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Cellular Response to Hyperosmotic Stresses

National Heart, Lung, and Blood Institute, Bethesda, Maryland

ABSTRACT

I. INTRODUCTION

II. PERTURBING EFFECTS OF HYPEROSMOLALITY

A. Cell Cycle Arrest

B. Apoptosis

C. DNA Damage

D. Oxidative Stress

E. Inhibition of Transcription and Translation

F. Mitochondrial Depolarization

III. HIGH NaCl: HYPERTONICITY

A. Many Cellular Components and Functions Are Affected by Hypertonicity

B. Cytoskeletal Rearrangement in Response to Hypertonicity

IV. ORGANIC OSMOLYTES ACCUMULATE IN RESPONSE TO HYPERTONICITY

A. Sorbitol

B. Betaine

C. Inositol

D. Taurine

E. GPC

V. ACTIVATION OF THE TRANSCRIPTION FACTOR TonEBP/OREBP BY HYPERTONICITY

A. TonEBP/OREBP mRNA and Protein Abundance

B. Dimerization of TonEBP/OREBP

C. Phosphorylation of TonEBP/OREBP

D. TonEBP/OREBP Nuclear Localization

E. TonEBP/OREBP Transactivational Activity

F. Interactions Between Proteins That Regulate TonEBP/OREBP

G. Inhibition of TonEBP/OREBP

H. DNA Breaks, ROS, and Cytoskeletal Perturbations as Regulators of TonEBP/OREBP

VI. ADAPTATION TO CHRONIC HYPERTONICITY

VII. SENSORS AND SIGNALERS OF HYPERTONICITY

VIII. HIGH UREA

A. Cellular Components and Functions Affected by High Urea

B. Counteraction of Urea Effects by GPC

C. Mutually Protective Effects of High Urea and High NaCl

IX. WHY IS OSMOTIC TOLERANCE OF RENAL MEDULLARY CELLS GREATER IN VIVO THAN IN CULTURE?

X. OSMOPROTECTION BY GENERAL STRESS PROTEINS IN CELL CULTURE AND IN VIVO

A. Gadd45

B. p53

C. Heat Shock Proteins

D. Heme oxygenase 1

XI. SUMMARY AND CONCLUSIONS

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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Our interest in hyperosmolality rose from consideration of the extraordinary osmotic stress that cells experience in the renal medulla. There, they are exposed to much higher interstitial levels of NaCl and urea than are cells anywhere else in the body, the high concentrations being associated with the role of the renal medulla in the urinary concentrating mechanism. Interstitial composition is estimated from that of plasma, since the two are closely related. At the tip of the renal inner medulla of antidiuretic male hamsters, representative concentrations in interstitial fluid (measured by micropuncture of vasa recta) are 697 meq/l sodium, 614 mM urea, and 1,744 mosmol/kgH₂O osmolality (189). In contrast, normal levels in peripheral plasma are much lower, namely, 128 meq/l sodium and 8 mM urea (199). High NaCl and urea are stressful to cells, altering their function or even killing them by apoptosis. In cell culture (198, 257), the apoptosis occurs at levels much lower than those that normally exist in the renal medulla. So, how do cells in the renal medulla survive? The answer, it turns out, is complex and becoming increasingly so. Many of the studies of osmotic stress have been with renal cells, especially renal medullary cells. However, the effects of osmotic stress have also been examined with many other mammalian cell types (Table 1), and numerous different changes have been observed in them. Some of the changes have been reported in a few or only one cell type. These could depend on the initial differentiated phenotype of the particular cells. However, some changes have been observed in a variety of cells, pointing to some general principles that may apply to all cells. The fact that cells not normally exposed to high NaCl and urea share these general responses suggests that hyperosmolality may be such a basic threat that all cells are prepared to confront it, whether they will ever experience it or not. In this review we concentrate mainly on renal epithelial cells, recognizing that osmotic stress has also been studied in many other cell types (Table 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Cellular adaptation to hypertonic stress.

	II. PERTURBING EFFECTS OF HYPEROSMOLALITY

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Acute elevation of NaCl or urea produces rapid arrest in all phases of the cell cycle. The cells in G₁ do not proceed to initiate DNA replication. Those in S stop replicating their DNA. Those in G₂ do not divide. The duration of the arrest depends on how high the NaCl or urea is. The arrest is shortest in G₁ and S, so cells accumulate in G₂ (198). By the time that the arrest ends, the cells are sufficiently adapted so that can they proliferate through many passages at the elevated osmolality. The rate of proliferation of adapted cells is directly related to the osmolality (198). When NaCl is elevated to 400–500 mosmol/kgH₂O, proliferation is the same as at 300 mosmol/kgH₂O, but at higher levels it decreases progressively. Finally, there is a limit as to how high a concentration the cells can withstand. Beyond this they die, as discussed in section IIB. Cell cycle arrest provides time for cells to adapt to higher osmolality by accumulation of compatible and counteracting organic osmolytes (see sects. IV and VIIIB) and increased expression of heat shock proteins (see sect. X). In the absence of such protective adaptation, the cells die.

DNA damage is a well-known cause of cell cycle arrest. High NaCl increases the number of DNA breaks in cells (see sect. IIC), and some of the same signals that arrest the cell cycle in response to DNA damage from other causes also play a role in high NaCl-induced cell cycle arrest. Delay in G₁ and S depends on activation of p53 following both high NaCl (72) and ionizing radiation (IR) (100). In the case of high NaCl, the delay in S is sensitive to caffeine, implying involvement of ATM/ATR kinases (72), which are targets of p53 (100). Delay in G₂ depends on activation of p38 following both high NaCl (74) and ultraviolet (UV) radiation (28).

Despite these similarities, there are major differences between the cell cycle delay caused by high NaCl and that from other causes. DNA breaks from the other causes are repaired during the cell cycle arrest, but those caused by high NaCl are not (see sect. IIC). Furthermore, the mechanisms differ. Phosphorylation of Chk1 is important for the cell cycle arrest induced by the DNA damage from other stresses (173), but high NaCl does not cause phosphorylation of Chk1 and, in fact, prevents its phosphorylation in response to DNA damage caused by UV or IR (73). Also, inhibition of Chk1 by UCN-01 prevents the cell cycle delay caused by other DNA damaging agents, but not the cell cycle delay caused by high NaCl (73). The question remains as to why the increased DNA breaks that persist in cells adapted to high NaCl do not activate cell cycle checkpoints. Possibly, they are modified so that they are invisible to the DNA damage sensors or the DNA damage response is inhibited.

The cell cycle arrest induced by high urea closely resembles that induced by high NaCl (198), but less is understood about its mechanism. Urea does not activate some of the DNA damage response proteins involved in the cell cycle arrest induced by high NaCl, such as p53 (72) and p38 (149, 251). High urea increases phosphorylation of ATM in HEK293 cells (124), but we do not know whether this is involved in the accompanying cell cycle arrest.

B. Apoptosis

Cell death results when acute hypertonicity exceeds a certain level. The level differs between cell lines, but is rather sharply defined in each. Above that level, massive cell death occurs within several hours. The dying cells exhibit classical features of apoptosis (25, 94, 198, 257). DNA condenses and apoptotic bodies appear, the DNA becomes fragmented with appearance of a "DNA ladder" in gel electrophoresis, the broken DNA ends are labeled in TUNEL assays, and phosphatidylserine becomes exposed at the cell surface. There are two basic pathways for apoptosis: an intrinsic (mitochondrial) pathway and an extrinsic (death receptor) pathway (132). There is evidence favoring both of these pathways as a cause of hypertonicity-dependent apoptosis. In favor of the intrinsic pathway is that hypertonicity causes a rapid mitochondrial dysfunction (see sect. IIF) that precedes apoptosis. Thus acutely exposing mIMCD3 cells to a lethal level of osmolality (700 mosmol/kgH₂O, NaCl added) rapidly depolarizes their mitochondria, decreases mitochondrial Bcl2/Bax ratio, and increases cell ADP/ATP ratio (197). However, the mitochondria do not release cytochrome c, and although their Bcl2/Bax ratio decreases, it also decreases at 500 mosmol/kgH₂O without any subsequent apoptosis. In favor of the extrinsic pathway is that acute exposure of HeLa cells to 900 mosmol/kgH₂O (sorbitol added) causes clustering and internalization of tumor necrosis factor- (TNF-) receptors (253). Also, increasing osmolality to 500 mosmol/kgH₂O by adding NaCl causes apoptosis in macrophages (U937) and B lymphocytes (LCL721), accompanied by increased production of TNF-, suggesting that they produce their own death ligand and self-destruct (162). Neutralization of the TNF- by a specific antibody protects the U937 cells from apoptosis, but it does not protect the LCL721 cells, indicating that the autogenous TNF- could be triggering the apoptosis in the former, but not in the latter. In another example, increasing the osmolality bathing primary rat hepatocytes to 405 mosmol/kgH₂O (NaCl added) causes trafficking of CD95 to the plasma membrane, accompanied by caspase-8 and -3 activation; however, this only amounts to a priming for apoptosis because full-blown apoptosis (DNA fragmentation and phosphatidylserine redistribution) does not occur unless exogenous CD95 ligand is also added (244). We conclude that, since hypertonicity has been observed to trigger components of both the intrinsic and extrinsic pathway to apoptosis in one type of cell or another, it is premature to conclude that either pathway is always involved.

The fact that both hypertonicity and apoptosis cause cells to shrink has led to recognition that the processes are linked. Thus apoptotic volume decrease (AVD) is an early event in apoptosis. It precedes cytochrome c release from mitochondria, and it is maintained until the cell fragments (184, 224). Inhibition of AVD prevents apoptosis from going to completion (184, 224). Regulatory volume increase (RVI) is the usual initial response to cell shrinkage by hypertonicity (311). During RVI, electrolytes and water are taken up into the cell, restoring its volume. An ion carrier (NKCC1) and ion exchangers (NHEs) are activated in RVI (Table 1 and Ref. 311). Although RVI occurs in cells shrunken by hypertonicity, it is impaired in cells undergoing an AVD initiated either by a death receptor ligand (Fas) or an by an inducer of mitochondria-mediated apoptosis (staurosporine) (185). Given that AVD is an early event in apoptosis and that RVI does not occur, it seemed possible that cell shrinkage induced by hypertonicity, if uncompensated by RVI, might by itself cause apoptosis (24, 25). This idea is supported by a number of observations. RVI is impaired in T lymphocytes, and they undergo apoptosis at a much lower level of tonicity than do cells proficient in RVI (25). CHO (PC120) cells, which are deficient in RVI because they lack NHE-1, are particularly susceptible to hypertonicity-induced apoptosis. Reconstituting them with NHE-1 restores RVI and protects them (185). Finally, inhibition of NHE by amiloride sensitizes HeLa (185) and renal tubular epithelial (RTC) cells (319) to hypertonicity-induced apoptosis. Taken together, these observations indicate that hypertonicity-induced shrinkage, if uncompensated by RVI, can cause apoptosis. What is lacking at this point is an understanding of how cell shrinkage activates the executioners of apoptosis. Although hypertonicity shrinks cells immediately, several hours elapse before the final stages of apoptosis are reached (25, 88, 185, 197). Interestingly, when cells are exposed to levels of hypertonicity that would be deadly if prolonged, returning the cells to normotonicity after a brief time (0.5–1 h) prevents apoptosis (24; Dmitrieva NI and Burg MB, unpublished results). Thus there must some additional processes between the initiation of hypertonicity and the point of no return from apoptosis. Those processes apparently include activation of Rac and p38, followed by phosphorylation and nuclear translocation of p53 (88).

C. DNA Damage

Acute elevation of osmolality from 300 to 500–600 mosmol/kgH₂O by adding NaCl to cells causes DNA strand breaks in mIMCD3 cells (73, 153), accompanied by cell cycle arrest in all phases of the cell cycle (see sect. IIA). The cell cycle arrest that is associated with most other causes of DNA damage provides a lull in DNA replication during which the DNA can be repaired (345). Thus it seemed logical that, during the transient cell cycle arrest that follows acute elevation of NaCl, the DNA breaks would be repaired. Surprisingly, they are not. Numerous DNA breaks persist in cells that have adapted to high NaCl and reentered the cell cycle (75). Despite these DNA breaks, the adapted cells proliferate rapidly, appear normal, and exhibit little, if any, apoptosis (42, 75, 259). Even more surprising, there are numerous DNA breaks in cells in the mouse renal inner medulla as long as its interstitial NaCl concentration remains at its normal high level (75).

High urea causes a different sort of DNA damage. Neither acute (153, 153) nor chronic (75) exposure of cells to urea causes DNA strand breaks. Furthermore, addition of urea does not significantly increase the number of breaks caused by high NaCl (75), implying that NaCl, alone, is responsible for the DNA breaks in the normal renal inner medulla. However, high urea does produce oxidative DNA damage, namely, 8-oxoguanine lesions (337). If 8-oxoguanine lesions are not repaired, they produce mutations during DNA replication (104). Thus renal medullary cells have DNA breaks caused by high NaCl and oxidative lesions caused by high urea. Nevertheless, the cells survive and function in vivo and, following adaptation, proliferate rapidly in culture.

Normally, DNA breaks attract and activate a protein complex that rapidly repairs the DNA. If the repair fails, the cells die by apoptosis. Cells deficient in components of the DNA repair system are very sensitive to extrinsically caused DNA damage and may not be viable because they cannot repair DNA breaks that occur during normal replication or transcription (173, 183, 320, 349). Remarkably, DNA breaks induced by high NaCl persist as long as NaCl remains high. Yet, despite the DNA breaks, apoptosis is not evident, and the cells proliferate rapidly in culture (75). Reducing NaCl back to normotonic levels in culture (73) and in vivo (75) results in rapid repair of the breaks, implying that high NaCl inhibits DNA repair. Direct proof for this came from measuring the rate of repair of luciferase reporter plasmids damaged by UV before they were transfected into cells. The repair is much faster at 300 mosmol/kgH₂O than when NaCl is added to 500 mosmol/kgH₂O (73, 75).

Although it is clear that hypertonicity induces DNA damage, the molecular basis is poorly understood. Hypertonicity increases expression or activity of several DNA damage response proteins including Gadd45 (see sect. XA), p53 (see sect. XB), ATM (see sect. V, D and E), and chk2 (264). Other DNA damage response proteins are not affected. Thus other genotoxic agents that induce DNA breaks cause phosphorylation of Chk1, signaling cell cycle arrest (27, 122), but high NaCl does not (73, 75, 264). The effect of high NaCl on some of the other DNA damage response proteins is controversial. Thus Mre11 exonuclease is a nuclear protein that normally binds rapidly to DNA breaks, beginning the process of repair (64), and histone H2AX becomes phosphorylated at DNA double-stranded breaks caused by a number of agents (90, 250, 309), both being prerequisite for successful DNA repair (45, 229). In some studies, high NaCl decreased the chromatin-bound fraction of Mre11 (76) and caused it to move from the nucleus to the cytoplasm, where it is unavailable to initiate DNA repair (73, 75). Also, phosphorylation of histone H2AX was not induced (73, 75). These aspects of the DNA damage response became activated immediately when NaCl was reduced. When osmolality was lowered from 500 to 300 mosmol/kgH₂O, Mre11 moved back into the nucleus, histone H2AX and Chk1 became phosphorylated, and the DNA breaks disappeared within a few hours. These results are consistent with the idea that high NaCl inhibits those particular components of DNA damage response or that the response to high NaCl modifies or camouflages DNA breaks so that they are not recognized by the usual DNA damage response system. However, in another study, Mre11 stayed in the nucleus after NaCl was increased and histone H2AX became phosphorylated (264). Why the results differ remains unresolved. The authors of the latter study (264) speculated that the transient H2AX phosphorylation that they observed indicated repair of any DNA double-strand breaks, but they did not actually measure the nature or extent of DNA damage. In summary, direct measurements demonstrate that high NaCl increases the number of DNA breaks both in cell culture and in vivo. However, the exact nature of the breaks, the mechanism by which they are induced, and the details of the cellular response remain to be settled.

The DNA-protein kinase (DNA-PK) complex, which contains Ku86, Ku70, and DNA-PKcs, contributes to repair of DNA double-strand breaks by nonhomologous end joining (NHEJ) (193, 222, 274). When cells that lack Ku86 are exposed to high NaCl, they have aberrant mitosis, slower growth, and increased chromatin fragmentation compared with wild type cells (76).

D. Oxidative Stress

Elevation of NaCl or urea causes oxidative stress (328, 337, 340, 347, 348, 352) with elevation of ROS (328, 337, 348). ROS can modify DNA bases by forming 8-oxoguanine (104) and can carbonylate proteins (170). Mutations result when DNA containing 8-oxoguanine lesions is replicated (104). Carbonylation affects protein stability and function, notably inhibiting enzyme activity (279). In cell culture, carbonylation occurs within 5 min and with addition of as little as 5 mM urea, which is the normal level in plasma. Since carbonylation increases as urea is raised over the range found in uremia (337), urea apparently can contribute directly to the carbonylation found in that condition. Strikingly, a high level of protein carbonylation is present in vivo in inner medullary cells (337), which are normally exposed to high NaCl and urea. Carbonylated proteins are not repaired. Instead, they undergo accelerated degradation by proteasomes and are replaced (169). At this point we do not know what the functional consequences are of urea- and NaCl-induced oxidation of renal medullary proteins, but it is of obvious interest to find out. Oxygen tension is low in the renal inner medulla because its countercurrent exchange system limits the access of oxygen. Glycolysis predominates there (9). The low oxygen tension and respiration may minimize the oxidative stress in this tissue.

ROS can be destructive, but they also have important signaling functions (reviewed in Ref. 87). Several observations support a role for ROS in signaling osmoprotective responses: 1) antioxidants that reduce superoxide suppress high NaCl-induced increases of both TonEBP/OREBP transcriptional activity and expression of BGT1 mRNA (see sect. VH)(347, 348). 2) The antioxidant N-acetyl-L-cysteine (NAC) prevents urea-induced increase of mRNA and protein abundance of the oxidative stress-responsive transcription factor Gadd153/CHOP (340). 3) NAC also prevents both high NaCl-induced phosphorylation of ERK1/2 and p38 and increase of COX-2 protein expression (328). 4) Lowering ROS production by mitochondrial inhibitors reduces high NaCl-induced increase of COX-2 (328).

E. Inhibition of Transcription and Translation

High NaCl reversibly inhibits synthesis of DNA, RNA, and protein in cultured cells (248). Synthesis recovers within 10 min if NaCl is lowered (248) and more slowly as cells adapt to sustained hypertonicity (235). The effect on DNA synthesis has already been discussed in section IIA. That on transcription and translation is discussed in what follows.

Hypertonicity rapidly and reversibly inhibits RNA synthesis in HeLa cells (248). Increasing osmolality to 450 mosmol/kgH₂O by adding NaCl inhibits synthesis of heterogeneous nuclear RNA (mRNA precursor), whereas higher concentrations of NaCl are necessary to inhibit synthesis of ribosomal and transfer RNA (232).

Hypertonicity also profoundly inhibits protein synthesis (248, 254). The inhibition is independent of the solute added (NaCl, KCl, or sucrose), occurs within several minutes, and reverses completely when normotonicity is restored. Inhibition was observed in HeLa cells (254), in chicken embryo fibroblasts (235), and in Madin-Darby canine kidney (MDCK) cells (58). Translation of globin mRNA by rabbit reticulocyte lysate in vitro is inhibited when osmolality is increased by adding NaCl, KCl, CH₃CO₂Na, or CH₃CO₂K, but not by adding the compatible osmolytes betaine or myo-inositol (26). Taken together, these results suggest that hypertonicity-induced increase of intracellular inorganic ions inhibits translation, but the inhibition abates as the inorganic ions are replaced by compatible organic osmolytes (see sect. IV) (26). Inhibition of translation could be responsible for the cell cycle arrest. The onset and ending of the cell cycle arrest (72) coincide temporally with the changes in protein synthesis (248, 254). A possible link is that cyclins are translationally regulated.

Hypertonicity reduces translation by inhibiting polypeptide chain initiation (248, 254, 313), whereas elongation and termination of those polypeptides already initiated generally proceed normally (254). Although hypertonicity inhibits translation of most cellular proteins, translation of some, such as heat-shock proteins, actually increases (see sect. XC) (209). The mechanism by which hypertonicity inhibits translation is not well understood, but there are some suggestive findings. Initiation factor eIF2 contributes to inhibition of translation by hypertonicity in reticulocytes and fetal liver nucleated erythroid progenitors (181). In those cells hypertonicity causes autophosphorylation and activation of heme-regulated inhibitor (HRI), which in turn phosphorylates and activates eIF2. The activated eIF2 inhibits translation (181). Other unidentified eIF2 kinases may also contribute (181). The lengths of untranslated regions (UTRs) in mRNAs and their secondary structure may also play a role. With the use of the rat preproinsulin II-coding sequence linked to the 5'-untranslated sequence of the message that encodes viral SV40, insertion of a hairpin barely affects translation in normotonic medium, but adding NaCl to increase osmolality by 240–300 mosmol/kgH₂O virtually abolishes translation (148). When a 136-nucleotide unstructured sequence is placed just upstream from the hairpin, translation in hypertonic medium is restored. Thus leader length and secondary structure can have opposing effects on translational efficiency under hypertonic conditions (148).

F. Mitochondrial Depolarization

Increasing osmolality by adding NaCl perturbs mitochondria in mIMCD3 (197) and Vero (60) cells, although the effects differ in detail. In the mIMCD3 cells, increasing osmolality to 700 mosmol/kgH₂O causes mitochondrial depolarization, but increasing it to only 500 mosmol/kgH₂O does not. Nuclei become condensed and caspase-9 activity increases at 700 mosmol, indicative of apoptosis, but this does not occur at 500 mosmol/kgH₂O. Electron microscopy does not reveal any mitochondrial structural changes at either of the elevated osmolalities. Although NADH exits the mitochondria at 700 (but not at 500) mosmol/kgH₂O, cytochrome c remains in the mitochondria at all osmolalities. In the Vero cells, high NaCl causes rapid mitochondrial depolarization, as in mIMCD3, but the effect occurs at a much lower osmolality. Increasing the osmolality by as little as 60 mosmol/kgH₂O depolarizes mitochondria in the Vero cells, whereas increasing it by 200 mosmol/kgH₂O does not in mIMCD3 cells. At 600 mosmol/kgH₂O mitochondria rapidly fragment in Vero cells, but the effect reverses when NaCl is lowered. In contrast, the mitochondria remain intact in mIMCD3 cells. High NaCl does not cause cytochrome c to exit from the mitochondria in either cell type. Given these results, it is not clear whether mitochondrial depolarization is a rapid response of viable cells to hypertonicity (Vero cells), or whether it is an early event in apoptosis (mIMCD3 cells). Possible mechanisms for the depolarization are reviewed in Reference 197. We conclude that high NaCl greatly perturbs mitochondria, but the significance of this effect and its mechanism remain uncertain.

	III. HIGH NaCl: HYPERTONICITY

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By definition, hypertonicity occurs when an added solute causes osmotic efflux of water from a cell and, thus, reduces cell volume. This requires a gradient of activity of the added solute across the cell membrane and semipermeability of the membrane. In accord with this definition, many experiments that test the effects of hypertonicity consist of acutely adding an impermeant solute, then observing the result over a period of minutes to hours. Since most cells have a high water permeability, water generally effluxes rapidly, and the osmotic shrinkage elevates the concentration of all intracellular components. As a result, macromolecular crowding increases, which greatly elevates the activity of cellular macromolecules (96). Also, intracellular ionic strength increases, which also affects most biochemical processes. Finally, the decreased volume is accompanied by alterations in the cytoskeleton. These are primary effects of hypertonicity, from which we presume other effects follow, although specifying the connections remains a challenge.

High NaCl is hypertonic, and NaCl is the principal determinant of tonicity of the extracellular fluid. Although cell membranes are permeable to Na, the Na⁺-K⁺-ATPase transports Na out of cells, which maintains the normal cell volume. In examining the effects of high NaCl, however, we also have to keep in mind that in addition to raising tonicity, high Na and Cl may also have specific effects. For example, elevated chloride specifically increases the expression of -Na⁺-K⁺-ATPase (43). Thus, although cellular changes caused by high NaCl may generally be due to hypertonicity, this is not necessarily so. For that reason, poorly permeating organic solutes like sorbitol, mannitol, or raffinose are often used to produce hypertonicity (Table 1). Of course, these also may have specific effects. Therefore, to establish definitively that a particular effect is caused by hypertonicity, hyperosmolality should be tested adding more than one different solute.

A. Many Cellular Components and Functions Are Affected by Hypertonicity

Table 1 lists 200 cellular components that change with osmolality, including mRNAs and proteins identified by Northern analysis, ribonuclease protection, RT-PCR, or Western analysis. The number of constituents that are affected by osmolality is too great to consider them all individually in detail. Also, caution should be exercised in interpreting the findings of those studies in which osmolality is increased by 300 mosmol/kgH₂O or more. That degree of hyperosmolality can lead to apoptosis (198, 257), so viability of cells is questionable under those circumstances. The different categories affected by tonicity (Table 1) include ion and water channels, transporters, heat shock proteins, other stress proteins, cellular and extracellular structural proteins, transcription factors and coactivators, kinases, phosphatases, other signaling proteins, enzymes, hormones, receptors, growth-related proteins, second messengers, and organic osmolytes. Determining the significance of any particular change requires additional information since it could represent an osmoprotective response, but it could also simply represent a dysfunction caused by hypertonicity. Many of these changes were discovered when an investigator added hypertonicity to the list of different stresses being applied to a component that initially was of interest for reasons unrelated to tonicity.

Screening methods have also been used to identify mRNAs and proteins affected by hypertonicity. The effect of hypertonicity on the abundance of cellular mRNAs in mIMCD3 cells was examined by using microarrays (294). When 200 mM mannitol or 100 mM NaCl is added for 6 h, 12% of 12,000 transcripts are downregulated by mannitol and NaCl, and 4% of transcripts are upregulated. As might be expected, the genes identified in this manner generally agree with those identified by other techniques (Table 1). The effect of hypertonicity on cellular proteins was examined by proteomic techniques. In one study (305) mIMCD3 cells were examined by two-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. Raising osmolality by 300 mosmol/kgH₂O (NaCl added) increases abundance of some proteins and decreases others. Several molecular chaperones are induced, such as _B-crystallin, two Hsp70 isoforms, and the osmotic stress protein (Osp94). Also, some isoforms of the translation elongation factors eEF2 and eEF1A1 are induced. With TLAH cells (70), osmolality was increased by 300 mosmol/kgH₂O for 35 h by adding NaCl. Proteins were examined by 2D-GE and MALDI-MS to determine changes in abundance; 25 proteins increase and 15 proteins decrease. Besides aldose reductase, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, -enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase increase. The cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V also increase, whereas tubulin and tropomyosins 1, 2, and 3 decrease. Vimentin, cytokeratin, and tropomyosin remained elevated in TAHL-NaCl cell lines adapted to high NaCl, as determined by Western analysis. The heat shock proteins -crystallin chain B, HSP70, and HSP90 and endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase decrease.

Since we do not have space to discuss all of these changes in detail, we will restrict ourselves to some that are known to be involved in osmoregulation and osmoprotection, including cellular structural proteins (see sect. IIIB), organic osmolytes (see sect. IV), stress proteins (see sect. X), the transcription factor TonEBP/OREBP (see sect. V), and the molecules that signal its activation (see sect. VII).

B. Cytoskeletal Rearrangement in Response to Hypertonicity

Hypertonicity induces rapid F-actin polymerization and remodeling of the actin cytoskeleton (35, 68, 322). However, the pattern of remodeling varies between cell types. In native rat MTAL cells (35) and glial cells (207), hypertonicity induces redistribution of F-actin from the cortical ring to a diffuse network of actin bundles, whereas in fibroblasts and HL60 cells the cortical actin ring becomes denser (68, 110). The assembly is induced by the combined action of small GTPases (Rac and Cdc42), an actin binding protein (cortactin), and a regulator of F-actin nucleation (the Arp2/3 complex), all of which form a complex with actin (68). Hypertonicity also causes rapid movement of myosin IIB to the cortical region, just beneath the plasma membrane, dependent in part on Rho kinase and p38 (230).

Integrins are a large family of heterodimeric transmembrane glycoproteins that attach cells to extracellular matrix proteins of the basement membrane. Integrin ₁₁ is a major collagen-binding receptor that is highly expressed in renal tubular cells. Hypertonicity increases integrin ₁ mRNA and protein in MDCK cells (269). Increased expression of this integrin is involved in hypertonicity-induced activation of the osmoprotective transcription factor TonEBP/OREBP (see sect. VH). Furthermore, inactivation of integrin-substratum interactions by using the integrin-binding peptide GRGDTP inhibits the hypertonicity-induced increase in glutamine transport that is mediated by phosphatidylinositol 3-kinase (PI3K) in skeletal muscle (180).

Phosphatidylinositol phosphate 5-kinase (PIP5K) kinase (322) contributes to hypertonicity-induced cytoskeletal rearrangement. Its lipid kinase activity catalyzes phosphorylation of inositol 4-phosphate (Ins-4-P) on the D5 position of the inositol ring, producing inositol 4,5-bisphosphate (Ins-4,5-P₂). PIP5KIs are predominantly cytosolic proteins that are recruited to plasma and organelle membranes through association with small GTPases such as Rho and Rac. Their lipid kinase activity is augmented by these associations and by integrin signaling. Hypertonicity increases Ins-4,5-P₂. siRNA knock down of PIP5K blocks both hypertonicity-induced increase of Ins-4,5-P₂ and cytoskeletal rearrangement. PIP5KI, being constitutively phosphorylated, is activated by Ser/Thr dephosphorylation. Hypertonicity reduces phosphorylation of PIP5K and increases its lipid kinase activity. The phosphatase inhibitor calyculin A decreases basal PIP5KI activity and blocks PIP5KI activation by hypertonicity. Thus hypertonicity induces a phosphatase that activates PIP5KI lipid kinase activity by dephosphorylating it, and the resultant increase of Ins-4,5-P₂ contributes to the ensuing cytoskeletal rearrangement.

The exact role of the cytoskeleton in osmoregulation remains speculative (77). It could function as a cell volume sensor, it could offer mechanical protection against the deleterious effects of excessive shrinkage, and it could be involved in signal transmission from osmosensors (whatever they may be) to osmoeffectors. A role in signal transmission is supported by evidence that hypertonicity induces assembly of an actin-associated protein complex containing a scaffolding protein (OSM), a GTPase (Rac), and members of a mitogen-activated protein (MAP) kinase cascade (MEKK3, MKK3, and p38) (303). p38 is a MAP kinase that contributes to signaling hypertonicity-induced activation of the osmoprotective transcription factor TonEBP/OREBP (see sect. VE). Activation of p38 by hypertonicity is inhibited if any of the components of the OSM complex is knocked out (303). These findings associate the actin cytoskeleton with a signaling molecule (p38) that contributes to osmoprotective transcription. However, the exact role of the actin cytoskeleton in the system remains to be established. Are tonicity-dependent actin cytoskeletal changes necessary to activate p38 or is the mere presence of the cytoskeleton sufficient? Also, p38 can be activated in many ways and convey many different signals. Is the OSM system the only one involved in that activation of p38 that contributes to TonEBP/OREBP activity? Is it the most important one? An added complexity is that cytoskeletal remodeling depends on p38 in MTAL cells (35), implying that there could be positive feedback.

	IV. ORGANIC OSMOLYTES ACCUMULATE IN RESPONSE TO HYPERTONICITY

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Cells often respond to osmotic shrinkage by RVI in which transporters are activated that mediate rapid uptake of inorganic ions, followed by an osmotic influx of water that restores cell volume. Many of the cellular components in Table 1 are involved in RVI. However, we will not consider RVI in detail here, referring instead to a general review (311). Although RVI increases cell volume back towards normal within minutes, the concentration of intracellular ions, and, consequently, the intracellular ionic strength remain abnormally high. This is an unhealthy state, since high ionic strength perturbs intracellular macromolecules (325).

Over a period of hours, however, compatible organic osmolytes accumulate in cells, gradually reducing the ionic strength back towards that in the normotonic state, while maintaining cell volume (Figs. 1 and 2). The benefit is that compatible organic osmolytes perturb less than do inorganic ions (325) because they stabilize proteins by interacting favorably with the protein backbone (282). The principal compatible organic osmolytes in renal medullary cells are sorbitol, glycine betaine (betaine), myo-inositol (inositol), taurine, and glycerophosphocholine (GPC) (8, 215). We previously reviewed (95) the evidence that hypertonicity increases the concentration of these organic osmolytes in renal cells both in vivo and in cell culture, and the mechanisms by which they are accumulated. To save space, we briefly summarize that earlier information and concentrate, instead, on more recent studies.

View larger version (17K): [in this window] [in a new window]   FIG. 2. High NaCl raises TonEBP/OREBP transcriptional activity via several signaling pathways, resulting in greater abundance of proteins involved in urinary concentration and in accumulation of organic osmolytes.

Sorbitol (95) is synthesized from glucose, catalyzed by aldose reductase (AR), and is metabolized to fructose, catalyzed by sorbitol dehydrogenase. Hypertonicity raises AR activity by increasing transcription of its gene, resulting in elevation of its mRNA and protein. Sorbitol dehydrogenase activity, on the other hand, is low in the renal cells that were examined and does not change significantly with tonicity. Furthermore, although hypertonicity increases AR mRNA in inner medullary collecting ducts (IMCDs) in vitro, it does not alter sorbitol dehydrogenase mRNA (106). Therefore, hypertonicity-induced increase of sorbitol apparently is entirely due to increased transcription of AR, leading to increased AR activity. TonEBP/OREBP is the transcription factor responsible for the hypertonicity-induced increase of AR. We consider osmotic regulation of TonEBP/OREBP in detail in section V.

AR is activated by other stimuli besides hypertonicity, and there are consequences of its activation besides osmoprotective synthesis of sorbitol. Although detailed consideration of other aspects of AR regulation and activity are beyond the scope of this review, it is worth noting that some of them are related to oxidative stress, which we have already seen is associated with osmotic stress (see sect. IID). For example, lipid aldehydes generated endogenously during the process of degradation of lipid peroxides contribute to cell damage associated with oxidative stress. 4-Hydroxynonenal (HNE), which is one of the most abundant and toxic lipid aldehydes, is efficiently detoxified by AR (278). AR mRNA and protein are elevated in areas of tissue destruction in inflamed arteries, colocalized with high levels of HNE, and in vitro exposure of mononuclear cells to HNE is sufficient to increase AR protein abundance (246). Similarly, HNE treatment of rat vascular smooth muscle cells induces AR mRNA (277). Oxidative stress can also activate AR posttranslationally. ROS, formed in the ischemic heart, activate AR by modifying its cysteine residues to sulfenic acids (133). Thus hypertonicity-induced increase of AR activity may affect and be affected by both the osmotic stress itself and the accompanying oxidative stress.

AR activation is not necessarily protective. AR activity is high in diabetes. This activity has been implicated in the microvascular complications of that disorder, and, as a result, there are major efforts to develop and use inhibitors of AR to minimize those complications (51). Furthermore, AR is activated during myocardial ischemic injury, and inhibitors of AR or sorbitol dehydrogenase protect rat hearts from ischemia-reperfusion injury (121). Adverse effects of increased traffic through the AR pathway have been attributed to elevation of the cytosolic NADH/NAD⁺ ratio.

B. Betaine

Betaine (95) is synthesized from choline both in liver and kidney. However, unlike sorbitol, hypertonicity does not significantly affect the rate of its synthesis. Rather, hypertonicity increases the number of transporters that carry it into the cells from the extracellular fluid. Betaine/GABA transporter (BGT1) is the osmoregulated betaine transporter (323). It couples transport of betaine to that of Cl and Na. Gradients of those ions energize the transport, providing a driving force that can concentrate betaine from micromolar levels in the plasma to millimolar levels within the cells. Hypertonicity elevates the transcription of the BGT1 gene, followed by increase in its mRNA and transport activity (302). Thus tonicity regulates betaine transporter activity by affecting BGT1 transcription. TonEBP/OREBP (see sect. V) is the transcription factor responsible for this effect.

BGT1 is regulated not only by transcription, but also by plasma membrane insertion (139). The relatively small amount of BGT1 that exists under normotonic conditions is mainly in the cytoplasm. Following hypertonicity, BGT1 is seen mainly in basolateral plasma membranes. Addition of the microtubule inhibitor nocodazole causes the additional BGT1 to remain in the cytoplasm, even following hypertonicity (139), and elevation of calcium causes internalization of BGT1, possibly mediated by PKC (138). Also, EGFP-BGT1, expressed in fibroblasts that lack endogenous BGT1, moves into plasma membranes following hypertonicity (139). Then, transport is upregulated without change in total abundance of BGT1, only its location. Interestingly, membrane insertion of existing EGFP-BGT is delayed for 3 h after tonicity increases, consistent with the need to synthesize accessory proteins for membrane insertion.

C. Inositol

Inositol (95) is synthesized by renal cells. Nevertheless, the inositol accumulated following hypertonicity is transported into the cells, not synthesized. In the absence of extracellular inositol, none is accumulated. Hypertonicity increases inositol transport by increasing the number of inositol transporters. Following its cloning, the transporter was named SMIT (sodium myo-inositol transporter)(160). SMIT couples transport of inositol to that of Na in a 1:2 ratio (109). The Na gradient energizes the transport, providing a driving force that can concentrate inositol from micromolar levels in the plasma to millimolar levels with the cells. Hypertonicity elevates the transcription of the SMIT gene, followed by increase in its mRNA and transport activity (324). Thus tonicity regulates inositol transporter activity by affecting SMIT transcription. TonEBP/OREBP (see sect. V) is the transcription factor responsible for this effect.

D. Taurine

Taurine content is regulated by tonicity in renal cells in vivo and in cell culture. Thus taurine in rat renal inner medullas is approximately twice as great when the rats are infused with high NaCl solutions (presumed to increase medullary interstitial NaCl) than when they are infused with low NaCl solutions (215). Taurine in MDCK cells doubles when tonicity is increased by adding raffinose (302). Accumulation of taurine could be due to increased synthesis or increased transport into cells. Of the two possibilities, hypertonicity-induced accumulation of taurine appears to be mainly via transport, rather than synthesis. The original evidence for transport was that hypertonicity (raffinose added) doubles taurine uptake into MDCK cells across their basolateral membranes (301). The taurine transporter that is involved was cloned by expression in Xenopus oocytes and named TauT (300). It is Na and Cl dependent, and hypertonicity increases its mRNA abundance in MDCK cells. In HepG2 cells, hypertonicity (high NaCl or sucrose) increases transcription of the TauT gene, the abundance of TauT mRNA, and the abundance of TauT protein, resulting in increased taurine uptake (126). This hypertonicity-induced increase in TauT transcription is regulated by the transcription factor TonEBP/OREBP (see sect. V) via ORE/TonE sites in the 5'-flanking region of the TauT gene (126). A possible role for Ca²⁺/calmodulin (CaM) kinase II in hypertonicity-induced activation of TauT was inferred from the action of its inhibitor, KN-93, in Caco-2 cells (261).

Taurine synthesis is from cysteine, catalyzed by the sequential actions of cysteine dioxygenase (CDO), which gives rise to cysteinesulfinate, and cysteinesulfinate decarboxylase (CSD), which decarboxylates cysteinesulfinate to hypotaurine. The synthesis is mainly in liver, from which it is released to the general circulation, a major portion being taken up by the kidneys and excreted. Although the enzymes for taurine synthesis are present in renal medullas, hypertonicity increases TauT mRNA much more in rat renal medullas than it does CDO and CSD mRNAs (22).

Taurine has many other sites of action besides the renal medulla (261). It regulates heart rhythm, contractile function, blood pressure, platelet aggregation, neuronal excitability, body temperature, learning, motor behavior, food consumption, eye sight, sperm motility, cell proliferation and viability, energy metabolism, and bile acid synthesis. At least some of these effects have been attributed to its effects on membranes and on protein phosphorylation. Thus it is conceivable that taurine that is accumulated by cells in response to hypertonicity affects them in other ways besides serving as a compatible organic osmolyte.

E. GPC

GPC (95) is abundant in cells of renal inner medullas, and it accumulates in renal cells in tissue culture in response to either high NaCl or high urea. It is synthesized from phosphatidylcholine (PC), then is broken down into choline and -glycerophosphate. The high NaCl-induced increase of GPC was studied both in isolated renal inner medullary collecting duct cells (14, 15) and in various cell cultures (156, 157, 332). In MDCK cells, high NaCl increases GPC synthesis and also reduces its degradation (156, 157). The synthesis from PC is catalyzed by a phospholipase activity that is increased by high NaCl. Similarly, phospholipase activity increases in inner medullas of rats when they are thirsted (331). Neuropathy target esterase (NTE) is a phospholipase B that catalyzes production of GPC from PC (333). In mIMCD3 cells (91), high NaCl raises NTE mRNA, beginning within 8 h, and NTE protein within 16 h. Di-isopropyl fluorophosphate (DFP), which inhibits NTE esterase activity, reduces the GPC accumulation, as does an siRNA that specifically decreases NTE protein abundance. In vivo, NTE protein expression is higher in the renal inner medulla than in the cortex. The lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1 –/– mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. Furthermore, the 20-h half-life of NTE mRNA is unaffected by high NaCl, but knock down of the transcription factor TonEBP/OREBP (see sect. V) by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA, showing that high NaCl elevates NTE mRNA through TONEBP/OREBP-mediated increase in its transcription. Transcriptional activity of TonEBP/OREBP requires binding to a specific DNA element, ORE/TonE (83, 287), whose consensus sequence is NGGAAAWDHMC(N) (83). Between bp –900 and –1 in the 5'-flanking region of NTE there are four DNA elements that fit this consensus. Thus high NaCl increases transcription of NTE, mediated by TonEBP/OREBP, and the resultant increase of NTE activity contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo.

In addition, high NaCl also reduces degradation of GPC by decreasing the activity of GPC-choline phosphodiesterase (GPC-PDE), the enzyme that degrades GPC to choline and -glycerophosphate (15, 157, 332), and the reduced degradation of GPC contributes to its increased abundance. The GPD-PDE that is involved is Gdpd5 (unpublished observations), which is a mouse homolog of bacterial glycerol diesterases. High NaCl reduces Gdpd5 mRNA abundance. In mIMCD3 cells at 300 mosmol/kgH₂O, reducing Gdpd5 abundance with a specific siRNA increases GPC. High NaCl reduces Gdpd5 mRNA by destabilizing it. In short, high NaCl inhibits degradation of GPC by decreasing the abundance of Gdpd5, a GPC-PDE that catalyzes breakdown of GPC.

One compatible organic osmolyte can substitute for another in protecting cells from hypertonicity. For example, addition of betaine to the medium, and its resultant uptake by the cells, largely replaces the decrease in sorbitol caused by AR inhibitors and restores the viability of the cells (205). Specific reduction of one organic osmolyte generally causes a compensating increase of the others (204). This is readily understandable, since the same transcription factor, TonEBP/OREBP (see sect. V), regulates betaine and inositol transporters and the enzymes that catalyze synthesis of sorbitol and GPC. GPC is exceptional in that its degradation is also regulated (see above). It is of interest that the degradation of GPC is also affected by the abundance of the other osmolytes. Thus increasing the level of betaine and inositol in MDCK cells reduces GPC by stimulating its degradation (158). Other factors have also been reported to affect GPC. In MDCK cells, caffeine increases GPC through ryanodine-inhibitable increase of cellular calcium and activation of phospholipase A₂ (140).

	V. ACTIVATION OF THE TRANSCRIPTION FACTOR TonEBP/OREBP BY HYPERTONICITY

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TonEBP/OREBP (also named NFAT5) is a member of the Rel family of transcriptional activators, as are nuclear factor B (NFB) and nuclear factor of activated T-cells (NFAT). Each TonEBP/OREBP target gene contains in its regulatory regions at least one DNA consensus motif called an osmotic response element (ORE) (83–85, 144, 346) or tonicity-responsive enhancer (TonE) (126, 187, 200, 245, 287).

As described in section IV, TonEBP/OREBP transactivates several genes that are responsible for tonicity-dependent accumulation of organic osmolytes including sorbitol, glycine betaine, inositol, taurine, and GPC (Fig. 2) (30, 91). TonEBP/OREBP also transactivates genes coding for heat shock protein 70 (HSP70) (317), vasopressin-activated urea transporters, UT-A1 (216) and possibly UT-A2 (161), as well as aquaporin-2 (AQP2) (113, 114). As described in section XC, HSP70 is a protein chaperone and is osmoprotective against high NaCl and high urea. UT-A1 and UT-A2 recycle urea, producing high concentrations in the renal inner medulla (255), which contributes to the corticomedullary osmotic gradient necessary for concentrating urine. AQP2 increases water permeability of the renal collecting duct, which increases urine concentration. Mice that overexpress a dominant negative form of TonEBP/OREBP in renal collecting tubule cells have impaired urinary concentrating ability, probably due to reduced expression of AQP-2 and UT-A (161).

TonEBP/OREBP knock-out or dominant negative expression in vivo has major renal and extrarenal consequences. Disruption of both TonEBP/OREBP alleles in mice results in embryonic lethality (101, 176). The few surviving homozygous TonEBP/OREBP null-mice have profound, progressive atrophy of the kidney medulla, coupled with impaired activation of the TonEBP/OREBP target genes AR, BGT1, and SMIT (176). Heterozygous mice exhibit defects in adaptive immunity, evidenced by lymphoid hypocellularity and impaired antigen-specific antibody response (101). Additionally, in culture, TonEBP/OREBP –/+ lymphocytes proliferate more slowly in hypertonic, but not normotonic media (101). Mice that overexpress a dominant negative form of TonEBP/OREBP in the lens develop nuclear cataracts soon after birth (308). Developing fiber cells in these lenses exhibit a physiological state similar to that produced by hypertonicity, including DNA strand breaks, activation of p53, and induction of checkpoint kinase (308).

In the following sections we analyze how the transcriptional activity of TonEBP/OREBP is controlled and the complex network by which hypertonicity signals the activation.

A. TonEBP/OREBP mRNA and Protein Abundance

Hypertonicity increases TonEBP/OREBP mRNA in MDCK (316), HeLa (143), and mouse inner medullary collecting duct (mIMCD3) cells (39). The increase is transient, peaking at 4–12 h depending on cell type. TonEBP/OREBP mRNA rises because of a transient increase in its stability, mediated by elements within its 5'-UTR (39). In mIMCD3 cells, TonEBP/OREBP mRNA is stabilized for 6 h after addition of NaCl, as measured by determining the rate of mRNA decrease after inhibiting transcription with actinomycin D (39). Stabilization by high NaCl is sufficient to explain the increase of TonEBP/OREBP mRNA without any change in transcription (39). Following increased mRNA abundance, TonEBP/OREBP protein increases. In combination with tonicity-dependent nuclear localization, discussed below, the elevated nuclear abundance of TonEBP/OREBP increases TonEBP/OREBP protein binding to OREs. The hypertonicity-induced increase in TonEBP/OREBP protein abundance and synthesis rate is approximately equal to the increase in mRNA abundance (316). Hypertonicity does not affect the rate of TonEBP/OREBP protein degradation (316).

B. Dimerization of TonEBP/OREBP

TonEBP/OREBP is a constitutive dimer, and dimerization is required for several aspects of its hypertonicity-dependent activation. The NH₂ terminus of TonEBP/OREBP contains two dimerization domains (177). TonEBP/OREBP forms stable dimers (or higher order oligomers) in solution (178) independent of tonicity (177). Dimerization of TonEBP/OREBP is required for DNA binding (177). In binding to DNA, TonEBP/OREBP forms a homodimer that completely encircles its DNA target (284). Constitutive dimerization of TonEBP/OREBP is also required for its "proper" phosphorylation when NaCl is high (166). Dimerization also is required for TonEBP/OREBP transactivation of downstream genes (177).

C. Phosphorylation of TonEBP/OREBP

TonEBP/OREBP becomes phosphorylated on serine and tyrosine residues within 30 min of addition of NaCl (65). Possible phosphorylation sites in TonEBP/OREBP isoform "c" (1,531 amino acids) are its 216 serines, 15 tyrosines, and 111 threonines. The exact role of this tonicity-dependent TonEBP/OREBP phosphorylation remains unclear because there has been no direct identification of the amino acids that are phosphorylated. However, single and multisite phosphorylation are known to regulate other transcription factors at multiple levels, including nuclear localization, DNA binding, and transactivation.

Serine/threonine phosphorylation is involved in TonEBP/OREBP binding to its DNA element. The binding, as measured using nuclear extracts and electromobility shift assays with an ORE probe, is eliminated by treatment with calf intestinal phosphatase (CIP), a general phosphatase, and PP1, a serine/threonine phosphatase, but not by treatment with PTPase, a phosphotyrosine phosphatase (2). Nevertheless, binding of immunoprecipitated TonEBP/OREBP is not prevented by CIP, indicating that its DNA binding does not require the actual phosphorylation of TonEBP/OREBP itself (65).

Phosphorylation generally regulates transactivation 1) by recruiting coactivator proteins that contact the general transcriptional machinery, 2) as a requirement for maximal transcriptional activity, or 3) as a requirement for the formation of stable homodimer-DNA complexes. High NaCl increases phosphorylation of TonEBP/OREBP within amino acids 548–1531, as indicated by phosphatase-inhibitable decrease of migration of that fragment in gels (86). TonEBP/OREBP amino acids 872–1271 contain a transactivation domain (TAD) that is activated by high NaCl (86). Serine/threonine kinase inhibitors reduce this high NaCl-dependent transactivational activity of TonEBP/OREBP (86), which could be due either to a direct effect on the phosphorylation state of TonEBP/OREBP, itself, or it could be mediated by a signaling partner or a transcriptional cofactor. An indirect effect is plausible, since no net tonicity-dependent change in phosphorylation of the TonEBP/OREBP TAD was detected (165), while a direct effect is supported by site-directed mutation of putative phosphorylation sites (124), as discussed below.

D. TonEBP/OREBP Nuclear Localization

To bind to their cognate DNA elements, transcription factors must enter the nucleus. Proteins the size of TonEBP/OREBP are too large (>50 kDa) to diffuse freely in and out through nuclear pores. Such large molecules require a nuclear localization sequence (NLS) to mediate binding to importin. The importin-cargo complex enters the nucleus by active transport through nuclear pores. There is a similar requirement for a nuclear export sequence (NES) and binding to exportin for large proteins to exit from the nucleus. The distribution of TonEBP/OREBP in the cell is tonicity dependent. In MDCK cells, HeLa cells and Jurkat cells at normotonicity (300 mosmol/kgH₂O), TonEBP/OREBP is distributed between cytoplasm and nucleus, but at 450–500 mosmol/kgH₂O, most of the TonEBP/OREBP is in the nucleus (143, 177, 201, 296). At 135–250 mosmol/kgH₂O in MDCK or HeLa cells, TonEBP/OREBP is mostly in the cytoplasm (296, 316), correlated with a decrease in its activity (86, 316). There is indirect evidence that TonEBP/OREBP may interact with a cytoplasmic binding protein. MG-132, a 26S proteasome inhibitor, blocks tonicity-dependent nuclear localization of TonEBP/OREBP in MDCK cells (318). This calls to mind the regulation of NFB (63). In the cytoplasm, IB binds to NFB blocking NFB nuclear import. During activation of NFB, IB becomes phosphorylated, then degraded in the proteasome, releasing NFB to move into the nucleus. Although a similar system could exist for regulation of TonEBP/OREBP, no TonEBP/OREBP cytoplasmic binding protein analogous to IB has been identified. MG-132 also blocks NaCl-induced nuclear localization in HepG2 cells, but not in COS-7 cells, indicating that the effect is cell type dependent (338). Cyclosporin A has a similar effect in MDCK cells (267), consistent with calcineurin dependence of nuclear localization.

Like the native protein, a recombinant TonEBP/OREBP expressing NH₂-terminal amino acids 1–547 localizes to the nucleus when NaCl is increased, indicating that the COOH terminus is not required for translocation (338). The NH₂ terminus of TonEBP/OREBP contains a series of amino acids (amino acids 199–216 of isoform "c") that fit with a consensus bipartite NLS (143, 179) in that there are two clusters of basic amino acids in tandem. However, the NLS of TonEBP/OREBP is actually monopartite. Only the first cluster of basic amino acids is necessary for its nuclear import (296). The NH₂ terminus of TonEBP/OREBP additionally contains two motifs that function in nuclear export (296). The NES at amino acids 8–15 (isoform "c") is a CRM1-responsive protein domain. Accordingly, leptomycin B, an inhibitor of the exportin CRM1, blocks nucleocytoplasmic shuttling of TonEBP/OREBP at 300 mosmol/kgH₂O (296). Disruption of this NES increases TonEBP/OREBP in the nucleus at 300 mosmol/kgH₂O, but hypotonicity can still cause nuclear export of this mutant, indicating that this NES is not responsible for nuclear export under hypotonic conditions (296). An auxiliary export domain (AED) is found at amino acids 132–156 (isoform "c") and is functional in nuclear export of TonEBP/OREBP under both normotonic and hypotonic conditions. Deletion of the AED causes nuclear retention of TonEBP/OREBP regardless of tonicity (296). However, the AED does not affect subcellular localization when fused to green fluorescent protein, indicating it does not function independently (296).

Activation of ataxia telangiectasia-mutated (ATM) kinase by high NaCl contributes to nuclear translocation of TonEBP/OREBP (338). In cells lacking functional ATM, nuclear translocation of native TonEBP/OREBP and of a recombinant NH₂-terminal construct (amino acids 1–547, which contains the NLS as well as the NES and AED) is reduced, and reconstituting the kinase in these cells restores the translocation (338). Thus ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP through interaction at sites within amino acids 1–547. The effect of ATM could be direct or it could be mediated by another factor in the signaling pathway that activates TonEBP/OREBP. Since transactivation domains can affect nucleocytoplasmic shuttling (238), it is possible that that ATM also modulates translocation via sites in TonEBP/OREBP-548–1531, which contains a tonicity-dependent transactivation domain as well as putative ATM consensus phosphorylation sites (86, 124).

Other kinases such as Fyn and PI-3K class 1A, which also are known to stimulate TonEBP/OREBP-dependent transcription, do not affect NaCl-dependent nuclear localization of TonEBP/OREBP (123, 142), but, as discussed below, each of these kinases does target the hypertonicity-sensitive TonEBP/OREBP transactivation domain.

E. TonEBP/OREBP Transactivational Activity

In addition to motifs that regulate nuclear localization and DNA binding, transcription factors also contain transactivation domains (TADs), which interact with and recruit a specific assemblage of proteins that forms an enhanceosome, and with the basal transcriptional complex which includes RNA polymerase. Transactivation of target genes by transcription factors is regulated by DNA binding, by association with cofactors, by heterologous transcription factors, and by posttranslational modifications. Transactivation of target genes by TonEBP/OREBP varies directly with extracellular NaCl concentration (86). TonEBP/OREBP has a high NaCl-dependent TAD within amino acids 1039–1249 (isoform "c") (86, 165). Additional TonEBP/OREBP TADs are located within amino acids 1–76 and 1363–1476, but these are not sensitive to tonicity (165). TonEBP/OREBP also has two modulation domains (amino acids 618–820 and 889–955) that potentiate the activity of the TADs but are not independently active in stimulating transcription. One of the modulation domains is responsive to tonicity (amino acids 618–820), and all of the domains act synergistically to transactivate target genes in response to hypertonicity (165).

Many regulators contribute to tonicity-dependent increase in TonEBP/OREBP transactivation of its target genes. Each is necessary for full TonEBP/OREBP activity, but no one of them is sufficient.

cAMP-dependent kinase (PKA) is a serine-threonine kinase that regulates many cellular processes, most commonly in response to increased intracellular cAMP. However, high NaCl activates PKAc without increasing intracellular cAMP (82). Chemical inhibition of PKAc or transfection with a PKAc dominant negative construct reduces hypertonicity-induced TonEBP/OREBP transcriptional activity, transactivation activity, and resultant accumulation of AR and BGT1 mRNAs (82). Conversely, overexpression of wild-type PKAc increases TonEBP/OREBP transactivation activity (82). Also, PKAc and TonEBP/OREBP reciprocally coimmunoprecipitate each other, indicative of physical association of the proteins (82).

p38 is a mitogen-activated protein kinase (MAPK). Hypertonicity activates p38 (111), contributing to increased TonEBP/OREBP activity (142, 210, 265). Thus inhibition of p38 by chemicals (142, 210, 265) or by transfection of a dominant negative construct (142) reduces 1) hypertonicity-induced activation of TonEBP/OREBP (142, 210), 2) the increase in its transactivational activity (142), and 3) the resultant increase of mRNA abundance of its transcriptional targets, AR and BGT1 (142, 265). The pathway for activation of p38 MAPK by osmotic stress is cell type-specific and can involve different combinations of the MAPK kinases, MKK3 and MKK6, as well as autoactivation (134). Hypertonicity causes MEKK3 activation (303) by autophosphorylation (89) and also activates MKK3 and MKK6 (62, 194). p38, itself, becomes autoactivated in response to hypertonicity, promoted by association with transforming growth factor--activated protein kinase 1-binding protein (TAB1) (134). Furthermore, some of the cellular p38 is contained in a multienzyme complex, attached to the scaffolding protein, OSM. Hypertonicity activates p38 in the complex, associated with hypertonicity-induced cytoskeletal rearrangement (see sect.