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Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA;
* A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia; and
Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
1Correspondence: Department of Anatomy, Physiology and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA. E-mail: narispe{at}usuhs.mil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendixREFERENCES |
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Key Words: heat shock proteins • polypeptide • lipid bilayer • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendixREFERENCES |
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2
3)
. Hsps are composed of a small family of polypeptides distributed across different subcellular compartments, including cytosol, nucleus, endoplasmic reticulum (ER), and mitochondria. They are classified according to their molecular weight (e.g., Hsp70, Hsp90 families) (1)
. Recent studies have shown that hsps placed in the extracellular environment activate target cells, particularly macrophages (M
) and antigen-presenting cells (APC) (4)
. Hsp70 and the glucose-regulated protein 94 (Grp94), an ER homologue of Hsp90, act as immune adjuvants by delivering priming antigen peptides to APCs (5)
. Interaction of Grp94, Hsp70, and Hsp60 with Ms and APCs results in maturation, activation, and release of cytokines (6)
. Several proteins have been proposed to act as specific receptors for hsps on the cell surface. These receptors include CD14, the toll-like receptor family (TLR4 and TLR2), and CD40 (7
8
9)
. Several scavenger receptors such as CD91 (
2-macroglobulin receptor) (10)
, low density lipoprotein receptor (LOX-1) (11)
, and macrophage scavenger receptor (Msr1) (12)
, have also been implicated in the interaction of hsps with Ms and APCs.
A novel property of the Hsp70 family (in particular, Hsp70 and Hsc70) is its capacity to interact with lipids. Earlier studies showed that Hsc70 was capable of inducing an ion conductance pathway in artificial lipid bilayers. The activity of this channel was regulated by ATP/ADP content in the medium (13)
. These observations were confirmed by demonstrating that Hsc70 and Hsp70 were capable of aggregating liposomes in a time- and concentration-dependent manner, also modulated by the presence of ATP or ADP (14)
. The characteristics of liposome-induced aggregation by Hsp70 and Hsc70 were different (14)
, suggesting these two proteins are not functionally identical. This finding is consistent with earlier studies indicating other functional differences between Hsc70 and Hsp70 despite their high degree of sequence homology. Hsp70 is induced by stress (e.g., heat shock) whereas Hsc70 is constitutively expressed in cells. The subcellular distribution of these two proteins is not identical. Moreover, Hsp70 has been found to bind to the 40S ribosomal subunit of translating ribosomes whereas Hsc70 interacts with nascent polypeptides emerging from ribosomes (15
, 16)
.
In the present study, we show that the interaction of Hsp70 and Hsc70 with PC12 cells is mediated by the presence of phosphatidyl serine (PS) on the cell surface. This interaction of Hsp70s with cells results in a decrease in cellular viability, which may be blocked by masking PS residues with annexin 5. These studies shed light on possible mechanisms of the interaction of Hsp70s with membranes and its biological consequences.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendixREFERENCES |
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Liposome preparations
Large, unilammelar liposomes were prepared using the extrusion method. Palmitoyl-oleoyl-phosphatidyl serine and palmitoyl-oleoyl-phosphatidylcholine (Avanti Polar Lipids, Inc., Alabaster, AL, USA), dissolved in CHCl3 (10 mg/mL), were mixed at the corresponding ratios and air dried under N2. The dried lipids were hydrated in a solution containing 250 mM NaCl and 25 mM TRIS-HCl to a final concentration of 10 mg of phospholipid/mL. For hydration, the phospholipid suspensions were kept at room temperature for 1 h (or at 4°C for 24 h) before being passed through an Avanti Mini-Extruder (Avanti Polar Lipids) apparatus. The suspension was passed 12 to 15 times through a 0.05 µm polycarbonate membrane to obtain a homogeneous size distribution of liposomes of 
50 nm.
Liposome aggregation assay
Aggregation of liposomes by the hsps was determined from the change in absorbance that follows the increase in turbidity of the liposome suspension. Absorbance was measured at 350 nm in a Hewlett Packard spectrophotometer and data were collected every 30 s. Aggregation reactions were performed at room temperature in 1 mm path-length optical glass cells. Standard assays were conducted in 300 mM sucrose-40 mM histidine-HCl, pH 6, in the presence of 0.5 mM MgCl2 and 1 mM CaCl2. Recombinant bovine Hsc70 (SPP-751) and human Hsp70 (SPP-755) free of contamination by nucleotides were purchased from Stressgen (Biotechnologies Co., Victoria, B.C., Canada). Finally, the aggregation reaction was initiated by addition of freshly prepared liposomes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendixREFERENCES |
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. We have demonstrated that Hsc70 and Hsp70 induce aggregation of liposomes by a process that is time and concentration dependent. The aggregation process was different for these two members of the Hsp70 family (14)
. These observations showed that Hsc70 and Hsp70 interact intimately with phospholipids. To investigate the relevance of PS in the interaction of Hsp70 or Hsc70 with membranes, we studied the effect of these proteins on the aggregation of liposomes of different PS and PC compositions. Figure 1
A, B shows curves of the liposome aggregation induced by Hsp70 or Hsc70 (10 µg/mL) of liposomes of different PS/PC compositions. Changes in the amount of PS in the liposome membrane affected the amplitude and aggregation rate in a concentration-dependent manner. To quantify these results, each aggregation curve was corrected for control values and fitted as described previously (17)
. Calculations performed to fit the curves assume three different kinetic steps: an initial nonlinear region (region I), where the time course of 
OD350 follows a first order kinetics; a linear region (region II), where d(OD)/dt is constant; and a third kinetic region (region III), where the optical density increases asymptotically slower to a saturation level. The respective calculations are provided in the Appendix. To illustrate the influence of PS on the interaction of Hsc70 and Hsp70 with the liposome membrane, we plotted the dependence of the ODIlim value, the limiting value of ODI at t 
(Fig. 1C
), and k1k2, the rate of the change in the linear region (Fig. 1D, E
), as a function of the percentage of PS, respectively. These parameters reflect the magnitude and the velocity of the binding of heat shock proteins to the phospholipids membrane.
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A small decrease in PS composition (10%) of the liposome membrane resulted in a remarkable decrease in aggregation induced by Hsc70 or Hsp70 (50% and 70%, respectively). Further decreases of PS concentration resulted in total inhibition of Hsp70/Hsc70-induced liposome aggregation. For both Hsc70 and Hsp70, the magnitude of ODlimI (Fig. 1C
) characterizing the protein adsorption to the liposome as a function of the PS content decayed steeply as the PS concentration decreased. The increase in PS content from 80 to 85% results in the increase of region I amplitude (binding of the protein to the liposome), as expected. The further increase in PS content (from 90 to 100%) is accompanied by a sharp increase in the rate of the step of liposome aggregation. Because of the turbidity increase corresponding to this step, region I becomes concealed (masked). As a result, of ODlimI values are underestimated. At high values of PS, it is difficult to estimate correctly the amplitude of region I, but not the initial slope, which may be estimated with high reliability. The rate of liposome aggregation, k1k2, was profoundly dependent on the concentration of PS, particularly for Hsp70 (Fig. 1D, E
), so when the PS content increases, the dependence of k1k2 on PS content is a proper measure of the increase in the rate of liposome aggregation.
Hsc70 and Hsp70 are toxic for surface PS-positive cells
The foregoing observations suggest that the interaction of Hsc70 and Hsp70 with lipids is PS specific. The next step was to investigate whether this interaction occurred with biological membranes. PS is usually present in the cytosolic side of cellular membranes and rarely present on the cell surface, except in apoptotic cells. We screened several cell lines for the presence of PS on the cell surface using annexin 5, a conventional reagent for the detection of this phospholipid in the surface of apoptotic cells. We found that under standard culture conditions, viable PC12 cells presented elevated PS levels on the cell surface. These cells were incubated with different concentrations of Hsc70 or Hsp70 and cell viability was measured by the XTT method. As shown in Fig. 2
, both Hsc70 (squares) and Hsp70 (circles) were toxic for PC12 cells in a concentration-dependent manner, with Hsc70 being significantly more toxic than Hsp70 at three different concentrations (P<0.1, P<0.02, and P<0.008, respectively).
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Reports have suggested that extracellular Hsp70 binds to the 2-macroglobulin receptor on the cell surface (5)
. To test whether the toxic effect of Hsp70 was due to interaction with this surface glycoprotein, PC12 cells were incubated with Hsp70 or Hsc70 in the presence or absence of 2-macroglobulin. The interaction of 1-macroglobulin with its receptor should prevent Hsp70 or Hsc70 from interacting with the membrane. As shown in Fig. 3
, no significant differences in the toxicity induced by Hsp70 or Hsc70 were observed in the presence or absence of 2-macroglobulin.
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Exogenous ATP or ADP modifies the toxic effect induced by Hsc70 and Hsp70
Earlier studies have pointed out that binding of nucleotides to Hsp70 modulates conformation of Hsp70 (18)
, the state of oligomerization (19)
, and interaction with phospholipids as studied by the liposome aggregation assay (14)
. On the assumption that the interaction of Hsp70 with the phospholipids in the cell membrane is the first step in the series of processes leading to Hsp70-induced toxicity, we evaluated how exogenous addition of ADP or ATP influenced the effect of Hsp70 on cell viability. PC12 cells were incubated in a medium containing Hsc70 (250 ng/mL) in the presence of ATP or ADP at a concentration of 1 or 2 mM (Fig. 4
). We found that these concentrations of nucleotides in the extracellular milieu were not toxic for PC12 cells (white bars). Incubation of Hsc70 with ATP or ADP (striped bars) significantly enhanced toxicity compared with cells incubated with Hsc70 alone (P<6x10–5). The effect of the nucleotides on Hsp70 toxicity differed from the one observed for Hsc70 (Fig. 5
). Addition of ADP significantly decreased (P<3x10–4) the toxicity expressed by Hsp70, whereas incubation with ATP significantly enhanced (P<2x10–4) Hsp70-mediated toxicity.
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The presence of PS exposed on the cell surface increases the sensitivity of cells to exogenous Hsc70 and Hsp70
To determine whether the interaction of Hsc70 and Hsp70 with cells was due to specific binding to PS on the cell surface, HeLa cells, which do not display PS on the cell surface, and PC12 cells were treated with Hsc70 and Hsp70 and later incubated with Alexa Fluor 488 conjugated annexin 5 and the impermeant propidium iodide (PI), then analyzed by flow cytometry. Annexin 5 specifically interacts with PS on the membrane and PI stains the nucleus of membrane-disrupted cells. Annexin 5, in the presence of calcium, has been shown to specifically bind to PS at the outer membrane leaflet of cells (20). PC12 cells displayed higher levels of Alexa Fluor 488 staining than HeLa cells (Fig. 6
A, B). A 73.4% of these cells were positive for the presence of PS on the cell surface, in contrast with HeLa cells, which showed a 90% of PS negative cells (Fig. 6C
). After incubation of PC12 or HeLa cells with Hsc70 or Hsp70 (2 µg/mL) for 24 h, the percentage of viable PC12 cells was reduced by 70%; neither Hsc70 nor Hsp70 significantly affected the viability of HeLa cells (Fig. 6D
).
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Toxicity induced by Hsc70 or Hsp70 is block by preincubation with annexin 5
To further substantiate that the interaction of Hsc70 and Hsp70 with cells was due to specific binding to PS on the cell surface, PC12 cells were preincubated with annexin 5 before addition of Hsp70 or Hsc70. In the presence of calcium, this anticoagulant protein binds specifically to PS at the outer membrane leaflet of cells. When the acidic phospholipid PS becomes externalized and available for detection, hsps and annexin 5 will compete by specifically interacting with PS. The percentage of viable cells was measured after 24 h of incubation. The toxicity shown by concentrations of 0.1 µg/mL for Hsp70 (Fig. 7
A) and Hsc70 (Fig. 7B
) on PC12 cells was significantly attenuated when 40 nM annexin 5 was simultaneously present in the media (P<5x10–6 compared with Hsc70 and Hsp70 alone). Annexin 5 was less effective in protecting cells as the hsp concentration increased to 0.2 µg/mL (P<0.02 vs. Hsc70 alone and P <0.06 vs. Hsp70 alone).
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Exogenous Hsp70 binding to PS precedes the apoptotic death of cells
Two different types of PC12 cells were found based on their response to exogenous Hsp70. In the first type, Hsp70 induces an increase in the number of annexin 5-positive cells; in the second, the number of annexin 5-positive cells is reduced upon incubation with Hsp70.Figure 8
A shows a flow cytometric analysis of a type of PC12 cell in which, after 2 days incubation with Hsp70 (0.5 µg/mL), the peak of the population of annexin 5-positive cells is shifted to higher fluorescence levels (top left). The distribution of intact cells shows that the percentage of PS-positive cells increased from 60% (control) to 80% after cells were exposed to Hsp70 (top right). This increase suggests a possible role for Hsp70 in the induction of preapoptosis in PC12 cells. We found that 50% of this type of cell is nonviable after 2 days incubation with Hsp70 (bottom left). Viable cells displayed a higher level of caspase 3/7 activities (as relative fluorescence light units, RFLU) compared with untreated cells (bottom right). The percentage of PS-positive cells, viability, and the increased level of caspase 3/7 activities 2 h after addition of the apoptosis inducer staurosporin (STS) (0.5 µg/mL) are shown for comparison.
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Figure 8B
shows the results from a type of PC12 cell that, after being exposed to exogenous Hsp70, showed a decrease in the detectable external PS. The peak of the population of annexin 5-positive cells after 2 days of Hsp70 (0.5 µg/mL) incubation was shifted to lower fluorescence levels relative to untreated cells (control, top left). The percentage of cells displaying PS on the surface membrane was reduced after exposure to Hsp70 from 60 to 37% (top right). This reduction suggests that Hsp70 binds and screens PS from interacting with the fluorescent conjugated annexin 5-Alexa Fluor 488. Exposing the cells for 4 h to STS increased the percentage of annexin 5-Alexa Fluor 488 positive cells to 90%. Results from the viability test performed in this type of cell (bottom left) revealed that these cells were more resistant as only 20% of the cells were nonviable after 2 days exposure to Hsp70. The percentage of viable cells after 4 h in the presence of STS was reduced to >50%, as shown for comparison in the same figure. The level of caspase 3/7 activities (in RFLU) from the population of PC12 cells 2 days after the addition of Hsp70 (0.2 and 1.0 µg/mL) to the culture medium remained significantly similar to the control (bottom right). However, the level of caspase 3/7 activities 2 h after the addition of STS (0.5 µg/mL) was dramatically increased.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendixREFERENCES |
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, 22)
. Moreover, Hsp70 has been detected in the surface of tumor cells (23)
. Using antibodies to different epitopes of Hsp70, it was established that part of Hsp70 was embedded within the plasma membrane with the C-end terminal of the polypeptide exposed to the extracellular milieu (24)
. Moreover, Hsc70 was found to form ion conductance pathways in artificial lipid bilayers (13)
. The potential interaction of Hsp70 with lipids was confirmed by their ability to aggregate liposomes (14)
. In the present study, we showed that the interaction of Hsc70 and Hsp70 with membranes is specific for the presence of PS. Moreover, the interaction of these proteins with cells containing PS on the outer part of the plasma membranes was toxic. This toxicity was concentration dependent and could be blocked by covering PS moieties with annexin 5. These studies echo additional reports indicating the interaction of other hsps with lipids (25
26
27
28)
.
Recent studies have shown that cells can be activated by exogenous hsps. This process apparently is mediated by the interaction of hsps with protein receptors on the cell surface. Indeed, several potential receptors for different hsps have been identified such as CD91, CD40, LOX-1, and Msr1 (7
, 10
11
12)
. Our observations illustrate that the interaction of hsps with cells should be expanded to include phospholipids as potential targets for Hsp70 and Hsc70. Cell viability was decreased upon incubation with Hsp70 and Hsc70. This effect was specific for the presence of PS on the cell surface. PS is not present on the cell surface under normal physiological conditions. However, the appearance of PS on the cell surface is an early marker of apoptosis. Thus, it is possible that the apoptotic cell death process may be accelerated by the presence of Hsp70 in the extracellular environment. Hsp70 could be present in the extracellular milieu after cell lysis. For example, expression of hsps is dramatically induced after ischemia/reperfusion injury. This injury also results in a large focus of necrosis. Prior studies have shown the release of Hsp70 from necrotic cells, but not from apoptotic cells (29)
. Thus, Hsp70 may be loaded into circulation after plasma membrane disruption following lysis by necrosis. Plasma levels of Hsp70 have been reported in the range of 15–27 ng/mL in individuals with coronary artery disease (30)
or trauma patients within 2 days of admission (31)
. Although these concentrations are an order of magnitude lower than that used in our study, the local concentration of Hsp70 that cells and organs are exposed to may be higher than plasma levels. This circulating Hsp70 may accelerate the apoptotic process in other cell types, such as lymphocytes and epithelial cells. We speculate that toxicity induced by Hsp70 is due to the formation of ion channels on the plasma membrane in a series of steps, as schematized in Fig. 9
. We hypothesize that Hsp70 oligomerizes prior to insertion into the lipid bilayer, which requires the presence of PS. In fact, analysis of the kinetics of liposome aggregation indicates that the insertion of Hsp70 within membranes containing high concentration of PS was very rapid. Moreover, oligomerization of Hsp70 has been well established in in vitro conditions (32
, 33)
. If this hypothesis were correct, cell death after induction of hsps would be expected since the cytosolic side of cellular membranes is rich in PS. On the contrary, expression of hsps is associated with cellular protection. This paradox could be explained by the assumption that Hsp70 is not free to be incorporated into membranes after synthesis under stressful conditions. Likely, Hsp70 is associated with target proteins, such as nascent polypeptides and other unfolded polypeptides, within the cells. Thus, oligomerization does not occur when Hsp70 is associated with other proteins. This argument could be used for Hsc70 that is present under normal physiological conditions. For example, 107 Hsc70 molecules have been estimated within a cell at any given time, a number that matches the concentration of ribosomes (107 particles) per cell that are actively engaged in translation. Thus, the ratio of Hsc70 and nascent polypeptides is at least 1:1. Upon experiencing stress, a large number of Hsp70 molecules are synthesized to deal with the large number of unfolded proteins that appear as a consequence of the stress. During recovery conditions after the insult, the number of possible targets (unfolded polypeptides) tends to decrease, shifting the equilibrium toward an excess of Hsp70 over unfolded polypeptides. In these conditions, this excess of Hsp70 tends to oligomerize and get incorporated into the membrane, thus opening new ion pathways. The opening of this new conductance results in cell death. In fact, overexpression of Hsp70 has been reported to be toxic for cells (34)
. Attempts to generate cell lines overexpressing Hsp70 have been unsuccessful (unpublished observations). That Hsp70 is potentially toxic for cells has been used to explain a self-limited mechanism for Hsp70 expression. Thus, attenuation of transcription (35
, 36)
, destabilization of Hsp70 mRNA, and reduced translation (37)
have been reported. In summary, our results confirm prior observations regarding the interaction of hsps with lipids and may explain the cellular toxicity observed by overexpression of hsps.
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Appendix |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendixREFERENCES |
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OD) in the liposome suspension, which is described by several kinetic regions, as it has been characterized for other protein-liposome interactions (17)
. A first nonlinear region, region I, corresponds to the binding of the protein to the liposomes. A second region displays an initial linear stage of liposome aggregation, region IIa, corresponding to the interaction of two liposome particles, followed by a nonlinear stage, region IIb, that represents the formation of aggregates of larger sizes. Regions IIa and IIb may be regarded as a nucleation step resulting in the formation of the seeding for aggregation. The increase in OD is proportional to the amount of these aggregated liposome particles and d(OD350)/dt is a measure of the rate of liposome aggregation. The optical density change continues into a third region, region III, which corresponds to the stage of aggregate growth. Finally, a decrease in absorbance, region IV, is observed for a much longer period, which corresponds to the precipitation of aggregated liposomes. Within the period of our experiments, we did not record this late region.
Depending on the experimental condition, some of the OD change curves in kinetic regions may be scarcely discernible. When the curve includes regions I, II, and III (as the example shown in Fig. 10
A for the case of Hsp70 and 95% PS), the first and second regions were fitted using the equation:
 | (1) |
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where the term ODI lim[1 – exp(–kt)] describes the initial nonlinear region and term k1[exp(k2t) – 1] describes the region where acceleration of the aggregation process occurs (region II). The parameters values were ODlimI = 0.0156 ± 0.0012, k = 0.162 ± 0.010 min–1, k1 = 0.036 ± 0.004, k2 = 0.038 ± 0.002 min–1, k1k2 = 0.00137 ± 0.00014 min–1.
The third region was fitted using the exponential function:
 | (2) |
where k3 is the rate constant of the first order, ODIII lim is the limiting value of OD at t , and t0 is the time value at which OD = 0. Parameter t0 characterizes the duration of lag period for region III. The parameters values were ODIII = 0.389 ± 0.008, k3 = 0.0122 ± 0.0004 min–1, t0 = 8.6 ± 0.2 min. The horizontal dotted line in Fig. 10A
corresponds to the ODIII value.
In the case of Hsc70 and 100% PS shown in Fig. 10B
, the initial nonlinear region was scarcely discernible. However, we took into consideration the contribution of this region in the OD value. To fit the curve, we used the equation:
 | (3) |
Fitting was started from t = 2.5 min. The following parameters values were obtained: ODlimI = 0.0027 ± 0.0002, k1 = 0.047 ± 0.002, k2 = 0.081 ± 0.002 min–1, k1k2 = 0.0038 ± 0.0002 min–1.
In this example, region III became strongly pronounced and we used Eq. 2
to fit this region of the curve. The parameter values were ODIII = 0.384 ± 0.001, k3 = 0.0300 ± 0.0001 min–1, t0 = 4.53 ± 0.04 min. Compared with the curve obtained at 95% PS, the value ODIII remains the same, the duration of lag period t0 (corresponding to the nucleation stage) decreases, and k3 (corresponding to the stage of the growth of nuclei) increases.
The liposome aggregation curve obtained in the case of Hsc70 and 80% PS (Fig. 10C
) illustrates an example where only region I and region IIa (the initial linear part of region II) are well defined. In this case, the following equation was used:
 | (4) |
The following values of parameters were obtained: ODlimI = 0.00168 ± 0.00005, k = 0.156 ± 0.010 min–1, k1k2 = 0.000043 ± 0.000001 min–1.
The rate of the initial linear part of region II, d(ODIIa)/dt, is numerically equal to the k1k2 product. Thus, parameter k1k2 may be estimated for all the curves.
Received for publication April 20, 2004. Accepted for publication July 6, 2004.
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