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Human Molecular Genetics Advance Access originally published online on April 27, 2006
Human Molecular Genetics 2006 15(11):1876-1883; doi:10.1093/hmg/ddl110
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Pharmacologic and genetic inhibition of hsp90-dependent trafficking reduces aggregation and promotes degradation of the expanded glutamine androgen receptor without stress protein induction

Monzy Thomas1, Jennifer M. Harrell2, Yoshihiro Morishima2, Hwei-Ming Peng2, William B. Pratt2 and Andrew P. Lieberman1,*

1Department of Pathology and 2Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA

* To whom correspondence should be addressed at: Department of Pathology, University of Michigan Medical School, 1301 Catherine, 4233 Medical Science 1, Ann Arbor, MI 48109, USA. Tel: +1 7349361887; Fax: +1 7347636476; Email: liebermn{at}umich.edu

Received March 3, 2006; Accepted April 17, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular chaperone hsp90 has emerged as an important therapeutic target in cancer and neurodegenerative diseases, including the polyglutamine expansion disorders, because of its ability to regulate the activity, turnover and trafficking of many proteins. For neurodegenerative disorders associated with protein aggregation, the rationale has been that inhibition of hsp90 by geldanamycin and related compounds activates heat shock factor 1 (HSF1) to induce the production of the chaperones hsp70 and hsp40 that promote disaggregation and protein degradation. However, we show here that geldanamycin blocks the development of aggregates of the expanded glutamine androgen receptor (AR112Q) of Kennedy disease in Hsf1–/– mouse embryonic fibroblasts where these chaperones are not induced. Geldanamycin is additionally known to inhibit hsp90-dependent protein trafficking and to promote proteasomal degradation of client proteins. Overexpression of the hsp90 cochaperone p23 also promotes AR112Q degradation, and inhibits both AR trafficking and AR112Q aggregation without altering levels of hsp70 or hsp40. The hsp90-dependent trafficking mechanism has been defined, and it is shown that key immunophilin (IMM) components of the trafficking machinery are present in polyglutamine aggregates in cell and mouse models of Kennedy disease. Our results indicate that inhibition of the hsp90-dependent trafficking mechanism prevents aggregation of the expanded glutamine androgen receptor, thereby opening a variety of novel therapeutic approaches to these neurodegenerative disorders.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hsp90 is an abundant molecular chaperone that controls the activity, turnover and trafficking of many proteins, in particular the mediators of signal transduction (1Go), and as such plays a critical role at regulatory points implicated in the pathogenesis of cancer and neurodegenerative diseases. Specific inhibition of hsp90 by drugs like geldanamycin and radicicol leads to the proteasomal degradation of hsp90-associated oncoproteins (2Go), and this is the basis for clinical trials of the hsp90 inhibitor 17-allylamino, 17-demethoxygeldanamycin (17-AAG) in cancer patients (3Go). The hsp90/hsp70-based chaperone machinery (1Go) that forms signaling protein•hsp90 heterocomplexes is also part of the cellular defense against unfolded proteins (4Go), and geldanamycin has been reported to inhibit protein aggregation in models of Huntington (5Go,6Go) and Parkinson diseases (7Go). The neuroprotective effect of geldanamycin is thought to be based on a different outcome of hsp90 inhibition. The chaperone machinery is a negative regulator of heat shock factor 1 (HSF1) (8Go) by maintaining it in an inactive apo-HSF1•hsp90 complex, and treatment of cells with geldanamycin induces an HSF1-dependent heat shock response (5Go–9Go). Because geldanamycin induces heat shock proteins, and overexpression of hsp70 and hsp40 inhibits aggregation of expanded polyglutamine proteins and {alpha}-synuclein (10Go–16Go), it has been proposed that geldanamycin alleviates the phenotype and the accumulation of misfolded proteins in models of Huntington and Parkinson diseases by inducing a stress response (5Go–7Go,16Go). However, this hypothesis has not been critically tested. If inhibition of hsp90 is to become a therapeutic approach for the treatment of protein aggregation neurodegenerative disorders, it is important to define the mechanistic basis for this effect.

Like Huntington disease, Kennedy disease, or spinal and bulbar muscular atrophy, is one of nine neurodegenerative disorders that results from the expansion of a CAG/glutamine tract in the coding region of otherwise unrelated genes (17Go). In Kennedy disease, an expanded glutamine tract near the amino terminus of the androgen receptor (AR) leads to hormone-dependent protein misfolding, aggregate formation in the cell cytoplasm and nucleus (18Go), and the predominant loss of lower motor neurons in the brainstem and spinal cord of the affected males (19Go). Recent studies showed that the hsp90 inhibitor 17-AAG prevents aggregation of the expanded glutamine AR and ameliorates motor neuron degeneration in a transgenic mouse model of the Kennedy disease (20Go). We sought to determine whether stress protein induction was required for this effect. Our results indicate that hsp90 inhibitors prevent aggregation of the expanded glutamine AR independent of a stress response, and instead, act by inhibiting hsp90 mediated retrograde trafficking. These data define an alternative mechanism of action for the hsp90 inhibitors, and suggest novel therapeutic strategies for treating protein aggregation neurodegenerative disorders.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We first sought to determine whether induction of a stress response was required for hsp90 inhibitors to repress ligand-dependent aggregation of the expanded glutamine AR. AR112Q expressed in wild-type or Hsf1–/– mouse embryonic fibroblasts (MEFs) at similar levels formed protein aggregates after treatment with the synthetic AR ligand R1881 (Fig. 1A and B). Aggregates first appeared several hours after the addition of ligand, were readily detectable after 6 h, and reached a maximal number after 16–24 h. Although Hsf1–/– MEFs do not exhibit a heat shock response, they constitutively express multiple heat shock proteins at levels equivalent to wild-type cells (21Go). Treatment of both wild-type and Hsf1–/– MEFs with either geldanamycin or radicicol blocked the ligand-dependent aggregation of AR112Q (Fig. 1A and B). These hsp90 inhibitors markedly increased the expression of hsp70 and hsp40 in wild-type but not in Hsf1–/– MEFs (Fig. 1C). Thus, the ability of the hsp90 inhibitors to block AR112Q aggregation was not dependent upon induction of a stress response and increased chaperone protein levels.


Figure 1101
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  		Figure 1. Hsp90 inhibitors prevent AR112Q aggregation without inducing a stress response in Hsf1 null MEFs. (A–C) Wild-type and Hsf1–/– MEFs expressing AR112Q were treated with 10 nM R1881 for 30 min at 4°C, then with 10 µM geldanamycin, 10 µm radicicol or vehicle control for an additional 30 min at 4°C. Incubation was continued for 8 h at 37°C. (A) AR was visualized by indirect immunofluorescence (green) in untreated cells, or following treatment with R1881 alone or R1881 plus geldanamycin. Nuclei were stained with DAPI. (B) Quantification of AR expressing cells with protein aggregates (mean±SD) after treatment with R1881 alone, or in combination with geldanamycin or radicicol. Inset shows western blot of HSF1 expression in wild-type and Hsf1–/– MEFs. Hsp90 serves as a loading control. (C) Lysates were examined by western blot for expression of AR, hsp90, the stress inducible form of hsp70 and hsp40. ß-tubulin serves as a loading control.

 
If induction of stress proteins was not necessary for the reduction of AR112Q aggregation by hsp90 inhibitors, then the basis for this effect must lie in a different hsp90 action. The dynamic assembly of hsp90 and its cofactors into heterocomplexes is required for retrograde, dynein-dependent movement of hsp90 client proteins such as the glucocorticoid receptor (GR) (Fig. 5A) (22Go,23Go). Geldanamycin and radicicol inhibit the translocation of several transcription factors, including glucocorticoid, androgen, and aryl hydrocarbon receptors and p53 (24Go). In this hsp90-dependent trafficking system, the hsp90-binding IMMs link client protein•hsp90 complexes to the dynein/dynactin motor proteins (22Go–24Go). Movement is inhibited by the overexpression of dynamitin (dyt) (22Go–24Go), a subunit of the dynein-associated dynactin complex that blocks dynein function by dissociating the motor protein from its cargoes (25Go).


Figure 1105
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  		Figure 5. hsp90-dependent trafficking system and therapeutic strategies to functionally disrupt it. Steroid hormone receptor•hsp90 heterocomplexes are linked by IMMs and dyt to dynein for retrograde movement along the microtubules (A). This model is based on studies of the GR, but is applicable to other hsp90 client proteins including the AR. Cytoplasmic dynein is the motor protein that processes along microtubules in retrograde movement to the nucleus. Dynein is a large multi-subunit complex (1.2 MDa) comprised of two heavy chains (HC), three intermediate chains (IC), and light chains (not shown). The IMM links to steroid hormone receptor-bound hsp90 via its TPR domain (solid black crescent), and links to dyt via its PPIase domain (dotted crescent). Proposed therapeutic strategies to functionally disrupt this hetercomplex and prevent retrograde movement include inhibition of hsp90 or HDAC6 (B), or use of bulky IMM ligands (C). Figure modified from Pratt et al. (24Go).

 
Aggresome formation is also dependent on dynein-mediated retrograde movement of proteins along the microtubules (26Go). Aggresomes form when coordinated protein disposal, mediated by protein chaperones, dynein-dependent trafficking and proteasomal degradation becomes overwhelmed (26Go–28Go). Aggresome formation by an amino-terminal fragment of the expanded glutamine AR is inhibited by nocodozole and by the overexpression of dyt (29Go), showing that dynein-mediated retrograde trafficking along microtubules is required. Thus, inhibition of trafficking by the hsp90 inhibitors could be an important mechanism by which aggregation of mutant AR is prevented (30Go). To test this hypothesis, we sought to inhibit hsp90-dependent movement and AR112Q aggregation by affecting hsp90 function in a novel manner without the use of inhibitors that induce heat shock proteins.

The ubiquitous hsp90 cochaperone p23 binds to the ATP-dependent conformation of hsp90 (31Go). When it is overexpressed, p23 stabilizes receptor•hsp90 heterocomplexes in vivo through its interaction with hsp90 (32Go). Yang and DeFranco (33Go) have shown that GR•hsp90 heterocomplex assembly/disassembly must be very dynamic for retrograde movement to occur. Overexpression of p23 makes this cycle much less dynamic (32Go), and slowed the rate of ligand-dependent GFP-GR translocation from a t1/2 of ~4 min in control cells to a t1/2 of ~20 min in cells overexpressing p23 (Fig. 2A). The rate of ligand-dependent AR translocation was also slowed by the overexpression of p23 in both HeLa and SH-SY5Y neuroblastoma cells (Fig. 2B and C). In contrast to geldanamycin and radicicol, overexpression of p23 did not induce a stress response in HeLa (Fig. 2D) or SH-SY5Y cells (data not shown).


Figure 1102
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  		Figure 2. p23 inhibits ligand-dependent movement of steroid hormone receptors to the nucleus. (A) Left panel shows GFP-GR visualized in 3T3 fibroblasts co-expressing p23 (+p23) or vector control (–p23) following treatment with 1 µM dexamethasone (+DEX) or ethanol (–DEX) for 10 min (left panel). Right panel shows the rate of GR nuclear translocation (mean±SEM) in 3T3 fibroblasts co-expressing p23 (filled boxes) or empty vector (open boxes). Differences significant at P=0.01 (*) or at P=0.05 (**). (B) Left panel shows AR24Q in HeLa cells co-expressing p23 (+p23) or vector control (–p23) following treatment with 10 nM R1881 (+R1881) or ethanol (–R1881) for 30 min. Right panel shows the rate of AR nuclear translocation (mean±SEM) in HeLa cells co-expressing p23 (filled boxes) or empty vector (open boxes). Differences significant at P<0.002 at 20 and 30 min. (C) AR24Q nuclear translocation (mean±SEM) in SH-SY5Y cells co-expressing p23 or empty vector. Differences significant at P<0.02 at 20 min. (D) Exogenously expressed p23 does not induce a stress response. Lysates from HeLa cells expressing hexahistidine-tagged p23 (lane 2), or following treatment with 10 µM geldanamycin or 10 µM radicicol (lanes 3, 4) for 8 h were compared with control cells (lane 1) for expression of hsp90, the stress inducible form of hsp70, hsp40 and exogenous p23 (anti-hexahistidine antibody). GAPDH serves as a loading control.

 
To determine whether p23 overexpression affects the aggregation of expanded glutamine AR, cells expressing AR112Q plus p23 or vector control were treated with ligand for 24 h. Overexpression of p23 in HeLa and SH-SY5Y cells significantly reduced AR112Q aggregation as detected by indirect immunofluorescence (Fig. 3A) and quantification (Fig. 3B and C). Separation of cell lysates into pelleted and soluble fractions after high-speed centrifugation showed that p23 overexpression resulted in a marked decrease of AR112Q in the pellet but not in the supernatant (Fig. 3D). The p23-dependent reduction in biochemically detectible AR112Q aggregates was inhibited by the treatment with proteasome inhibitor MG132 (Fig. 3E). As p23 forms a multiprotein complex with hsp90 and the expanded glutamine AR (Fig. 3F; 20Go), our data suggest that p23 stabilization of the AR112Q•hsp90 heterocomplex inhibits AR112Q aggregation and maintains a soluble form of the receptor that can be degraded by the proteasome. Consistent with this interpretation, p23 overexpression significantly decreased glutamine length-dependent caspase 3 activation by an amino-terminal AR112Q fragment (Fig. 3G).


Figure 1103
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  		Figure 3. p23 inhibits AR112Q aggregation and promotes its degradation by the proteasome. (A) HeLa cells expressing AR112Q plus hexahistidine-tagged p23 or vector control were treated with 10 nM R1881 for 24 h. AR (green) and exogenous p23 (red, anti-hexahistidine antibody) were visualized by indirect immunofluorescence, and nuclei were stained with DAPI. (B, C) Quantification of AR expressing HeLa (B) or SH-SY5Y cells (C) with protein aggregates (mean±SD). Differences significant at P<0.005. (D) Protein lysates were prepared from HeLa cells co-expressing AR112Q plus p23 (+p23) or vector control (–p23) 24 h after treatment with 10 nM R1881. Lysates were separated into supernatant (lanes 1 and 3) and pelleted fractions (lanes 2 and 4) by centrifugation (20 000 g, 15 min) and analyzed by western blot for AR expression. (E) HeLa cells co-expressing AR112Q and p23 were treated with 10 nM R1881 plus 10 µM MG132 or vehicle control for 16 h. Lysates were separated into supernatant (lanes 1 and 3) and pelleted fractions (lanes 2 and 4), and analyzed by western blot for AR expression. (F) Cytosol prepared from HeLa cells expressing AR112Q and p23 was immunoadsorbed with non-immune (NI) or anti-AR (immune, I) IgG. Co-immunoprecipitated proteins were visualized by western blot using antibodies against AR, hsp90 and p23. (G) HeLa cells were co-transfected with truncated AR16Q or AR112Q plus p23 or control vector. Caspase 3 activity was determined 48 h post-transfection. Data are mean±SD from one of three similar experiments. Overexpression of p23 significantly decreased caspase activation by AR112Q (P=0.024).

 
Hsp90-dependent movement utilizes the hsp90-binding IMMs such as FKBP52 and protein phosphatase 5 (PP5), an IMM homolog. These proteins link hsp90-bound client proteins to the dynein/dynactin motor complex (22Go–24Go). We next checked if FKBP52 and PP5 concentrated with dynein in AR112Q aggregates. Dynein, PP5 and FKBP52 co-localized with AR112Q aggregates in cell culture (Fig. 4A), consistent with the entry of AR112Q into aggregates as trafficking complexes. Similar findings were made in a knock-in mouse model of Kennedy disease in which 113 CAG repeats were targeted to exon 1 of the AR gene (34Go). In mutant male mice, a subset of intranuclear aggregates of the expanded glutamine AR in skeletal muscle contained PP5 and hsp90 (Fig. 4B).


Figure 1104
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  		Figure 4. Localization of IMMs in AR112Q aggregates. (A) HeLa cells expressing AR112Q were treated with 10 nM R1881 for 24 h. Co-localization of AR with dynein (p a–d), PP5 (p e–h) and FKBP52 (p i–l) was determined by indirect immunofluorescence. Nuclei were stained with DAPI. (B) Immunohistochemical detection, used to circumvent background autofluorescence, demonstrates intranuclear aggregates (arrows) containing AR, PP5 and hsp90 in skeletal muscle of 18-month-old male AR113Q knock-in mice. (Original magnification 400x).

 
We have shown here that geldanamycin and radicicol prevent ligand-dependent aggregation of the expanded glutamine AR in cells that do not express HSF1. Thus, under conditions where pharmacologic inhibition of hsp90 does not cause a stress response, aggregation is nevertheless inhibited. Geldanamycin and radicicol also inhibit hsp90-dependent AR movement (30Go), and we propose that it is inhibition of the dynein-dependent trafficking that accounts for the block of aggregation. Overexpression of the hsp90 cochaperone p23 inhibits receptor translocation without inducing stress proteins and also inhibits AR112Q aggregation. AR112Q aggregates contain IMMs (FKBP52 and PP5) that link hsp90 to the dyenin/dynactin motor protein complex. We were unable to dissociate inhibition of hsp90-mediated trafficking from enhanced proteasomal degradation of AR, indicating that these two processes are mechanistically linked and that both are dependent upon molecular chaperones.

Our observations indicate that inhibition of trafficking may be a productive therapeutic approach to the treatment of neurodegenerative diseases caused by the proteins with expanded glutamine tracts (Fig. 5). One approach is to exploit the geldanamycin/radicicol-type of hsp90 inhibitors that are being developed as anti-cancer drugs. A second approach might be through the use of histone deacetylase 6 (HDAC6) inhibitors. Aggresomes formed by CFTR-{Delta}F508, the most common allele causing cystic fibrosis, are not formed in cells with siRNA knockdown of HDAC6 because of a failure to load polyubiquitinated, misfolded proteins onto the dynein motor for transport to aggresomes (35Go). Targeted inhibition of HDAC6 leads to acetylation of hsp90 and disruption of its function (36Go), and specific HDAC6 inhibitors are being developed (37Go). A third approach might be to disrupt the IMM connection to dynein. Both biochemical and genetic studies have shown that the peptidylproline isomerase (PPIase) domain of the IMM links it to the dynein/dynactin motor complex and that disruption of this linkage inhibits receptor trafficking (22Go–24Go). The immunosuppressive drug FK506 binds specifically in the PPIase site of the IMM without inhibiting trafficking (24Go), but the addition of bulky substituents that permit FK506 binding to the IMM might inhibit the linkage to dynein and disrupt both trafficking and aggregation by polyglutamine tract proteins. Both FK506 derivatives of this sort (38Go) and bivalent FK506 compounds (39Go) that may inhibit aggregation are being developed, albeit for different purposes and with different rationales for their use.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
NIH 3T3 fibroblasts and HeLa cells were purchased from the American Type Culture Collection, Hsf1–/– MEFs (21Go) were from Dr Ivor Benjanmin (University of Utah). Phenol red-free Dulbecco's modified Eagle's medium was from Life Technologies, and charcoal-stripped calf serum was from Hyclone. Trans-Fast transfection kit was from Promega and Fugene 6 was from Roche. JJ3 monoclonal IgG against p23 was provided by Dr David Toft (Mayo Clinic, Rochester, MN, USA). Plasmids encoding AR24Q and AR112Q were from Dr Kenneth Fischbeck (N.I.H.). Plasmids encoding amino-terminal fragments AR16Q and AR112Q (40Go) were from Diane Merry (Thomas Jefferson University). p23 expression plasmid was generated by PCR amplification of full-length human cDNA, subcloned into the expression vector pcDNA4/HisMax-C (Invitrogen). Antibodies against AR (N-2O), hsp90 and ß-tubulin were from Santa Cruz. Anti-hsp40 and an antibody specific for the stress inducible form of hsp70 (SPA-812) were from Stressgen. UPJ56 antiserum against FFKBP52 was from Dr Karen Leach (Pfizer), and PP5 antiserum was from Dr Michael Chinkers (University of South Alabama). Anti-dynein antibody, radicicol, DHT and MG132 were from Sigma. FITC and Texas red conjugated secondary antibodies were from Jackson Immuno Research. R1881 was from Perkin Elmer.

DNA transfection
NIH 3T3 fibroblasts were grown on coverslips to ~50% confluency, and transfected using Trans-Fast. HeLa and SH-SY5Y cells were grown in 35 mM dishes and transfected using Fugene 6. Media was removed after 18 h, and cells were washed and then re-fed with phenol red-free DMEM with 10% charcoal-stripped calf serum for an additional 24 h. Prior to use, charcoal-stripped serum was delipidated by adding 1 g of fumed silica (Sigma) per 100 ml and then mixing overnight at 4°C. The next day, the fumed silica was removed by centrifugation at 4000g for 5 min and the resulting supernatant was filter sterilized.

Protein expression analysis
Cells were treated as indicated, washed with PBS and lysed in RIPA buffer. Protein lysates were separated into supernatant and pelleted fractions by centrifugation at 20 000g for 15 min at 4°C. Protein samples were electrophoresed through a 10% SDS-polyacrylamide gel and transferred to Immunobilon-P membranes using a semi-dry transfer apparatus. Immunoreactive proteins were detected by chemiluminescence.

For immunofluorescence, cells were grown on coverslips or chambered slides. Following fixation and staining, cells were visualized using a Zeiss Axioplan 2 imaging system. AR aggregation was scored by determining the percentage of transfected cells with visible protein aggregates. For each experiment and each condition, more than 100 transfected cells were scored in a blinded manner in triplicate. Fluorescence images were captured using a Zeiss LSM 510 confocal microscope under a 63x water immersion objective. For immunohistochemistry, skeletal muscle from AR113Q knock-in mice (34Go) was frozen in liquid nitrogen-chilled isopentane and cut in 7 µM thick sections. Staining with primary antibodies was visualized using a Vectastain ABC kit (Vector Laboratories).

For isolation of AR112Q•hsp90•p23 complexes, HeLa cells transiently expressing AR112Q and p23 were harvested 48 h post-transfection. Cytosol (120 µl) prepared in 10 mM Hepes, pH 7.35, 1 mM EDTA, 20 mM molybdate containing Complete Mini (1 tablet/10 ml) and 1 mM PMSF was immunoadsorbed for 2 h at 4°C with 4 µg of non-immune (NI) rabbit IgG or purified rabbit IgG (N-20) against the AR. The immunopellets were washed four times with 10 mM TES, pH 7.6, 50 mM NaCl, 4 mM EDTA, 10% glycerol, resolved on 12% SDS-polyacrylamide gels and transferred to an Immobilon-P membrane. The membrane was probed with 1 µg/ml N-20 for AR, 1 µg/ml AC88 for hsp90, and 0.1% JJ3 mouse ascites for p23. The immunoblots were then incubated a second time with the appropriate [125I]-conjugated counterantibodies for autoradiography.

Steroid hormone receptor translocation
We scored steroid hormone receptor translocation as described previously (22Go,30Go), using a value of four for nuclear fluorescence much greater than cytoplasmic fluorescence, three for nuclear fluorescence greater than cytoplasmic fluorescence, two for nuclear fluorescence equal to cytoplasmic fluorescence, one for nuclear fluorescence less than cytoplasmic fluorescence, and zero for nuclear fluorescence much less than cytoplasmic fluorescence. The reported translocation scores represent the means±S.E. from three experiments in which more than 50 cells per condition per experiment were scored.

Cell death assay
We determined caspase 3 activity 48 h post-transfection with truncated AR16Q or AR112Q by measuring cleavage of the fluorescent substrate DEVD-AFC using the ApoTarget caspase-3/CPP 32 fluorometric protease assay kit (Biosource International). Fluorescence intensity was measured using a Fluoroskan Ascent FL fluorometer (Thermo Electron).

Statistical analysis
We analyzed the data by unpaired t-tests to determine the significance of differences in aggregation and Bonferroni t-tests to evaluate differences in translocation.


   ACKNOWLEDGEMENTS
 
The authors thank Drs David Toft, Kenneth Fischbeck, Karen Leach, Zhigang Yu and Michael Chinkers for providing reagents used in this work, Dr Ivor Benjamin for Hsf1–/– MEFs, Dr Diane Robins for review of their manuscript, the University of Michigan Microscopy and Image Analysis Laboratory for assistance with confocal imaging, and Elizabeth Horn for the preparation of the figures.

This work was supported by a Beeson Career Development Award from the American Federation for Aging Research and the National Institutes of Health (K08 AG024758 to A.P.L.), by a grant from the Muscular Dystrophy Association (#3393 to A.P.L.), and by National Institutes of Health grant CA28010 (to W.B.P.). H.M.P. was supported by Grant ES08365 from the National Institutes of Health.

Conflict of Interest statement: The authors have no conflicts of interest to disclose.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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