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• W2056164078 abstract "The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion. The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion. Acute ischemic renal failure is characterized by detachment of proximal tubule epithelial cells from the substratum, permitting backleak of glomerular filtrate and intra-tubular obstruction (1Brady H.R. Brenner B.M. Clarkson M.R. Lieberthal W. Brenner B.M. The Kidney. 6th Ed. 1. W. B. Saunders, Philadelphia, PA2000: 1201-1262Google Scholar, 2Noiri E. Gailit J. Sheth D. Magazine H. Gurrath M. Muller G. Kessler H. Goligorskyx M.S. Kidney Int. 1994; 46: 1050-1058Abstract Full Text PDF PubMed Scopus (106) Google Scholar, 3Racusen L.C. Clin. Exp. Pharmacol. Physiol. 1998; 25: 273-275Crossref PubMed Scopus (23) Google Scholar). ATP depletion, an in vitro model of ischemia, disrupts the cytoskeleton, causing filamentous actin to be replaced by macromolecular aggregates of short actin polymers (4Sogabe K. Roeser N.F. Davis J.A. Nurko S. Venkatachalam M.A. Weinberg J.M. Am. J. Physiol. 1996; 271: F292-F303PubMed Google Scholar, 5Borkan S.C. Wang Y.H. Lieberthal W. Burke P.R. Schwartz J.H. Am. J. Physiol. 1997; 272: F347-F355PubMed Google Scholar, 6Herget-Rosenthal S. Hosford M. Kribben A. Atkinson S.J. Sandoval R.M. Molitoris B.A. Am. J. Physiol. 2001; 281: C1858-C1870Crossref PubMed Google Scholar). In intact organisms (3Racusen L.C. Clin. Exp. Pharmacol. Physiol. 1998; 25: 273-275Crossref PubMed Scopus (23) Google Scholar, 7Racusen L.C. Fivush B.A. Li Y.L. Slatnik I. Solez K. Lab. Investig. 1991; 64: 546-556PubMed Google Scholar) and in culture (3Racusen L.C. Clin. Exp. Pharmacol. Physiol. 1998; 25: 273-275Crossref PubMed Scopus (23) Google Scholar), viable cells that originate from the proximal tubule have been harvested after an ischemic insult, suggesting that cell death is not a prerequisite for detachment. The tri-partite structure composed of integrins, the cytoskeleton, and cell-cell contact sites permit epithelial cells to adhere to the extracellular matrix. Attachment is directly mediated by the interaction between α and β integrins, transmembrane proteins that reside on the basolateral surface, and arginine-glycine-aspartic acid (RGD) residues of extracellular matrix proteins (8Simon E.E. Liu C.H. Das M. Nigam S. Broekelmann T.J. McDonald J.A. Am. J. Physiol. 1994; 267: F612-F623PubMed Google Scholar). Adhesion is stabilized by the actin cytoskeleton by anchoring to the cytosolic domain of integrins (9Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (431) Google Scholar) as well as to cell-cell contact sites (10Martin-Bermudo M.D. Brown N.H. J. Cell Sci. 2000; 113: 3715-3723Crossref PubMed Google Scholar). This cytoskeleton-integrin connection is formed and regulated by the focal adhesion complex, which is located at the basolateral region of the epithelial cell (11Mueller S.C. Kelly T. Dai M.Z. Dai H.N. Chen W.T. J. Cell Biol. 1989; 109: 3455-3464Crossref PubMed Scopus (68) Google Scholar, 12Giannone G. Jiang G. Sutton D.H. Critchley D.R. Sheetz M.P. J. Cell Biol. 2003; 163: 409-419Crossref PubMed Scopus (223) Google Scholar). This complex, comprised of talin, vinculin, paxillin, focal adhesion kinase, Src, and other proteins, is crucial for regulating cell attachment. Stress-induced alterations in the activity and/or intracellular distribution of focal adhesion complex proteins cause detachment (13Gailit J. Colflesh D. Rabiner I. Simone J. Goligorsky M.S. Am. J. Physiol. 1993; 264: F149-F157PubMed Google Scholar). During ischemia in vivo or ATP depletion in vitro, many cytoskeletal-associated component proteins are either denatured or form large macromolecular aggregates (5Borkan S.C. Wang Y.H. Lieberthal W. Burke P.R. Schwartz J.H. Am. J. Physiol. 1997; 272: F347-F355PubMed Google Scholar, 14Aufricht C. Lu E. Thulin G. Kashgarian M. Siegel N.J. Van Why S.K. Am. J. Physiol. 1998; 274: F268-F274PubMed Google Scholar, 15Hinshaw D. Armstrong B.C. Burger J. Beals T. Hyslop P. Am. J. Pathol. 1988; 132: 479-488PubMed Google Scholar, 16Kabakov A.E. Gabai V.L. Experientia. 1993; 49: 706-713Crossref PubMed Scopus (41) Google Scholar, 17Molitoris B.A. Dahl R. Geerdes A. Am. J. Physiol. 1992; 263: F488-F495PubMed Google Scholar, 18Shelden E.A. Borrelli M.J. Pollock F.M. Bonham R. J. Am. Soc. Nephrol. 2002; 13: 332-341Crossref PubMed Scopus (18) Google Scholar). Following exposure to solvents, toxins, heat, or ATP depletion, the increased burden of detergent-insoluble proteins contributes to cell dysfunction and impairs recovery (16Kabakov A.E. Gabai V.L. Experientia. 1993; 49: 706-713Crossref PubMed Scopus (41) Google Scholar, 19Hightower L.E. Cell. 1991; 66: 191-197Abstract Full Text PDF PubMed Scopus (955) Google Scholar, 20Kabakov A.E. Budagova K.R. Latchman D.S. Kampinga H.H. Am. J. Physiol. 2002; 283: C521-C534Crossref PubMed Scopus (65) Google Scholar, 21Hendrick J.P. Hartl F.U. FASEB J. 1995; 9: 1559-1569Crossref PubMed Scopus (209) Google Scholar). Perturbations in protein conformation correlate with their solubility in non-selective detergents such as Triton X-100 (14Aufricht C. Lu E. Thulin G. Kashgarian M. Siegel N.J. Van Why S.K. Am. J. Physiol. 1998; 274: F268-F274PubMed Google Scholar, 17Molitoris B.A. Dahl R. Geerdes A. Am. J. Physiol. 1992; 263: F488-F495PubMed Google Scholar, 20Kabakov A.E. Budagova K.R. Latchman D.S. Kampinga H.H. Am. J. Physiol. 2002; 283: C521-C534Crossref PubMed Scopus (65) Google Scholar). Intracellular proteins that denature or aggregate during ATP depletion are likely targets for rescue by molecular chaperones (20Kabakov A.E. Budagova K.R. Latchman D.S. Kampinga H.H. Am. J. Physiol. 2002; 283: C521-C534Crossref PubMed Scopus (65) Google Scholar, 22Rassow J. von Ahsen O. Bomer U. Pfanner N. Trends Cell Biol. 1997; 7: 129-133Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 23Beissinger M. Buchner J. Biol. Chem. 1998; 379: 245-259PubMed Google Scholar). Structural proteins that comprise the cytoskeleton or cell adhesion sites are likely to interact with molecular chaperones because: 1) disruption of these cellular sites is one of the earliest morphologic features of ischemia (24Van Why S.K. Mann A.S. Ardito T. Siegel N.J. Kashgarian M. Am. J. Physiol. 1994; 267: F75-F85Crossref PubMed Google Scholar, 25Molitoris B.A. Am. J. Physiol. 1991; 260: F769-F778PubMed Google Scholar, 26Fish E.M. Molitoris B.A. Am. J. Physiol. 1994; 267: F566-F572PubMed Google Scholar, 27Shelden E.A. Weinberg J.M. Sorenson D.R. Edwards C.A. Pollock F.M. J. Am. Soc. Nephrol. 2002; 13: 2667-2680Crossref PubMed Scopus (18) Google Scholar) and ATP depletion (5Borkan S.C. Wang Y.H. Lieberthal W. Burke P.R. Schwartz J.H. Am. J. Physiol. 1997; 272: F347-F355PubMed Google Scholar, 28Kroshian V.M. Sheridan A.M. Lieberthal W. Am. J. Physiol. 1994; 266: F21-F30PubMed Google Scholar); 2) ischemia increases the content of detergent-insoluble cytoskeletal-associated proteins (14Aufricht C. Lu E. Thulin G. Kashgarian M. Siegel N.J. Van Why S.K. Am. J. Physiol. 1998; 274: F268-F274PubMed Google Scholar, 16Kabakov A.E. Gabai V.L. Experientia. 1993; 49: 706-713Crossref PubMed Scopus (41) Google Scholar, 17Molitoris B.A. Dahl R. Geerdes A. Am. J. Physiol. 1992; 263: F488-F495PubMed Google Scholar, 20Kabakov A.E. Budagova K.R. Latchman D.S. Kampinga H.H. Am. J. Physiol. 2002; 283: C521-C534Crossref PubMed Scopus (65) Google Scholar, 29Gabai V.L. Kabakov A.E. Mosin A.F. Tissue Cell. 1992; 24: 171-177Crossref PubMed Scopus (24) Google Scholar); and 3) ischemia promotes the formation of large protein aggregates (5Borkan S.C. Wang Y.H. Lieberthal W. Burke P.R. Schwartz J.H. Am. J. Physiol. 1997; 272: F347-F355PubMed Google Scholar, 16Kabakov A.E. Gabai V.L. Experientia. 1993; 49: 706-713Crossref PubMed Scopus (41) Google Scholar, 26Fish E.M. Molitoris B.A. Am. J. Physiol. 1994; 267: F566-F572PubMed Google Scholar). In contrast to the pathologic changes in the actin cytoskeleton and cell-cell contact sites that accompany ATP depletion, alterations in focal adhesion complex proteins are poorly characterized. Intracellular proteins that are denatured or aggregate during ATP depletion are likely targets for rescue by molecular chaperones (20Kabakov A.E. Budagova K.R. Latchman D.S. Kampinga H.H. Am. J. Physiol. 2002; 283: C521-C534Crossref PubMed Scopus (65) Google Scholar, 22Rassow J. von Ahsen O. Bomer U. Pfanner N. Trends Cell Biol. 1997; 7: 129-133Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 23Beissinger M. Buchner J. Biol. Chem. 1998; 379: 245-259PubMed Google Scholar, 50Mifflin L.C. Cohen R.E. J. Biol. Chem. 1994; 269: 15710-15717Abstract Full Text PDF PubMed Google Scholar). Hsp72, a known cytoprotectant protein, is induced by renal ischemia in vivo (30Emami A. Schwartz J.H. Borkan S.C. Am. J. Physiol. 1991; 260: F479-F485PubMed Google Scholar) and acts as a molecular chaperone, binding and repairing non-native proteins (20Kabakov A.E. Budagova K.R. Latchman D.S. Kampinga H.H. Am. J. Physiol. 2002; 283: C521-C534Crossref PubMed Scopus (65) Google Scholar, 23Beissinger M. Buchner J. Biol. Chem. 1998; 379: 245-259PubMed Google Scholar, 31Hartl F.U. Martin J. Curr. Opin. Struct. Biol. 1995; 5: 92-102Crossref PubMed Scopus (154) Google Scholar). Although the chaperone function of hsp72 is well characterized, its role in protecting proteins involved in cell attachment has not been previously described. The present study evaluated the hypothesis that hsp72 protects cell attachment, at least in part, by interacting with proteins that comprise the focal adhesion complex. ATP depletion caused hsp72 and paxillin (an adaptor protein that localizes to the focal adhesion plaque) to shift from the Triton X-100-soluble protein fraction (containing primarily cytosolic proteins) to the Triton X-100-insoluble pool (containing cytoskeletal and other structural proteins). In addition, ATP depletion caused the reversible re-distribution of talin, vinculin, and paxillin from focal adhesion plaques into the cytosol. Loss of focal adhesion staining was associated with cell detachment. Prior heat stress prevented the loss of paxillin from the detergent-soluble protein pool, increased paxillin extractability from the detergent-insoluble pool, enhanced paxillin staining in focal adhesion plaques during recovery from ATP depletion, and increased cell attachment (p < 0.05). Both heat stress and ATP depletion increased the interaction between paxillin and hsp72, suggesting that protein repair facilitates the re-entry of paxillin into the focal adhesion plaque during recovery from ATP depletion. Selective overexpression of hsp72 also increased its interaction with paxillin and reproduced the protective effect of prior heat stress on the reformation of focal adhesion plaques. These studies suggest that hsp72 prevents focal adhesion protein denaturation and increases the release of focal adhesion components from detergent-insoluble protein aggregates. By preventing primary protein denaturation and secondarily, by increasing the content of functional proteins available for re-assembly of focal adhesions, hsp72 exerts cytoprotective effects on cell attachment. All reagents were obtained from Sigma unless otherwise indicated. Opossum kidney cells were obtained from the American Type Culture Collection (ATCC CRL-1840) and grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Cells were used within 72 h of achieving confluence as assessed by visual inspection with a phase-contrast microscope. In opossum kidney cells, ATP content was reduced by exposure to 1 h of glucose-free medium (catalog 5030) containing sodium cyanide (5 mm) and 2-deoxy-d-glucose (5 mm). This maneuver causes equivalent reductions in ATP content to <10% of the baseline value within 10 min in both control and previously heated renal epithelial cells (32Wang Y.H. Borkan S.C. Am. J. Physiol. 1996; 270: F1057-F1065PubMed Google Scholar). Dulbecco's modified Eagle's medium containing 10 mm glucose without metabolic inhibitors was added to initiate recovery. Parallel medium changes were made in controls using glucose-containing Dulbecco's modified Eagle's medium. To induce hsp72, opossum kidney cells were heated to 43 ± 0.5 °C for 45-60 min in a temperature-regulated incubator followed by incubation at 37 °C for 12-16 h (32Wang Y.H. Borkan S.C. Am. J. Physiol. 1996; 270: F1057-F1065PubMed Google Scholar). Cells were co-infected with adenoviruses containing wild-type human hsp72 and green fluorescent protein (AdTR5/hsp70-GFP) expressed on separate cistrons and a tetracycline-regulated promoter (AdCMV/tTA) kindly provided by Dick Mosser (University of Guelph, Ontario Canada) as recently described (33Mao H. Li F. Ruchalski K. Mosser D.D. Schwartz J.H. Wang Y. Borkan S.C. J. Biol. Chem. 2003; 278: 18214-18220Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This maneuver increases hsp72 content to a level compared with that of heat stress (33Mao H. Li F. Ruchalski K. Mosser D.D. Schwartz J.H. Wang Y. Borkan S.C. J. Biol. Chem. 2003; 278: 18214-18220Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Control cells were co-infected for 24 h at 37 °C with 3 × 107 plaque-forming units/35-mm2 Petri dish of AdTR5/GFP and AdCMV/tTA. Infection efficiency for both viruses was >95% as estimated by direct visualization of GFP. 1The abbreviations used are: GFP, green fluorescent protein; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid. After 24 h exposure to either adenovirus, cells were subjected to ATP depletion as described above. Whole Cell Lysate—Harvested cells were re-suspended in cell lysis buffer (described below) containing a protease mixture (10 units/ml). Cells were sonicated and then centrifuged at 10,000 × g for 5 min at 4 °C. The supernatant was designated as the whole cell lysate. Triton X-100 Extraction—Monolayers of cells in individual, 60-mm2 culture dishes were incubated for 30 min on a rocker at 4 °C with 0.4 ml of Triton X-containing CSK extraction buffer containing (in mm): NaCl, 50; sucrose, 300; PIPES, 10; MgCl2, 3; and Triton X-100 (0.5% v/v) and protease inhibitor mixture (10 units/ml) at pH 7.40. After centrifugation (13,000 rpm × 15 min at 4 °C), the supernatant (the Triton X-soluble protein fraction) was harvested. The pellet (the Triton X-insoluble protein fraction) was sonicated at 4 °C in 0.1 ml of SDS immunoprecipitation (IP) buffer containing (in mm): Tris-HCl, 10; EDTA, 0.5; DTT, 0.5; with DNase, 0.1 mg/ml; RNase, 0.1 mg/ml), a protease inhibitor mixture (10 units/ml) and SDS (1%). Protein components of the focal adhesion plaque including paxillin (catalog number 05-417), talin, (catalog number 05-385), and vinculin (catalog number 05-386) were examined using commercially available antibodies (Upstate Biotechnologies, Lake Placid, NY). Hsp72 (catalog number SPA-810, StressGen Biotechnologies, Victoria, British Columbia, Canada) and hsp27 (Santa Cruz catalog number SC-1049) were detected with mouse monoclonal antibodies specific for these proteins. Immunoreactive proteins were detected with a horseradish peroxidase-based enzyme-linked chemiluminescence system as previously described (33Mao H. Li F. Ruchalski K. Mosser D.D. Schwartz J.H. Wang Y. Borkan S.C. J. Biol. Chem. 2003; 278: 18214-18220Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Immunodetected protein bands were quantified using NIH Image Quant Software after scanning the blot with a densitometer (Hewlett-Packard, Desk Scan II). Species-specific Cy3-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used for all immunohistochemical studies in cells fixed with 2% paraformaldehyde after permeabilization with 1% sodium dodecyl sulfate as previously reported (5Borkan S.C. Wang Y.H. Lieberthal W. Burke P.R. Schwartz J.H. Am. J. Physiol. 1997; 272: F347-F355PubMed Google Scholar). As previously reported (33Mao H. Li F. Ruchalski K. Mosser D.D. Schwartz J.H. Wang Y. Borkan S.C. J. Biol. Chem. 2003; 278: 18214-18220Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 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, 55Wang Y.H. Li F. Schwartz J.H. Flint P.J. Borkan S.C. Am. J. Physiol. 2001; 281: C1667-C1675Crossref PubMed Google Scholar), aliquots of samples obtained from the whole cell lysates or Triton X-100-soluble and -insoluble protein extracts were subjected to co-IP. Samples were diluted to 1 mg/ml with IP buffer (in mm): NaCl, 150; Tris-HCl, 10; EDTA, 5; EGTA, 1; pH 7.40) to which Triton X-100 (0.1%) and a protease inhibitor mixture (10 units/ml; catalog number 539131; Merck Biosciences AG, Germany) were added. Apyrase and EDTA prevent ATP and Mg-mediated release of potential binding partners from hsp72 (33Mao H. Li F. Ruchalski K. Mosser D.D. Schwartz J.H. Wang Y. Borkan S.C. J. Biol. Chem. 2003; 278: 18214-18220Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 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). After centrifugation (14,000 × g for 5 min at 4 °C), 250-500 μg of total protein of supernatant was “pre-cleared” for 1 h with non-immune serum (10 μl/mg protein) obtained from the same host species as the primary antibody. Supernatant was incubated overnight at 4 °C with either anti-paxillin (Santa Cruz, catalog number SC-5574), anti-hsp27 (Stressgen, catalog number SPA-803), or anti-hsp72 (Stressgen, catalog number SPA-810) antibody titrated to permit equivalent yields of paxillin under each experimental condition (8 μg/mg of protein/ml of IP buffer). Immobilized protein G-agarose was added to the solution during the final 2 h of incubation. An agarose pellet was obtained by centrifugation and then washed for 5 min with high stringency buffer (0.1% sodium dodecyl sulfate, 1% deoxycholic acid, 0.5% Triton X-100, 20 mm Tris-HCl, 120 mm NaCl, 25 mm KCl, 5 mm EDTA, 5 mm EGTA, 0.1 mm DTT (“HS-B”)) with 1 m sucrose, pH 7.5. Samples were then washed in a high salt buffer (HS-B + 1 m NaCl) followed by a final wash in low salt buffer (2 mm EDTA, 0.5 mm DTT, 10 mm Tris-HCl, pH 7.5). Samples were mixed with 2× sample buffer in preparation for SDS-PAGE. Blots were then probed for paxillin, hsp27, and hsp72 as described above. Protein concentrations were determined from a colorometric dye binding assay (BCA Assay, Pierce, Rockford, IL) and expressed in milligrams of protein per milliliter. The mean number ± S.E. of cells containing at least 10 focal adhesion plaques was assessed in 4-7 random fields of ATP depleted in two separate experiments by an observer blinded to the experimental conditions. The mean number of detached cells was determined in at least three randomly selected fields of subconfluent monolayers (85-90% confluent) 30 min after 1 or 1.5 h of ATP depletion by an observer blinded to the experimental conditions. Minimal apoptosis (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) or necrosis (32Wang Y.H. Borkan S.C. Am. J. Physiol. 1996; 270: F1057-F1065PubMed Google Scholar) have been observed at this time point in renal epithelial cells subjected to transient ATP depletion. Data are expressed as the mean ± S.E. Experimental groups were compared using an unpaired, two-tailed Student's t test. A p value of <0.05 was considered significant. Analysis was performed with standard statistical software (Excel, Microsoft Corp., Santa Monica, CA). Effect of ATP Depletion and Prior Heat Stress on Cell Attachment—After transient ATP depletion followed by 30 min recovery, numerous detached cells were visible by routine contrast microcopy. After 1 or 1.5 h of ATP depletion, ∼25 and 41% of cells, respectively, were detached (Fig. 1). Prior heat stress significantly reduced the number of detached cells to 9 and 13%, respectively, after both periods of injury (p < 0.05 versus control at each time point). In the absence of ATP depletion, only 4-5% of cells were detached in control or heat-stressed cells (data not shown; p > 0.05). Effect of ATP Depletion and Prior Heat Stress on Focal Adhesion Integrity—Several components of focal adhesion complex were examined by immunohistochemical analyses. ATP depletion caused a marked re-distribution of vinculin, talin, and paxillin from the adhesion plaque into the cytosol. In control, focal vinculin (Fig. 2A) and talin (Fig. 2B) staining in adhesion plaques were visualized. Immediately after ATP depletion, however, virtually no intact adhesion plaques (i.e. vinculin or talin staining) could be observed. After 30 min recovery, many adhesion plaques were again visualized. Similar results were obtained in control cells stained with antibody directed against paxillin, another protein that localizes to the focal adhesion (Fig. 3A). At baseline, both control and previously heat-stressed cells exhibited an identical pattern of paxillin staining in focal adhesions (Fig. 3A, left upper panel versus left lower panel). As described for both vinculin and talin, no focal adhesions were seen immediately after 1 or 1.5 h of ATP depletion in either control or heated cells (images not shown). After 30 min recovery from transient ATP depletion, paxillin staining was more abundant in heat-stressed cells (Fig. 3A, right lower versus right upper panels). To quantify this difference, the number of cells with at least 10 visible focal adhesion plaques was counted before, during, and after ATP depletion (Fig. 3B). At baseline, >10 focal adhesion plaques were observed in virtually all control and previously heated cells. Immediately after 1 h ATP depletion, no adhesion plaques were visible in either experimental group. After 15 or 30 min recovery, however, less than 5% of control cells exhibited ≥10 focal adhesion plaques. In contrast, >10 focal adhesions were visible in nearly 30% of previously heated cells (p < 0.05 versus control at both time points). Because all three focal adhesion proteins exhibited a similar pattern of re-distribution following ATP depletion, paxillin was selected as a marker of focal adhesion integrity in subsequent studies.Fig. 3Effect of prior heat stress on paxillin in focal adhesions following ATP depletion. A, control (upper panels) and previously heated (lower panels) cells were examined at baseline and after 1 h of ATP depletion followed by 30 min recovery. Focal adhesions were visualized using antibody directed against paxillin. B, the number of control (open bars) and previously heated (solid bars) cells with visible focal adhesion plaques was determined as described under “Experimental Procedures.” Open bars, data represent the mean ± S.E. Some S.E. bars are too small to be seen. *, p < 0.05 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of Transient ATP Depletion on Triton X-100-soluble and -insoluble Protein—In cells subjected to 1 h ATP depletion followed by 30 min recovery, a progressive, reciprocal shift in the relative amounts of Triton X-100-soluble and -insoluble total cell protein was observed (Fig. 4). At baseline, ∼28% of total cell protein was insoluble. Insoluble protein increased to 35% after 1.5 h ATP depletion and represented almost half (48%) of the total cell protein following 30 min recovery. These data demonstrate that the absence of ATP alters protein conformation and precipitates aggregate formation. Effect of Prior Heat Stress on the Triton X-100 Solubility of Paxillin—Compared with control, the relatively rapid recovery of paxillin staining suggested that heat stress exerted a cytoprotective effect on the re-assembly of focal adhesions. To evaluate this hypothesis, the detergent solubility of paxillin was examined. In control, ATP depletion resulted in the progressive shift of paxillin from the detergent-soluble to the -insoluble protein fraction (Fig. 5A). This shift was significantly reduced by heat stress (fig 5B; p < 0.05), suggesting that heat-inducible proteins protect paxillin from ATP depletion-mediated denaturation. In addition to preventing paxillin denaturation, prior heat stress could promote focal adhesion reassembly by enhancing paxillin release from the detergent-insoluble protein pool. To evaluate this hypothesis, the content of immunoreactive paxillin was serially assessed in Triton X-100-insoluble protein fractions obtained from control and previously heated cells subjected to ATP depletion as outlined (Fig. 6A). In this in vitro assay, various maneuvers were used to extract paxillin from the Triton X-100-insoluble protein fraction. The content of immunodetectable paxillin was used to estimate the amount of paxillin available to reassemble focal adhesions. In the absence of ATP (+ ayprase) or the presence of ATP and magnesium (“ATP + Mg”), virtually no paxillin could be detected at any time point before, during, or after ATP depletion in the detergent-insoluble protein fraction (Fig. 6B, upper portions of the top and middle panels). The combination of ATP, magnesium, high temperature (100 °C), and a reducing agent (DTT) resulted in modest paxillin release during recovery from ATP depletion (upper portion of panel C). The addition of prior heat stress to each of these maneuvers markedly increased paxillin release under all experimental conditions (lower portion of panels A-C). Heat stress was most effective for extracting paxillin from the detergent-insoluble protein fraction. Effect of ATP Depletion on the Triton X-100 Solubility of Hsp72—Because hsp72 has been reported to bind and repair non-native proteins, the detergent solubility of hsp72 was serially examined. At baseline, hsp72 was detected in both the Triton X-100-soluble and -insoluble fractions (Fig. 7A). Prior heat stress markedly increased hsp72 content in both the detergent-soluble and -insoluble protein fractions. ATP depletion (1 h) and recovery (30 min) resulted in a marked, reciprocal shift in immunoreactive hsp72 from the detergent-soluble to the detergent-insoluble protein fraction (Fig." @default.
• W2056164078 created "2016-06-24" @default.
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• W2056164078 date "2004-04-01" @default.
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• W2056164078 title "Hsp72 Interacts with Paxillin and Facilitates the Reassembly of Focal Adhesions during Recovery from ATP Depletion" @default.
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