Hypertonic induction of COX-2 expression in renal medullary epithelial cells requires transactivation of the EGFR

Hongyu Zhao, Wei Tian, Cynthia Tai, and David M. Cohen

Division of Nephrology and Hypertension, Oregon Health and Science University and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201

Submitted 21 January 2003 ; accepted in final form 30 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypertonic stress increases expression of cyclooxygenase-2 (COX-2) in renal medullary epithelial and interstitial cells. Because hypertonic COX-2 expression is, in part, sensitive to inhibition of the ERK MAPK, an effector of activated receptor tyrosine kinases such as the EGF receptor, we investigated a role for this receptor in signaling to COX-2 expression. Hypertonic stress increased COX-2 expression at the mRNA and protein levels at 6 and 24 h of hypertonic treatment. Two potent, specific inhibitors of the EGF receptor kinase, AG-1478 and PD-153035, abrogated this effect. These inhibitors also blocked the ability of hypertonic stress to increase PGE2 release; in addition, they partially blocked tonicity-dependent phosphorylation of ERK but not of the related MAPKs, JNK or p38. Pharmacological inhibition of ERK activation partially blocked tonicity-dependent COX-2 expression. Hypertonic induction of COX-2 was likely transcriptionally mediated, as NaCl stress increased luciferase reporter gene activity under control of the human COX-2 promoter, and this effect was also sensitive to inhibition of the EGF receptor kinase. Metalloproteinase action is required for transactivation of the EGF receptor. Pharmacological inhibition of metalloproteinase function blocked tonicity-inducible COX-2 expression. Furthermore, the effect of hypertonicity on COX-2 expression was also evident in the EGF-responsive Madin-Darby canine kidney and 3T3 cell lines but was virtually absent from the EGF-unresponsive (and EGF receptor null) Chinese hamster-derived CHO cell line. Taken together, these data indicate that hypertonicity-dependent COX-2 expression in medullary epithelial cells requires transactivation of the EGF receptor and, potentially, ectodomain cleavage of an EGF receptor ligand.

hypertonicity; heparin-binding epidermal growth factor; kidney; cylooxygenase


HYPERTONICITY IS A fundamental, phylogenetically ubiquitous environmental stressor. In mammals, few tissues are exposed to wide ranges of ambient tonicity; one exception is the renal medulla where osmolality may exceed 1 osmol/kgH2O. A number of genes have been described whose expression is upregulated by hypertonic stress in renal medullary cells both in vitro and in vivo. Most are regulated by activation or synthesis of the tonicity-responsive transcription factor, TonEBP/NFAT5, and its subsequent interaction with its cognate cis-acting element, the tonicity enhancer element/osmotic response element (TonE/ORE; reviewed in Ref. 18).

Cyclooxygenases (COX) are oxidoreductases that catalyze the conversion of membrane arachidonic acid to PGH2, a precursor of all prostaglandins, thromboxanes, and prostacyclins (39). There are at least two [and perhaps more (6)] isoforms of COX; COX-1 is constitutively and nearly ubiquitously expressed, whereas the inducible COX-2 isoform is primarily expressed in kidney and brain. COX-2, as a key mediator of inflammation, may be upregulated by a variety of cell activators, including mitogens, hormones, and environmental stressors (3, 21). Although ambient tonicity has long been known to influence prostaglandin synthesis in the renal medulla (11, 12) and gastrointestinal tract (2, 26), the ability of hypertonicity to increase expression of COX-2 was first noted in liver macrophages (51). In the kidney medulla, upregulated COX-2 (but not COX-1) expression was described in rodent models of chronic salt loading and water restriction (48, 49); in vitro, experimental hypertonic stress correspondingly increased COX-2 but not COX-1 expression in a collecting duct cell line (47, 48).

The signaling mechanism through which hypertonicity increases COX-2 expression is of great interest. Yang et al. (47) observed MAPK dependence of this phenomenon in cultured cells derived from the inner medullary collecting duct; specifically, p38, ERK, and JNK axes were all implicated, either pharmacologically or through a dominant negative approach. In renal medullary interstitial cell and cortical thick ascending limb models (perhaps most consistent with the in vivo pattern of renal COX-2 expression; see Ref. 20), tonicity also regulated COX-2 expression but did so in an NF-{kappa}B-dependent fashion (7, 19).

In an unbiased screen, we identified COX-2 as one of the genes most prominently upregulated by hypertonicity in the renal medullary mIMCD3 cell model (41). Because other tonicity-inducible phenomena are potentially dependent on transactivation of the EGF receptor (EGFR) kinase (e.g., Refs. 24, 35, and 38) and because at least partial dependence on the EGFR kinase effector, ERK, had previously been reported for tonicity-inducible COX-2 expression in this epithelial cell model (47), we investigated the role of the EGFR kinase in tonicity-dependent COX-2 expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture conditions and reagents. mIMCD3 (34), 3T3, Madin-Darby canine kidney (MDCK), and Chinese hamster ovary (CHO) cells were maintained and passaged in DMEM-F-12 medium supplemented with 10% FBS, as previously described (8). Cells were treated after achieving confluence, and supplemental solute was applied at ~200 mosM (200 mM urea vs. 100 mM NaCl). All reagents were purchased from Sigma unless otherwise specified. The following pharmacological inhibitors were used: AG-1295 (100 nM-1 µM; Calbiochem); AG-1478 (100 nM; Calbiochem); PD-153035 (100 nM); N-{DK-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl}-L-3-(2'-naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI; 3–10 µM); doxycycline (100 µM); PD-98059 (50 µM); and U-0126 (10 µM). Inhibitors were applied 30 min before solute or ligand treatment, unless otherwise indicated; all inhibitors were present for the duration of the solute or ligand treatment interval (additional 5 min-16 h, depending on assay).

Biochemical and molecular biological assays. Immunoblot analysis was performed as previously described (8, 50) using anti-COX-2 primary antibody (no. 160106; Cayman Chemical) at 1:1,000 and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Pierce) at 1:4,000 dilution. Anti-phosphorylated (P)-ERK (recognizing phosphorylated Thr202/Tyr204 of ERK1/2), anti-P-JNK (recognizing phosphorylated Thr183/Tyr185 of JNK1/2), anti-P-p38 (recognizing phosphorylated Thr180/Tyr182 of p38), and anti-P-EGFR (recognizing phosphorylated Tyr1068) were purchased from Cell Signaling Technologies and used in accordance with the manufacturer's directions; 60 kDa anti-heat shock protein (HSP60) was from Santa Cruz Biotechnologies. Anti-phosphokinase immunoblots were controlled with subsequent or parallel immunoblotting with the corresponding anti-kinase antibody. In no instance did kinase expression change during the brief intervals (0–30 min) over which kinase-dependent events were explored. Stable transfection and luciferase reporter gene analyses were performed as previously described (9) using sequences from –724 to +7 of the murine COX-2 promoter subcloned into pXP2 (43). Briefly, mIMCD3 cells were transfected with the indicated plasmid via lipofection and subjected to selection pressure with G418. A pool of clones was generated in this fashion and used in aggregate over several passages with equivalent results in each passage. As the plasmid was integrated, there was no normalization for transfection efficiency. In transient transfection experiments performed in parallel, the degree of induction by hypertonicity was less pronounced (data not shown), consistent with the nature of the COX-2 minimal promoter (5). Induction was more apparent after normalization for cotransfected {beta}-galactosidase reporter gene; however, normalization of studies of this sort via cotransfection is problematic because of a general transcriptional inhibitory effect of hypertonicity (e.g., see Ref. 41). Still other "housekeeping" genes, such as actin, are immediate-early genes and are susceptible to the nonphysiological superinduction (15) that accompanies a tonicity-dependent decrement in protein synthesis (Ref. 10 and references therein). Depicted data are means ± SE of at least three separate experiments (see legends for Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9), with the exception of immunoblot data wherein a representative figure is shown. Statistical significance was ascribed to P < 0.05 via t-test (VassarStats; http://vassun.vassar.edu/~lowry/VassarStats.html). For RNase protection assay to detect COX-2 mRNA, murine kidney poly(A)+ mRNA was subjected to RT-PCR using primers COX-1411 (5'-TGTACAAGCAGTGGCAAAGG-3') and COX-1726 (5'-GCTCGGCTTCCAGTATTGAG-3') to generate a 316-bp partial cDNA, which was T/A-subcloned into pCR4. The resultant plasmid was confirmed by sequencing (Vollum Institute for Advanced Biomedical Research, Oregon Health and Science University); clone CT005 exhibited identity with murine COX-2 in BLAST analysis (http://www.ncbi.nlm.nih-.gov/BLAST/). This plasmid was linearized with NotI, and transcribed with T3 RNA polymerase to generate a specific antisense COX-2 riboprobe. ELISA for PGE2 production (R&D Systems) was performed according to the manufacturer's directions, using 10-fold dilutions of cell-conditioned medium.



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Fig. 1. Hypertonic NaCl increases cyclooxygenase (COX)-2 mRNA expression. Effect of the indicated concentration of supplemental NaCl on COX-2 mRNA abundance at 6 and 24 h of treatment, as assessed via RNase protection assay in renal medullary mIMCD3 cells. Open arrowhead, COX-2-protected antisense riboprobe; filled arrowhead, actin control; P, undigested probe-only lane. Figure is representative of 2–3 experiments (depending on condition).

 


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Fig. 2. Pharmacological inhibitors of the EGF receptor (EGFR) kinase block tonicity-dependent COX-2 expression at the mRNA and protein levels. Effect of control treatment (C) or supplemental NaCl (N; 100 mM x 6 h) on COX-2 mRNA (A) or protein (B) abundance in the presence or absence of pretreatment (100 nM x 30 min) with the EGFR kinase inhibitors PD-153035 (+PD) or AG-1478 (+AG), as assessed via RNase protection assay (A) or anti-COX-2 immunoblot (B) in renal medullary mIMCD3 cells. Open arrowhead, COX-2-protected antisense riboprobe (A) or immunoreactive COX-2; filled arrowhead, actin control (A). P, undigested probe-only lane. C: effect of EGF (10 nM) or hypertonic stress (NaCl; 100 mM) for the indicated interval (in min) on tyrosine phosphorylation (on residue Y1068) of the EGFR, as assessed through anti-phosphorylated (P)-EGFR immunoblotting. In Figs. 2, 3, 4, 5, 6, 7, 8, 9, the molecular mass (in kDa) of ladder proteins is depicted on left. Data in A, B, and C are representative of 2–3, 3, and 2 such experiments, respectively (depending on condition).

 


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Fig. 3. Hypertonic stress increases reporter gene activity driven by the COX-2 promoter. Reporter gene assay of mIMCD3 cells stably transfected with a construct encoding the luciferase (Luc) reporter gene driven by 0.7 kb of the human COX-2 promoter, and then subjected to the indicated solute stress for the indicated interval, in the presence or absence of the EGFR kinase inhibitor PD-153035. Data are means ± SE of 3–4 separate experiments (depending on condition), with determinations performed in triplicate for each experiment. Data for each experiment were normalized to control treatment in the absence of PD-153035. Effect of PD-153035 was not tested under conditions represented by urea (200 mM) and NaCl (50 and 150 mM). P < 0.05 with respect to control (*) and with respect to the same treatment in the absence of PD-153035 ({dagger}).

 


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Fig. 4. Inhibition of the EGFR kinase blocks tonicity-dependent PGE2 generation. Effect of supplemental NaCl (100 mM x 6 or 16 h) on PGE2 release in cell culture medium in the presence or absence of pretreatment (100 nM x 30 min) with PD-153035, as quantitated via anti-PGE2 RIA in renal medullary mIMCD3 cells. Data are means ± SE of 3 separate experiments, with determinations performed in triplicate for each experiment. P < 0.05 with respect to control (*) and with respect to the same treatment in the absence of PD-153035 ({dagger}).

 


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Fig. 5. Inhibition of the EGFR kinase blocks tonicity-dependent phosphorylation of ERK, but not JNK or p38. Effect of supplemental NaCl (100 mM x 10, 30, or 360 min) on phosphorylation of ERK, JNK, or p38 in the presence or absence of pretreatment (100 nM x 30 min) with PD-153035, as quantitated via anti-P-MAPK immunoblotting; open arrowheads, migration of relevant MAPK; molecular mass markers are indicated on left. JNK migrated as two bands at ~46 and 54 kDa, consistent with published reports. The band migrating at 42 kDa on the anti-P-JNK most likely represented nonspecific binding to activated ERK. Figure is representative of 2–3 experiments (depending on condition).

 


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Fig. 6. Hypertonicity-inducible COX-2 expression is sensitive to inhibition of MEK1/2. Anti-COX-2 immunoblot depicting effect of control treatment (C) or treatment with the indicated solutes (N, 100 mM NaCl; M, 200 mM mannitol; U, 200 mM urea) for 6 h and in the presence of the MEK1/2 inhibitors PD-98059 (50 µM) and U-0126 (10 µM). Figure is representative of 2 experiments.

 


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Fig. 7. Hypertonicity-inducible COX-2 expression is sensitive to inhibition of metalloproteinases. Anti-COX-2 immunoblot depicting effect of control treatment (C) or hypertonic NaCl [100 mM NaCl (N)] for 6 h and in the presence of the metalloproteinase inhibitors N-{DL-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl}-L-3-(2'-naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI; 3 and 10 µM) and doxycycline (100 µM; Doxy). Because DMSO may influence COX-2 expression, the effect of DMSO vehicle (Veh) alone, in percentages (vol/vol) corresponding to diluent requirements for TAPI (0.07% for TAPI at 3 µM and 0.2% for TAPI at 10 µM), is also depicted. Figure is representative of 2–3 experiments (depending on condition).

 


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Fig. 8. Hypertonicity-dependent COX-2 regulation is HSP60 independent. Effect of control treatment or hypertonic stress (100 mM NaCl for the indicated interval) on HSP60 abundance, as measured by anti-HSP60 immunoblotting of mIMCD3 whole cell detergent lysates. Figure is representative of 2 experiments.

 


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Fig. 9. Tonicity-dependent COX-2 upregulation is absent from an EGFR kinase-null cell line. A: effect of EGF treatment (10 nM x 5 or 30 min) on ERK phosphorylation in the EGFR-positive cell lines 3T3 and MDCK and in the EGFR-null Chinese hamster ovary (CHO) cell line. B: effect of hypertonic stress (100 mM NaCl x 6 h; N) and urea stress (200 mM x 6 h; U) on COX-2 expression in the EGFR-positive cell lines 3T3 and MDCK and in the EGFR-null CHO cell line. Figure is representative of 2–3 experiments (depending on condition).

 


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Consistent with the data of others, hypertonic stress (50–150 mM NaCl) increased COX-2 expression at the mRNA level at 6 and 24 h of treatment, as assessed via RNase protection assay (Fig 1). There was no effect on control (actin) mRNA expression in response to these stimuli.

Because we have observed other potentially tonicity-dependent signaling events to be mediated via transactivation of the EGFR, we determined the effect of pharmacological inhibition of the EGFR on tonicity-dependent COX-2 expression. Two highly specific and potent EGFR kinase inhibitors, PD-153035 and AG-1478, both dramatically inhibited tonicity-dependent COX-2 mRNA expression (Fig. 2A).

Because upregulation at the mRNA level in response to hypertonic stress is frequently nonspecific, a consequence of so-called "superinduction" in the setting of the inhibition in protein synthesis that often accompanies hypertonic stress (Ref. 10 and references therein), we sought to determine the effect on COX-2 expression at the protein level. Again, consistent with the data of others, hypertonic stress dramatically increased COX-2 expression at the protein level (Fig. 2B), suggesting physiological relevance. Because the degree of cell confluence may influence signaling events, experiments were performed in parallel using subconfluent cells with comparable results (data not shown). As in the mRNA studies, both of the EGFR inhibitors markedly decreased the tonicity-dependent COX-2 upregulation (Fig. 2B).

We sought to confirm that the degree of hypertonicity used in this model was sufficient to activate the EGFR kinase. Hypertonic NaCl (100 mM) induced tyrosine phosphorylation of this receptor (on residue Tyr1068) to a degree comparable to that induced by the bona fide EGFR ligand EGF (Fig. 2C).

To address the upregulation of COX-2 expression in a more mechanistic fashion, we examined the effect of hypertonicity on reporter gene activity of an expression plasmid encoding luciferase and under control of the proximal ~0.7 kb (–724 to +7, relative to the transcriptional start site) of the human COX-2 promoter (43). When stably transfected in mIMCD3 cells, the reporter gene exhibited approximately fivefold greater activity after 6 h of hypertonic stress (100 mM NaCl; Fig 3). This effect was also dose dependent within the range of 50–150 mM NaCl. The effect of urea was much more modest (27% increase) but achieved statistical significance. The tonicity-dependent upregulation was partially blocked when cells were pretreated with the EGFR kinase inhibitor PD-153035 (Fig. 3). The effect of the impermeant solute mannitol was equivalent to that of NaCl in this assay, as was its sensitivity to PD-153035 (data not shown). Because the most widely recognized tonicity-responsive enhancer element is TonE, we sought this element in the COX-2 promoter. Using the reported canonical sequence for TonE (YGGAANNNYNY; see Ref. 31), we were unable to identify this cis-acting element in the proximal 0.7 kb of the COX-2 5'-flanking sequence (data not shown).

We assessed PGE2 generation as an index of COX-2 function. Hypertonic NaCl increased PGE2 release in cell supernatants in a time-dependent fashion, and this effect was blocked by EGFR kinase inhibition (Fig. 4). Of note, the large error bar in the 16-h treatment in the absence of PD-153035 is a consequence of the variability in maximal upregulation by hypertonicity (i.e., 3-fold vs. 20-fold) in these unnormalized data; in all experiments, the ordinal relationship was identical.

ERK and JNK have been implicated in COX-2 regulation, particularly in medullary epithelial cells (47) where they may function downstream of EGFR in a signaling cascade. We therefore assessed the effect of EGFR kinase inhibition on activation of all three of the principal MAPK families by hypertonic stress. As anticipated and as previously shown in this model, hypertonicity increased ERK activation as assessed via anti-P-ERK immunoblotting (Fig. 5). Consistent with our earlier observation (52), EGFR kinase inhibition markedly blunted the effect of tonicity on ERK activation. In contrast, there was virtually no effect of EGFR kinase inhibition on tonicity-dependent JNK or p38 phosphorylation.

Because ERK appeared to be a principal effector of EGFR in this and other contexts, we tested the effect of pharmacological inhibition of ERK activation on tonicity-inducible COX-2 expression. We used two different mitogen/extracellular signal-regulated kinase (MEK) inhibitors (and inhibitors of ERK activation), PD-98059 and U-0126, in part to permit discrimination between the anticipated ERK1/2-dependent phenomenon and a potential ERK5-dependent phenomenon (23) recently reported in another model (17). Both MEK inhibitors substantially blocked the effect of NaCl on this end point, consistent with a role for MEK (and hence ERK) activation in tonicity-responsive COX-2 expression. Of note, the inhibitors were equally effective when applied at 50 µM for PD-98059 and 10 µM for U-0126, strongly suggesting that ERK5 was not playing a major role (23). Consistent with our observations with tonicity-dependent COX-2 transcription, the effect of hypertonic mannitol vis-à-vis COX-2 expression was equivalent to that of equiosmolar NaCl, and this effect was equally sensitive to MEK inhibition (Fig. 6).

EGFR activation generally occurs via metalloproteinase-dependent cleavage of an EGF-like ectodomain that then acts in an autocrine or juxtacrine fashion (reviewed in Ref. 16). To test this possibility in preliminary fashion in the present context, we investigated the ability of two metalloproteinase inhibitors to abrogate the effect of tonicity on COX-2 expression. The metalloproteinase inhibitor TAPI, which blocks ectodomain cleavage of the EGFR ligand, heparin-binding (HB)-EGF (28), inhibited tonicity-dependent COX-2 expression in a dose-dependent fashion, whereas vehicle exerted no effect (Fig. 7). The nonspecific metalloproteinase inhibitor doxycycline (e.g., Refs. 14 and 40) also blocked the effect, albeit to a lesser extent (consistent with its reported efficacy). We were unable to use the specific hydroxamate-based metalloproteinase inhibitors (e.g., ilomastat) because the vehicle concentration required by these relatively insoluble compounds (i.e., DMSO at 0.5–2%) was sufficient to interfere with COX-2 expression (data not shown). In support of our alternative approach, a recent report documented functional equivalence between TAPI and a hydroxamate-based inhibitor in at least one context (42).

HSP60 expression induces COX-2 expression (4), and hypertonic stress has been associated with increased expression of HSPs in renal epithelial cells (10). We investigated the possibility that HSP60 may mediate the effect of hypertonicity on COX-2 expression. Abundant HSP60 expression was detected in mIMCD3 cells (Fig. 8); however, this level was unaffected by hypertonic stress or by the presence of EGFR kinase inhibitors.

Last, we sought an EGFR-null model in which to test for the presence of tonicity-dependent signaling. Although the EGFR is nearly ubiquitous among cultured cell lines, CHO cells reportedly lack this tyrosine kinase (29). We compared CHO cells with two additional cell lines known to express the EGFR: 3T3 cells and the renal epithelial MDCK cell line. We first assessed the ability of EGF to activate ERK in each cell line. As anticipated, EGF induced a marked increase in ERK phosphorylation in both the 3T3 and MDCK cells at 5 and 30 min of treatment but produced no effect in the CHO cells (Fig. 9A). This confirmed the EGF nonresponsiveness of the CHO line. With this validated model in hand, we next assessed the ability of hypertonic stress to upregulate COX-2 expression in each cell line. Similar to the effect in mIMCD3 cells, hypertonic stress dramatically increased COX-2 expression in both 3T3 and MDCK cells (Fig. 9B); in marked contrast, however, there was essentially no effect in the EGFR kinase-null CHO cells. For comparison, we also determined the effect of the medullary solute, urea, on COX-2 expression in each of these models. Like NaCl, urea produced no effect in CHO cells. The urea effect was also much less prominent than that of hypertonic NaCl in both 3T3 and MDCK cells. These data were consistent with the relatively modest effect of urea vis-à-vis the COX-2 promoter (Fig. 3). Of note, introduction of an expression vector encoding EGFR in CHO cells failed to recapitulate tonicity-inducible COX-2 expression. We infer that EGFR expression is necessary but not sufficient in this context; CHO cells may lack either the EGFR ligand (i.e., HB-EGF) or the relevant ligand-directed metalloproteinase, both of which are required for an intact signaling module.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We extend the observations of Yang et al. (47), who reported MAPK dependence of renal medullary epithelial cell COX-2 expression in response to hypertonic stress. We found that two potent and highly specific inhibitors of the EGFR kinase blocked tonicity-dependent upregulation of COX-2 mRNA expression, protein expression, transcription, and PGE2 synthesis. This signaling event was independent of HSP60 expression, which induces COX-2 in at least one model (4). The inhibitors of EGFR kinase partially blocked tonicity-dependent ERK activation, as would be expected from the data of Yang et al. (47), but failed to significantly influence JNK or p38 phosphorylation. Pharmacological inhibition of metalloproteinase action similarly blocked COX-2 expression. Furthermore, the EGF-unresponsive (and EGFR null; see Ref. 29) CHO cell line failed to exhibit the tonicity-dependent COX-2 expression evident in the EGF-responsive 3T3, MDCK, and mIMCD3 cell lines. In aggregate, these data strongly suggest that the EGFR kinase mediates the effect of hypertonicity on COX-2 expression in the renal medullary cell model and that ectodomain cleavage of an EGFR ligand may be required.

Although medullary COX-2 expression and action likely play a pivotal role in renal physiology, particularly in response to dehydration, much of this effect has been attributed to actions of the medullary interstitial cells and adjacent cells of the macula densa and cortical thick ascending limb (reviewed in Ref. 20). Nonetheless, recent reports have emphasized expression and regulation of COX-2 in other renal epithelial cells in vitro (13, 47, 48), particularly in the context of hypertonic stress (47, 48).

It is of interest that Yang et al. (47) did not detect an effect of the tyrosine kinase inhibitors, tyrphostin-A23 (AG-18) and tyrphostin-A51 (AG-183), on tonicity-dependent COX-2 expression, although they did note an inhibitory effect of the general tyrosine kinase inhibitor genistein (47). We, in contrast, found a profound effect in the mIMCD3 cell line at the mRNA and protein level and at the level of PGE2 production. The study of Yang et al. (47) was performed using the mIMCD-K2 cell line (25), whereas the present study employed the mIMCD3 line (34). Tyrphostin-A23 and tyrphostin-A51 exhibit IC50 for EGFR kinase of 40 µM and ~800 nM, respectively, and were applied at 10 µM (47); it is conceivable that insufficient cellular levels were achieved, particularly for tyrphostin-A23. While the present studies were being completed, Guo et al. (17) reported the EGFR dependence of COX-2 expression in a rat intestinal epithelial cell model in response to the peptide hormone gastrin, in further support of this mode of regulation.

Importantly, these are not the first data implicating a role for the EGFR in transactivating tonicity-dependent signaling. Rosette and Karin (35) reported aggregation of, and signaling by, the EGFR using extreme hypertonic stress in a cultured cell line, although specific genetic targets of this process were not examined. In contrast, King et al. (24) reported activation of the EGFR by osmotic shock in the absence of receptor dimerization. Sheikh-Hamad and co-workers (38) described tonicity-dependent association of the following three proteins: the osmotically inducible CD9 (36), {beta}1-integrin (37), and the EGFR ligand HB-EGF. A related phenomenon was observed recently in normal human renal tissue (32). Moreover, expression of HB-EGF appears to be itself osmotically regulated (1, 27). Interestingly, HB-EGF and CD9 also interact with A disintegrin and metalloprotease domain 10 (also known as Kuzbanian), and this metalloproteinase may mediate the ectodomain shedding and autocrine action of EGFR ligands such as HB-EGF (46). This ternary association is enhanced by G protein-coupled receptor activation (46), a phenomenon that has not been observed in the context of hypertonic stress.

The EGFR ligand conferring transactivation in the present context is unknown. Potential ligands, including EGF, amphiregulin, epiregulin, transforming growth factor-{alpha}, and HB-EGF, all exist as precursors with a single membrane-spanning domain and exhibit autocrine or juxtacrine action after cleavage by one or more metalloproteinases (reviewed in Ref. 33). Our data using the metalloproteinase inhibitors TAPI and doxycycline support a role for this mechanism in tonicity-dependent COX-2 regulation, but they do not permit discrimination among potential EGFR agonists. Neutralizing antibodies specific for EGFR and EGFR ligands have seen widespread use in other models, but none are effective in murine systems; we have thus far been unable to identify a human renal epithelial cell line suitable for studying this phenomenon.

In our studies, the proximal ~0.7 kb of the murine COX-2 promoter was sufficient to confer tonicity responsiveness to a heterologous reporter gene. Although lacking a canonical tonicity-responsive TonE element, this gene region contains several cis-acting elements validated in other contexts. For example, a cAMP-response element (CRE) is instrumental in the COX-2 response to endotoxin, as are tandem CCAAT/enhancer-binding protein sites (22, 30, 43); the CRE site, however, is also responsive to platelet-derived growth factor (44), in a JNK-dependent fashion. An NF-{kappa}B binding site was implicated in the COX-2 response to tumor necrosis factor-{alpha} (45). Although most of these data were acquired in heterologous expression systems, studies have emerged using more native models. For example, both in cultured medullary interstitial cells exposed to hypertonicity (19) and in cultured thick ascending limb cells exposed to low-salt or low-chloride conditions (7), the NF-{kappa}B site mediated COX-2 transcriptional induction. Our data clearly support a role for EGFR kinase signaling in tonicity-dependent upregulation of COX-2 expression in the well-studied mIMCD3 model; whether this effect requires the CRE [as might be suggested by earlier data with another agonist of a receptor tyrosine kinase, PDGF (44)], the NF-{kappa}B element [consistent with the interstitial cell and thick ascending limb models (19)], or still another element, it clearly occurs in a TonE-independent fashion. Consistent with these data, EGFR kinase inhibition failed to influence TonE- and TonEBP-dependent transcription (52).

In summary, we have shown that the well-described ERK-dependent upregulation in COX-2 expression accompanying hypertonic stress in medullary epithelial cells is mediated via transactivation of the EGFR. The EGFR agonist mediating this effect and the additional upstream activating events remain obscure.


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52494, the American Heart Association, and the Department of Veterans Affairs.


    ACKNOWLEDGMENTS
 
We thank Dr. H. Herschman for the kind gift of the COX-2 promoter in pXP2.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. Cohen, Mailcode PP262, Oregon Health & Science Univ., 3314 S. W. US Veterans Hospital Rd., Portland, OR 97201 (E-mail: cohend{at}ohsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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