Heat Shock Protein 27 Associates with Basolateral Cell Boundaries in Heat-Shocked and ATP-Depleted Epithelial Cells
Eric A. Shelden*,
Michael J. Borrelli,
Fiona M. Pollock* and
Rita Bonham*
*Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan; and Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan.
Correspondence to Dr. Eric A. Shelden, Assistant Professor, Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616. Phone: 734-764-0271; Fax: 734-763-1166; E-mail: sheldencommat;umich.edu
ABSTRACT. Heat stress alters epithelial barrier function, andheat stress preconditioning protects epithelial function frominjury. Hsp27 is a small stress protein that has previouslybeen shown to modulate actin assembly. Thus, by regulating actinfilaments associated with cell junctions, hsp27 could alterepithelial function. To begin to address this hypothesis, theregulation and distribution of a human hsp27-green fluorescencefusion protein (EGFPhHsp27) that is expressed in cultured renalepithelial cells was assessed. EGFPhHsp27, like the endogenoushsp27, associated with the cytoskeleton in heat-stressed andchemically ATP-depleted cells, and both proteins were regulatedsimilarly. Confocal microscopy of intact and detergent-lysedcells revealed novel distribution patterns in which EGFPhHsp27associated with basolateral, but not apical, cell borders ininjured cells. Double labeling studies revealed EGFPhHsp27 andactin filament colocalization in ATP-depleted cells. However,during heat shock, granules of EGFPhHsp27 were found at sitesof cell-cell contact and in the cell body, but colocalizationwith actin was not apparent. Thus, heat stress and ATP depletioninduce distinct patterns of hsp27 redistribution in epithelialcells, and sites of cell-cell and cell-substrate attachmentare unique in their ability to recruit hsp27 during injury.The association of EGFPhHsp27 with basolateral cell boundariessupports a potential role for hsp27 in protection or regulationof epithelial cell-cell and cell-substrate attachments.
Heat shock preconditioning enhances renal epithelial cell survival(1) and protects barrier function (2,3) from subsequent moresevere injury. Several mechanisms have been investigated toexplain this phenomenon, including inhibition of apoptotic cascades(4) and expression of high molecular weight stress proteinshsp70 (1,5–8) and hsp90 (9,10). However, alteration tothe cytoskeleton underlies epithelial injury by many mechanisms(11,12). Thus, actin-associated proteins responsive to thermalstress could also regulate epithelial injury or repair. Onesuch protein is the mammalian small heat shock protein, hsp27.Nonphosphorylated, but not phosphorylated, hsp27 behaves likean actin-capping protein that is capable of inhibiting actinpolymerization in vitro (13,14), and altered hsp27 levels orexpression of hsp27 phosphorylation site mutants induce changesin cellular actin polymer content and actin-dependent behavior(15,16). On the basis of these findings, previous investigatorshave suggested that hsp27 phosphorylation can enhance actinfilament polymerization and cytoskeletal stability by reducingits ability to inhibit actin polymerization (17–19).
Although the above model of hsp27 function is consistent within vitro and in vivo data, it is not clearly supported by thereported intracellular distribution of hsp27 in stressed andinjured cells. For example, the hypothesis predicts that hsp27associates with actin filaments in unstressed cells but maybe released during injury. Instead, hsp27 is predominately foundin the cytosolic fraction of unstressed cells and associateswith the detergent-insoluble cell fraction, or cytoskeleton,in response to various injuries (20–23). Constitutiveassociation of hsp27 with the apical microvillar border of proximaltubule epithelial cells and redistribution of hsp27 to intracellularcompartments during injury have been observed in renal tissues(20,24). However, hsp27 in unstressed cells was detergent soluble,and association of hsp27 with specific cellular structures wasnot detected during injury. Thus, there is agreement that thefactors that regulate hsp27 distribution and its associationwith the cytoskeleton are critical aspects of hsp27 function,but neither the mechanisms that mediate hsp27 redistributionnor their significance are fully understood.
In this study, we examined the distribution of a human wild-typehsp27-green fluorescence protein (EGFPhHsp27) in control andinjured epithelial cells using two- and three-dimensional confocalimaging techniques. Novel and distinct distribution patternsof total and detergent-insoluble EGFPhHsp27 were detected, includingcolocalization of EGFPhHsp27 with actin-filament arrays at basolateralcell boundaries after ATP depletion but not heat shock. Biochemicalstudies confirm that the EGFPhHsp27 fusion protein and endogenouscanine hsp27 are regulated in a similar manner under these conditions.The association of EGFPhHsp27 with basolateral sites of cell-celland cell-substrate attachment suggests that hsp27 could directlyaffect the function or regulation of cell junctions and cell-substrateattachments.
Cell Culture
MDCK cells were purchased from American Type Culture Collection(Manassas, VA) and cultured as described elsewhere (25). Anexpression vector for EGFPhHsp27 was produced by cloning a humanwild-type hsp27 cDNA into a commercial vector (EGFP-C1; Clontech,Palo Alto, CA). MDCKs were transfected using lipofectamine (InvitrogenLife Technologies, Carlsbad, CA) according to the manufacturer’sinstructions. Transfected cells were cultured in Dulbecco’smodified Eagle medium (DMEM) that contained 600 µg/mlG418 sulfate (Invitrogen Life Technologies) for 4 wk and werethen inspected by fluorescence microscopy. Cells were trypsinizedbriefly, and a fluorescence colony (MDCKe27) was subcloned byscraping with a sterile pipette tip while removing approximately100 µL of medium. Expression and integrity of the EGFPhHsp27fusion protein was confirmed by Western blotting using antibodiesthat recognize human hsp27 (SPA-800; StressGen Biotechnologies,Victoria, BC, Canada). An MDCK cell line that was stably transfectedwith the empty cloning vector was used for control studies.
For biochemical studies, equal volumes of a trypsinized cellsuspension were plated into wells of a 24-well plate. For microscopy,MDCKe27 cells were mixed with parental cells at a ratio of 1:5and plated onto 22-mm coverslips in Petri dishes. Cells werecultured 2 d after plating to allow monolayer formation. ForATP depletion experiments, medium was removed and cells wereincubated for 1 h at 37°C in saline containing 10 mM 2-deoxyglucose and 1 µM antimycin A (reagents were purchasedfrom Sigma Chemical Co., St Louis, MO, except as indicated).In other studies using similar methods, actin filament alterationsin cultured renal epithelial cells similar to that describedin the present study (see Results) correlated with a reductionin ATP level to <10% of their initial values (26,27). Forheat shock studies, culture medium was replaced with DMEM mediumcontaining 20 mM HEPES (pH 7.3). Cultures were sealed with parafilmand placed in a water bath (Fisher Scientific, IsoTemp 202;Fisher Scientific, Pittsburgh, PA) for 30 min at 37°C (controls),42°C (mild heat shock), or 45°C (severe heat shock).Trypan blue exclusion studies after overnight incubation at37°C indicated that about 80% of cells survived the severeheat shock and that negligible cell death occurred after ATPdepletion or mild heat shock (not shown).
Cell Fractionation and Western Blotting
Treated and control cells were rinsed with ice-cold phosphate-bufferedsaline (PBS) and then harvested on ice in 500 µl of detergentlysis buffer (DLB: 20 mM Tris, 150 mM NaCl, 1 mM ethylenediaminetetraaceticacid [EDTA], 1 mM ethyleneglycotetraacetic acid [EGTA], 0.5%Triton X100, pH 7.4) by vigorous scraping. Lysed cells werecentrifuged at 16,000 x g for 5 min at 4°C. Supernatantswere incubated with 2 volumes of ethanol overnight at -20°Cand then recentrifuged. Both pellets were dissolved in 50 µlof sample buffer (2 mM EDTA, 70 mM sodium dodecyl sulfate [SDS],20% glycerol, 20 mM dithiothreitol, 0.05% bromophenol blue,20 mM Tris, pH 8.0) followed by brief sonication and boilingfor 2 min. Equal sample volumes were loaded and resolved on13% acrylamide gels using a Miniprotean II system (BioRad Laboratories,Hercules, CA), and proteins were transferred to nitrocellulosemembranes using a BioRad Mini Trans-Blot. Membranes were probedwith human hsp27-specific antibodies (SPA-800; Stressgen) ora monoclonal antibody (8A7, gift of Dr. M. J. Welsh, Universityof Michigan) detecting the conserved crystallin domain of hsp27and B-crystallin (28) and peroxidase-labeled secondary antibodies(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).Blots were developed with a chemiluminescence detection system(Amersham Pharmacia Biotech, Inc., Piscataway, NJ) using Kodakx-ray film, and films were digitized by using a flatbed scanner(ScanJet 4C; Hewlett Packarad, Avondale, PA).
Analysis of hsp27 Phosphorylation
Previously described methods for detection of changes in hsp27phosphorylation by isoelectric focusing (IEF) gel electrophoresis(29) were used here. To analyze total cell hsp27, cells wererinsed with ice-cold PBS and suspended in 50 µl of extractionbuffer (EB: 9 M urea, 2% NP-40, 67 mM (ß-D)-glycerophosphate,50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodiumorthovanadate, 2% 2-mercaptoethanol, 1 mM EDTA, pH 7.6). Foranalysis of triton-insoluble hsp27, cells were scraped in 500µl of ice-cold DLB that contained protease and phosphataseinhibitors and centrifuged at 16,000 x g for 4 min, and pelletswere dissolved in 50 µl of EB. IEF gels containing ampholytes(pH range, 5 to 7; Sigma Chemical Co) were prepared by usingthe Bio-Rad 111 Mini-IEF Cell. Equal volumes of sample werefocused at 100 V for 15 min, 200 V for 15 min, and 450 V for90 min. Proteins were transferred to nitrocellulose membraneby using a semi-dry transfer apparatus (Model FB-SDB-2020; FisherScientific), and hsp27 was detected as described above for Westernblotting.
Fluorescence Microscopy
Cells on glass coverslips were rinsed with PBS and then fixedeither with or without a 2-min pretreatment with DLB in PBSthat contained 4% paraformaldehyde for 30 min (all at 21°C).Some preparations were also incubated with 0.2 µg/ml rhodaminephalloidin for 15 min. Anti ZO-1 immunostaining was conductedby using a monoclonal antibody (MAB1520; Chemicon InternationalInc., Temecula, CA) and Texas-red conjugated secondary antibodies(Southern Biotechnology Associates, Inc., Birmingham, AL) asdescribed elsewhere (25). Preparations were mounted by usingProlong (Molecular Probes Inc., Eugene, OR) and sealed withnail polish. Alternatively, cells were cultured on membraneswith 3-µm pores in MilliCell chambers (Sigma ChemicalCo.) and then lysed, fixed, and cryopreserved overnight in PBSthat contained 20% sucrose. The membrane was excised with arazor blade, incubated in PBS that contained 20% sucrose and30% Tissue Freezing Medium (Electron Microscopy Sciences, Washington,PA), and then frozen in an ethanol/dry ice bath. Using a cryotome(Frigocut N 2800, Reichert-Jung, Germany), 7-µm-thicksections were cut perpendicular to the membrane and mountedon Superfrost slides (Fisher Scientific). Imaging was conductedwith a Zeiss LSM510 confocal microscope (Carl Zeiss Inc., Thornwood,NY) with a 60 x 1.2 NA water immersion lens. Confocal pinholeswere adjusted to an Airy disk diameter of 1, generating images0.14 µm/pixel (X and Y dimensions) and 0.2 µm/plane(Z dimension). Dual-labeled specimens were imaged by using amultiscan mode in which the 512 individual scan lines of animage were scanned twice, illuminating and imaging each of thetwo fluorescence probes sequentially instead of simultaneously.This method eliminated the tendency of the signal from one fluorescenceprobe to corrupt images of the other probe.
EGFPhHsp27 and hsp27 Redistribution in MDCKs by Heat Shock and ATP Depletion
In control parental MDCKs, hsp27 is detected in the detergent-soluble(Cnt-S), but not -insoluble (Cnt-P), cell fraction (Figure 1A).ATP depletion (1 h) and severe heat shock (30 min at 45°C)cause a dramatic increase in detergent-insoluble hsp27 and adecrease in detergent-soluble hsp27, but little change in hsp27solubility is detected in cells after mild heat shock (30 minat 42°C). In MDCKs that express EGFPhHsp27 (cell line MDCKe27),the fusion protein (>43 kD) is also predominately solublein control cells and cells after mild heat shock. Detergent-insolubleEGFPhHsp27 is detected after 1 h of chemical ATP depletion or30 min of severe, but not mild, heat shock (Figure 1B). Relativelymore EGFPhHsp27 than canine hsp27 is retained in the solublefraction in these experiments, perhaps because excess EGFPhHsp27is expressed by transfected cells. Figure 1C shows this blotafter reprobing with a monoclonal antibody (8A7) that recognizesEGFPhHsp27, the endogenous canine hsp27, and a protein of approximately22 kD (B-crystallin). Normal association of endogenous hsp27and B-crystallin with the detergent-insoluble cell fractionoccurs in cells expressing EGFPhHsp27. Thus, canine hsp27 andEGFPhHsp27 are specifically detected by immunoblotting withappropriate antibodies, and the solubility of EGFPhHsp27 andcanine hsp27 is regulated similarly by MDCKs. Overexpressionof EGFPhHsp27 may saturate the ability of the triton-insolublecell fraction to recruit EGFPhHsp27, but it does not otherwiseappear to alter hsp27 regulation.
Figure 1. Redistribution of endogenous canine hsp27 and a human hsp27-green fluorescent protein fusion protein in MDCKs. (A) Western blot of detergent-insoluble (P) and -soluble (S) cell fractions isolated from parental MDCKs after no treatment (Cnt), 1 h of ATP depletion (-ATP), or heat stress for 30 min at 42°C (42C) or 45°C (45C) probed with a primary antibody specific for canine hsp27. Hsp27 coprecipitates with detergent-insoluble proteins in ATP-depleted cells and cells subject to heat stress at 45°C but not in controls or cells heat stressed at 42°C. (B) Western blot of cell fractions isolated from MDCKs expressing EGFPhHsp27 probed with a primary antibody specific for human hsp27, showing similar results. (C) Western blot of cell fractions isolated from MDCKs expressing EGFPhHsp27 probed with a primary antibody that recognizes hsp27 and B-crystallin.
Posttranslational Modification of Total and Triton-Insoluble hsp27 in MDCKs
Phosphorylation of hsp27 can be detected as altered migrationof hsp27 in IEF gels (28,30–32). Three isoelectric isoformsof canine hsp27 occur; the most basic of which is nonphosphorylated.Singly and doubly phosphorylated isoforms are progressivelymore acidic (32). In our studies, untreated and ATP-depletedparental MDCKs expressed a single predominate isoform (presumablynonphosphorylated). Cells show increasing amounts of two moreacidic isoforms after mild and severe heat shock (Figure 2A).Triton-insoluble pellets isolated from cells after ATP depletionor severe heat shock contain hsp27 isoforms representative ofthose observed in the total cell pool (Figure 2B, lanes 2 and4). As expected, little hsp27 is detected in pellets that havebeen isolated from untreated cells or cells subject to mildheat shock (Figure 2B, lanes 1 and 3). Overall IEF behaviorof EGFPhHsp27 in MDCKe27 cells is similar to that observed forcanine hsp27 in parental MDCKs (Figure 2, C and D), althoughthe specific IEF points of EGFPhHsp27 and canine hsp27 isoformsare not identical (not shown). Most EGFPhHsp27 in untreatedand ATP-depleted MDCKe27 cells focuses at a single location,and a single more acidic isoform is also detected in heat-shockedcells (Figure 2C). Again, triton-insoluble EGFPhHsp27 (Figure 2D)displays an IEF migration pattern that is indistinguishablefrom that of total cell samples (Figure 2C). Only one phosphorylatedisoform of EGFPhHsp27 is detected, which is consistent withthe results of earlier studies that examined phosphorylationof an exogenous human hsp27 in nonhuman cell lines (15). Together,these data indicate that EGFPhHsp27 and the endogenous caninehsp27 are regulated similarly in MDCKs under our experimentalconditions.
Figure 2. Isoelectric focusing patterns of endogenous canine hsp27 and a human hsp27-green fluorescent protein fusion protein in MDCKs. (A) Isoelectric focusing (IEF) gel of total cell protein in MDCKs. The most basic hsp27 isoform (presumably nonphosphorylated) predominates in control (Cnt) and ATP-depleted (-ATP) cells. More acidic (phosphorylated) isoforms are detected in heat-stressed cells (42C, 45C). (B) IEF gel showing detergent-insoluble canine hsp27 in MDCKs. (C) IEF gel showing the isoelectric focusing pattern of total cell EGFPhHsp27 in MDCKe27 cells. A single isoform is seen in control and ATP-depleted cells, and a more acidic isoform is also detected in heat-stressed cells. (D) IEF gel showing the isoelectric focusing pattern of detergent-insoluble EGFPhHsp27. Gels are oriented so that the most basic proteins are at the bottom of each image. Blots were probed antibodies that recognize canine hsp27 (A and B) or are specific for human hsp27 (C and D).
EGFPhHsp27 Localization in MDCKs during Heat Stress or Chemical ATP Depletion
To specify the localization of hsp27 in epithelial cells, totalcell EGFPhHsp27 distribution was first examined in MDCKe27 cellsthat were fixed without detergent lysis. In untreated cells(Figure 3A) and cells after mild heat shock (Figure 3B), homogeneousdistribution of EGFPhHsp27 within the cell body is observed.Decreased fluorescence intensity at cell boundaries after mildheat shock probably reflects changes in cell shape (flattening)and reduced cell-cell attachment. After severe heat shock, thedistribution of EGFPhHsp27 is much less homogeneous (Figure 3C).Fluorescence granules of EGFPhHsp27 are seen throughoutthe cell and at sites of cell-cell contact (Figure 3C, arrow).Finally, after ATP depletion (Figure 3D), association of EGFPhHsp27with sites of cell-cell contact is much more apparent than incells after heat shock (Figure 3, compare C with D). EGFPhHsp27is also excluded from the nucleus under all experimental conditions.To provide internal negative controls for fluorescence, theseexperiments were conducted by using cocultures of MDCKe27 andparental MDCK cells (see Methods). The shape of fluorescencecell boundaries in Figure 3 (A, C, and D) indicates the presenceof nonfluorescence parental MDCKs in these images. No fluorescencewas detected in cultures of parental MDCKs alone (not shown).
Figure 3. Localization of EGFPhHsp27 in intact cells. (A) Homogeneous distribution of EGFPhHsp27 is seen in control cells. (B) Largely homogeneous EGFPhHsp27 is seen in cells incubated at 42°C for 30 min before fixation. (C) Cytoplasmic aggregates and some recruitment of EGFPhHsp27 to sites of cell-cell contact (arrow) is seen in cells incubated at 45°C for 30 min before fixation. (D) Recruitment of EGFPhHsp27 to sites of cell-cell contact is highly evident in cells after 1 h of chemical ATP depletion. Scale bar, 20 µm.
To determine the location of detergent-insoluble EGFPhHsp27,cells were detergent-lysed before fixation and then examined.Little EGFPhHsp27 is seen in untreated cells after detergentlysis (Figure 4A), which confirms that EGFPhHsp27 is solublein nonstressed cells. After mild heat shock, detergent-insolubleEGFPhHsp27 is detected at low levels in a novel distributionpattern that consists of small granules at sites of cell-cellcontact (Figure 4B, middle and green). Actin filament arraysare associated with cell boundaries in this optical section(Figure 4B, left and red) but are not clearly associated withEGFPhHsp27. In ATP-depleted cells, detergent-insoluble EGFPhHsp27is strikingly associated with cell boundaries; granules arealso seen throughout the cell body (Figure 4C). In these cells,colocalization with phalloidin-stained actin is evident, butonly at cell boundaries (Figure 4C, yellow). EGFPhHsp27 withinthe cell body (Figure 4C, green) shows little association withphalloidin-stained structures. Finally, cells also retain moreEGFPhHsp27 after severe heat shock (Figure 4D) than after mildheat shock, and EGFPhHsp27 is found in larger and more distinctgranules throughout the cell and at sites of cell-cell contact.Intriguingly, comparison of actin and EGFPhHsp27 distributionsat sites of cell-cell contact reveals that granules of EGFPhHsp27(Figure 4D, arrows) alternate rather than colocalize with areasof high phalloidin staining. In an overlay image, the resultingcodistribution pattern resembles a string of alternating redand green beads along sites of cell-cell contact (Figure 4D).For all studies, cells without phalloidin staining were alsoexamined, yieldeding identical EGFPhHsp27 distributions (notshown).
Figure 4. Localization of detergent-insoluble EGFPhHsp27 and actin. Dual-channel confocal optical sections show actin (left panel and red in the overlay image) and detergent-insoluble EGFPhHsp27 (middle panel and green in the overlay image). (A) Control cells exhibit little detergent-insoluble EGFPhHsp27. (B) Small localized regions containing detergent-insoluble EGFPhHsp27 are seen at sites of cell-cell contact in cells heat stressed at 42°C. (C) Detergent-insoluble EGFPhHsp27 in ATP-depleted cells showing aggregates throughout the cell body and strong colocalization with actin filament arrays at sites of cell-cell contact (yellow). (D) Detergent-insoluble EGFPhHsp27 in cells heat stressed at 45°C throughout the cell body and at discrete sites associated with cell-cell contacts. Scale bar, 10 µm.
To test whether the EGFP protein alone could be detergent-insolublein some conditions, MDCKs stably expressing EGFP were examined.Intact cells fixed without detergent lysis show fluorescencethroughout the cell body and nucleus (Figure 5A). Control cells(Figure 5, B and E) and cells subject to either 1 h of chemicalATP depletion (Figure 5, C and F) or severe heat shock (Figure 5, D and G)were detergent lysed, fixed, and double labeledwith fluorescence phalloidin. No detergent-insoluble EGFP wasdetected after any experimental treatment (Figure 5 B through D).Alterations in actin filament organization, such as formationof actin aggregates in ATP-depleted cells (Figure 5F), and areduction in basal actin filament bundles after heat stress(Figure 5G) were observed, which is consistent with observationsthat have been made by previous investigators using similarprotocols (26,33).
Figure 5. The enhanced green fluorescent protein alone does not remain in epithelial cells after detergent lysis. (A) Untreated MDCKs stably expressing EGFP were fixed without detergent lysis, showing homogeneous EGFP distribution. (B through D) Detergent-insoluble EGFP is not detected in untreated cells (B) or cells after ATP depletion (C) or severe heat shock (D). Also detected in these double-labeled cells is actin filament organization in untreated cells (E) and cells after ATP depletion (F) or severe heat shock (G) showing typical alterations. Scale bar, 10 µm.
The relationship between actin and detergent-insoluble EGFPhHsp27was determined in three dimensions by using optical sectioningmethods. Figure 6 is a series of confocal images taken with0.2-µm changes in focal position of the junction betweentwo cells heat shocked at 42°C. Granular structures thatcontain detergent-insoluble EGFPhHsp27 are indicated by arrowsin the most basal optical section (but individual granules maybe most apparent in a neighboring image plane). These granulesare only visible in panels 1 to 3. In panels 6 through 13, asecond, more apical site that shows EGFPhHsp27 is seen. Thesegranules are smaller and more uniform than those observed atthe basal surface. The inset shows a model of the distributionof basal coarse granules (1) and finer granules at lateral andapical sites of cell-cell contact (2).
Figure 6. Distribution of detergent-insoluble EGFPhHsp27 in epithelial cells after mild heat shock. Fourteen confocal images of a single cell-cell contact were obtained with a 0.2-µm focal change between images. Granules of EGFPhHsp27 associated with the boundary between the two cells are indicated with arrows in the first (most basal) image. A more apical site containing EGFPhHsp27 is also seen in higher images. Inset shows a diagram of EGFPhHsp27 distribution at basal (1) and apical (2) sites of cell-cell contact. Scale bar, 5 µm.
The greater amounts of EGFPhHsp27 that were detected in ATP-depletedand severely heat-shocked cells compared with untreated cellsand cells subject to mild heat shock allowed double labelingand computer-assisted reconstruction of XZ images. Figures 7A and 7Bshow two examples of XZ images that are oriented so theimage plane passes vertically through a site of cell-cell contact(the orientation of the image plane is illustrated in Figure 7B).Actin (red and lower gray scale image) typically appearedas large globular structures containing little or no detectableEGFPhHsp27 (upper gray scale image). Occasionally, verticalcolumns of actin are seen (Figure 7B, arrows) that also lackEGFPhHsp27. In ATP-depleted cells, detergent-insoluble EGFPhHsp27is found along the lateral sites of cell-cell contact and atthe basal surface of cells (Figure 7, C and D), but little EGFPhHsp27is observed at the apical cell surface (the plane of sectionis illustrated in Figure 7D). In contrast, filamentous actinis present at both the apical and basolateral cell surfaces(Figure 7C, red). Arrows in Figure 7C mark the most apical extentof the EGFPhHsp27 fluorescence that is associated with sitesof lateral cell-cell contact. In a separate experiment, ATP-depletedMDCKe27 cells were also stained with antibodies to reveal thedistribution of the tight junction protein ZO1. Detergent-insolubleEGFPhHsp27 at the basolateral cell border does not extend abovethe level of apical tight junctions (Figure 7D, arrows). Finally,previous studies have shown that fluorescence objects can displaydifferent apparent intensities of fluorescence at differentorientations with respect to the optical axis (34). To addressthis potential problem, MDCKe27 cells exposed to chemical ATPdepletion and severe heat shock were detergent lysed, fixed,and cryosectioned perpendicular to the substrate plane. BasolateralEGFPhHsp27 is observed in cryosectioned cells after ATP depletion(Figure 7E) and is particularly bright at the most apical siteof cell-cell contact (arrows). In contrast, no similar distributionof EGFPhHsp27 is observed at sites of cell-cell contact afterheat shock (Figure 7F).
Figure 7. Three-dimensional distribution of actin, Z01, and detergent-insoluble EGFPhHsp27 in epithelial cells after severe heat shock or ATP depletion. (A and B) Two examples of XZ images through sites of cell-cell attachment after sever heat shock showing actin (red and lower gray-scale images) and EGFPhHsp27 (green), showing lack of colocalization. (B) Illustration of the plane of section for panels A and B. (C) XZ image of ATP-depleted MDCKe27 cells showing colocalization of actin (red and lower gray-scale images) and EGFPhHsp27 (geen) at basolateral but not apical cell borders. (D) Double labeling of ATP-depleted MDCKe27 cells showing EGFPhHsp27 (green) and Z01 (red). Localization of EGFPhHsp27 is restricted to sites below tight junctions. (D) Plane of section for images shown in panels C through F. (E) Cryosection showing XZ distribution of EGFPhHsp27 in ATP-depleted MDCKe27 cells without optical sectioning. (F) Cryosection showing EGFPhHsp27 in severely heat-shocked MDCKe27 cells. Scale bar, 10 µm.
The most significant new finding of this study is evidence ofthe direct and specific association of hsp27 with sites of epithelialcell-cell and cell-substrate contact in injured epithelial cells.Because association of EGFPhHsp27 with lateral cell boundariesis seen in cells fixed before lysis (Figure 3) as well as afterlysis, this association represents recruitment of hsp27 fromthe cellular pool to these sites and is not a result of washingaway detergent-soluble protein from homogeneously distributedhsp27. A relationship between hsp27 and the regulation of cell-celland cell-substrate attachment has been suggested by studiesin which changes in hsp27 expression or phosphorylation correlatewith altered cell adhesion, junctional integrity, and cell migration(reviewed in (18). However, our results provide the first directevidence that hsp27 is a component of cell-cell and cell-substrateattachments in injured cells. Our results also confirm and extendthose of a previous study indicating that up to one third ofcellular hsp27 can associate with a basolateral membrane cellfraction in vascular endothelial cells (17). However, thereare notable differences between the previous and current results.For example, studies conducted by Piotrowicz and Levin (17)showed constitutive association of hsp27 with the basolateralmembrane in endothelial cells, and the abundance of membrane-associatedhsp27 did not change when cells were stimulated with phorbolester. In contrast, EGFPhHsp27 is found in our studies at sitesof epithelial cell-cell and cell-substrate contact during hyperthermiaor ATP depletion, but not in untreated cells. The previous studyalso did not specifically address whether membrane-associatedhsp27 was a component of the triton-insoluble cell fraction,and because the previous study used only biochemical assays,the subcellular localization pattern of membrane-associatedhsp27 was not determined. Our work extends the previous studyby showing that basolateral EGFPhHsp27 is a component of thedetergent-insoluble cell fraction and that discrete granularstructures found at apical and basal sites of epithelial cell-cellcontact are specific sites of hsp27 association in cells duringmild thermal injury. Sites of both cell-cell and cell-substrateattachment show colocalization of actin and hsp27 in ATP-depletedcells in our studies.
The intracellular distribution of hsp27 was also previouslyexamined in renal epithelial cells subject to ischemia/ATP-depletionusing immunofluorescence staining methods. Studies examininghsp27 distribution in sectioned rat kidney tissues providedevidence for the colocalization of hsp27 with actin filamentarrays, most notably at or near the apical microvillar borderin unstressed renal proximal convoluted tubule epithelial cells(17,21, but see (24 for an exception). Redistribution of hsp27accompanied ischemic injury, resulting in loss of hsp27 fromthe apical compartment and its association with the triton-insolublecell fraction. Thus, the previous and current studies supportthe view that hsp27 redistribution and differential regulationof its association with the cytoskeleton accompany renal cellinjury. However, there are also findings that are unique tothe present study. For example, in contrast to our own results,association of hsp27 with specific actin filament arrays wasnot detected during injury in previous studies, and hsp27 detectedat the apical microvillar border of unstressed cells did notassociate with the detergent insoluble cell fraction. It isof interest to consider the basis of these differences. Previousinvestigators examined hsp27 in intact renal proximal tubules.We examined cultured MDCK epithelial cells, a cell line derivedfrom canine distal convoluted tubule epithelial cells. MDCKsare more resistant to some types of cell injury than are epithelialcells of proximal tubule origin (35). Thus, the associationof hsp27 with sites of cell-cell and cell-substrate attachmentobserved in our studies may partially account for the observedgreater resistance to injury of distal versus proximal convolutedtubule epithelia. Alternatively, our results may reflect a relativelypoor state of differentiation of MDKCs in culture, and our cellcultures may model hsp27 function during development or repairof epithelial tissues, rather than the condition of fully differentiatedcells. Differences between the results of our own and previousstudies using immunostaining methods may indicate that highlyantigenic hsp27 domains are important to the association ofhsp27 with the triton-insoluble cell fraction or cell junctionsand are unavailable for antibody binding in intact cells. Finally,cells expressing the EGFPhHsp27 fusion protein in our studieslikely express higher than normal overall levels of hsp27. Thus,the cellular response of MDCKe27 cells to heat shock and ATPdepletion in our studies may model that of preconditioned cellsexpressing high levels of hsp27 before injury. In contrast,preconditioning of renal tissues was not used in the in vivostudies conducted by Aufricht et al. (20) or Smoyer et al. (29).Further studies examining hsp27 redistribution in cells subjectto injury after heat stress preconditioning or with no preconditioningwill likely resolve this issue. However, the differences inresults between the present and previous studies may indicatethat association of hsp27 with basolateral cell boundaries isan element of cellular cytoprotection rather than a responseto initial cellular injury.
Although our results clearly demonstrate alteration in the abilityof epithelial cell-cell and cell-substrate attachment to recruitregulatory factors during thermal and ATP depletion-inducedinjury, these results only address the function of hsp27 ifthe EGFPhHsp27 fusion protein accurately mimics the behaviorof endogenous hsp27. We believe there is ample evidence thatthis is the case. Borrelli has shown that expression of EGFPhHsp27provides thermal protection to A549 human lung carcinoma andL929 murine fibroblast cells (Michael J. Borrelli, personalcommunication, March 2001). Here, we demonstrate that EGFPhHsp27and canine hsp27 associate with cytoskeleton similarly in MDCKsafter chemical ATP depletion and severe heat shock. However,neither protein is detected significantly in pellets obtainedfrom untreated cells or cells after mild heat shock (Figure 1).Our studies also reveal similar overall changes in the regulationof canine hsp27 and the EGFPhHsp27 fusion protein in responseto heat stress and ATP depletion (Figure 2). Although EGFPhHsp27is only singly phosphorylated in MDCKs in response to thermalinjury, this is consistent with previous work showing that nontaggedhuman hsp27 is predominately singly phosphorylated in responseto heat shock in a Chinese hamster cell line (15). Finally,we show that expression of the EGFPhHsp27 fusion protein inMDCKs does not disrupt normal regulation of canine hsp27 (Figure 1C).Thus, there is no evidence that addition of the EGFP tagalters the function of the human hsp27 protein in our studies.
In summary, our results indicate that EGFPhHsp27 is an accuratemodel of hsp27 function in renal epithelial cells and that studiesof the intracellular distribution of EGFPhHsp27 are an importantcomplement to immunostaining studies. The novel distributionpatterns of total and detergent-insoluble EGFPhHsp27 proteinobserved in our studies provide new evidence for a specificassociation of hsp27 with structures mediating cell-cell andcell-substrate attachment, and they suggest that hsp27 couldplay a role in the regulation and function of these structuresduring injury.
Acknowledgments
These studies were supported by grants from the National KidneyFoundation of Michigan, Inc., the National Institute of EnvironmentalHealth and Safety (NIH R01 ES11196–01), and the NationalInstitute on Aging (NIH R03-AG19847–01) to E. A. S. anda grant from the National Cancer Institute (NIH R01 CA71650)to M. J. B. We thank Drs. R. Benndorf and J. M. Weinberg forcritical reading of this manuscript and helpful comments, Dr.S. A. Ernst for access to his cryotome, and Ms. J. Edwards forinstruction in its use.
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Received for publication August 23, 2001.
Accepted for publication October 20, 2001.
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Top
Abstract
Introduction
B-crystallin (28) and peroxidase-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Blots were developed with a chemiluminescence detection system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) using Kodak x-ray film, and films were digitized by using a flatbed scanner (ScanJet 4C; Hewlett Packarad, Avondale, PA). 
