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EDITORIAL FOCUS
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
THE ABILITY OF AN ANIMAL CELL to move is essential for life. Cell locomotion occurs during development, in response to injury, and during regeneration and/or remodeling of existing tissue. It also occurs under pathological conditions, examples being cancer cell metastasis and cardiac hypertrophy and angiogenesis. Clearly, cell motility is a complex process involving many different systems, including, for example, the cytoskeleton, signal transduction molecules, molecular motors, extracellular matrix proteins, and ion channels, to name a few. All of these processes have to be integrated and coordinated in a precise fashion in order for directed and efficient movement to occur.
Grifoni et al. (11a) report that one specific chaperone, the constitutive form of the 70-kDa heat shock protein (Hsc70), interacts with acid-sensing ion channel 2 (ASIC2) to effect its delivery to the plasma membrane. What is even more interesting is that this ion channel protein somehow influences the migratory ability of vascular smooth muscle cells (VSMCs). Obviously, because VSMC migration is essential for vascular remodeling following injury, or surgical intervention, these findings will have significant clinical implications. The way in which ASIC2 modulates migration of cells is not known. One might think that because during injury the tissue microenvironment becomes acidic and because ASIC2 is activated by protons, the presence of ASIC2 would induce a cation current that would electrically couple the extracellular acidity to signal-increased cellular motility. But no; when ASIC2 is present in the plasma membrane, migration decreases.
What then is the role of ASIC2? Transcriptional splicing generates two ASIC2 gene products, ASIC2a and -2b. Whereas rodent ASIC2a generates an acid-sensitive current when expressed, ASIC2b fails to do so (12). However, both ASIC2a and -2b have been proposed as modulators of ASIC1 current, decreasing the sensitivity to pH, the desensitization rate, and facilitating the effect of other modulators (1). In glial tumors, human ASIC2b is a negative regulator of the glioma cation conductance (3). Thus changes in the membrane expression of ASIC2 may influence already resident ion channels and thus regulate cellular membrane potential.
What is the evidence, then, for coupling membrane potential to cell migration? Several studies have shown that TTX, an inhibitor of voltage-gated Na+ channels, also blocks migration and metastasis in cancer cells (7). The inhibition of the epithelial Na+ channel (ENaC) is associated with membrane hyperpolarization and decreased migration in bovine corneal epithelial cells (5). The resting membrane potential of pulmonary VSMCs is more depolarized in proliferating versus growth-arrested cells (15). These studies suggest that membrane depolarization increases migration. However, in intestinal epithelial cells, the reverse seems to be true, in that the enterocyte migration was associated with hyperpolarization (8, 14). It has been suggested that increased expression and activity of K+ channels in these cells increases the inward driving force for Ca2+, which is essential for cytoskeletal remodeling required for migration (14). Increased Ca2+ entry could also be a consequence of membrane depolarization due to opening of voltage-sensitive Ca2+ channels. In smooth muscle cells, Ca2+ is required for the calmodulin-dependent activation of myosin light chain kinase and the subsequent phosphorylation of myosin. Contraction of myosin provides traction during movement, allowing VSMCs to migrate (9).
Intriguingly, VSMCs are extremely plastic cells, with the ability to change significantly and reversibly their phenotype to noncontractile, migratory cells from stationary contractile cells (2). VSMC phenotype switching, which is a characteristic of several cardiovascular diseases, and such processes as restenosis following angioplasty, is accompanied by changes in ion channel expression, notably of a large-conductance K+ channel (BK) and a voltage sensitive Ca2+ channel, Cav1.2. It has been proposed that the loss of the BK channel is complemented by increased expression of a second K+ channel, KCa3.1, and that increased expression of transient receptor potential (TRP) channels compensates for the loss of voltage-gated Ca2+ channels (2). The net effect of these changes would be cell hyperpolarization and perhaps increased Ca2+ entry via the TRP channels. However, it is important to consider that an altered expression of multiple ion channels has been reported when VSMCs change phenotype. These changes include voltage-gated Na+ channels, other K+ and Ca2+ channels, anion channels, and transporters. It should also not be forgotten that an appropriate intracellular ionic milieu is required for migration and that this includes a suitable cell volume; hence, water transport is also an important element of migration.
What part does ASIC2 play in this scheme? Other work from Drummond's laboratory has reported that the silencing of ASIC2 was associated with a small stimulation in chemotactic migration, suggesting that ASIC2 may inhibit migration like in glial tumor. In contrast, small-interfering (si)RNA silencing of ASICs 1, 2, and 3 inhibited wound healing (11). This result may reflect differences in other factors, potentially secreted by the cells in response to damage. Consistent with this result, drugs known to inhibit ASICs such as benzamil and amiloride also inhibited migration (10). In the present study, Grifoni et al. (11a) report that by using a chemical chaperone, glycerol, they were able to increase cell surface expression of ASIC2. In the presence of glycerol, the migration of the model VSMC line A10 was significantly inhibited, while at the same time, surface expression of ASIC2 increased threefold. This effect of glycerol was abolished by siRNA silencing of ASIC2. Similar results with chemical chaperones have been reported for both the mutant form of cystic fibrosis transmembrane conductance regulator (CFTR) in which the phenylalanine at position 508 is deleted (
F508CFTR) and human ASIC2 (18, 19, 22). Both F508CFTR and human ASIC2 also specifically interact with Hsc70, a cytosolic chaperone (17, 18, 20). Similarly, Grifoni et al. now also show that Hsc70 regulates surface expression of ASIC2 in A10 cells; as with glycerol, the silencing of Hsc70 is associated with increased surface ASIC2 and decreased migration.
If ASIC2 is a regulator of VSMC migration, as the present study suggests, the question now becomes, What keeps this protein trapped in the cytosol? Previous studies of CFTR trafficking showed that glycerol and a transcriptional activator, sodium 4-phenylbutyrate, could rescue misfolded F508CFTR and deliver it to the membrane (18, 22) and that the rescue was due to the downregulation of the molecular chaperone, Hsc70 (17, 18). Furthermore, degradation of F508CFTR was a consequence of ubiquitination by the Hsc70 associated cochaperone, carboxyl terminus of heat shock protein 70-interacting protein (CHIP) (13, 21), which targeted F508CFTR for degradation by the proteasome. As would be predicted, the inhibition of CHIP also rescued F508CFTR and was associated with functional expression of F508CFTR at the plasma membrane (21). Although our laboratory has similarly reported that ASIC2 expression is associated with reduced migration of glioma cells and that this can be rescued both by glycerol and by downregulation of Hsc70 (19, 20), we have not identified a misfolding mutation in ASIC2; rather, an overexpression of Hsc70, a characteristic of cancer cells (16), may underlie ASIC2 retention in this case. The knockdown of Hsc70 may also increase the residence time of ASIC2 at the cell surface by interfering with endocytosis (6). We have also shown that the interaction between Hsc70 and ASIC2 is highly specific; Hsc70 does not bind the related ASIC1, for example. However, little is known about chaperone expression in migrating VSMCs. One recent study has reported that tyrosine phosphorylation of several chaperones is an early event in VSMC activation, although the relative levels of expression were not compared (4). It is possible that activating VSMCs by exposure to chemotactic agonists reduces surface expression of ASIC2 by influencing expression and/or binding activity of Hsc70, thereby increasing migration. Further studies are clearly required to illuminate this aspect of VSMC behavior. Moreover, a characterization of the VSMC membrane currents with and without ASIC2 present would go a long way in deciphering the mechanisms underlying the influence of ASIC on VSMC migration.
FOOTNOTES
Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, 1918 University Blvd., MCLM 704, Birmingham, AL 35294-0005 (e-mail: benos{at}uab.edu)
REFERENCES
F508-CFTR. Am J Physiol Cell Physiol 278: C259–C267, 2000.F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase. J Cell Biol 167: 1075–1085, 2004.
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