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Am J Physiol Heart Circ Physiol 294: H2022-H2030, 2008. First published February 29, 2008; doi:10.1152/ajpheart.01271.2007
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Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration

Samira C. Grifoni, Susan E. McKey, and Heather A. Drummond

Department of Physiology and Biophysics and the Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi

Submitted 31 October 2007 ; accepted in final form 28 February 2008


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies suggest members of the degenerin (DEG)/epithelial Na+ channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes.

vascular smooth muscle cell migration; ion channel; degenerin proteins; acid-sensing ion channel; heat shock protein 70; interference ribonucleic acid

CELLULAR MIGRATION CONTRIBUTES to many biological processes, including embryogenesis, metastasis, and inflammation (6, 16, 18, 35). In the cardiovascular system, vascular smooth muscle cell (VSMC) migration is a key event during vascular development and in response to many vascular pathophysiological disorders, such as hypertension, diabetes, atherosclerosis, and vascular injury (20, 28, 29, 32). The mechanisms regulating VSMC migration are not well understood; however, ion channels play an important role (34). Our laboratory recently identified the involvement of a novel class of proteins, the degenerin (DEG)/epithelial Na+ channel (ENaC)/acid-sensing ion channel (ASIC), in VSMC migration (12, 13).

DEG/ENaC/ASIC proteins are part of a large, evolutionarily conserved, protein family. Members of this family participate in diverse functions, including Na+ transport, touch sensation, learning, taste, acid sensation, and vascular reactivity (8, 14, 15, 19). Two groups of DEG/ENaC/ASIC proteins have been identified in mammals: ENaC and ASIC. ENaC and ASIC proteins form non-voltage-gated cation channels, primarily conducting Na+ and Ca2+. Recently, we and others (5, 12, 13, 38) have demonstrated that ENaC and ASIC members of this protein family participate in migration of epithelial, glial, and VSMCs.

In previous investigations, we found most, but not all, ENaC/ASIC proteins play a positive regulatory role in VSMC migration (12, 13). However, we found that one family member, ASIC2, might play an inhibitory role. We found that suppression of ASIC2 using small-interfering RNA (siRNA) produced a weak (+4%) stimulation of migration in response to a chemical attractant (referred to as chemotactic migration). However, the effect was too small to conclude a negative regulatory role (13). We also observed a perinuclear-staining pattern for ASIC2 protein in VSMCs, suggesting that ASIC2 proteins are retained in a perinuclear intracellular pool. We reasoned that increasing cell surface ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration. The chemical chaperone glycerol has been used by others to increase surface expression of ion channels by stabilizing protein conformation, increasing the rate of protein refolding, and accelerating oligomeric protein conformation and assembly (3, 26, 33, 36–38, 40). Therefore, we used the chemical chaperone glycerol as a tool to enhance ASIC2 expression at the cell surface and help determine if ASIC2 plays a negative regulatory role in VSMC migration.

Other investigators have shown that glycerol-stimulated surface expression of ENaC and ASIC2 proteins is mediated by inhibition of a certain heat shock protein, Hsc70 (26, 31, 39). However, whether Hsc70 interacts with ASIC2 to regulate chemotactic migration is unknown. Therefore, the second aim of the study was to determine if Hsc70 inhibits cell surface ASIC2 expression and regulates migration. Data presented in this investigation demonstrate that cell surface expression of ASIC2 inhibits VSMC migration and Hsc70 inhibits basal ASIC2 expression. When the inhibitory effect of Hsc70 on ASIC2 expression is removed, ASIC2 cell surface expression is upregulated, which in turn inhibits VSMC migration.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. VSMC (A10 cells; ATCC; Manassas, VA) were grown at 37°C, 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were regularly passaged to maintain exponential growth. Studies were performed with cells at passage 1–10.

RT-PCR. To determine if Hsc70 transcripts are expressed in VSMCs, we used RT-PCR. Total RNA was isolated from cultured VSMC cells using the RNA STAT-60 kit (Tel-Test, Friendswood, TX), DNase treated using the TURBO DNA-free protocol (Ambion, Austin, TX), and reverse transcribed using oligo(dT) primers and AMV reverse transcriptase (Promega, Madison, WI). Oligonucleotides directed to Hsc70 5'-CAGAATCCCAAGATCCAGA-3' and 5'-GTGACATCCAAGAGCAGCAA-3' were used to amplify a 168-bp product. All reactions were preheated (Robocycler; Stratagene, La Jolla, CA) to 95°C for 3 min, then cycled 30 times at 95°C for 30 s, and annealed for 30 s at 55°C and 72°C for 42 s. PCR reactions in which reverse transcriptase was omitted served as a negative control. PCR products were separated using gel electrophoresis, visualized with ethidium bromide, and sequenced to confirm identity.

Glycerol treatment. VSMCs were pretreated with 100–500 mM of glycerol added to DMEM for 24 h at 37°C. Control samples did not receive glycerol supplementation.

Live/dead viability/cytotoxicity assay. The effect of glycerol on VSMC viability was determined using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes), according to the manufacturer's instructions. This two-color fluorescence cell viability assay is based on the simultaneous determination of live and dead cells with two fluorescence probes. Calcein AM, a probe for intracellular esterase activity, labels live cells, whereas ethidium homodimer-1, a probe for loss of plasma membrane integrity, labels dead cells. For this assay, VSMCs were cultured on coated glass slides as subconfluent monolayers. VSMCs were treated with 500 mM glycerol for 48 h. Cells treated with normal media alone and 70% methanol served as controls for live and dead cells, respectively. Cells were examined using a fluorescence confocal microscope. Images of four to six randomly chosen fields of view were obtained for each condition. Data were represented as the average percent of live cells per field of view.

siRNA. We used siRNA technology to suppress ASIC2 and Hsc70 expression in VSMCs. We have used this method previously to silence ENaC/ASIC expression (7, 12–14). For migration assays, VSMCs were plated and allowed to grow to 90% confluence before transfection with siRNA molecules. Validated siRNA molecules, directed to ASIC2 or Hsc70 (5–100 nM; ASIC2, ID no. 197232; and Hsc70, ID no. 201028), were obtained from Ambion. As a negative control, we used a nontargeting siRNA control molecule that activates the RNA-induced silencing complex (RISC, catalog no. D 001210-2; Dharmacon, Lafayette, CO). siRNA molecules were transfected in the cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Following a 4-h incubation, cultures were supplemented with growth media for 72 h before study. Quantification of siRNA suppression of ASIC2 and/or Hsc70 expression was completed with Western blotting analysis.

Protein isolation. Cultured VSMC were plated on T75 flasks and grown to 90% confluence. For total cellular protein isolation, cells were lysed by scraping in 500 µl of 2x Laemmli sample buffer and then incubated at room temperature for 20 min (12, 13). For cytosol protein isolation, we used a protein extraction kit (BioVision, Mountain View, CA) according to the manufacturer's protocol. For isolation of cell surface proteins, we used a Pierce Biotinylation Kit (Pierce, Rockford, IL).

Anti-ASIC2 antibody. Antibodies to the COOH-terminal peptide sequence in ASIC2 (CVPLQTALGTLEEIA) were generated in rabbits (Affinity Bioreagents, Golden, CO). The antibodies were epitope affinity purified and tested for reactivity and specificity using 1) enzyme-linked immunosorbent assay, 2) Western blotting in cultured VSMC with antigen blockade, and 3) Western blotting and immunofluorescence in ASIC2 wild-type and null mouse tissues.

Western blotting. After protein isolation, cell lysates (40 µl) were separated using standard electrophoresis procedures as described previously (12). After being transferred to nitrocellulose membranes, blots were rinsed in PBS, blocked in Odyssey blocking buffer (LI-COR, Lincoln, NE) for 1 h at room temperature, and then incubated with rat monoclonal anti-Hsc70 (1:10,000; Abcam, Cambridge, MA), rabbit anti-ASIC1 (1:1,000; Chemicon), affinity-purified rabbit anti-ASIC2 C-term (1:1,000), or rabbit anti-ASIC3 (1:1,000; Chemicon) antibodies overnight at 4°C. The membranes were probed with mouse anti-β-actin (1:10,000; Abcam) as a loading control. Membranes were incubated with IR700-conjugated donkey anti-rabbit IgG or IR700-conjugated donkey anti-rat and IR800-conjugated donkey anti-mouse IgG (1:2,000; Rockland Immunologicals). Antibody labeling was visualized using the Odyssey Infrared Scanner (LI-COR), which allows for the simultaneous detection for two fluorophores. Fluorescence intensity analysis was performed using Odyssey software (LI-COR). ASIC2 and Hsc70 protein levels were normalized to β-actin to control for variations in sample loading. For antigen blockade studies, antibodies were preincubated with antigen (1:10) overnight and centrifuged before incubation with membrane.

Coimmunoprecipitation. Cytosol fractions were incubated with 75 µg of rat anti-Hsc70 monoclonal antibody, 50 µg mouse anti-Hsp70 monoclonal antibody, 50 µg of rat anti-Grp94 antibody, rabbit anti-calnexin antibody (dilution 1:50), 50 µg of rabbit anti-Grp78, or 10 µg mouse anti-Hsp90 monoclonal antibody (Abcam) for 4 h at room temperature on a rotator, followed by the addition of protein G agarose beads. Beads were washed with lysis buffer two times. Samples were resuspended with 100 µl of 1x Laemmli sample buffer and heated at 95°C for 5 min. Standard electrophoresis and Western blotting detection for ASIC proteins followed. Specificity of the antibodies for immunoprecipitation was demonstrated by substitution of nonimmune mouse, rat, or rabbit IgGs (Jackson Immunoresearch Laboratories) for the primary antibodies.

Immunofluorescence cell staining. Cultured cells were plated on fibronectin coated glass slides, grown for 24 h, rinsed with PBS, and then fixed in 4% paraformaldehyde for 10 min. After fixation, samples were rinsed with PBS and then blocked with 5% normal donkey serum (NDS) in PBS for 1 h. Monoclonal rat anti-Hsc70 (1:200; Abcam) was used for immunostaining. All samples were colabeled with mouse anti-{alpha}-smooth muscle actin (1:200; Sigma Chemicals, St. Louis, MO). Samples were incubated with primary antibodies plus 5% NDS in PBS overnight at 4°C. The following day, the samples were rinsed with PBS and then exposed to secondary antibody [Alexa 488-conjugated donkey anti-mouse IgG (1:1,000; Molecular Probes, Eugene, OR) and Cy-3-conjugated donkey anti-rat F(ab')2 (1:100; Jackson Immunologicals, West Grove PA)] in 5% NDS for 1 h. Samples were examined using a fluorescence confocal microscope (TCS-SP2; Leica Microsystems, Exton, PA), and images were prepared in PhotoShop (Adobe Systems, San Jose, CA).

Chemotactic migration assay. We evaluated VSMC migration in response to a chemotactic stimulus using modified Boyden chambers (Costar Transwell inserts, 6.5 mm diameter, 8.0 µm pore size) as described previously (12, 13). Platelet-derived growth factor-bb (PDGF-bb, 0.05 µg/ml; RDI, Flanders, NJ) was used as chemoattractant. PDGF-bb-stimulated migration was quantified as the average number of cells identified from four fields of view per insert. All samples were run in triplicate, and each experiment was performed at least three times. The data were normalized as percent of control samples.

Statistical analysis. All data are expressed as means ± SE. Groups were compared using ANOVA followed by the Student-Newman-Keul's posttest. Values of P ≤ 0.05 were considered statistically significant.


   RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Enhanced ASIC2 protein expression stimulated by glycerol inhibits VSMC migration. Using Western blot analysis of cytosol fraction proteins (Fig. 1A), we observed that treatment with the chemical chaperone glycerol (500 mM) increased ASIC2 protein expression in VSMCs nearly threefold (Fig. 1B). Incubation with corresponding antigen was used to determine antibody specificity. Antibody binding was blocked or inhibited by preincubation with excess antigen (Fig. 1A, bottom). We also observed that glycerol treatment improved detection of ASIC2 at the cell surface (Fig. 1C). Interestingly, the presence of a faster migrating product than observed in the cytosol was also observed after glycerol treatment. In addition, chemotactic migration was inhibited following treatment with glycerol (100 or 500 mM, Fig. 1D). A cell viability assay was performed to determine if glycerol-induced VSMC migration inhibition was due to its cytotoxic effect. Glycerol (500 mM) did not affect VSMC viability. Representative images (Fig. 2A) and group data (Fig. 2B) are shown. To determine if glycerol inhibits VSMC migration by increasing ASIC2 protein expression, we used siRNA to silence ASIC2 expression. As shown in Fig. 3, A and B, delivery of ASIC2 siRNA significantly suppressed expression of ASIC2 protein when compared with RISC-transfected controls by ~90%. Furthermore, silencing ASIC2 expression reversed the inhibitory effect of glycerol on PDGF-bb-stimulated VSMC migration (Fig. 3C), suggesting that increases in glycerol-mediated cytosol and cell surface protein expression of ASIC2 inhibit VSMC migration.


Figure 1
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  		Fig. 1. Glycerol treatment enhances expression of acid-sensing ion channels (ASIC2) in cultured vascular smooth muscle cells (VSMCs) and inhibits VSMC migration. A: Western blotting detection of ASIC2 in cytosolic VSMC lysates following treatment with glycerol. β-Actin loading control is shown below the corresponding ASIC2 blot. Antibody labeling is blocked when the antibody is incubated with excess antigen (bottom). B: fluorescence intensity analysis of ASIC2 cytosolic expression shown in A. C: Western blot detection of ASIC2 from surface-biotinylated proteins following with and without glycerol treatment. Biotinylated proteins were pulled down using streptavidin beads and represent the cell surface fraction. D: glycerol treatment inhibits VSMC migration in response to platelet-derived growth factor (PDGF)-bb. Data are means ± SE; n = 6 experiments. *Significantly different from control, P < 0.05.

 

Figure 2
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  		Fig. 2. Effect of glycerol on VSMC viability. Representative images (A) and group data (B) are shown. Metabolically active cells stained with calcein AM appear green (left), and dead cells stained with ethidium homodimer-1 (EthD-1) appear red (middle). Merged immunofluorescence is shown on right. Methanol (MeOH, 70%) was used as a control for dead cells. Group data show that 500 mM glycerol does not alter viability compared with vehicle. Data are means ± SE; n = 4–6. *Significantly different from control, P < 0.05.

 

Figure 3
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  		Fig. 3. Silencing ASIC2 expression by small-interfering RNA (siRNA) approach reverses the inhibitory effect of glycerol treatment on VSMC migration. A: Western blot detection of ASIC2 in whole VSMC lysates following transfection with ASIC2 siRNA. Delivery of ASIC2 siRNA downregulates ASIC2 protein expression in cultured VSMCs compared with nontargeting siRNA [RNA-induced silencing complex (RISC)] controls. β-Actin loading control is shown below the corresponding ASIC2 blot. A: representative Western blot. B: quantitative data. C: ASIC2 suppression reverses the inhibitory effect of glycerol treatment on PDGF-bb-stimulated VSMC migration. Data are means ± SE; n = 5–6. *Significantly different from RISC control, P < 0.05.

 
Expression of Hsc70 in cultured VSMCs. To determine if Hsc70 might regulate ASIC2 expression in VSMCs, we first wanted to determine whether transcripts and proteins are expressed in cultured VSMCs. Therefore, we used RT-PCR, immunoblotting, and immunolabeling (Fig. 4). RT-PCR analysis detected transcript expression of Hsc70 in cultured VSMCs (Fig. 4A). By Western blotting and immunolabeling, we determined that VSMCs express Hsc70 proteins. As shown in Fig. 4B, punctate cytoplasmic expression of Hsc70 proteins was detected in cultured VSMCs. Hsc70 was detected near the predicted molecular mass of 73 kDa (Fig. 4C).


Figure 4
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  		Fig. 4. Heat shock protein 70 (Hsc70) interacts with ASIC2 protein in cultured VSMCs. A: representative image of RT-PCR detection of Hsc70. The presence (RT+) and absence (RT–) of reverse transcriptase is indicated. B: representative images of Hsc70 protein expression by immunolabeling in cultured VSMC. {alpha}-Actin was used as a marker for VSMCs. C: Western blotting detection of Hsc70 in cytosol lysates from control and glycerol-treated VSMCs. β-Actin loading control is shown below the corresponding Hsc70 blot. D: group fluorescence intensity analysis of samples in C; n = 3. E: ASIC2, but not ASIC1 or ASIC3, coimmunoprecipitates with Hsc70. Cytosol proteins were immunoprecipitated with anti-Hsc70 rat monoclonal antibody as described in EXPERIMENTAL PROCEDURES. Rat IgG was used as a negative control.

 
Glycerol treatment on Hsc70 protein expression in VSMCs. To evaluate whether glycerol could modify Hsc70 protein expression, we performed Western blotting in both control and glycerol-treated VSMCs. As shown in Fig. 4D, glycerol treatment tended to decrease Hsc70 protein by ~20% (P = 0.09). To test whether ASIC2 and Hsc70 proteins associate in VSMCs, we used coimmunoprecipitation. As shown in Fig. 4E, ASIC2 coimmunoprecipitates with Hsc70, and this suggests Hsc70 and ASIC2 associates under control conditions in VSMCs. Importantly, our data suggest that Hsc70 may specifically associate with ASIC2, since this chaperone does not seem to interact with ASIC1 or ASIC3 in cultured VSMCs (Fig. 4E).

Association between ASIC2 and other chaperone proteins. We also investigated if ASIC2 associates with other chaperones in VSMCs. Our results demonstrate that ASIC2 does not associate with Hsp70, Grp94, Grp78, calnexin, and Hsp90 (Fig. 5, A–E).


Figure 5
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  		Fig. 5. Coimmunoprecipitation of chaperones with ASIC2 in cultured VSMCs. Western blotting detection of ASIC2 from cytosol proteins following immunoprecipitation with anti-Hsp70 mouse monoclonal, anti-Grp94 rat monoclonal, anti-calnexin rabbit polyclonal, anti-Grp78 rabbit polyclonal, or anti-Hsp90 mouse monoclonal antibodies, as described in EXPERIMENTAL PROCEDURES. Specificity of the antibodies for immunoprecipitation was demonstrated by substitution of ChromoPure nonimmune IgGs for the primary antibodies.

 
Hsc70 siRNA downregulates Hsc70 expression and upregulates ASIC2 expression. As expected, delivery of Hsc70 siRNA significantly suppressed expression of Hsc70 protein when compared with RISC-transfected controls by ~80% (Fig. 6, A and B). Silencing of Hsc70 expression was also accompanied by upregulation of cytoplasmic ASIC2 expression (~40%), as shown in Fig. 6, C and D. More importantly, Hsc70 silencing increased detection of ASIC2 at the cell surface (Fig. 6E). Interestingly, as observed after glycerol treatment (Fig. 1C), the presence of a faster-migrating product than observed in the cytosol was observed after Hsc70 silencing. Product labeling was blocked by antigen coincubation, suggesting the smaller protein product is ASIC2.


Figure 6
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  		Fig. 6. siRNA-induced Hsc70 silencing upregulates ASIC2 expression and inhibits VSMC migration. A: Western blotting detection of Hsc70 in whole VSMC lysates. Delivery of Hsc70 siRNA downregulates Hsc70 protein expression in cultured VSMCs compared with nontargeting siRNA (RISC) controls. β-Actin loading control is shown below the corresponding Hsc70 blot. B: group fluorescence intensity analysis of Hsc70 expression. C: Western blotting detection of ASIC2 in whole VSMC lysates after Hsc70 silencing upregulates ASIC2 protein expression in cultured VSMCs compared with nontargeting siRNA (RISC) controls. β-Actin loading control is shown below the corresponding ASIC2 blot. D: fluorescence intensity analysis of ASIC2 expression. E: inhibition of Hsc70 increases detection of ASIC2 in surface-biotinylated proteins. Antibody labeling is blocking following incubation with excess antigen. F: suppression of Hsc70 inhibits migration in response to PDGF-bb. Furthermore, silencing ASIC2 expression almost completely reversed the inhibitory effect of specific Hsc70 gene silencing on PDGF-bb-stimulated VSMC migration. Data are means ± SE; n = 3–4. *Significantly different from RISC control, P < 0.05.

 
Silencing Hsc70 expression inhibits VSMC migration. As shown in Fig. 6F, silencing Hsc70 expression significantly inhibited PDGF-stimulated migration by ~80% when compared with RISC-transfected controls. In contrast, ASIC2 suppression alone enhanced chemotactic migration by ~3%, a finding consistent with our previous study (13). Furthermore, silencing ASIC2 expression reversed the inhibitory effect of specific Hsc70 gene silencing on PDGF-bb-stimulated VSMC migration. Taken together, these data suggest that, when the inhibitory effect of Hsc70 on ASIC2 expression is removed, ASIC2 expression is upregulated, which in turn inhibits VSMC migration.


   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Migration of VSMCs contributes to the pathophysiology of many vascular disorders, such as hypertension, diabetes, atherosclerosis, and restenosis after coronary angioplasty (20, 28, 29, 32). Ion transport plays a prominent role in cell migration (34). Others and we (5, 12, 13, 38) recently demonstrated that ENaC/ASIC channels play an important role in cellular migration in diverse cell types, including glial, epithelial, and VSMC. A recent investigation from our laboratory suggested that ASIC2 might play an inhibitory role in VSMC migration. We found that suppression of ASIC2 expression, using siRNA, caused a small (3–4% increase) but statistically significant increase in chemotactic migration. However, the functional effect of ASIC2 inhibition on migration was too small to confidently conclude a physiologically significant negative regulatory role. We also observed a perinuclear-staining pattern for ASIC2 protein in VSMCs, suggesting that the protein may be retained in a perinuclear intracellular pool (13). These two findings raised the possibility that the functional effect of ASIC2 expression on chemotactic migration was small because there was very little protein at the surface membrane. We reasoned that increasing surface expression of ASIC2 might unmask its importance as a negative regulator of chemotactic migration. We used glycerol to increase surface expression of ASIC2 since previous studies have shown that glycerol increases surface expression of ASIC2 and the closely related ENaC proteins (11, 30, 38). Furthermore, we wanted to determine if Hsc70 prevents surface expression of ASIC2 in VSMCs, since previous studies have shown that glycerol increases surface expression of ASIC/ENaC channels by inhibiting Hsc70. There are two major findings of this study. First, increasing surface expression of ASIC2 inhibits VSMC migration. Second, Hsc70 protein interacts with ASIC2 to prevent cell surface expression; when the inhibitory effect of Hsc70 on ASIC2 expression is removed, ASIC2 surface expression increases, which inhibits VSMC migration.

How does glycerol increase surface expression of ASIC2 protein? Glycerol has been shown to increase cell surface expression of several ion channels; however, the precise mechanisms by which glycerol elicits proper maturation of proteins remain unclear (11, 30, 31, 38). One possible mechanism involves molecular chaperones heat shock proteins (11, 30, 38). The heat shock protein chaperone system is involved in 1) folding and trafficking of newly synthesized proteins in the cells and 2) degradation by lysosome and proteasome pathways (4, 10, 21). One heat shock protein, Hsc70, a constitutively expressed member of the 70-kDa heat shock protein family, has been identified as an important negative regulator of ion channel trafficking. Hsc70 is a key chaperone involved in lysosomal and proteasomal degradation of proteins. The Hsc70 cochaperone CHIP (carboxyl-terminus of Hsc70-interacting protein) interacts with Hsc70 to recognize improperly formed proteins for proteasomal degradation (22). Prolonged interaction of a target channel with Hsc70 increases the likelihood that the channel will be targeted for ubiquitination and intracellular degradation by the proteasome (31). Previous studies have shown that glycerol inhibits Hsc70 expression and thus enhances cell surface expression of ENaC and ASIC2 in epithelial and glial cells (31, 39). Similarly, in our investigation, we found that glycerol inhibits Hsc70 expression and increases ASIC2 surface expression in VSMCs. We also demonstrate in this study that ASIC2 specifically interacts with Hsc70 in cultured VSMCs. Furthermore, our evidence indicates that reductions in Hsc70 expression likely mediate increases in surface ASIC2, since Hsc70 silencing with siRNA increases surface ASIC2 detection. Therefore, even though we were just able to detect a very small decrease in Hsc70 expression following glycerol treatment, it is possible that glycerol stimulates surface expression of ASIC2 by inhibition of Hsc70 interactions, which would decrease the rate of intracellular degradation of ASIC2. However, it is unknown whether Hsc70 regulates ASIC2 degradation via lysosomal or proteasomal pathways.

Why is ASIC2 retained in the cytoplasm? It is unclear why ASIC2 is intracellularly retained in cultured VSMCs; however, our findings suggest that Hsc70 mediates this, at least in part. This localization pattern contrasts with freshly dissociated VSMCs where ASIC2 tends to be localized at or near the surface membrane (9). VSMCs have a unique characteristic known as phenotype plasticity [that is the ability to change its phenotype in response to changes in local environmental cues, including mechanical influences (pressure, flow, strain), growth factors/inhibitors, cell-cell and cell-matrix interactions, and various inflammatory mediators (25, 27)]. Evidence suggests that migrational ability is linked to VSMC phenotype. The "contractile" state is characterized by low rates of proliferation and migration while the "synthetic" state is characterized by increased protein production, proliferation, and migration rates. Freshly dissociated cells tend to have heterogeneous phenotypes, i.e., contractile, synthetic, and in-between; however, most tend to be in the contractile state (27). The culturing environment is associated with a phenotypic shift, where contractile VSMC switch to the synthetic state. Thus cultured VSMCs tend to be in the synthetic state and have stronger migrational ability (27). Thus ASIC2 localization may be dependent on the status of the cell. It is interesting that ASIC2, a negative regulator of migration, is expressed near the membrane of VSMCs that do not migrate (i.e., in vivo; see Ref. 9). Yet, ASIC2 membrane expression is almost undetectable in cells that have a strong ability to migrate. It is tempting to speculate that ASIC2 localization may be an important determinant in VSMCs migrational ability, and a shift in ASIC2 localization away from the membrane may allow the VSMC to migrate. Alternatively, it could just be coincidence. The mechanisms underlying this localization pattern are unknown. We speculate that the culturing environment itself may be responsible for the change in localization. Factors such as hormonal supplements, loss of stabilizing extracellular matrix proteins, protein misfolding, and imbalance of proteins regulating trafficking and degradation are likely involved in signaling the changes.

Does ASIC2 form a homomeric or heteromeric channel? In other expression systems, ASIC2 can form a homomeric channel or interact with other ASIC and ENaC proteins to form a heteromeric channel (1, 19, 23). Based on our findings, we cannot determine if ASIC2 forms a homomeric channel. However, three lines of evidence suggest that interactions between ASIC2 and other DEGs are possible. First, ENaC proteins ({alpha}, β, and {gamma}) and ASIC1 and ASIC3 are expressed in these VSMCs (12, 13). Second, chemotactic migration is inhibited when expression of {alpha}-, β-, or {gamma}ENaC or ASIC1 and ASIC3 are individually silenced using siRNA, suggesting all proteins are required for migration (12, 13). Third, ENaC proteins biochemically interact with ASIC proteins in glial cells and other systems; however, this interaction has not been determined in cultured VSMCs (1, 23). A recent study from our laboratory suggests that, under normal cell culture conditions, there is very little interaction between ASIC2 and ASIC1 or ASIC3: colocalization among ASIC2 and ASIC1 or -3 was <1.5% in the cytoplasm (13). This finding suggests that there is very little interaction between ASIC2 and ENaC proteins under control conditions. It is unknown if inhibition of Hsc70 expression alters the colocalization of ASIC2 with other DEGs.

How does ASIC2 contribute to VSMC migration? It is not clear how ASIC2 regulates migration. It is possible that increased surface expression correlates with increased channel activity that leads to activation of a negative regulatory pathway. Alternatively, increases in ASIC2 expression could disrupt activity of a heteromultimeric ENaC/ASIC channel, so increased surface ASIC2 could inhibit ion transport. Vila-Carriles et al. (39) and others (1, 24) have addressed this latter possibility. Collectively, these studies demonstrate that ASIC proteins form heteromeric channels that participate in glial cell migration, and the inclusion of ASIC2 inhibits inward Na+ current and disrupts migration. Our previous finding that there is very little colocalization between ASIC2 and ASIC1 or ASIC3 under control conditions where VSMCs have a strong ability to migrate would be consistent with the hypothesis that the absence of ASIC2 interactions with other DEG subunits enhances channel/migration activity.

An illustration of how Hsc70 may regulate ASIC2 expression to control migration is shown in Fig. 7. This possible scenario is based on the findings presented here and those of Vila-Carriles et al. (38, 39). We hypothesize that, under normal cell culture conditions, Hsc70 is likely expressed in excess of ASIC2. Excess Hsc70 promotes ASIC2-Hsc70 interactions, which prevents trafficking of ASIC2 to the cell surface membrane and favors ASIC2 targeting for sequestration and/or degradation. The absence of cell surface ASIC2 may permit activity of heteromeric ENaC/ASIC channels at the surface that play an important role in VSMC migration. However, inhibition of Hsc70 disrupts interactions between ASIC2 and Hsc70 and increases ASIC2 availability for membrane targeting. Incorporation of ASIC2 in the heteromeric channel inhibits the channel's activity and prevents VSMC migration. Thus controlling ASIC2 surface expression may be a means for the VSMC to regulate migration. Allowing ASIC2 to reach the surface, and possibly interact with other ENaC/ASIC protein, may provide a brake on the cells' migrational ability that can be released when environmental conditions signal a need to migrate.


Figure 7
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  		Fig. 7. Illustration of a potential mechanism for Hsc70 regulation of cell surface ASIC2 expression and VSMC migration. This illustration is based on our findings and those of others. We hypothesize that, under normal conditions, Hsc70 may be expressed in excess of ASIC2. Excess Hsc70 may promote ASIC2-Hsc70 interactions and prevent trafficking of ASIC2 to the surface membrane and target ASIC2 for sequestration and/or degradation. The lack of surface ASIC2 may permit activity of heteromeric ENaC/ASIC channels at the surface that are important in VSMC migration. However, inhibition of Hsc70 disrupts interactions between ASIC2 and Hsc70 and increases ASIC2 availability for membrane targeting. Incorporation of ASIC2 in the heteromeric channel inhibits the channels' activity and prevents VSMC migration.

 
Heat shock proteins in VSMC migration. Other than the findings presented here, evidence of Hsc70 involvement in VSMC migration is lacking. However, heat shock protein families have been implicated in the pathogenesis of atherosclerosis, which involves the migration of several cell types, including VSMCs. Unfortunately, no data are available on the role of Hsc70 in the pathogenesis of atherosclerosis. Because our findings suggest that Hsc70 is an important regulator of VSMC migration, they raise the possibility that Hsc70 may be an important target for VSMC migration following vascular injury.

Many ion channels, such as members of the DEG/ENaC/ASIC family of proteins, undergo regulated trafficking as a means of controlling their plasma membrane density (2); however, relatively little is known about the regulation of ASIC protein processing, trafficking, and stability at the plasma membrane. ENaC and ASIC proteins are subjected to quality control checkpoints during which misfolded proteins may be degraded, resulting in a decreased number of channels expressed at the cell surface. Molecular chaperones, including heat shock proteins, have recently been shown to play a role in this quality control process (11, 17, 26, 39). In summary, we provide evidence that Hsc70 plays an important role in ASIC2 trafficking in VSMCs. Downregulation of Hsc70 increases ASIC2 expression at the cell surface, which inhibits VSMC migration. Thus ASIC2 may act as a negative regulator of VSMC migration, and either the ion channel itself or its regulatory pathways may provide a target for potential therapeutic use in disease states in which VSMC migration plays a critical pathophysiological role.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work supported by National Institutes of Health Grants HL-51917, HL-071603 (H. Drummond), and HL-086996 (H. Drummond) and by American Heart Association Gratns AHA0725322B (S. Grifoni) and AHA0655305B (H. Drummond).


   ACKNOWLEDGMENTS
 
We thank our laboratory colleagues for excellent discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. A. Drummond, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216 (e-mail: hdrummond{at}physiology.umsmed.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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