Hyposmotic Stress Induces Cell Growth Arrest via Proteasome Activation and Cyclin/Cyclin-dependent Kinase Degradation*

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-depend-ent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G 1 /S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition pre-vented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome were then cultured for 6 h inhyposmotic media containing the same inhibitors used during the preincubation. Proteins were isolated after 6 h followed by immunoblotting with antibodies directed to cell cycle- related proteins. Electron Microscopy— Cell lines were cultured under hyposmotic con- ditions for 48 h followed by collection of the floater cells, washing with PBS (pH 7.4) then fixing with 0.1 M sodium phosphate buffer (pH 7.2) containing 2% glutaraldehyde (60 min, 4 °C). Alternatively, asynchro-nously growing cells were collected by trypsinization followed by a similar glutaraldehyde fixation. Further processing of the fixed cells was done as described previously (40). Cells were examined and photo- graphed using a Philips CM-12 transmission electron microscope. and synthesis, in cell leads to apoptosis cell cycle catastrophe formation

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G 1 /S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.
The cell cycle involves a meticulously ordered series of events that control defined cell cycle stage check points and ultimate cell division. These events are tightly regulated by the expression and degradation, activation and inactivation, and subcellular localization of cyclins and cyclin-dependent kinases (Cdks) 1 (1)(2)(3). Cyclins associate with, and activate, Cdks and are periodically synthesized then degraded during cell cycle progression, whereas cellular Cdk levels tend to remain in excess throughout the normal cell cycle (1)(2)(3)(4)(5). In mammalian cells, Cdk4 and Cdk6 associate with D-type cyclins and regulate G 1 cell cycle phase progression. Cdk2 associates with the E-and A-type cyclins, and the respective complexes control G 1 /S transition and S phase progression, respectively (1)(2)(3)(4)(5). Cdc2 (also termed Cdk1) and the B-type cyclin form a cytoplasmic complex at the G 2 /M phase checkpoint, which translocates to the nucleus just prior to nuclear envelope breakdown during prophase. Abnormal sequestering of cyclin B1/Cdc2 complexes in the cytoplasm leads to perturbation of cell cycle progression (6 -8).
Most mammalian cells have developed compensatory mechanisms to respond to changes in the osmolarity of the surrounding medium, which allow them to re-establish homeostasis of osmotically disturbed aspects of cell structure and function (9,10). These mechanisms are necessary, because osmotic stress may severely compromise eukaryotic cell function due to the importance of maintaining homeostasis of inorganic ions as a prerequisite for normal progression of metabolic processes. Disruption of such mechanisms can give rise to a diverse group of disease states and their complications, and several disease states are known to be associated with fluctuations in extracellular osmolarity (11)(12)(13)(14)(15). When cells are exposed to an osmotic stress, they generally respond by regulation of their cell volume, in association with reorganization of their cytoskeletal networks, particularly the actin-associated cytoskeletal elements, to accommodate the surrounding conditions (16 -21). Hyperosmotic stress also causes cell growth arrest as reported for murine kidney cells, in association with stress-activated protein kinase activation (22). Hypo-or hyper-osmotic conditions activate a variety of kinases, including stress-and mitogen-activated kinases (23)(24)(25)(26)(27), Janus tyrosine kinases (28), and Rho kinase (29) among others. The mechanism of hyperosmotic stress-induced growth arrest is unknown, but hyperosmotic stress does induce proteasome-mediated degradation of cyclin D1 in the lymphoma cell line Granta-519 (30).
Two correlations led us to test the hypothesis that hyposmotic stress may have a significant effect on cell growth. These correlations include the established link between cell growth and stress-associated kinases (31)(32)(33), as well as the link between these kinases and osmotic changes (10,(23)(24)(25)(26)(27). Here we show that hyposmotic stress results in the arrest of cell growth in colonic and pancreatic cultured cell lines. This growth arrest leads to cell cycle catastrophe, with or without apoptosis, depending on the cell line tested. The mechanism of the growth arrest involves proteasome activation with subsequent degradation of several cyclins and Cdks.

EXPERIMENTAL PROCEDURES
Cell Culture and Hyposmotic Treatment-Three human colon (Caco2, HRT18, HT29) and two pancreas (AsPC-1, PaCa-2) carcinoma cell lines were used (American Type Culture Collection, Manassas, VA). All the cell lines were cultured at 37°C, in 5% CO 2 humidified incubators. Hyposmotic treatment was done by dilution of the culture medium with distilled water (1:1) to a final osmolarity of 150 mosM. As a control, cells were cultured in iso-osmotic medium by diluting the medium with an equal volume of distilled water containing 0.15 M sodium chloride (24,26). Floater cells were collected after centrifugation of the culture medium, and adherent cells were collected after trypsinization. Cell death was assessed by trypan blue staining. All data are presented as the mean Ϯ S.D. Student's paired t tests were used for statistical analysis.
Detection of Apoptosis and Cell Cycle Analysis-Monoclonal antibody (mAb) M30 "CytoDEATH antibody" (Roche Molecular Biochemicals, Indianapolis, IN), which recognizes a keratin 18 (K18) fragment generated after caspase digestion, was used for immunoblotting of cell lysates. M30 reactivity is indicative of cells undergoing apoptosis (34,35). A rabbit polyclonal anti-keratin polypeptide 18 (K18), Ab 4668, was generated against an N-terminal K18 peptide ( 26 RPVSSAASVYAGA 38 ) as described previously (36). This Ab was used to detect the intact and caspase-generated N-terminal K18 fragment (35). Formation of DNA fragments was assessed after genomic DNA isolation (using a Qiagen kit) then separation using 1.5% agarose gels. For cell cycle analysis, the total cells (floater and adherent) were collected, washed with phosphate-buffered saline (PBS), then fixed with 70% ethanol (stored at Ϫ20°C) in PBS. Cell DNA content was measured using a FACScan after treatment with RNase A (40 g/ml) and staining with propidium iodide (20 g/ml) as described previously (37).
Metabolic Labeling of Cells-Cells were cultured for 5 h in iso-or hypo-osmotic medium. The cell culture media was then replaced by isoor hypo-osmotic methionine-free labeling media after rinsing twice with the same labeling media. [ 35 S]Methionine (100 Ci/ml) was added, and the cells were further cultured for one additional hour (total culture time ϭ 6 h in the presence of iso-or hypo-osmotic media). Labeled cells were washed with cold PBS and solubilized with 1% Nonidet-P40 (Nonidet P-40) in PBS containing 5 mM EDTA. After measurement of protein concentration using the BCA method (Pierce, Rockford, IL), protein synthesis was estimated by measuring the incorporated radioactivity per microgram of protein.  Fig. 1 were cultured in hypotonic (H) or isotonic (I) control conditions for 24 h. A, the numbers of floater (dark bars) and adherent (light bars) cells were counted. Note that most of the cells remain adherent after exposure to hypotonic conditions. B, in a separate but similar experiment to that shown in panel A, the percent cell death in the proportionately small floater cell populations was determined. C, genomic DNA was purified from the floater cells, and an equal amount of DNA from each cell line was resolved using a 1.5% agarose gel, stained with ethidium bromide followed by visualization using a UV trans-illuminator (subpanel a). The lower subpanel b shows a lighter exposure of the high molecular weight dense DNA bands (as compared with subpanel a) to demonstrate that the ladder formation noted in HRT18 cells is not related to sample loading. H, hypotonic; I, isotonic.
were then cultured for 6 h in hyposmotic media containing the same inhibitors used during the preincubation. Proteins were isolated after 6 h followed by immunoblotting with antibodies directed to cell cyclerelated proteins.
Electron Microscopy-Cell lines were cultured under hyposmotic conditions for 48 h followed by collection of the floater cells, washing with PBS (pH 7.4) then fixing with 0.1 M sodium phosphate buffer (pH 7.2) containing 2% glutaraldehyde (60 min, 4°C). Alternatively, asynchronously growing cells were collected by trypsinization followed by a similar glutaraldehyde fixation. Further processing of the fixed cells was done as described previously (40). Cells were examined and photographed using a Philips CM-12 transmission electron microscope.

Cell Growth Arrest upon Exposure to an Hyposmotic
Stress-To assess the effect of hyposmotic culture conditions on cell proliferation, three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines were exposed to hypotonic medium for different times as indicated (Fig. 1). As compared with isotonic control medium, the hyposmotic culture conditions were associated with a reduced total cell number (floater and adherent) in all the cell lines beginning at 6 h ( Fig.  1). The reduced cell numbers upon culturing in hyposmotic conditions reached statistical significance (Student's t test, p Ͻ 0.05) at different time points depending on the tested cell lines (HRT18 and AsPC-1 cells at 6 and 12 h, respectively; HT29, Caco2, and PaCa-2 cells at 24 h). Notably, the growth arrest was reversible, after switching to isotonic control medium, in the five cell lines up to 24 h after exposure to the hyposmotic treatment (not shown). In addition and as may be expected, the isotonic control medium has a 50% dilution of serum and other normal culture medium factors, which can result in up to a 30% decrease in growth rate (depending on the culture duration and the cell line, not shown). However, for all the experiments shown we used the isotonic control medium for appropriate comparison with the hyposmotic conditions.
Hyposmosis-induced Cell Growth Arrest Leads to Apoptosis in HRT18 Cells-We examined the effect of the hyposmosisinduced cell-growth arrest by determining if this arrest was accompanied by cell death. Most of the cells (Ͼ90%) remain attached to the tissue culture dish even after 24 h of exposure to hyposmotic conditions ( Fig. 2A). The viability of the adherent cells, as determined by trypan blue exclusion, was Ͼ95% (not shown) whereas the viability of the floater cells was Ͼ60% in all the cell lines tested except for HRT18 where Ͼ80% of the floater cells were dead (Fig. 2B). The percent death of floating cells increased significantly upon hypotonic exposure (p Ͻ 0.05) in HRT18 (28% to 86%) and Caco2 (17% to 36%) cells but not in other cell lines. Genomic DNA analysis of the floater cells showed ladder formation only in HRT18 cells (Fig. 2C), which is consistent with apoptosis, whereas none of the adherent cells in the five cell lines manifested any DNA ladder formation (not shown). The DNA ladder formation occurs only in the floater cells isolated from HRT18 cells grown under hyposmotic conditions but not cells grown under isotonic conditions (Fig. 2C, compare lanes 2 and 6). The apoptotic cell death of a fraction of HRT18 cells that have been exposed to hyposmotic conditions was confirmed by the caspase-mediated formation of the two K18 fragments, p29 and p43 (Fig. 3). The two keratin-degradation fragments were detected only in HRT18 cells, and the stoichiometry of the p29 fragment (which is generated after formation of p43 (35)), as compared with uncleaved K18, increases with increasing exposure time to hyposmotic conditions (compare Fig. 3B with 3A). Therefore, the results of Figs. 1-3 indicate that hyposmotic stress causes minimal cell death and suggest that such stress leads primarily to growth arrest with some cell death occurring primarily in HRT18 cells via apoptosis.
Hyposmotic Stress Results in a Stage-independent Cell Cycle Growth Arrest-The rapid effect of hyposmotic stress (e.g. within 6 h) on cell growth suggested that this stress could act via a generalized and/or a cell cycle stage-specific arrest. We tested these possibilities by cell cycle analysis after culturing cells in the presence or absence of hyposmotic conditions. As shown in Fig. 4, the colonic cell lines (Caco2, HRT18, and HT29) manifested a preferential accumulation of cells at the G 1 /S interface after 6 h of culture, whereas the two pancreatic cell lines (AsPC-1 and PaCa-2) had a more generalized arrest. Cell cycle analysis after 12, 24, or 48 h of culture in hyposmotic conditions manifested a similar trend for all the five cell lines (not shown) thereby indicating that the growth arrest was not necessarily related to a single cell cycle stage per se, and that Equal amounts of protein from each sample were separated on 10% SDS-PAGE followed by transfer to membranes, then blotting with mAb M30 (which recognizes the K18 p43 fragment but not intact K18) or Ab 4668 (which recognizes intact K18 and its p29 N-terminal fragment). The p43 species corresponds to a K18 fragment that is generated after caspase cleavage of a short 3-kDa peptide from the C terminus of K18, whereas p29 is generated after a second cleavage of p43 (35).  Figs. 1-3 were cultured in hypo-or iso-osmotic conditions for 6 h. Floater cells were collected and pooled with the adherent cells (isolated by trypsinization). After washing with PBS, cells were fixed then processed for cell cycle analysis as described under "Experimental Procedures." In some cases, the total percentage of cells (i.e. G 0 /G 1 ϩ S ϩ G 2 /M) was slightly less than 100 (e.g. Caco2 cells) due to the presence of cell fragments or cells with DNA content of Ͻ2n or Ͼ4n, respectively. more than one stage can be impacted depending on the cell line examined.
We also examined the ultrastructural features of the five cell lines after hyposmotic stress-induced growth arrest. For this, floater cells were collected from each cell line after 48 h of hyposmotic treatment then examined by transmission electron microscopy. As shown in Fig. 5, the ultrastructural features included formation of: variable-sized vacuoles in all the cell lines (panels a-f), nuclear condensation (panels b-f), multiple nuclei (panels c, d), and giant cells (panels a, d, e), and nuclear fragments (panel b). These features were found primarily in the floater cells ( Fig. 2A and not shown) and were present in all of the cell types, although nuclear fragmentation was noted only in some of the HRT18 cells, which is consistent with some HRT18 cells undergoing apoptosis as noted in Figs. 2C and 3. Overall, these morphological changes and the cell cycle arrest and cell cycle analysis data suggest that hyposmosis causes a global cell cycle catastrophe.
Reduction of Cell Cycle Protein Levels after Hyposmotic Treatment-Because the cell cycle is controlled by expression and activation of several cyclins and Cdks, we asked whether their expression levels changed after cell exposure to a hyposmotic environment. As shown in Fig. 6, the expression level of the cell cycle-related proteins Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 were dramatically reduced in all the cell lines after hyposmotic treatment in a time-dependent fashion. However, this effect was not generalized in that some protein levels such as cyclin E and keratin 8 remained unaltered. Some cyclins and Cdks showed a profound rapid degradation/turnover (e.g. Cdc2 and cyclin B1 in HRT18 cells) whereas others showed more gradual degradation (Cdc2 in Caco2 cells) (Fig. 6). Although the expression levels of individual cyclins/Cdks varied during isotonic conditions among the cell lines, the overall effect for most but not all of the cell cycle-related proteins was either rapid or a gradual degradation/turnover. Therefore, the observed cell cycle arrest upon exposure to hyposmotic conditions is likely due to decreased levels of several cell cycle-related proteins.

Activation of the Proteasome System Is Required for Hyposmosis-induced Degradation of Cell Cycle-related Proteins and
for Cell Growth Arrest-The decreased levels of several cell cycle-related proteins in response to hyposmotic conditions implies that these altered protein levels result from a decreased biosynthetic rate and/or from increased degradation. Assess-

FIG. 6. Effect of hyposmotic stress on cell cycle-related protein levels.
Cells were cultured in iso-(I) or hypo-(H) osmotic medium for 6 (A) or 24 h (B), followed by isolation of a total cellular protein homogenate by solubilization of the isolated cells (floater and adherent) in reducing sample buffer. Equal amounts of protein from each sample were separated on 10% acrylamide gels followed by blotting using antibodies to Cdc2, cyclin B1, Cdk2, cyclin E, Cdk4, cyclin D3, and K8. Note the dramatic and progressive decrease in the steady-state levels in most but not all of the cell cycle-related proteins. Equal protein loading was confirmed by the similar observed levels of K8. ment of the protein synthetic rate after hyposmotic stress showed that the overall protein synthetic rate decreased to 28 -53% of the basal isotonic-condition rates (Fig. 7). This decreased synthetic rate does not, however, account for the dramatic decrease in some of the cell cycle-related proteins that became essentially undetectable within 6 h of exposure to hyposmotic conditions (e.g. Fig. 6A, see cyclin B1 and D3 and Cdk4 in HRT18 cells). Hence, protein degradation is also a likely major factor in the hyposmosis-induced increased turnover of the cell cycle-related proteins. To test this hypothesis, we examined the effect of proteasome and other protease inhibitors on the expression level of several cell cycle-related proteins in the presence or absence of 6-h exposure to a hyposmotic treatment. As shown in Fig. 8, lactacystin (a specific proteasome inhibitor) and ALLN (a nonspecific proteasome inhibitor) strongly inhibited the hyposmosis-induced reduction of cell cycle-related protein levels. In contrast, PD150606 (calpain inhibitor), chloroquine (lysosomal acidification inhibitor), and the pepstatin A/leupeptin (lysosome protease inhibitors) mixture did not have a significant effect on cyclin and Cdk expression levels.
Given the above findings, we examined whether proteasome FIG. 7. Hyposmotic conditions partially inhibit overall protein biosynthesis. The five colonic and pancreatic cell lines were cultured in iso-or hypo-osmotic medium for 5 h then labeled for 1 h using [ 35 S]methionine as described under "Experimental Procedures." After washing off the labeling medium, cells were solubilized with 1% Nonidet P-40 in PBS containing 5 mM EDTA. Aliquots of the detergent lysate were counted, and the counts were normalized to the amount of solubilized protein. were seeded into cell culture dishes and incubated overnight in normal medium at 37°C. Cells were then incubated in normal medium containing lactacystin (5 or 10 M) or 0.1% Me 2 SO (DMSO, as a vehicle control). After 2 h, the medium was changed to hypo-or iso-osmotic medium (Ϯ lactacystin), and the cells were cultured at 37°C for 20 h then counted. The histograms show mean Ϯ S.D. based on three independent experiments. Asterisks highlight p values Ͻ 0.05 when comparing the hyposmotic treatments with lactacystin to those without. inhibition influenced hyposmosis-induced cell growth arrest. To do so, we compared the cell number in the presence or absence of lactacystin, under isotonic or hypotonic conditions. As shown in Fig. 9, lactacystin resulted in a slight decrease in cell number when cells were grown in isotonic control conditions, which may be related to the known effect of proteasome inhibitors as apoptosis-inducing agents in a variety of cell lines (e.g. Refs. 41,42). In contrast, lactacystin reversed the hyposmosis-induced growth inhibition when tested in HT29 and AsPC-1 cells, reaching statistically significant reversal at doses of 10 M for HT29 cells and 5 and 10 M for AsPC-1 cells (Fig.  9). Doses of Ͼ10 M were ineffective (not shown), likely due to overriding of the growth inhibitory effect of such compounds at the higher doses. These findings lend further strong support to the conclusion that hyposmosis-induced growth arrest is due in large part to proteasome activation and subsequent degradation of several key cell cycle-related proteins.

Effect of Changes in Extracellular Osmosis on Cell Growth-
Due to the high water permeability of the mammalian plasma membrane, an imbalance between the osmolarity of the cytoplasm and the external environment will immediately lead to a redistribution of water and to a subsequent change in intracellular osmolarity and cell volume (9). Most cell types respond immediately to osmotic changes by activating plasma membrane transport pathways leading to net accumulation (upon hyperosmotic stress) or loss (upon hyposmotic stress) of osmotically active intracellular solutes (11,43). The mechanism(s) whereby altered intracellular osmolarity interferes with physiological mammalian cell functions such as proliferation and differentiation is(are) poorly understood. Osmotically induced growth arrest has been observed in several organisms (44), including bacteria (45)(46)(47)(48) and yeast (49,50), and appears to be necessary for recovery from osmotic stress. Here we demonstrate for the first time that hyposmotic conditions induce reversible cell growth arrest within 6 -24 h in all five human cell lines that we tested (Fig. 1).
Similar results of cell growth arrest were demonstrated in a murine kidney cell line after exposure to hyperosmotic conditions (22). The mechanism of this growth arrest is not known, but it correlates with induced expression of the so-called growth arrest and DNA damage-inducible (GADD) proteins GADD45 and GADD153 (22). It is speculated that the biologi-cal significance of cell growth arrest relates to allowing the arrested cells sufficient time for adaptive responses to be activated, and to switch mitotic energetic resources to cell protective systems to cope with the osmotic stress (10).
Growth Arrest Leads Cells to Undergo Apoptosis or Cell Cycle Catastrophe-Several studies point to the intimate association between apoptosis and cell cycle control (51,52). In the present study, the hyposmosis-induced apoptosis was verified by formation of genomic DNA fragments (Fig. 2C), and by the caspase-mediated degradation of K18 (Fig. 3). Among the five cell lines we tested by exposing to hypotonic conditions, apoptotic death was observed only in HRT18 cells. The hyposmosis-associated partial G 1 /S arrest of HRT18 cells, and the subsequent apoptosis of a small subpopulation of these cells are consistent with previous studies showing that growth arrest (e.g. at G 1 phase) can lead cells to undergo apoptosis (e.g. Refs. 53,54). Caco2 and HT29 cells were also partially arrested at the G 1 phase of the cell cycle by hyposmotic treatment (Fig. 4), but no apoptotic changes were noted in these cells. The reason for the lack of apoptosis in the remaining cell lines may be related to differences in susceptibility to apoptosis. Interestingly, inhibition of phosphatidylinositol 3-kinase by the compound LY294002 protected from cytosine arabinoside-induced apoptosis (55), so hyposmosis-induced degradation of related kinases may provide similar protection. In addition, the severity of the hyposmotic stress may also play a role in the induction of apoptosis. For example, apoptosis but not cell growth was studied and noted in several cell lines, including HT29 and AsPC-1 cells, under severe hyposmotic conditions (84 mosM) (56) that we did not test in our cell systems.
Our results show that hyposmotic conditions result in growth arrest and cell cycle catastrophe (also called mitotic catastrophe) that may in some cell types lead to apoptosis as well. The features of generalized cell cycle arrest (Figs. 1 and  4), formation of giant and polyploid cells (Fig. 5) in the absence of significant cell death, are consistent with cell cycle catastrophe (57). Cell cycle/mitotic catastrophe can occur via multiple mechanisms, including modulation of checkpoint kinases (58) or adapter proteins (59), and has also been noted upon administration of anti-tumor drugs such as 5-fluorouracil (60). However, the growth arrest that we observe is independent of the formation of the giant and polypoid cells per se, because these cells are found in small numbers (primarily within the small Cell cycle-related proteins, including cyclins and cyclin-dependent kinases (Cdks), play crucial roles in controlling cell cycle progression. When cells are exposed to extracellular hyposmotic conditions, the expression level of several cell cycle-related proteins is reduced via activation of the proteasome degradation pathway and via a global decrease in protein synthesis. The decreased protein synthesis may also be related to proteasome activation and potential degradation of enzymes that regulate translation or mRNA stability, although this possibility was not tested. Hyposmosis-induced proteasome activation, and decreased protein synthesis, result in cell growth arrest, which subsequently leads to apoptosis and cell cycle catastrophe with formation of multinucleated giant cells depending on the cell type.
subgroup of floater cells) and because cell cycle protein degradation involved the adherent cells.
Hyposmosis-mediated Activation of the Proteasome as a Mechanism of Growth Arrest-The findings of Figs. 8 and 9 indicate that activation of the proteasome pathway, and subsequent degradation of key cell cycle-related proteins, are largely responsible for the hyposmosis-induced cell growth arrest. Our studies add hyposmotic stress as a novel new mechanism for proteasome activation and also strongly implicate this degradation pathway in causing cell cycle arrest upon exposure of cells to hypotonic conditions (Fig. 10). The proteasome clearly plays an essential role in modulating the steadystate levels of a variety of proteins (61). To that end, degradation of cyclins, Cdks inhibitors, tumor suppressor proteins, and proto-oncogene products by the proteasome pathway highlights the important role of the proteasome in regulating cell growth (61). In addition, proteasome-mediated degradation of several individual proteins upon exposure to hypertonic stress has been reported for cyclin D1 (30), the insulin-regulated IRS-2 protein (62), and aquaporin-1 (63). In the case of hypotonic stress, it appears that several proteins become degraded upon hyposmotic exposure. Our hypothesis is that hyposmotic stress results in a generalized activation of the proteasome, with subsequent degradation of individual proteins that may be modulated by their: (i) steady-state half-life at the time of exposure to the stress, (ii) cellular localization, or (iii) posttranslational modification state or whether they are bound to an associated protein that could affect their turnover.
The degradation of several cyclins and Cdks, among other likely proteasome substrates, provides a molecular explanation for the observed growth arrest upon hyposmotic exposure. For example, Cdk4/cyclin D are responsible for progression through the G 1 cell cycle phase, so their decreased protein levels after hyposmotic exposure supports the observed accumulation of Caco2, HRT18, and HT29 cells at the G 1 cell cycle stage (Fig. 4). Cdk2 and Cdc2/cyclin B, which are necessary for the S and G 2 /M transitions, respectively, also undergo degradation upon exposure to hypotonic conditions, which supports the cell cycle stage-independent nature of the growth arrest that we observed. Interestingly, cyclin E was relatively resistant to degradation ( Fig. 6) even though it is an established substrate for the ubiquitin-proteasome pathway (64). Although the precise reason for the observed cyclin E stability remains to be defined, the observation that mice lacking the ubiquitin ligase Skp2 manifest accumulation of cyclin E and polyploidy (65) raises the possibility that Skp2 and/or a related protein may be involved.
Our working hypothesis (Fig. 10) is that hyposmotic stress results in growth arrest via proteasome activation and subsequent degradation of several cell cycle-regulating proteins. Growth arrest may also be related to the observed decrease in protein synthesis, which, in turn, affects the steady-state level of important cell cycle-related proteins. In addition, protein steady-state levels may be affected by mRNA stability, which can be rapidly degraded through AU-rich elements in the 3Јuntranslated region upon proteasome activation (66).