Mitogen-Activated Protein Kinase Cascades and the Signaling of Hyperosmotic Stress to Immediate Early Genes

Comp. Biochem. Physiol. Vol. 117A, No. 3, pp. 291–299, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00266-6

Mitogen-Activated Protein Kinase Cascades and the Signaling of Hyperosmotic Stress to Immediate Early Genes David M. Cohen Division of Nephrology, Oregon Health Sciences University and Portland V.A. Medical Center, Portland, OR 97201, U.S.A. ABSTRACT. Among prokaryotes and lower eukaryotes, the threat of exposure to hyperosmotic stress is ubiquitous. Among higher eukaryotes, in contrast, only specific tissues are routinely exposed to marked hypertonicity. The mammalian renal medulla, the prototypical example, is continually subjected to an elevated solute concentration as a consequence of the renal concentrating mechanism. Until recently, the investigative focus has concerned the effects of diverse solutes on the regulation of genes essential for the adaptive accumulation of osmotically active organic solutes. Recent and sweeping developments elucidating the molecular mechanisms underlying stress signaling to the nucleus have focused interest on earlier events in the response to hyperosmotic stress. Such events include the transcriptional activation and post-translational modification of transcriptional activating proteins, a large subset of which represent the protein products of so-called immediate early genes. This review highlights developments in the understanding of stress signaling in general and hypertonic stress signaling in particular in both yeast and higher eukaryotic models. The relationship between hyperosmotic stress signaling and the transcription and activation of immediate-early gene transcription factors is explored. comp biochem physiol 117A;3:291–299, 1997.  1997 Elsevier Science Inc. KEY WORDS. Hypertonicity, transcription, signal transduction, review, mitogen-activated protein kinase, urea, sodium chloride, kidney, Egr-1

INTRODUCTION In recent years, remarkable progress has been made in understanding how externally applied environmental cell stressors signal from the cell membrane to the nucleus. This review provides an overview of developments implicating diverse kinase cascades in transducing stress signals in general and hyperosmotic stress in particular into nuclear trans-activating events in yeast and higher eukaryotes. The specific contributions of these observations to the understanding of hyperosmotic stress-inducible immediate early gene (IEG) (Table 1) transcription and trans-activation are emphasized. In addition, wherever possible, the response of kidney cells to hypertonicity is emphasized, because the mammalian renal medulla represents one of the few anatomical sites routinely (and physiologically) exposed to an elevated and fluctuating ambient osmolality. Regardless of the inciting stimulus, two principal features characterize an IEG response. After cell stimulation (e.g., Address reprint requests to: D.M. Cohen, M.D., Division of Nephrology, PP262, Oregon Health Sciences University, 3314 S.W. US Veterans Hospital Rd., Portland, OR 97201. Tel. (503) 220-8262 x6654, Fax (503) 7217810, e-mail cohen.david [email protected]. Received 7 September 1995; accepted 10 February 1996.

with a mitogen or cell stressor), upregulation of IEG mRNA is rapid (occurring within minutes) and transient. In addition, this response at the mRNA level is insensitive to a global inhibition of protein synthesis. Therefore, the protein products of IEGs represent the first round of gene expression after cell stimulation. A subset of these IEGs encode transcription factors—proteins capable of directly or indirectly interacting with specific regulatory sequences in noncoding regions of DNA and thereby altering the rate of transcription of the associated gene. There are several families of IEG transcription factors, including the c-fos, c-jun and Egr families (64), and the number of members of each family, as well as the number of families, is continually increasing. IEG transcription factors tend to be inducible transcription factors; their expression at the mRNA level is temporally governed by exogenous stimuli. (Basal transcription factors, in contrast, regulate the constitutive expression of gene products and are generally not subject to wide fluctuations in abundance.) As inducible transcription factors, IEG protein products are ideally poised to initiate a coordinated cascade of adaptive gene expression in response to a given stimulus. Hence, there has been abundant interest in elucidating the specific profile of IEG transcription factors inducible by each of a broad array of stimuli

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TABLE 1. Definitions of abbreviations used in the text and

functional role (e.g., MAPK) of the listed protein Abbreviation

Definition (Functional Role)

TABLE 2. Phylogenetic conservation of the principal components of the hyperosmotic signaling pathway in prokaryotes, yeast and mammals

Conservation of Osmotic Signaling CSBP ERK HOG1 IEG JNKK MAPK MAPKK MAPKKK MEK MEKK MKK3 PBS2 SAPK/JNK SEK SHO1 SLN1 SSK1 SSK2/22

Cytokine-stimulated anti-inflammatory drug binding protein (mammalian HOG1 homologue, also p38, a MAPK) Extracellular signal-regulated kinase (a MAPK) High-osmolarity glucose gene (a yeast MAPK) Immediate-early gene jun kinase kinase (also SEK, a MAPKK) Mitogen-activated protein (MAP) kinase MAP kinase kinase MAP kinase kinase kinase MAPK, or ERK, kinase (a MAPKK) MEK kinase (a MAPKKK) MAP kinase kinase 3 (a MAPKK) Yeast osmoresponsive MAPKK Stress-activated protein kinase/jun kinase (a MAPK) SAPK, or ERK, kinase (also JNKK or MKK4, a MAPKK) Yeast transmembrane osmosensor (activated by hyperosmolarity) Yeast transmembrane histidine kinase osmosensor (inhibited by hyperosmolarity) Yeast response regulator and inhibitory SLN1 substrate Yeast osmoresponsive MAPKKK

Tonicity sensor MAP kinase Transcription factor/substrate

Bacteria

Yeast

Mammals

EnvZ

SLN1 SHO1 HOG1

?

— OmpR

?

ERK SAPK/JNK CSBP/p38 TCF (Elk-1) c-jun

? denotes a component whose presence has not been demonstrated or confirmed.

digm established by the prokaryotic sensor, the membraneassociated sensing protein is also a histidine kinase. Upon sensing a decrease in tonicity, the protein transfers a phosphate in cis from an amino terminal His residue to an Asp residue located in the carboxy terminal ‘‘receiver domain.’’

(both in vitro and in vivo) with the presumption that such information will provide insight into patterns of downstream gene expression. Before an elaboration on the role of IEGs in the higher eukaryotic cell response to hyperosmotic stress is undertaken, it is instructive to examine the yeast response to environmental hypertonicity; in this way, parallels between respective signaling pathways can readily be appreciated. HYPEROSMOTIC STRESS RESPONSE: YEAST Tonicity Sensor In enteric bacteria, osmoregulation utilizes a so-called ‘‘twocomponent’’ pathway: the membrane-associated environmental osmolality sensor, EnvZ (a histidine kinase), interacts with and modulates the activity of a transcriptional regulatory protein, OmpR (14) (Table 2). Recently, an analogous tonicity sensing system was identified in the yeast Saccharomyces cerevisiae (47). In response to environmental hyperosmotic stressors, yeast increases synthesis and retention of the organic solute glycerol (Fig. 1). Maeda and coworkers cloned the putative tonicity sensor, SLN1, as well as downstream elements in the signaling cascade (v.i) by complementation of mutant osmosensitive yeast strains. SLN1 had previously been identified in a search for yeast mutants sensitive to a defect in proteolysis; however, its function had remained obscure (52). Following the para-

FIG. 1. Cascade of convergent activation events in the re-

sponse of the yeast S. cerevisiae to hyperosmotic stress. Filled arrows represent activation events; open arrows represent inhibitory events. Extracellular high osmolarity inhibits SLN1 and activates SHO1; both events independently result in activation of the MAPKK, PBS2 (46,47).

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The same phosphate group may instead be transferred in trans to an Asp residue in an analogous ‘‘receiver domain’’ on the yeast cytosolic protein, SSK1. Phosphorylation inactivates SSK1, inhibits downstream signaling events and thereby inhibits glycerol synthesis. A second putative transmembrane osmosensor, SHO1, has recently been described by the same group (46). In contrast to SLN1, SHO1 does not appear to signal through histidine phosphorylation (v.i.). Kinase Signaling Although the direct association between SLN1 and SSK1 phosphorylation states and downstream events is incompletely understood, the SLN1 sensor functions upstream of a yeast osmoresponsive mitogen-activated protein kinase (MAPK) signaling pathway (3) (Fig. 1). MAPK signaling pathways or cascades are extraordinarily highly conserved transducers of receptor and postreceptor events to the nucleus (15,49). Although named for MAPK, they include multiple kinase steps both upstream and, potentially, downstream of MAPK activation. In species from yeast to mammals, these cascades are characterized by sequential activation of a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase. MAP kinases are proline-directed kinases; they phosphorylate serine and threonine residues embedded in a proline-rich consensus sequence in substrate proteins. In contrast to phosphorylation by MAP kinases, catalytic activation of MAP kinases requires both threonine and tyrosine phosphorylation by a MAPKK, a so-called dual specificity protein kinase (Fig. 1). MAPKK is, itself, activated by MAPKKK through serine phosphorylation (29). In the yeast hyperosmotic stress response, the MAPK role is filled by the yeast osmoresponsive MAPK homologue, HOG1, whereas the MAPKK role is assumed by the yeast MAPKK homologue, PBS2 (3) (Fig. 1). Both enzymes are required for yeast cell growth in the presence of high osmolarity (3). The recently identified proteins, SSK2 and SSK22, serve as upstream MAPKKK homologues, which transduce the signal from SSK1 to the MAPKK, PBS2 (46). SHO1, the more recently described transmembrane osmosensor, appears to directly interact with PBS2, bypassing the need for an intermediary MAPKKK step. The interaction is likely mediated via a Src homology 3 (SH3) domain integral to the SHO1 protein; deletion of this domain abolishes the interaction with PBS2 (46). Of note, multiple ‘‘parallel’’ MAP kinase signaling pathways have been described in yeast, of which the HOG1 pathway represents but one. Consistent with the general model of MAPK activation, HOG1 undergoes rapid tyrosine phosphorylation in response to hyperosmotic stress (3). Coupling of HOG1 activation to events leading to adaptive glycerol accumulation are less clear. HOG1 may indirectly activate transcription from the yeast high osmolarity re-

FIG. 2. Diagrammatic representation of the three principal

‘‘parallel’’ MAP kinase pathways in cells of higher eukaryotes. Boxes indicating the respective MAPK homologues are shaded. Heavy vertical arrows represent phosphorylation or activation events. Dashed lines indicate ‘‘cross-talk’’ between pathways (see text).

sponse element (STRE) present in the promoters of multiple potentially stress-responsive yeast genes (1,57). To date, there has been no report of a histidine kinase osmosensing protein in higher eukaryotes (Table 1). It is conceivable that osmoregulation in yeast, with their rigid cell wall, may be more akin to that of prokaryotes than to that of cells of higher eukaryotes. Nonetheless, there is a remarkable degree of conservation between the yeast and higher eukaryotic response to hyperosmotic stress. HYPEROSMOTIC STRESS RESPONSE: HIGHER EUKARYOTES Three Potentially Stress-Responsive Kinase Cascades In higher eukaryotes, three distinct potentially stressresponsive families of MAP kinases have been identified (Fig. 2). Extracellular signal-regulated kinases (ERKs) transduce signals from growth factors (and their receptor tyrosine kinases) to a family of transcriptional activating proteins, the ternary complex factors (TCFs). Stress-activated protein kinase/jun kinase (SAPK/JNK) phosphorylates and activates the transcription factor c-jun in response to diverse cellular stressors. Thekinase p38/CSBP, in response to primarily hyperosmotic stress, phosphorylates another kinase implicated in the phosphorylation of a small heat shock protein. In addition to the multiplicity of MAPK activities, at least three distinct MAPKK activities have been identified in higher eukaryotes (Fig. 2). These include MEK, MKK3 and

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SEK; each displays a characteristic profile of substrate specificity among the MAPK homologues (v.i.). This specificity appears to be governed by the presence of a unique consensus sequence in each of the MAP kinases: ERKs are activated through phosphorylation at Thr-Glu-Tyr, SAPK/JNKs at Thr-Pro-Tyr and p38/CSBP at Thr-Gly-Tyr (18). In contrast to MAPK and MAPKK, thus far only two distinct MAPKKK activities, Raf and MEKK, have been described. Substrate preferences of both MAPKKK and MAPKK have led to the establishment of a widely cited model wherein separate but ‘‘parallel’’ MAP kinase cascades are activated in response to distinct cellular stressors (Fig. 2). These three pathways are described in turn, although there is mounting evidence for substantial interaction among them. ERK PATHWAY. The first identified and best-studied MAPK pathway in higher eukaryotes involves the MAP kinases, ERK1 and ERK2 (15). ERK activation is achieved in numerous models through phorbol ester treatment or interaction of a peptide growth factor with its cognate receptor tyrosine kinase. The primary signal is then transduced from the cell membrane in either a Ras-dependent or -independent fashion to achieve activation of Raf, the MAPKKK of the ERK pathway (Fig. 2). The precise mechanism of Rasinduced Raf activation has engendered controversy; recent evidence suggests that activated Ras serves merely to target and anchor Raf to the cell membrane where an additional activation event is required (38,63). Activated Raf phosphorylates and activates MEK, which, in turn, activates ERK1 and ERK2. Putative substrates (and therefore potential effectors) of activated ERKs include cytoplasmic phospholipase A2 (44) and the kinases p90rsk (19) and MAPKAP kinase 2 (62). Importantly, activated ERKs can also translocate from the cytoplasm to the nucleus (6,25,41) and phosphorylate TCF (v.i.) (28) and c-myc (58). In addition to being positively regulated by phosphorylation, the ERKs can be negatively regulated by dephosphorylation. Two nuclear vanadate-sensitive ERK-specific dual specificity phosphatases, MKP1 (2,66) and PAC1 (73), dephosphorylate ERKs at their activating Tyr and Thr phosphorylation sites. Interestingly, both phosphatases, as immediate-early genes, are transcriptionally inducible by the very stimuli that may result in ERK activation. ERKs are also negatively regulated in diverse models through activation of cAMP-dependent protein kinase. This inhibitory effect appears to occur at the level of the MAPKKK, Raf (reviewed in 29), potentially via an interruption in the Ras– Raf interaction by Raf serine phosphorylation (75). As expected, synthetic dominant negative mutants of Raf also inhibit ERK activation (56). In addition, the ERK pathway may be negatively regulated by the okadaic acid-sensitive phosphatase PP2A, an effect that appears to be mediated through dephosphorylation events at multiple levels including MEK and ERK (reviewed in 29). SAPK /JNK PATHWAY. Stress-activated protein kinases comprise a second distinct family of MAP kinases. Cloned

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independently as JNK (17) and SAPK (36), the SAPK/ JNKs, unlike the ERKs, are generally poorly responsive to mitogens and phorbol esters (36). Rather, as their name implies, they are activated by numerous stressors (Fig. 2) including endotoxin and the pro-inflammatory cytokines (TNF-α, IL-1), UV light, heat shock and protein synthesis inhibitors (36). Like other MAP kinases, SAP kinases require both threonine and tyrosine phosphorylation for activation (17). The SAPK-specific MAPKK homologue responsible for this activation, SEK, was recently identified (55,78). (SEK was also independently cloned as MKK4 [18] and JNKK [43]). Although originally described as a SAPKactivator, SEK can also directly activate the osmotically responsive MAPK, p38 (v.i.) (43), but apparently not the ERKs. Accordingly, a synthetic dominant negative SEK mutant inhibits SAPK activation by stressors but fails to affect the ERK pathway (55). SEK is in turn phosphorylated and activated by the MAPKKK, MEKK (51,78). MEKK activation does not appear to ultimately result in activation of the ERK cascade but rather only of the SAPK/JNK cascade (51,78). The principal substrate of the JNKs is c-jun, which undergoes regulated phosphorylation in response to JNK activation (v.i.). p38/CSBP PATHWAY. The third family of potentially stress-responsive MAP kinase homologues are the mammalian homologues of the yeast osmoresponsive MAPK, HOG1. In contrast to HOG1, however, the higher eukaryotic homologues have generally been identified using stressors other than hyperosmolality, suggesting considerable cross-talk among the ‘‘parallel’’ kinase pathways. Higher eukaryotic homologues of HOG1 have been independently identified as murine p38 (27), cytokine-suppressive antiinflammatory drug binding protein (37), human pp40 (21) and amphibian Mpk2 (54). That they indeed comprise a functional homologue of HOG1 is underscored by the observation that p38 can complement a HOG1-deficient mutant yeast strain and restore osmotic tolerance (27). p38 is phosphorylated and activated by the recently identified and cloned p38-specific human MAPKK homologue, MKK3 (18). In vitro, MKK3 tyrosine- and threonine-phophorylates p38 but not ERK or JNK (18). When transfected into COS cells, MKK3 was itself activated by both osmotic stress and the ‘‘SAPK-like’’ stressors, UV light and inflammatory cytokines (18). It had previously been recognized that p38 was a potential substrate for the SAPK-specific MAPKK, SEK, but the physiological significance of this was unknown. Interestingly, Derijard et al. (18) also showed that p38 may, at least in vitro, represent a better substrate for SEK than does SAPK/JNK; the implications are discussed in greater detail (v.i.). The only physiological p38/CSBP substrate identified to date is the kinase MAPKAP kinase 2, which in turn phosphorylates the small heat shock proteins (21,54). The identification of specific pharmacological inhibitors of p38 (37) will likely aid dissection of its relative contribution to other downstream events in stress responses.

MAPK Cascades and Hyperosmotic Stress

Kinase Cascades in Hyperosmotic Stress Although the model outlined above suggests that the principal kinase activated by hyperosmotic stress in mammalian cells is the p38/CSBP and although there exists ample experimental evidence to support this thesis [e.g., (18,27)], the unfolding picture is far less clear. In fact, there is evidence supporting a role for each of the three principal MAPK pathways in transducing the signal of hyperosmotic stress. In the renal epithelial MDCK cell model, two lines of evidence support a role for ERKs. In hyperosmotically stressed cells, Itoh and coworkers (30) observed a prompt upregulation of MAPK activity after treatment with hypertonic NaCl or raffinose. Similarly, Terada and coworkers (68) noted Raf activation, followed temporally by ERK activation. In both studies, activation of ERKs (as opposed to other MAP kinases) was inferred from the upregulation of the myelin basic protein-phosphorylating activity of a kinase of the appropriate molecular mass for the ERKs. In addition to the ERKs, evidence also implicates JNKs in the osmotic response. Hyperosmotic stress increased activation of JNK1 in mammalian CHO cells, and osmotic shock of yeast cells resulted in both phosphorylation and activation of transfected (mammalian) JNK1 (22). In addition, JNK1, like p38, could complement a HOG1-deficient yeast strain and restore osmotic tolerance (27). In further support of appreciable cross-talk with regard to hyperosmotic stress and MAPK signaling, not only does osmotic stress activate multiple kinase pathways but the osmotically responsive MAPK, p38, may be activated by nonosmotic stimuli, such as those typically associated with JNK1 activation (e.g., cytokines and heat shock). Although this may represent a direct effect, it is more likely a consequence of SEK-induced activation of p38 (18). Therefore, multiple nonosmotic signals can activate the osmotically responsive MAPK, p38, and multiple MAP kinases besides p38 can be activated by osmotic stressors. TRANSCRIPTION FACTOR SUBSTRATES OF HIGHER EUKARYOTIC MAP KINASES Cellular stressors mediate many of their effects through MAPK activation; MAPKs, in turn, mediate many of their effects through activation of transcription factors. Two examples that have received the greatest attention include: TCF activation by ERK and c-jun activation by SAPK/JNK. TCF Mitogen-inducibility of the IEG c-fos is a consequence of the presence, in the c-fos promoter, of a consensus element of the form CC(A/T)6GG. This motif, called the serumresponse element (SRE), is bound by a nuclear protein, the serum response factor (SRF). This binding occurs in a constitutive fashion in many models; in addition, SRF does not appear to undergo regulated phosphorylation in response to a mitogenic stimulus [reviewed in (33)]. Transcriptional

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regulation under the control of the SRE is apparently a result of an inducible interaction between the SRE:SRF complex and an additional protein factor, a member of the TCF family (including proteins Elk-1 and SAP-1). In contrast to SRF, TCFs undergo rapid phosphorylation on multiple carboxy-terminal serine residues and are thereby activated in response to mitogenic stimulation (48,79). The identical sites are phosphorylated by the ERKs in vitro (reviewed in 33). In addition, evidence from the use of activating and dominant-negative synthetic mutants of MEK and ERK suggest that ERK activation is essential for TCF activation in vivo (31,34). Therefore, TCF appears to be a principal effector of ERK function. [Importantly, it has recently been demonstrated that SAPK/JNK may also activate TCFs (74)]. c-jun Analogous with ERK-induced phosphorylation and activation of TCF, SAPK/JNK phophorylates and activates the transcription factor and IEG, c-jun. c-jun (and its homologues) can either homodimerize or heterodimerize with c-fos (and its homologues) to constitute the DNA binding protein and transcriptional activator, activating protein-1 (AP-1). AP-1 activity is rapidly unregulated in response to diverse stimuli including mitogens and phorbol esters—a consequence of increased transcription and translation of c-fos and c-jun, as well as post-translational modification of the protein products of these genes. In this latter fashion, c-jun activation is governed by phosphorylation of five regulatory sites [reviewed in (33)]. Functionally, c-jun is composed of an amino terminal activation domain and a carboxy terminal DNA binding domain. Amino terminal phosphorylation increases transcriptional activation by c-jun without influencing its DNA binding activity (60,61). JNK specifically phosphorylates these same serine residues in vitro (17), whereas the ability of ERK to phosphorylate these sites remains controversial (reviewed in 33). In addition, the c-jun heterodimerization partner, ATF-2, is also an effective substrate for activated SAPK/JNK in vitro and in vivo (45,72). Furthermore, there is evidence for a unique proline-directed MAP kinase, p88, that phosphorylates the transcriptional activation domain of the jun heterodimerization partner, c-fos (16). HYPEROSMOTIC STRESS AND SPECIFIC TRANSCRIPTION FACTORS The Model of the Mammalian Renal Medulla As alluded to above, there are two principal means by which trans-activating activity of IEG transcription factors may be regulated. IEG transcription factors may themselves be rapidly induced at the transcriptional level in response to a given stimulus, resulting in increased IEG mRNA synthesis with subsequent translation of this mRNA into new potentially active IEG protein. The prototypical example is the

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upregulation of c-fos transcription after mitogenic stimulation (26). Alternatively, a preformed transcription factor protein may remain in an inactive state (either constitutively bound to its cognate DNA consensus sequence or in an off-DNA state) and undergo prompt post-translational modification (e.g., phosphorylation) in response to a given stimulus. Both types of mechanisms likely take place in the response of cells of higher eukaryotes to hyperosmotic stressors; however, there is at present no direct evidence for the role of activation through post-translational modification. One can be inferred, however, from the unequivocal activation of JNK by hyperosmolality in CHO cells, with the likely attendant phosphorylation and activation of c-jun, as described above. In contrast, the role of hyperosmotic stress in activating transcription of potential IEGs has been examined directly; the model has been the epithelial cell of the mammalian renal medulla. As a consequence of the renal concentrating mechanism, the mammalian renal medulla is one of very few higher eukaryotic tissues routinely exposed to an elevated and fluctuating ambient osmolality. Excess osmolality in the renal medullary interstitium is comprised largely of NaCl and urea. Eukaryotic cells exposed to hyperosmotic stressors (in vitro and in vivo) accumulate osmotically active organic solutes, or organic osmolytes, to counteract net water efflux (23). Recent elegant work has described the cloning of several cDNAs encoding proteins instrumental in the synthesis or transport of organic osmolytes in renal epithelial cells (24,35,70,76). Although these genes appear to be transcriptionally activated by hyperosmotic stressors such as NaCl or raffinose (59,71,77), none has yet been demonstrated to be an IEG. Therefore, it is likely that upstream transcriptional and protein synthetic events are required for enhanced expression of these later gene products. In addition, none of the MAPK cascades have yet been implicated in the transcriptional regulation of these genes in response to hyperosmotic stress. Hyperosmotic Stress and IEG Expression in Renal Epithelial Cells In a screen of diverse putative transcription factors, it was observed that hyperosmotic NaCl (100 mM) reproducibly increased mRNA expression of the IEG transcription factor, c-fos, as well as Egr-1, in renal epithelial cells in culture (13). Egr-1 (also known as zif268, Krox-24, NGFI-A and TIS8) (7,39,42,50,65) is a zinc-finger containing nuclear protein that activates transcription from promoters containing its GC-rich consensus sequence (4,8,40). The kinetics of c-fos and Egr-1 induction by NaCl were similar to those observed after mitogen stimulation, with peak mRNA expression occurring at 30 min. A number of possible explanations for NaCl-inducible IEG expression were explored. There was no demonstrable toxic effect of the solute and there was no effect on cell viability. Hyperosmotic NaCl

did, however, globally inhibit protein synthesis (as quantitated by tritiated leucine incorporation) (13). Consistent with this observation, NaCl treatment failed to increase Egr-1 protein expression, an obvious prerequisite for transcriptional activation by Egr-1. IEGs are often ‘‘superinduced’’ at the mRNA level in the presence of an inhibition in protein synthesis, a consequence of both enhanced transcription and prolonged mRNA half life. It was to this phenomenon that NaCl-inducible Egr-1 mRNA expression was attributed. In addition to NaCl, the other principal renal medullary solute, urea, was examined. Unlike NaCl, urea is believed to be readily membrane-permeant and fails to engender a marked change in cell volume. Nonetheless, urea is a potent competitive and noncompetitive inhibitor of enzyme function and can be present in the molar range in the mammalian renal medulla as a consequence of the renal concentrating mechanism. In marked contrast to NaCl, hyperosmotic urea upregulated Egr-1 and c-fos mRNA expression with the same rapid kinetics and did so without globally affecting protein synthesis (12). The half-maximal effect on mRNA expression occurred at approximately 30–50 mM urea with a maximal effect at 200 mM urea—concentrations in mammals that are unique to the renal medulla. Interestingly, urea-inducible Egr-1 mRNA expression was observed in three cell lines of renal epithelial origin: MDCK, LLC-PK1 and mIMCD3 cells (a clonal transformed cell line isolated from terminal inner medullary collecting ducts of mice transgenic for the SV-40 large T antigen [53]). The phenomenon was absent, however, from renal nonepithelial cells (e.g., glomerular mesangial cells) and from epithelial nonrenal cells (e.g., T84 human colon carcinoma cells), as well as other cell types such as C6 glioma cells and bovine aortic endothelial cells (12). Hyperosmotic Urea and Egr-1 Transcription Urea-inducible Egr-1 expression was pursued as a model of renal epithelial cell-specific urea-inducible gene expression. Using the mIMCD3 cell line, it was determined that urea increased Egr-1 mRNA abundance through enhanced transcription. Specifically, urea had no effect on mRNA stability by actinomycin D chase experiments (12), whereas it increased transcription as determined by nuclear run-off analysis (9). Importantly, urea also increased Egr-1 immunoreactivity by Western analysis, as well as de novo Egr-1 protein synthesis, as demonstrated by Egr-1 antiserum immunoprecipitation of lysates prepared from cells metabolically labeled with 35 S-methionine. In addition, this increase in Egr-1 protein was associated with enhanced binding to a radiolabeled oligonucleotide consisting of the Egr-1 consensus DNA binding site (9). Furthermore, this enhanced DNA binding resulted in increased trans-activation by the Egr-1 protein product, as demonstrated by transient transfection with a luciferase reporter gene driven by tandem

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repeats of the Egr-1 DNA binding site (10). Together these data indicated that hyperosmotic urea increased transcription of the Egr-1 gene and that this increased transcription was accompanied by increased synthesis of and trans-activation by the Egr-1 protein. The mechanism underlying urea-inducible Egr-1 transcription was further examined. The Egr-1 genomic 5′ flanking sequence, cloned by multiple investigators (5,7, 32,69), contains numerous putative regulatory elements. These include multiple SREs, two AP-1 sites (potentially bound by fos :jun heterodimers), as well as Sp1 and CRE sites. Recent evidence has shown that Egr-1 transcription by urea is mediated through the multiple SREs and adjacent TCF-interaction sites called Ets motifs (11). Both models of transcription factor regulation discussed above are operative in urea-inducible stress: urea-induced post-translational modification of the transcription factor TCF followed by TCF-induced transcription (and ultimately translation) of the Egr-1 gene. The identity of ‘‘downstream’’ targets of urea-inducible transcription factors remains to be established. Candidate effector genes include numerous growth factors and growth factor receptors, as well as diverse other genes known to contain the Egr-1 binding site within their regulatory sequences. The role that members of the MAPK family play in these and other hyperosmotic stress-inducible signaling events remains unclear. The potential involvement of TCF, alluded to above, implicates members of the ERK family—known activators of the TCFs. In addition to c-fos and Egr-1, it remains likely that previously undescribed transcription factors will play a role in the renal epithelial cell response to hyperosmotic stress. Recent elegant work has demonstrated the presence of novel putative consensus elements in the promoters of several hyperosmotically responsive genes (20,67). These elements confer solute inducibility when cloned upstream of either minimal or heterologous promoters and appear to interact in a sequence-specific fashion with DNA binding activities present in nuclear extracts prepared from hyperosmotically stressed renal epithelial cells. Exciting developments such as these bode well for continued rapid progress in elucidating the molecular mechanisms of transcriptional regulation and kinase signaling in the renal cellular response to hyperosmotic stress.

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