Title: Hsp27 Inhibits Bax Activation and Apoptosis via a Phosphatidylinositol 3-Kinase-dependent Mechanism
Abstract: Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism. Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism. Hsp27, a member of the small heat shock protein family, is induced by stress and protects against heat shock, oxidative stress, hypertonic stress, and other forms of cellular injury in numerous cell types including neurons (1Akbar M.T. Lundberg A.M. Liu K. Vidyadaran S. Wells K.E. Dolatshad H. Wynn S. Wells D.J. Latchman D.S. de Belleroche J. J. Biol. Chem. 2003; 278: 19956-19965Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 2Benn S.C. 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Apoptotic signal transduction pathways converge at the mitochondrion to cause membrane permeabilization, an event regulated by mutually antagonistic members of BCL-2 protein family that includes Bcl-2 and Bax (11Bernardi P. Petronilli V. Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). In renal epithelial cells, as in other cell types, the balance between death and survival is determined by the ratio of these apoptosis-stimulating and suppressing BCL-2 proteins (12Korsmeyer S.J. Shutter J.R. Veis D.J. Merry D.E. Oltvai Z.N. Semin. Cancer Biol. 1993; 4: 327-332PubMed Google Scholar). Renal ischemia in vivo (13Wolfs T.G. de Vries B. Walter S.J. Peutz-Kootstra C.J. van Heurn L.W. Oosterhof G.O. Buurman W.A. Am. J. Transplant. 2005; 5: 68-75Crossref PubMed Scopus (40) Google Scholar) as well as exposure to metabolic inhibitors in vitro causes mitochondrial membrane injury and Bax activation in epithelial cells (14Ruchalski K. Mao H. Li Z. Wang Z. Gillers S. Wang Y. Mosser D.D. Gabai V. Schwartz J.H. Borkan S.C. J. Biol. Chem. 2006; 281: 7873-7880Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar). In healthy cells, Bax exists as a 21-kDa cytosolic monomer. After a conformational change in both the carboxyl and amino termini, Bax forms toxic oligomers, translocates to the mitochondrial outer membrane (16Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (549) Google Scholar), and either forms de novo pores or opens existing mitochondrial membrane channels that release pro-apoptotic proteins such as cytochrome c and apoptosis-inducing factor (16Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (549) Google Scholar, 17Hsu Y.T. Youle R.J. J. Biol. Chem. 1998; 273: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 18De Giorgi F. Lartigue L. Bauer M.K. Schubert A. Grimm S. Hanson G.T. Remington S.J. Youle R.J. Ichas F. FASEB J. 2002; 16: 607-609Crossref PubMed Scopus (233) Google Scholar, 19Adachi M. Higuchi H. Miura S. Azuma T. Inokuchi S. Saito H. Kato S. Ishii H. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: 695-705Crossref PubMed Scopus (97) Google Scholar). Leakage of pro-apoptotic mediators normally sequestered in the intramembranous mitochondrial space results in activation of caspase-dependent and independent pathways that ultimately precipitate cell death (11Bernardi P. Petronilli V. Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 20Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar). Recent evidence suggests that Bax activation is regulated by site-specific serine phosphorylation by kinases known to mediate apoptosis. Specifically, serine phosphorylation by Akt, a potent anti-apoptotic serine/threonine kinase, inactivates Bax (21Gardai S.J. Hildeman D.A. Frankel S.K. Whitlock B.B. Frasch S.C. Borregaard N. Marrack P. Bratton D.L. Henson P.M. J. Biol. Chem. 2004; 279: 21085-21095Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar), whereas serine phosphorylation at another site by glycogen synthase kinase 3β (GSK3β), 2The abbreviations used are: GSK3β, glycogen synthase kinase 3β; PI3-kinase, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; p-Ser, phosphoserine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AIF, apoptosis-inducing factor. an Akt substrate, promotes Bax activation and apoptosis (22Linseman D.A. Butts B.D. Precht T.A. Phelps R.A. Le S.S. Laessig T.A. Bouchard R.J. Florez-McClure M.L. Heidenreich K.A. J. Neurosci. 2004; 24: 9993-10002Crossref PubMed Scopus (325) Google Scholar). Taken together, these reports suggest that stressors that inactivate Akt and activate GSK3β promote Bax activation by a dual mechanism. Several laboratories have investigated the mechanism of Hsp27-mediated cytoprotection. Specifically, Hsp27 inhibits caspase 3 and 9 activation and reduces apoptosome formation (8Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (376) Google Scholar, 23Garrido C. Bruey J.M. Fromentin A. Hammann A. Arrigo A.P. Solary E. FASEB J. 1999; 13: 2061-2070Crossref PubMed Scopus (447) Google Scholar, 24Bruey J.M. Ducasse C. Bonniaud P. Ravagnan L. Susin S.A. Diaz-Latoud C. Gurbuxani S. Arrigo A.P. Kroemer G. Solary E. Garrido C. Nat. Cell Biol. 2000; 2: 645-652Crossref PubMed Scopus (840) Google Scholar). However, each of these protective effects operates downstream of mitochondrial membrane injury and cannot explain the observation by multiple investigators that Hsp27 inhibits cytochrome c release after pro-apoptotic stress (8Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (376) Google Scholar, 23Garrido C. Bruey J.M. Fromentin A. Hammann A. Arrigo A.P. Solary E. FASEB J. 1999; 13: 2061-2070Crossref PubMed Scopus (447) Google Scholar, 24Bruey J.M. Ducasse C. Bonniaud P. Ravagnan L. Susin S.A. Diaz-Latoud C. Gurbuxani S. Arrigo A.P. Kroemer G. Solary E. Garrido C. Nat. Cell Biol. 2000; 2: 645-652Crossref PubMed Scopus (840) Google Scholar, 25Concannon C.G. Orrenius S. Samali A. Gene Expr. 2001; 9: 195-201Crossref PubMed Scopus (189) Google Scholar). Despite these intriguing reports, the mechanism by which Hsp27 antagonizes mitochondrial injury and prevents apoptosis is not understood. Hsp27 has been closely associated with Akt. However, most reports emphasize the effect of Akt on the phosphorylation and activation of Hsp27 rather than vice versa (26Konishi H. Matsuzaki H. Tanaka M. Takemura Y. Kuroda S. Ono Y. Kikkawa U. FEBS Lett. 1997; 410: 493-498Crossref PubMed Scopus (237) Google Scholar, 27Suga H. Nakajima K. Shu E. Kanno Y. Hirade K. Ishisaki A. Matsuno H. Tanabe K. Takai S. Akamatsu S. Kato K. Oiso Y. Kozawa O. Arch. Biochem. Biophys. 2005; 438: 137-145Crossref PubMed Scopus (8) Google Scholar). At least in neutrophils, Hsp27 and Akt co-exist in a large multiprotein complex, suggesting that Akt and Hsp27 regulate one another (28Wu R. Kausar H. Johnson P. Montoya-Durango D.E. Merchant M. Rane M.J. J. Biol. Chem. 2007; 282: 21598-21608Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Despite their apparent co-localization in these cells, direct evidence that Hsp27 modifies Akt activity has not been shown. This prompted us to speculate that Hsp27 inhibits Bax-mediated mitochondrial membrane injury by promoting the activation of phosphatidyl inositol 3 kinase (PI3-kinase), a major upstream regulator of Akt. In the present study we report that Hsp27 expression reduces mitochondrial membrane injury and improves cell survival after stress, whereas Hsp27 down-regulation has the opposite effect on these parameters. Hsp27 expression enhances PI3-kinase activity, promotes Akt-Bax interaction, and inhibits Bax activation, oligomerization, and translocation to mitochondria. Importantly, each of the protective effects ascribed to Hsp27 is prevented by the addition of a PI3-kinase inhibitor. We propose that Hsp27-mediated regulation of PI3-kinase is responsible for the potent protective effects of Hsp27 on the outer mitochondrial membrane during stress. Materials and Reagents—All reagents were purchased from Sigma-Aldrich unless otherwise specified. Cell Culture—Previously characterized proximal tubular epithelial cell lines derived from the immortalized mouse; (29Sinha D. Wang Z. Price V.R. Schwartz J.H. Lieberthal W. Am. J. Physiol. Renal Physiol. 2003; 284: 488-497Crossref PubMed Scopus (61) Google Scholar), from the normal human kidney (HK-2; ATCC, catalog #CRL-2190), or human embryonic kidney cells (293) were maintained as previously described (29Sinha D. Wang Z. Price V.R. Schwartz J.H. Lieberthal W. Am. J. Physiol. Renal Physiol. 2003; 284: 488-497Crossref PubMed Scopus (61) Google Scholar). Metabolic Stress—Cells were incubated for 15 min to 2 h at 37 °C in glucose-free medium (Dulbecco's modified Eagle's medium, Invitrogen 23800-014) that contained 5 mm sodium cyanide and 5 mm 2-deoxy-d-glucose as previously described by our laboratory (see Schwartz and co-workers (30Li F. Mao H.P. Ruchalski K.L. Wang Y.H. Choy W. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2002; 283: 917-926Crossref PubMed Scopus (66) Google Scholar) and Ref. 31Schwartz J.H. Shih T. Menza S.A. Lieberthal W. J. Am. Soc. Nephrol. 1999; 10: 2297-2305Crossref PubMed Google Scholar). This maneuver results in apoptosis due to the rapid, persistent ATP depletion to <10% of base-line ATP content and is reversible with the removal of cyanide and the addition of exogenous glucose (32Wang Y.H. Borkan S.C. Am. J. Physiol. 1996; 270: F1057-F1065PubMed Google Scholar). In control, parallel medium changes were performed using Dulbecco's modified Eagle's medium. Selective Hsp27 Expression—Wild type human Hsp27 expression was increased in renal cells using a previously characterized adenoviral construct (33Martin J.L. Hickey E. Weber L.A. Dillmann W.H. Mestril R. Gene Expr. 1999; 7: 349-355PubMed Google Scholar). Control cells were infected with empty vector (AdTR5/GFP). Infection efficiency was >90–99% as determined by direct visualization of GFP in cells infected with 40–100 multiplicity of infection. Cells were infected with adenovirus for 16 h at 37 °C in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum (FBS) followed by a 24-h washout period during which the medium was replaced with fresh Dulbecco's modified Eagle's medium containing 10% FBS. Increased Hsp27 expression was confirmed by immunoblot analysis and was titrated to approximate the level of expression detected after sublethal heat stress (43.0 °C × 45 min (34Wang Y. Knowlton A.A. Christensen T.G. Shih T. Borkan S.C. Kidney Int. 1999; 55: 2224-2235Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar)) followed by 16 h of recovery (Fig. 1A). RNA Interference—HK-2 cells were transected with siRNA against Hsp27 (sc-29350) or control siRNA (sc-37007) from Santa Cruz Biotechnology (Santa Cruz, CA) as per the manufacturer's protocol. Briefly, subconfluent (50–60%) HK-2 cells grown in antibiotic free medium were transfected using siRNA Transfection Medium (sc-36868) and Transfection Reagent (sc-29528) at an siRNA concentration of 10 nm. After 8 h, cells were washed and cultured for 72 h in complete medium. Mitochondrial Membrane Injury/Cytosolic Cytochrome c and Apoptosis-inducing Factor (AIF) Leakage—To assess mitochondrial membrane injury, the leakage of mitochondrial cytochrome c and AIF was measured by immunoblot analysis of cytosolic fractions using digitonin permeabilization as previously reported by us (30Li F. Mao H.P. Ruchalski K.L. Wang Y.H. Choy W. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2002; 283: 917-926Crossref PubMed Scopus (66) Google Scholar, 35Borkan S.C. Emami A. Schwartz J.H. Am. J. Physiol. 1993; 265: F333-F341PubMed Google Scholar, 36Ruchalski K. Mao H. Singh S.K. Wang Y. Mosser D.D. Li F. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2003; 285: C1483-C1493Crossref PubMed Scopus (82) Google Scholar). Co-localization of Bax and the Mitochondria with Confocal Microscopy—Subconfluent, live cells grown on glass coverslips were incubated with Mitotracker Green-FM (600 nm; Molecular Probes), a mitochondrial specific marker, for 30 min at 37 °C. Cells were then fixed in methanol (4 °C for 20 min), exposed to an anti-Bax (N-20) antibody (Santa Cruz, #sc-493), detected with a Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), and routine immunohistochemistry was performed (30Li F. Mao H.P. Ruchalski K.L. Wang Y.H. Choy W. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2002; 283: 917-926Crossref PubMed Scopus (66) Google Scholar). Confocal microscopy was used to localize Bax and Mitotracker Green using an Olympus confocal microscope (FV300-IX70) with a 60× UPlan apochromatic objective (Olympus, Melville, NY). Images were processed using Fluoview software. Immunoblot Analysis and Immunoprecipitation—Immunoblot analysis was performed as described previously (31Schwartz J.H. Shih T. Menza S.A. Lieberthal W. J. Am. Soc. Nephrol. 1999; 10: 2297-2305Crossref PubMed Google Scholar). Commercially available antibodies were used to detect Hsp27 (1:1000 dilution, StressGen Biotechnologies, Victoria BC, Canada, catalog #SPA-803 or Santa Cruz Biotechnology, catalog number sc-1048), apoptosis-inducing factor (1:250 dilution, Santa Cruz Biotechnology, catalog #sc-13116), cytochrome c (1:1000, Research Diagnostics, clone 6H2.B4), caspase-3 (1:1000 dilution, Cell Signaling, #9665), cleaved caspase-3 (1:1000 dilution, Cell Signaling, #9664), activated Bax (6A7; 1:1,000 dilution; Trevingen, Inc., Gaithersburg, MD, catalog #2281-MC-100), total Bax (5B7; 1:500 dilution; Pharmingen, catalog #556467), β-actin (1:750 dilution, Abcam, Inc., Cambridge, MA, catalog #ab6276-100), AKT/p-Ser473-AKT (1:1000 dilution, Cell Signaling, #9272/#9271), PI3-kinase p85 (1:1000 dilution, Santa Cruz, catalog #423), GSK3β/p-Ser9-GSK3β (1:1000 dilution, Cell Signaling, catalog #9315 and 9336), β-actin (Sigma catalog A5316), glyceraldehyde-3-phosphate dehydrogenase (N-14; Santa Cruz, catalog #sc-20356), and β-tubulin (1:1500 dilution, Sigma, catalog #A5441). Secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) were used in combination with a chemiluminescence detection method (Amersham Biosciences) to visualize specific protein bands. For co-immunoprecipitation, 7.5 × 106 cells were harvested in buffer containing 150 mm NaCl, 10 mm Tris HCl, 5 mm EDTA, 1 mm EGTA, 1% Triton X-100, 0.5% Nonidet P-40, with protease inhibitors at pH 7.4 as previously described by our laboratory (30Li F. Mao H.P. Ruchalski K.L. Wang Y.H. Choy W. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2002; 283: 917-926Crossref PubMed Scopus (66) Google Scholar). Lysates were centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatants were collected. Samples containing 400 μg of total protein were incubated with 5 μg of antibody directed against Hsp27 (Santa Cruz, anti-goat or StressGen, anti-rabbit), PI3-kinase p85a (Santa Cruz), active Bax (Trevingen, Gaithersburg, MD, clone YTH-6A7), or total Bax (Lab Vision/Neomarkers, Fremont, CA, clone 5B7) overnight at 4 °C. Immunoprecipitates were collected by the addition of protein G- or A-agarose (ImmunoPure Immobilized Protein G or A, Pierce) to each sample followed by incubation at 4 °C for 1 h. Complexes were harvested by centrifugation and washed 4× with lysis buffer or phosphate-buffered saline. Finally, the beads were resuspended in 1× SDS sample buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol) and heated at 100 °C for 10 min before separation by SDS-PAGE and immunoblot analysis. In selected experiments cells were lysed in either Tris-buffered saline or 2% CHAPS lysis buffer, and the identical buffer was then used in all subsequent steps as described above. Cell Viability—Viability was assessed with a modified colorimetric technique (3-(4,5 dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay) that is based on the ability of live cells to convert MTT, a tetrazolium compound, into purple formazan crystals as described by our laboratory (37Lieberthal W. Triaca V. Koh J.S. Pagano P.J. Levine J.S. Am. J. Physiol. 1998; 275: F691-F702PubMed Google Scholar). Chemical Cross-linking—Cell extracts were prepared in 1× Tris-buffered saline containing protease and phosphatase inhibitors by subjecting 2.5 × 106 cells to lysis with one freeze-thaw cycle in a dry-ice-ethanol bath and 37 °C water bath, respectively. Lysates were then purified by centrifugation at 12,000 × g for 10 min at 4 °C. Samples containing 50 μg of protein were suspended for 30 min at room temperature in 50 μl of freshly dissolved disuccinimidyl tartrate (DST) with a 6.4-Å spacer arm (Pierce) in DMSO to achieve a final concentration of 2 mm. DMSO without DST was added to control. The reactions were then quenched by the addition of 15 mm Tris, pH 7.4. The samples were prepared for immunoblot analysis as described above and were probed with an anti-Bax antibody directed against total Bax (mouse monoclonal, Clone 5B7, Lab Vision/Neomarkers). PI3-kinase Assay—Cell extracts harvested in the presence of Nonidet P-40 were obtained from human kidney (HK-2) cells infected with adenovirus containing either Hsp27 or empty adenovirus that were incubated overnight with an antibody directed against the non-catalytic PI3-kinase p85 subunit (Santa Cruz) followed by a 1-h incubation with protein A/G-agarose. The immunoprecipitates were washed 3 times with lysis buffer then twice in buffer that contained 10 mm Tris·HCl, 100 mm NaCl, and 1 mm EDTA, pH 7.5, then once in PI3-kinase assay buffer that contained 20 mm Tris·HCl, 100 mm NaCl 10 mm, MgCl2, 0.1 mm EGTA, 100 mm vanadate, 20 mm ATP, 200 mm adenosine, pH 7.5. After the last wash, 10 μl of sonicated, PI substrate (l-α-phosphatidylinositol, Sigma, 1 mg/ml in 10 mm HEPES, pH 7.5) was added to each sample, and the samples were incubated for 10 min on ice. The reaction was carried out at 25 °C for 20 min by adding 40 ml of PI3-kinase assay buffer containing 10 μCi of [γ32P]ATP and then quenched with 100 ml of 1 m HCl. Phospholipids were extracted once with 200 μl of CHCl3/MeOH (1:1) (8000 rpm × 3 min) using a bench-top centrifuge, and then the lower (organic) phase was transferred to a fresh Eppendorf tube and extracted twice with 160 μl of 1 m HCl/CH3OH (1:1 vol:vol). The organic phase was dried under 100% nitrogen gas and re-suspended in 20 μl of CHCl3/MeOH (1:1 vol:vol). Phosphorylated products were resolved on oxalate-impregnated silica 60 plates that were wetted with 1.2% potassium oxalate mixed (1:1 vol:vol) with CHCl3/MeOH and 4 m NH4OH (9:7:2) for 2 h, the gel was air-dried, and then autoradiography was performed. Radioactive bands representing PI3-kinase were separated and quantified using a liquid scintillation analyzer (Packard-PerkinElmer Life Sciences). Densitometry and Statistical Analysis—After digitizing the immunoblot image (Hewlett-Packard, Desk Scan II), band densities were quantified using NIH ImageQuant Software. Data are expressed as the means ± S.E. Comparison of two groups was performed using a two-tailed Student's t test. Results involving more than one group were compared using 2-way analysis of variance and were then analyzed with the Fisher post hoc test. A result was considered significant if p < 0.05. Hsp27 Expression Regulates Cell Survival After Metabolic Stress—In renal tubular epithelial cells, non-lethal heat stress (43 °C × 45 min with 16 h recovery; Fig. 1A, third lane)or infection with 40 multiplicity of infection adenovirus containing human Hsp27 (first lane) exhibited a similar, marked increase in Hsp27 content compared with either uninfected cells (fourth lane) or cells infected with adenovirus containing empty vector (second lane). To reproduce the physiologic cellular response to stress, Hsp27 expression was increased by adenovirus to the same level as that afforded by heat stress in all subsequent experiments. In contrast, siRNA directed against Hsp27 markedly decreased steady state Hsp27 content compared with non-sense siRNA (Fig. 1B). Importantly, metabolic stress did not itself induce Hsp27 within the time frame examined in our studies (Fig. 1C). Apparent differences in the amount of immunoreactive Hsp27 detected in control (first lane, Fig. 1A), non-sense siRNA (first lane, Fig. 1B), and in normal cells at base line (first lane, Fig. 1C) are due to differences in exposure time that were optimized for each study. To assess its effect on survival, Hsp27 content was manipulated in epithelial cells after 1–1.5 h of metabolic stress, an insult that causes apoptosis (14Ruchalski K. Mao H. Li Z. Wang Z. Gillers S. Wang Y. Mosser D.D. Gabai V. Schwartz J.H. Borkan S.C. J. Biol. Chem. 2006; 281: 7873-7880Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 32Wang Y.H. Borkan S.C. Am. J. Physiol. 1996; 270: F1057-F1065PubMed Google Scholar). Only 50% of control (empty vector) survived after stress (Fig. 1D). Hsp27 expression increased cell survival after stress to 87 ± 6%% (p < 0.05; n = 3). In contrast, Hsp27 knockdown with specific siRNA decreased survival by almost 50% compared with non-sense siRNA (38 ± 6 versus 71 ± 3%, p < .05; n = 4). Differences in relative cell survival between empty vector and non-sense siRNA are due to differences in the duration of stress (1 h empty vector versus 1.5 h non-sense siRNA) that were intentionally selected to optimize cytoprotection or injury caused by changes in Hsp27 expression. Hsp27 Prevents Mitochondrial Membrane Injury and Caspase 3 Activation—Metabolic stress resulted in the progressive leakage of mitochondrial cytochrome c and AIF into the cytosol of cells exposed to digitonin (Fig. 2A) as previously reported by us (36Ruchalski K. Mao H. Singh S.K. Wang Y. Mosser D.D. Li F. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2003; 285: C1483-C1493Crossref PubMed Scopus (82) Google Scholar). Compared with control, Hsp27 up-regulation markedly decreased mitochondrial cytochrome c and AIF leakage both during and after stress. In addition, enhanced Hsp27 expression decreased stress-induced activation of caspase 3 (Fig. 2B), a downstream consequence of cytochrome c leakage. In this study the decrease in pro-caspase 3 content parallels cytochrome c leakage and is a reliable estimate of caspase activity in these cells (30Li F. Mao H.P. Ruchalski K.L. Wang Y.H. Choy W. Schwartz J.H. Borkan S.C. Am. J. Physiol. Cell Physiol. 2002; 283: 917-926Crossref PubMed Scopus (66) Google Scholar). Taken together, these results demonstrate that Hsp27 ameliorates stress-induced mitochondrial membrane injury and caspase activation, hallmarks of apoptosis. Hsp27 alters Bax activation, oligomerization, and translocation to mitochondria. Because Bax is a primary cause of stress-induced mitochondrial membrane injury after metabolic stress (14Ruchalski K. Mao H. Li Z. Wang Z. Gillers S. Wang Y. Mosser D.D. Gabai V. Schwartz J.H. Borkan S.C. J. Biol. Chem. 2006; 281: 7873-7880Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar, 16Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (549) Google Scholar, 38Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), the effect of Hsp27 on Bax activation was examined. Compared with base line, metabolic stress markedly increased active Bax in cell lysates as assessed by a 6A7 epitope-specific antibody (Fig. 3A, upper panel) without changing total Bax content (bottom panel). Hsp27 expression markedly reduced conformational Bax activation during and after metabolic stress (Fig. 3A). In contrast, RNA interference-mediated Hsp27 knockdown resulted in a marked increase in activated Bax after stress (Fig. 3B, upper panel) without affecting total Bax (bottom panel). Hsp27 expression also reduced the formation of toxic Bax oligomers during and after stress (Fig. 3C). In the absence of stress, however, neither the detergent-free buffer nor the chemical cross-linker caused Bax oligomerization in normal or Hsp27-expressing cells. In intact cells, dual channel confocal microscopy was used to co-localize Bax with mitochondria, visualized as overlap between active Bax, stained in red with cy3, and