Laboratory of Physiology, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
THE CYTOSKELETON, so the textbooks say, consists of three fiber types: actin-containing microfilaments, intermediate filaments, and microtubules. One of the functions assigned to the cytoskeleton is to preserve cell shape and to protect against mechanical stress. Because changes in cell volume deform cells and may even lead to cell disruption in the case of excessive swelling, one is intuitively inclined to link the cytoskeleton with cell volume regulation. Problems arise, however, when one starts probing for the precise role of the cytoskeleton in cell volume regulation: is the cytoskeleton a volume sensor or a volume effector, or does it transmit signals from volume sensor to effector? The study by Di Ciano-Oliveira et al., the current article in focus (Ref. 5, see p. C555 in this issue), lifts the veil on the role of Rho, a small GTPase that regulates the actin cytoskeleton, in the cellular response to hyperosmotic stress.
There are three basic sets of observations in the study by Di Ciano-Oliveira et al. (5). First, using LLC-PK1 cells, they clearly show that cell shrinkage induced by hyperosmotic stress results in a fast activation of Rho that is proportional to the rise in extracellular osmolarity and that reverses upon returning to isotonicity. Second, the increase in myosin light chain (MLC) phosphorylation during hyperosmotic stress is mainly (but not exclusively) caused by Rho kinase, one of the downstream effectors of Rho. Third, hyperosmotic activation of the Na+-K+-Cl cotransporter (NKCC), which requires MLC phosphorylation (9, 17), is independent of Rho kinase.
To fully appreciate the implications of these findings, we should consider how the cytoskeleton can participate in cell volume regulation. A first possibility is that the cytoskeleton functions as a cell volume sensor. Changes in cell volume, either swelling or shrinking, entail changes in cellular architecture, membrane stress, and/or folding which, in principle, can be detected by the cytoskeleton. For example, Ingber (6) has proposed the concept of cellular tensegrity, in which actin microfilaments connected to focal adhesions exert a continuous tension that is counteracted by microtubules acting as struts. In principle, cell swelling or shrinkage could alter the force balance between microfilaments and microtubules, thereby generating an intracellular signal, e.g., by (in)activating one of the signaling proteins in focal adhesions (cellular mechanochemistry) (7). However, Di Ciano-Oliveira et al. (5) show that an increase in intracellular ionic strength under isovolumic conditions, and hence without any change in cellular tensegrity, is perfectly capable of activating Rho. Conversely, a decrease in intracellular ionic strength in the absence of cell swelling, and hence without any mechanical stress on the cytoskeleton, suffices to activate volume-regulatory anion channels (VRACs) in vascular endothelial cells (20). These data do not completely rule out the cytoskeleton as a volume sensor, but they do point out that cells can detect volume changes via signals (ion concentrations, ionic strength, macromolecular crowding, etc.) that are independent of the cytoskeleton.
Second, the cytoskeleton could play an effector role in cell volume responses, e.g., by offering mechanical protection against the deleterious effects of excessive swelling or shrinkage. In Dictyostelium discoideum, hyperosmotic stress induces a redistribution of myosin to the cortical cytoskeleton, which confers mechanical resistance against cell shrinkage (11). In a previous paper, Di Ciano et al. (4) provided evidence for a hypertonicity-induced accumulation of F-actin and cortactin in the cortical cytoskeleton that is at least partially mediated by Rac and Cdc42, two other small GTPases. Although not formally addressed in their present paper, it is very well possible that the Rho/Rho kinase-dependent MLC phosphorylation contributes to the shrinkage-induced remodeling of the cortical cytoskeleton and, hence, to mechanical protection. However, for mammalian cells it is not clear whether rearrangement of the cytoskeleton plays a prominent role in preserving cell and/or membrane integrity during hypo- or hypertonic stress. Indeed, mammalian cells typically protect themselves against swelling or shrinking by compensatory volume responses that neutralize the initial volume change (12). For example, hypotonically swollen cells activate a series a membrane transporters (K+ channels, Cl channels, organic osmolyte pathways), which allows the efflux of osmolytes followed by osmotic water loss, thereby restoring the initial cell volume (regulatory volume decrease, RVD). Conversely, shrunken cells try to regain their initial volume by the accumulation of osmolytes (K+, Cl, taurine, myo-inositol, sorbitol, etc.) followed by water influx (regulatory volume increase, RVI). In this perspective, cytoskeletal rearrangements may offer mechanical protection during the initial phase of volume perturbation, but there is probably no long-term requirement for mechanical protection in view of the volume compensation.
Finally, the cytoskeleton could be involved in signal transmission from volume sensor (whatever this may be) to volume effectors such as the transporters and channels that mediate RVD or RVI. A fundamental question is how the cytoskeleton can contribute to signaling in general. Depending on the mode of engagement of the cytoskeleton, one can think of two scenarios: active signaling vs. permissive support. Active signaling means that cytoskeletal proteins (actin, myosin, etc.) and/or their regulators (e.g., Rho GTPases) are an integral part of the signaling cascade. In other words, signal transmission requires a change in the "activity state" of the cytoskeleton (for a striking example of active signaling via the cytoskeleton, albeit not in volume regulation, see Ref. 14). Such an active role contrasts with a permissive support function. For example, the cytoskeleton may provide the infrastructure for signal transmission, e.g., by assembling signal transduction complexes or by defining local microdomains for signal transduction (8). However, in this scenario the cytoskeleton is not actively engaged in the signaling process. Whatever the signaling role of the cytoskeleton may be, much attention has been paid in this context to Rho, Rac, and Cdc42. These small GTPases act as molecular switches that regulate the turnover and assembly of actin filaments, and they organize actin filaments in higher order structures such as stress fibers, lamellipodia, or filopodia (1). All three GTPases stabilize actin filaments and promote actin polymerization, but they differ in how they organize the actin cytoskeleton. Rho activation leads to MLC phosphorylation, actomyosin interaction, and formation of stress fibers. In contrast, Rac and Cdc42 inhibit stress fiber formation but induce the assembly of actin complexes underneath the plasma membrane, either sheets of actin filaments parallel with the plasma membrane (lamellipodia) in the case of Rac or finger-like protrusions (filopodia) in the case of Cdc42. However, apart from regulating the actin cytoskeleton, Rho, Rac, and Cdc42 can also activate other signal transduction pathways such as MAP kinases (1).
Let us now return to the study by Di Ciano-Oliveira et al. (5) and the role of Rho, Rho kinase, and MLC phosphorylation in volume signaling. It has been known for some time that hypertonic shrinkage leads to MLC phosphorylation and, concomitantly, an activation of NKCC (9, 17). Moreover, inhibitors of myosin light chain kinase (MLCK) counteract the hyperosmotic activation of NKCC, suggesting a causal link between MLCK/MLC phosphorylation and NKCC activation (9, 10, 17). Di Ciano-Oliveira et al. now clearly show that hyperosmotic stress results in a Rho/Rho kinase-dependent MLC phosphorylation that, as discussed above, could contribute to cytoskeletal remodeling. However, pharmacological inhibition of Rho kinase with Y-27632 does not prevent the shrinkage-induced activation of NKCC, whereas inhibition of MLCK with ML-7 does. This mirrors a recent observation on the regulation of the Na+/H+ exchanger (NHE), another RVI effector, in Ehrlich ascites tumor cells (19). Hypertonicity-induced activation of NHE requires MLCK activity, but it is independent of Rho kinase activity. The converse (Rho kinase dependent, MLCK independent) is true for the translocation of myosin to the cortical cytoskeleton during cell shrinkage (19). Thus, at least for the cell types in these studies (LLC-PK1 and Ehrlich ascites tumor), the myosin phosphorylation pathways seem to exert pleiotropic effects during cell shrinkage. The Rho/Rho kinase axis mediates cytoskeletal rearrangements (MLC phosphorylation, myosin translocation), but it is not required for the activation of NKCC or NHE, which, in contrast, depends on MLC phosphorylation via MLCK. Di CianoOliveira et al. rationalize these seemingly disparate data by invoking two different pools of myosin, one regulated by Rho/Rho kinase and another by MLCK. Both myosin pools are activated by cell shrinkage, but apparently with a different purpose. MLCK-regulated myosin seems to be a component of the signal transduction pathway that activates membrane transporters such as NKCC and NHE (signaling function). In contrast, Rho/Rho kinase controlled myosin could exert an effector function, e.g., by providing mechanical protection during shrinkage. Although attractive, this model should be treated with some caution. First, it relies on the relative specificity of the pharmacological tools, Y-27632 to selectively block Rho kinase and ML-7 to selectively block MLCK. Second, the myosin pools are functionally defined, but it is not clear how they are spatially distributed in the cell. Third, the upstream signals that activate Rho and MLCK during shrinkage have yet to be identified.
Irrespective of the precise details of this model, Rho, Rho kinase, and MLCK seem to be actively engaged in the cellular response triggered by shrinkage. But what about their role in cell swelling? The Rho/Rho kinase/MLC phosphorylation pathway is required for the swelling-induced activation of VRAC in vascular endothelial cells (15, 16) and in NIH/3T3 fibroblasts (18). However, constitutively active Rho cannot activate VRAC, and there is no activation of Rho during cell swelling (3). These data are consistent with a permissive role for the Rho/Rho kinase/myosin phosphorylation pathway during cell swelling: it is required for proper activation of VRAC, but it does not actively participate in the signaling process.
To conclude, Rho, Rho kinase, and MLC phosphorylation are strongly implicated in cell volume responses, during both cell swelling and cell shrinkage. However, Rho is only part of the cytoskeleton-cell volume story. Indeed, for an integrated view we have to incorporate the effects of Rac and Cdc42. Indeed, Rac and Cdc42 are activated during both cell shrinkage and cell swelling, which results in the de novo formation of actin patches under the plasma membrane (2, 4, 13). Whether this represents a protective mechanical response against cell volume changes or, alternatively, whether this is part of a signaling pathway for RVI/RVD transporters remains to be elucidated.
Address for reprint requests and other correspondence: J. Eggermont,
Laboratory of Physiology, K. U. Leuven, Campus Gasthuisberg O&N, 3000
Leuven, Belgium (E-mail:
Jan.Eggermont{at}med.kuleuven.ac.be).
REFERENCES
1. Bishop AL and Hall A. Rho GTPases and their effector proteins. Biochem J 348: 241255, 2000.[ISI][Medline]
2. Carton I, Hermans D, and Eggermont J. Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts. Am J Physiol Cell Physiol. In press.
3. Carton I,
Trouet D, Hermans D, Barth H, Aktories K, Droogmans G, Jorgensen NK, Hoffmann
EK, Nilius B, and Eggermont J. RhoA exerts a permissive effect on
volume-regulated anion channels in vascular endothelial cells. Am J
Physiol Cell Physiol 283:
C115C125, 2002.
4. Di Ciano C, Nie
Z, Szaszi K, Lewis A, Uruno T, Zhan X, Rotstein OD, Mak A, and Kapus A.
Osmotic stress-induced remodeling of the cortical cytoskeleton. Am
J Physiol Cell Physiol 283:
C850C865, 2002.
5. Di Ciano-Oliveira C, Sirokmány G, Szászi K, Arthur WT, Masszi A, Peterson M, Rotstein OD, and Kapus A. Hyper-osmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation. Am J Physiol Cell Physiol 285: C555C566, 2003.
6. Ingber DE.
Tensegrity I. Cell structure and hierarchical systems biology. J
Cell Sci 116:
11571173, 2003.
7. Ingber DE.
Tensegrity II. How structural networks influence cellular information
processing networks. J Cell Sci
116: 13971408,
2003.
8. Janmey PA.
The cytoskeleton and cell signaling: component localization and mechanical
coupling. Physiol Rev 78:
763781, 1998.
9. Klein JD and
O'Neill WC. Volume-sensitive myosin phosphorylation in vascular
endothelial cells: correlation with Na-K-2Cl cotransport. Am J
Physiol Cell Physiol 269:
C1524C1531, 1995.
10. Krarup T,
Jakobsen LD, Jensen BS, and Hoffmann EK.
Na+-K+-2Cl cotransport in Ehrlich
cells: regulation by protein phosphatases and kinases. Am J Physiol
Cell Physiol 275:
C239C250, 1998.
11. Kuwayama H, Ecke M, Gerisch G, and Van Haastert PJ. Protection against osmotic stress by cGMP-mediated myosin phosphorylation. Science 271: 207209, 1996.[Abstract]
12. Lang F, Busch
GL, Ritter M, Volkl H, Waldegger S, Gulbins E, and Haussinger D.
Functional significance of cell volume regulatory mechanisms.
Physiol Rev 78:
247306, 1998.
13. Lewis A, Di
Ciano C, Rotstein OD, and Kapus A. Osmotic stress activates Rac and Cdc42
in neutrophils: role in hypertonicity-induced actin polymerization.
Am J Physiol Cell Physiol 282:
C271C279, 2002.
14. Miralles F, Posern G, Zaromytidou AI, and Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113: 329342, 2003.[ISI][Medline]
15. Nilius B, Prenen J, Walsh MP, Carton I, Bollen M, Droogmans G, and Eggermont J. Myosin light chain phosphorylation-dependent modulation of volume-regulated anion channels in macrovascular endothelium. FEBS Lett 466: 346350, 2000.[ISI][Medline]
16. Nilius B, Voets
T, Prenen J, Barth H, Aktories K, Kaibuchi K, Droogmans G, and Eggermont
J. Role of Rho and Rho kinase in the activation of volume-regulated anion
channels in bovine endothelial cells. J Physiol
516: 6774,
1999.
17. O'Donnell ME,
Martinez A, and Sun D. Endothelial Na-K-Cl cotransport regulation by
tonicity and hormones: phosphorylation of cotransport protein. Am J
Physiol Cell Physiol 269:
C1513C1523, 1995.
18. Pedersen SF,
Beisner KH, Hougaard C, Willumsen BM, Lambert IH, and Hoffmann EK. Rho
family GTP binding proteins are involved in the regulatory volume decrease
process in NIH3T3 mouse fibroblasts. J Physiol
541: 779796.,
2002.
19. Pedersen SF and Hoffmann EK. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells. Exp Cell Res 277: 5773., 2002.[ISI][Medline]
20. Voets T,
Droogmans G, Raskin G, Eggermont J, and Nilius B. Reduced intracellular
ionic strength as the initial trigger for activation of endothelial
volume-regulated anion channels. Proc Natl Acad Sci
USA 96:
52985303, 1999.