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Departments of Pediatrics and Pathology, Yale University School of Medicine, New Haven, Connecticut.
Correspondence to Dr. Scott K. Van Why, Department of Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, WI 53226-0509. Phone: 414-456-4180; Fax: 414-456-6539;
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E-mail: svanwhy@mcw.edu
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Introduction |
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Hsp25/27 is involved in regulation of actin dynamics and can protect against cytoskeletal breakdown from oxidative stress (14,15). Cytoskeletal disruption is an early and central process in ischemic renal injury, and renal ischemia both induces and causes a redistribution of Hsp25 in renal epithelia (1,3). Moreover, the pattern of Hsp25 redistribution suggests specific interactions between Hsp25 and actin during the early postischemic reorganization of the cytoskeleton (3). An attractive hypothesis, then, is that small Hsp (sHsp) might interact with the cytoskeleton after ischemic renal injury and that increased expression of sHsps might be able to protect renal epithelia from specific manifestations of sublethal injury.
The purpose of the present study was to determine whether Hsp25/27 interacts with actin during or after ATP depletion in renal epithelia and whether this interaction might be associated with protection against manifestations of cell injury from energy deprivation. Renal epithelia were transfected to overexpress labeled Hsp27. During ATP depletion, the labeled Hsp27 closely associated with actin as demonstrated by fluorescence resonance energy transfer. Overexpression of the labeled Hsp27 limited the disruption of Na,K-ATPase attachment to the cytoskeleton in response to energy deprivation, but it had no effect on ezrin. Protection was not attributable to preservation of cellular ATP levels during the energy deprivation. Thus, cytoprotection of renal epithelia afforded by Hsp25/27 is selective and may depend on the specific function and distribution of this stress protein.
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Materials and Methods |
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Cell Injury
Confluent cells containing the Hsp27-GFP construct, GFP alone, as well as the parent LLC-PK1 cells were subjected to ATP depletion in parallel using antimycin A and 0 mg/dl D-glucose in substrate-free media as described previously (16) for 2 h or for 4 h. Additional sets of cells from each group were subjected to 2 h of ATP depletion as above, followed by washes and reintroduction of normal growth media for 4 h of recovery. Non-injured controls for each experimental group were cells from each respective clone, grown in parallel, that underwent equivalent washes in substrate-free incubation media containing 100 mg/dl D-glucose without antimycin A. Cellular ATP levels were determined in each experimental group using methods previously described (16).
Fluorescence Resonance Energy Transfer (FRET)
Experimental injury and recovery were performed on cells from each group that were grown in eight-chambered Lab-Tek slide chambers. Cells were fixed at the end of the experiment by removal of culture media and immersion in 4% formaldehyde in PBS, pH 7.4, for a minimum of 1 h at room temperature. Cells were then washed three times in PBS and stored cold until used.
F-actin distribution in the cells was examined with rhodamine phalloidin. The cells were washed and blocked with PBS and 0.1% BSA for 15 min. They were then covered with 100 µl of rhodamine phalloidin diluted 1:300 in PBS, 0.1% BSA, and allowed to label for 30 min at room temperature. The cells were then washed three times over 30 min with PBS, and the coverslips were mounted on slides using Vectashield antifade mounting media.
The cells were viewed on an Olympus Fluoview Scanning Laser Confocal microscope equipped with separate shutter controlled Argon Ion (blue excitation) and Krypton (green excitation) lasers that are fiberoptically coupled to the scanning head. The microscope was used with an Olympus UPLAPO 60X oil immersion and utilizing the smallest confocal aperture setting. Cells were imaged at a mid-cellular plane to assess actin organization with the cortical cytoskeleton. This was identified by the presence of nuclei in the plane. The criterion for establishing Resonance Energy Transfer was adapted from the methodology of Uster and Pagano (17). To calibrate the system for energy transfer, one well of cells that expressed Hsp27-GFP but unlabeled for rhodamine was placed on the stage and imaged with both lasers. The blue (GFP) channel photo-multiplier gain and offset were set to give a bright high-quality image. The green (rhodamine) channel was set to show only a very low degree of background fluorescence originating from bleed-through of GFP to the rhodamine channel. Another well that was labeled with rhodamine phalloidin was also imaged, but it lacked the green fluorescence protein. This was to demonstrate that the blue laser did not provide enough off-peak excitation to the rhodamine phalloidin to be detected. Double-labeled cells were imaged in the mid-cellular plane with both lasers exciting the specimen, and the co-localization was documented. The green laser excitation was then removed by manually closing its shutter; no change in image level in the cells was made. The image resulting from the resonance energy transfer was then recorded. Fluorescence intensity of phalloidin staining and FRET was determined using Scion software similar to methods previously described (24). Background fluorescence was subtracted, and threshold functions were applied to the original image to remove extraneous fluorescence. Maximum brightness was set at 255, with a minimum set at two. Pixel intensity of phalloidin staining or FRET was determined for five fields for each experimental condition. Each field contained approximately 20 cells.
Assessment of Cell Injury
Disruption of interactions between the cytoskeleton and selected associated proteins was used to assess sublethal injury. The detergent extractability of two cytoskeletal-associated proteins, the basolaterally located, integral membrane protein Na,K-ATPase and the apically located protein ezrin was assayed for each cell line. Changes in cytoskeletal attachment and redistribution of these proteins as an early and consistent manifestation of sublethal renal epithelial cell injury from ischemia, ATP depletion, or hypoxia has been rigorously characterized (1,16,27,29). Cells from each experimental condition underwent Triton X-100 extraction as described previously (16). The supernatant containing the detergent-soluble proteins was removed, and the insoluble pellet containing the cytoskeletal-associated proteins was resuspended in an equal volume of extraction buffer. Equal aliquots from each fraction were dissolved in phoresis sample buffer (18) and stored at -20°C.
Western Blot Analyses
Expression of the endogenous Hsp25 and the transfected Hsp27-GFP was assessed in each clone after lysis of cells in 150 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, and 1 mM phenyl-methylsulfonyl fluoride. Protein determinations were performed, and equal amounts of total cell protein from the lysates underwent SDS-PAGE as described previously (3). To detect the endogenous Hsp25, primary antibody developed against rodent Hsp25 was used (Stressgen, Victoria, BC). To detect the transfected GFP-tagged Hsp27, primary antibody specific for human Hsp27 was used (Stressgen).
To assess shift between detergent-soluble and detergent-insoluble fractions of the cytoskeletal-associated proteins Na,K-ATPase and ezrin, equal volume aliquots from both fractions from cell extracts underwent SDS-PAGE and detection with primary antibodies for Na,K-ATPase (16,18) and for ezrin (Sigma). For all Western blots, a chemiluminescent assay was applied using secondary antibodies and reagents as recommended by the manufacturer (Supersignal West Pico; Pierce, Rockford, IL). Densitometry was performed as described previously (18) on immunoblots for Na,K-ATPase that contained samples from either 2 h or 4 h of ATP depletion along with the parallel non-injured cells from the same day for both the Hsp27-GFP transfectants and the GFP transfectant control cells. The percent of soluble Na,K-ATPase was determined for each individual condition. Response to injury was then expressed as the change in the percent of soluble enzyme during ATP depletion for each transfectant relative to its paired, uninjured control. Difference in Na,K-ATPase solubilization was considered statistically significant if P < 0.05 by ANOVA.
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The right column contains micrographs of the same cells studied using FRET. The cells were excited at 488 nM (blue) and analyzed at 600 nM. The fluorescence transfer is pseudocolored white. In the uninjured control cells, there is no fluorescence transfer between the two fluorochromes, indicating no evident interaction between Hsp27-GFP and F-actin. However, after 2 h of ATP depletion, FRET between the two proteins (pseudocolored white) is found to coincide prominently with the pattern of disorganized aggregates of F-actin filaments within the cell body. In addition, FRET shows Hsp27 interaction with F-actin at lateral cell boundaries at 2 h of ATP depletion. In cells transfected with GFP alone, there was no FRET to rhodamine phalloidin decorated actin (not shown). Therefore, with 2 h of ATP depletion, Hsp27 is closely associated with the disorganized F-actin filaments within the cell body and with actin filaments at the lateral cell margins. By 4 h of recovery after ATP depletion, association between the two proteins detectable by FRET persists in some cells but appears less prominent. Where Hsp27 interaction with actin persists, it appears to be primarily with the residual aggregates of actin in the cell body. Though somewhat less pronounced than at 2 h of ATP depletion, at 4 h of recovery, FRET between Hsp27 and cortical actin at the lateral cell margins persists.
To determine whether the apparent decrease in FRET at 4 h of recovery compared with 2 h of ATP depletion is related to changes in actin staining, intensity of the FRET was determined relative to the F-actin fluorescence for each experimental condition. Figure 3a shows quantification of phalloidin staining during and after ATP depletion relative to uninjured control cells. At 2 h of ATP depletion, phalloidin staining was increased to 136 ± 15% of control values. This increase in phalloidin staining of F-actin during ATP depletion is consistent with that previously reported in the same model of cell injury using EYFP-labeled actin (24). By 4 h of recovery after ATP depletion, F-actin staining had returned to control levels (97 ± 8%). Figure 3b shows FRET expressed as percent of phalloidin fluorescence for the same experimental condition. Though total FRET between Hsp27-GFP and phalloidin-decorated actin was decreased at 4 h recovery compared with during ATP depletion, when adjusted for level of phalloidin fluorescence in the same sections, the level of FRET did not change between 2 h of ATP depletion and 4 h of recovery.
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Each cell line was subjected to both 2 h and 4 h of ATP depletion. At the end of the interval, the cells underwent detergent extraction and aliquots from both the detergent-soluble (S) and detergent-insoluble pellet (P) were examined for Na,K-ATPase (n = 4 for each condition). The parent cell line showed the previously described (1,16) typical redistribution of Na,K-ATPase into detergent-soluble extracts from cells during ATP depletion. The change in detergent extractability of Na,K-ATPase during ATP depletion relative to paired, uninjured cells of both transfectants was determined for both clones, the Hsp27-GFP cells and its control GFP-transfected cells. Baseline soluble Na,K-ATPase did not differ significantly between Hsp27-GFP and GFP alone cells under non-injury conditions (n = 8 for each, total of 4 paired controls for 2-h and 4-h ATP depletion experiments). At 4-h ATP depletion, no significant difference was found between Hsp27-GFP and GFP transfectants. However, at 2-h ATP depletion, Hsp27-GFP cells had a significantly smaller change in soluble enzyme compared with GFP transfectants. Figure 4a shows a representative immunoblot from the two transfectants at 2-h ATP depletion relative to paired, uninjured cells. Figure 4b shows densitometry for change in soluble Na,K-ATPase at 2-h ATP depletion relative to paired, uninjured controls (n = 4 for each). At 2-h ATP depletion, the change in percent of soluble Na,K-ATPase was significantly less in Hsp27-GFP transfectants (19.6 ± 6.8) compared with GFP transfectants (74.3 ± 9.0; P = 0.001).
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Figure 6 shows ATP levels under each experimental condition for each clone expressed as a percent of its respective level in control conditions. After both 2-h and 4-h of treatment with antimycin-A and substrate deprivation, ATP levels in the GFP transfectants were higher than the Hsp27-GFP or the parent LLC-PK1 cells. So the limitation of Na,K-ATPase solubilization at 2 h of metabolic inhibition attributable to Hsp27 transfection was not associated with preservation of cellular ATP levels. After 2 h of treatment and 3 h of recovery in normal media, ATP levels had recovered to near baseline levels in all three groups.
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Discussion |
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We transfected the proximal tubule cell line LLC-PK1 with the human isoform Hsp27. The vector contained an insert for enhanced green fluorescence protein (GFP) to track the transfected protein and to determine whether it associates with actin during or after ATP depletion. To assess functional effects of the transfected expression of the construct, transfectants containing the vector only (GFP alone), were studied as the control. Transfection of the cells with the construct resulted in prominent expression of Hsp27-GFP without concomitant reduction in expression of the endogenous isoform in these cells compared with either parent LLC-PK1 or GFP cells. The total amount of the small Hsp was therefore increased in the Hsp27 transfectants. This allowed for determining functional effects of transfected overexpression of Hsp27 along with defining interactions with actin.
Fluorescence resonance energy transfer detects close spatial association between two separately labeled proteins. Two fluorochrome labels are chosen that have different excitation and emission spectra. The first, donor, fluorochrome is excited at a wavelength that does not overlap with the excitation spectrum of the second, acceptor, fluorochrome. However, donor fluorescence emission coincides with the excitation spectrum of the acceptor fluorochrome such that if the two fluorochromes are in close proximity (and hence the labeled proteins of interest), energy is transferred between the donor and acceptor molecules and fluorescence at the emission wavelength of the acceptor molecule is then found. If no fluorescence of the second acceptor fluorochrome occurs, the labeled proteins of interest are not closely associated. Efficient energy transfer occurs only if the two molecules are less than 70 Angstrom apart (21,22).
Using this technique, we found little interaction between the transfected Hsp27 and phalloidin-decorated actin under standard growth conditions (uninjured cells). This does not preclude the participation of endogenous Hsp25 in the regulation of actin dynamics as occurs in other types of cells (14). With ATP depletion, on the other hand, distinct fluorescence at the acceptor emission wavelength indicates that the labeled Hsp27 is closely associated with actin. This confirms our findings in vivo that suggested direct interaction of Hsp25/27 with actin shortly after renal ischemia (3). The disruption of the actin cytoskeleton by ATP depletion may then require recruitment of additional Hsp25/27 to assist in the processing of actin, either through its actin capping activity or through its protein chaperone function (14,23). We studied a single clone of cells with labeled Hsp27 alongside the transfectant GFP control; therefore, the results presented here need be interpreted conservatively. However, placing our findings in the context of two recent complementary studies that use an approach similar to ours is informative. ATP depletion–induced changes in actin organization and distribution were studied in a population of LLC-PK cells stably transfected with labeled actin, and changes in actin and Hsp27 distribution were studied in a clone of MDCK cells stably transfected with labeled Hsp27 (24,28). The recent report using labeled actin to study dynamic changes in actin during and following ATP depletion is particularly illuminating to our findings. LLC-PK cells stably transfected with EYFP-actin showed that ATP depletion caused EYFP-actin to accumulate in aggregates that co-localized with F-actin. During ATP repletion, EYFP-actin showed less aggregated pattern and reformation of stress fibers and the cortical actin network (24). We also saw changes consistent with this carefully defined dynamic alteration in actin distribution. By FRET analysis, Hsp27-GFP during ATP depletion appears to interact specifically both with the disorganized aggregates of F-actin as well as with filamentous actin at lateral boundaries of cell-cell contact. The latter finding is consistent with localization of Hsp27 found in the clone of MDCK cells stably transfected with labeled Hsp27 and subjected to ATP depletion (28). During recovery when normal cellular ATP levels are restored, the association of Hsp27-GFP with actin appears diminished compared with that seen during ATP depletion. Nevertheless, Hsp27-GFP during recovery appears still to interact predominantly with the residual aggregated actin. When total FRET was adjusted for fluorescence intensity of the phalloidin-stained actin, we found that net FRET to phalloidin was unchanged between 2-h ATP depletion and 4-h recovery. When considered in the setting of the changes in total F-actin staining and organization that we found and as previously reported (24,28), our findings with FRET suggest that Hsp27 interacts with disrupted or disorganized actin both during ATP depletion and as recovery progresses. Hsp27-GFP has little or no interaction with actin in non-injured control cells; but along with its distinct association with aggregated actin in the cell body, Hsp27 is further associated with filamentous actin at lateral cell boundaries during ATP depletion. The latter finding at the lateral cell boundary persists but appears less prominent during recovery. The interaction of Hsp27 with actin in cells injured by energy depletion, then, seems to be a dynamic process. This suggests a role for Hsp27 in the reorganization of actin into its normal arrangement during recovery from ATP depletion.
That Hsp25/27 interacts with actin in ATP depleted renal epithelia is in itself a significant finding. Whether the overexpression of this stress protein and its association with actin might protect renal epithelia from generalized or specific manifestations of injury was an additional question we sought to answer by studying two distinct cytoskeleton associated proteins. The sodium pump is a basolaterally located, integral membrane protein that is attached to the cytoskeleton under normal circumstances, but it dissociates and redistributes to apical domains with ischemia in vivo and ATP depletion in cultured renal epithelia (1). Previous studies have shown a close relationship between changes in Na,K-ATPase distribution and induction of the stress response and have suggested that stress proteins may be active in processing this enzyme during cellular recovery (16,25). In addition, ischemic preconditioning associated with Hsp25 induction prevented the expected redistribution of Na,K-ATPase during subsequent ischemic insults as determined by both immunohistochemistry and detergent extractability (26). Moreover, a direct, specific effect of Hsp25 on the preservation of Na,K-ATPase attachment to the cytoskeleton was suggested because addition of anti-Hsp25 antibody in vitro blocked the salutary effect contributed by preconditioning (26). Located in a separate subcellular domain from the sodium pump, ezrin is a microvillar protein that links the apical plasma membrane to the cytoskeleton. Hypoxia causes dissociation of ezrin from the cytoskeleton, which contributes to subsequent loss of the apical brush border (27). As expected, we found that ATP depletion caused dissociation of both Na,K-ATPase and ezrin from the cytoskeleton as manifested by increased detergent extractability. Overexpression of Hsp27 did not completely prevent, but nevertheless significantly reduced solubilization of Na,K-ATPase in response to 2-h ATP depletion compared with the GFP transfectant control cells, but it did not have significant effect at 4-h ATP depletion. So with respect to this manifestation of cellular injury, the cytoprotection provided by Hsp27 appeared to be overcome by longer intervals of ATP depletion.
In contrast, Hsp27-GFP had no effect on detachment of ezrin from the cytoskeleton during energy depletion. This difference in effect of Hsp27 on these two cytoskeletal-associated proteins may be due to the specific location and function of this stress protein in this form of cellular injury. In a similar model of epithelial cell injury, labeled Hsp27 migrated and co-localized with phalloidin-stained actin mainly in lateral domains at cell boundaries, with little labeled Hsp27 seen at the apical surface (28). Furthermore, the mechanism by which ezrin becomes dissociated from the cytoskeleton appears to differ from how the sodium pump becomes dissociated. Na,K-ATPase distribution is altered with primary disruption of the underlying actin-based cytoskeleton (1). In contrast, ezrin becomes dissociated primarily by changes in its phosphorylation state (27,29). Hsp25/27, then, may not be able to prevent ezrin from dephosphorylating and detaching from the cytoskeleton in energy-depleted cells. However, with translocation to the necessary subcellular domain and direct interaction with actin, Hsp25/27 may preserve cytoskeletal integrity in that particular domain and thereby limit Na,K-ATPase detachment.
We determined cellular ATP levels under each condition in each cell line to determine whether the observed cytoprotection might be attributable to preservation of cellular energetics. We found no relationship between cellular ATP levels and cytoprotection. Thus, preservation of cellular energy levels does not appear to be the mechanism by which Hsp27 protects these cells from this form of insult.
The present study demonstrates that Hsp27 interacts with actin during and after ATP depletion and that interaction is associated with protection against a select, specific manifestation of renal cell injury. We speculate that Hsp25/27 may have several actions in renal epithelia injured by energy depletion. Our finding that Hsp27 associates with actin fibers in the lateral domain during and after ATP depletion, which is consistent with recent similar findings of Hsp27 redistribution (28), along with our finding of associated selective protection of Na,K-ATPase attachment to the cytoskeleton, suggests that this stress protein may preserve and help restore cell architecture in this region. Our additional observation that Hsp27 closely associates with aggregates of actin within the cell body during and after ATP depletion, which is consistent with the recently well-characterized accumulation of actin aggregates in this region (24,28), suggests an additional separate function for this stress protein. Hsp27 may be interacting with disorganized aggregates of actin to prevent these proteins, altered by ATP depletion, from proceeding to a metabolic dead-end (23). That Hsp27 may serve these two separate roles is consistent with previously described multifunctional properties of Hsp25/27 that include actin-capping activity to prevent actin disruption (14,15) and activity as a protein chaperone through interactions with aggregates of disorganized or disrupted proteins (23).
Although overexpression of the labeled Hsp27 limited one aspect of injury from energy deprivation, Na,K-ATPase detachment from the cytoskeleton, it had no apparent effect on ezrin detachment from the cytoskeleton. Furthermore, the protection afforded by Hsp27 was not complete, especially with more severe injury. Our findings indicate that specific Hsp may have selective effects on manifestations of cell injury that depend on the unique function and intracellular location of a particular stress protein after ischemia. The association of Hsp27 with actin predominantly in disorganized cytoplasmic aggregates and as separately reported in basolateral domains (28) is consistent with a specific effect in those areas rather than a global effect on cytoskeletal-associated proteins throughout the cell. Though this small Hsp may have several functions, the specificity of its actions under different states of cell stress would explain why it, like other Hsp, does not provide uniform protection against all severity of insult and all manifestations of injury. Cytoprotection and tolerance to injury from preconditioning are likely provided by several mechanisms (30,31). Stress proteins, then, are one of several effectors that may prevent progressive injury or augment recovery.
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