERK (pErk), and total Akt were examined by Western blot analysis after treatment with 2 µM antimycin A (AA) for 1 h (A) or 5 mM 2-deoxy-D-glucose for 2 h (B) followed by growth factor stimulation for 15 min. A, antimycin A treatment for 1 h suppressed NRG, EGF, and serum signaling. B, 2 h of 2-deoxy-D-glucose treatment suppressed ERK and Akt signaling activated by NRG and fetal calf serum but had no clear effect on EGF-dependent ERK activation. The blots are representative of four separate experiments. C, ATP recovery was induced by washing in 1,000 mg/liter D-glucose Dulbecco's modified Eagle's medium for 30 min prior to stimulation by NRG-1{beta}. ErbB2 expression did not increase with recovery, and NRG-1{beta} activation of pAkt was not restored by ATP recovery. Blots are representative of three separate experiments. Con, control. pJNK, phosphorylated JNK; pAMPK, phosphorylated AMPK.

 
Physiological Stress Causes ErbB2 Degradation—To examine whether physiological metabolic stress can activate Hsp90 client protein degradation, we examined the effect of electrical pacing and {beta}-AR on ErbB2 expression (Fig. 5). Both {beta}-AR stimulation and mechanical stimulation increase oxygen consumption and ATP utilization (15), and we have shown previously under these conditions that myocytes are insensitive to neuregulin 1-{beta} cytoprotection (11). Neither pacing alone (p > 0.05) nor {beta}-AR stimulation (NE plus prazosin at 100 nM) (p > 0.05) altered ErbB2 expression. However the combination caused marked ErbB2 degradation (Fig. 5B; #, p < 0.05; *, p < 0.01), explaining the resistance to NRG cytoprotection under these conditions.



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  		FIG. 5.
Metabolic stress causes ErbB2 degradation. A, ErbB2, Akt, and actin expression were examined by immunoblot in myocytes electrically stimulated or left quiescent for 4 h in the absence (0 µM) or presence of NE (0.1 and 1 µM). The blots are representative of three separate experiments. B, ErbB2 expression was analyzed by densitometry and normalized to untreated myocytes without (W/O) pacing. ErbB2 expression decreased in ARVM electrically paced and treated with a pharmacologic concentration of NE. #, p < 0.05; *, p < 0.01; n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our present findings demonstrate a role for Hsp90 in regulating the stability of ErbB2 and other Hsp90 client proteins in relation to cellular energy charge. This observation is consistent with the recently published report on the relative affinity of Hsp90 for ATP versus ADP (5) and the well known importance of ATP binding and hydrolysis by Hsp90 in the formation and function of protein heterocomplexes (16, 17). This result may provide some insight into the physiological role of Hsp90 chaperone activity, placing Hsp90 into a group of proteins that sense energy charge and modulate growth factor responsiveness.

We have focused our attention on how ATP depletion alters Hsp90 stabilization of ErbB2, a member of the ErbB receptor tyrosine kinase family that plays a key role in cell proliferation, differentiation, survival, and migration and has emerged as an important contributing factor in tumorigenesis (18). In the heart, ErbB2 is critical for cardiac development (19) as well as for the maintenance of cardiac function postnatally (20). Our findings that ErbB2 expression in cardiac myocytes is stabilized by Hsp90 and that degradation is mediated by the CHIP/Hsp70/proteosomal pathway is predictable based upon in vitro work with cell lines (9, 21). However to our knowledge this is the first demonstration of this mechanism in a cell expressing endogenous levels of ErbB2 and Hsp90, as well as its activation by metabolic stress in any cell type.

This is only one of the mechanisms for ErbB2 degradation, as both EGF- and antibody-induced ErbB2 degradation are mediated through the recruitment of the ubiquitin ligase Cbl to ErbB2 autophosphorylated at Tyr-1112 (22, 23). The kinase activity of ErbB2 is essential for Cbl-dependent ubiquitinylation and down-regulation. In contrast, ErbB2 down-regulation induced by the Hsp90 inhibitor GA requires the kinase domain but not the kinase activity of ErbB2, and the C-terminal tail carrying the Cbl-binding site is dispensable (23). Thus, it appears that distinct mechanisms are involved in ligand-associated ErbB2 degradation versus that induced by the disruption of Hsp90 chaperone activity.

We found that the sensitivity of Hsp90 client proteins differs in response to metabolic stress, with some proteins showing earlier evidence of degradation than others. For both conditions of metabolic stress, the rank order of sensitivity appears to be ErbB2 > Raf-1 > Akt. One explanation for this finding is that Hsp90 client protein interactions have differential sensitivity to ATP/ADP ratios. In contrast to the effect of GA, Hsp70 expression did not increase with ATP depletion. Thus, HSF-1 and the classic heat shock response are not activated by ATP depletion in cardiac myocytes, in contrast to what has been observed in kidney cells (24). ErbB2/Hsp90 dissociation occurred earlier in the course of ATP depletion than activation of the metabolic sensor AMPK (25, 26), indicating that a relatively subtle degree of metabolic stress is sufficient to disrupt Hsp90 chaperone activity.

We found that {beta}-AR stimulation in the presence of electrical pacing caused ErbB2 degradation, similar to pharmacologic ATP depletion. Both stimuli increased myocyte ATP utilization and oxygen consumption. We have shown previously that electrically pacing rat myocytes at physiological frequencies (5 Hz) has no effect on cell survival but sensitizes myocytes to the pro-apoptotic effect of {beta}-AR stimulation (11). Moreover, paced myocytes in the presence of high [NE], a state of metabolic stress, are insensitive to the pro-survival effect of NRG. Our finding that metabolic stress induces ErbB2 degradation may explain this NRG insensitivity. We speculate that the "ATP-sensing" activity of Hsp90 may be the underlying mechanism by which these metabolically stressed myocytes regulate their responsiveness to exogenous growth factor stimulation.

The lack of induction of a classic heat shock response by metabolic stress/ATP depletion in cardiac myocytes is an interesting finding that deserves further investigation. Pharmacologic disruption of Hsp90 chaperone activity is clearly sufficient to induce activation of HSF-1 and up-regulate Hsp70 expression. This difference may explain why metabolic stress would appear to sensitize cells to apoptosis, whereas GA and radicicol protect against cell death (27–29); Hsp70 overexpression alone is sufficient to protect against ischemia and other cytotoxic stresses (30). A more detailed understanding of the requisite step in HSF-1 activation, beyond dissociation from Hsp90, may lead to strategies to induce cardioprotection under clinically relevant conditions of metabolic stress.

Overall, these data are consistent with an ATP sensing activity for Hsp90 that functions to regulate growth and survival signaling in relation to cellular energy charge. As Hsp90 is ubiquitously expressed, we suspect that a similar sensitivity of Hsp90 client proteins to ATP depletion will be found in cell types other than cardiac myocytes. In support of this hypothesis, we found that an ErbB2 overexpression mammary carcinoma cell line showed evidence of ErbB2 degradation when treated with antimycin A.2 We propose that the "ATP sensing activity" of Hsp90 may play an important role in the regulation of tissue growth both physiologically (e.g. fetal development) as well as pathologically (e.g. tumorigenesis). Hsp90 stabilizes a number of oncogenes that regulate organ and organism growth, including pp60v-src protein-tyrosine kinase (31), c-Raf-1 serine/threonine kinase (32), and the estrogen receptor (33). Our data predict that under conditions of continuous high levels of ATP (e.g. obesity), Hsp90 stabilization is increased, potentiating growth pathways. Inversely, perhaps disruption of Hsp90 chaperone function is one mechanism by which caloric restriction and exercise suppress oncogenesis (34–37).


   FOOTNOTES
 
* This study was funded by National Institutes of Health Grant HL68144 and a grant from the Juvenile Diabetes Research Foundation (to D. B. S.). This work was presented in part at the American Heart Association Annual Scientific Sessions, November 9–12, 2003 in Orlando, Florida. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Whitaker Cardiovascular Inst. and Center for Molecular Stress Response, Boston University Medical Center, 650 Albany St., Boston, MA 02118. Tel.: 617-6388071; Fax: 617-414-1719; E-mail: douglas.sawyer{at}bmc.org.

1 The abbreviations used are: Hsp, heat shock protein; AMPK, AMP-activated protein kinase; ARVM, adult rat ventricular myocyte; {beta}-AR, {beta}-adrenergic receptor; CHIP, carboxyl terminus of Hsc70-interacting protein; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GA, geldanamycin; HSF, heat shock factor; JNK, c-Jun N-terminal kinase; NE, norepinephrine; NRG, neuregulin. Back

2 X. Peng, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Lin Zhong, Keith Tornheim, and Nathan LeBrasseur for helpful discussions. We also thank Khaleque Md-Abdul and Nicholas Grammatikakis for technical advice.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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