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发表于 2009-4-21 13:42 |只看该作者 |倒序浏览 |打印

作者：Hongyu Zhao, Wei Tian, Cynthia Tai, and David M. Cohen作者单位：Division of Nephrology and Hypertension, Oregon Health and ScienceUniversity and the Portland Veterans Affairs Medical Center, Portland, Oregon97201
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      【摘要】1 E/ O  ^# `6 c: r; `$ Y
   Hypertonic stress increases expression of cyclooxygenase-2 (COX-2) in renalmedullary epithelial and interstitial cells. Because hypertonic COX-2expression is, in part, sensitive to inhibition of the ERK MAPK, an effectorof activated receptor tyrosine kinases such as the EGF receptor, weinvestigated a role for this receptor in signaling to COX-2 expression.Hypertonic stress increased COX-2 expression at the mRNA and protein levels at6 and 24 h of hypertonic treatment. Two potent, specific inhibitors of the EGFreceptor kinase, AG-1478 and PD-153035, abrogated this effect. Theseinhibitors also blocked the ability of hypertonic stress to increasePGE 2 release; in addition, they partially blockedtonicity-dependent phosphorylation of ERK but not of the related MAPKs, JNK orp38. Pharmacological inhibition of ERK activation partially blockedtonicity-dependent COX-2 expression. Hypertonic induction of COX-2 was likely transcriptionally mediated, as NaCl stress increased luciferase reporter geneactivity under control of the human COX-2 promoter, and this effect was alsosensitive to inhibition of the EGF receptor kinase. Metalloproteinase actionis required for transactivation of the EGF receptor. Pharmacologicalinhibition of metalloproteinase function blocked tonicity-inducible COX-2expression. Furthermore, the effect of hypertonicity on COX-2 expression wasalso evident in the EGF-responsive Madin-Darby canine kidney and 3T3 cell lines but was virtually absent from the EGF-unresponsive (and EGF receptornull) Chinese hamster-derived CHO cell line. Taken together, these dataindicate that hypertonicity-dependent COX-2 expression in medullary epithelialcells requires transactivation of the EGF receptor and, potentially,ectodomain cleavage of an EGF receptor ligand. * e1 U! D0 `7 K
      【关键词】 hypertonicity heparinbinding epidermal growth factor kidney cylooxygenase: d. N7 j* @2 \' o+ G! B( u
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HYPERTONICITY IS A fundamental, phylogenetically ubiquitous environmental stressor. In mammals, few tissues are exposed to wide ranges ofambient tonicity; one exception is the renal medulla where osmolality mayexceed 1 osmol/kgH 2 O. A number of genes have been described whoseexpression is upregulated by hypertonic stress in renal medullary cells bothin vitro and in vivo. Most are regulated by activation or synthesis of thetonicity-responsive transcription factor, TonEBP/NFAT5, and its subsequentinteraction with its cognate cis -acting element, the tonicityenhancer element/osmotic response element (TonE/ORE; reviewed in Ref. 18 ).: O, n; {& H* {+ O; o

Cyclooxygenases (COX) are oxidoreductases that catalyze the conversion ofmembrane arachidonic acid to PGH 2, a precursor of allprostaglandins, thromboxanes, and prostacyclins( 39 ). There are at least two[and perhaps more ( 6 )] isoformsof COX; COX-1 is constitutively and nearly ubiquitously expressed, whereas theinducible COX-2 isoform is primarily expressed in kidney and brain. COX-2, asa key mediator of inflammation, may be upregulated by a variety of cellactivators, including mitogens, hormones, and environmental stressors( 3, 21 ). Although ambient tonicityhas long been known to influence prostaglandin synthesis in the renal medulla( 11, 12 ) and gastrointestinal tract( 2, 26 ), the ability ofhypertonicity to increase expression of COX-2 was first noted in livermacrophages ( 51 ). In the kidney medulla, upregulated COX-2 (but not COX-1) expression was described inrodent models of chronic salt loading and water restriction( 48, 49 ); in vitro, experimentalhypertonic stress correspondingly increased COX-2 but not COX-1 expression ina collecting duct cell line( 47, 48 ).  l! ~( S' t( g$ B3 P3 {9 d

The signaling mechanism through which hypertonicity increases COX-2expression is of great interest. Yang et al.( 47 ) observed MAPK dependenceof this phenomenon in cultured cells derived from the inner medullarycollecting duct; specifically, p38, ERK, and JNK axes were all implicated,either pharmacologically or through a dominant negative approach. In renalmedullary 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 regulatedCOX-2 expression but did so in an NF- B-dependent fashion( 7, 19 ).8 q9 h. ?0 X" ?- e
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In an unbiased screen, we identified COX-2 as one of the genes mostprominently upregulated by hypertonicity in the renal medullary mIMCD3 cellmodel ( 41 ). Because othertonicity-inducible phenomena are potentially dependent on transactivation ofthe EGF receptor (EGFR) kinase (e.g., Refs. 24, 35, and 38 ) and because at leastpartial dependence on the EGFR kinase effector, ERK, had previously beenreported for tonicity-inducible COX-2 expression in this epithelial cell model( 47 ), we investigated the roleof the EGFR kinase in tonicity-dependent COX-2 expression.

MATERIALS AND METHODS# X5 h7 X' Z) Q; v$ H; \2 y

Cell culture conditions and reagents. mIMCD3( 34 ), 3T3, Madin-Darby caninekidney (MDCK), and Chinese hamster ovary (CHO) cells were maintained andpassaged in DMEM-F-12 medium supplemented with 10% FBS, as previouslydescribed ( 8 ). Cells weretreated after achieving confluence, and supplemental solute was applied at 200 mosM (200 mM urea vs. 100 mM NaCl). All reagents were purchased fromSigma unless otherwise specified. The following pharmacological inhibitorswere used: AG-1295 (100 nM-1 µM; Calbiochem); AG-1478 (100 nM; Calbiochem)

D-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 beforesolute or ligand treatment, unless otherwise indicated; all inhibitors werepresent 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 primaryantibody (no. 160106; Cayman Chemical) at 1:1,000 and goat anti-rabbithorseradish peroxidase-conjugated secondary antibody (Pierce) at 1:4,000dilution. Anti-phosphorylated (P)-ERK (recognizing phosphorylatedThr 202 /Tyr 204 of ERK1/2), anti-P-JNK (recognizingphosphorylated Thr 183 /Tyr 185 of JNK1/2), anti-P-p38(recognizing phosphorylated Thr 180 /Tyr 182 of p38), andanti-P-EGFR (recognizing phosphorylated Tyr 1068 ) were purchased from Cell Signaling Technologies and used in accordance with themanufacturer's directions; 60 kDa anti-heat shock protein (HSP60) was fromSanta Cruz Biotechnologies. Anti-phosphokinase immunoblots were controlledwith subsequent or parallel immunoblotting with the corresponding anti-kinaseantibody. In no instance did kinase expression change during the briefintervals (0-30 min) over which kinase-dependent events were explored.Stable transfection and luciferase reporter gene analyses were performed aspreviously described ( 9 ) usingsequences from -724 to  7 of the murine COX-2 promoter subcloned intopXP2 ( 43 ). Briefly, mIMCD3cells were transfected with the indicated plasmid via lipofection andsubjected to selection pressure with G418. A pool of clones was generated inthis fashion and used in aggregate over several passages with equivalentresults in each passage. As the plasmid was integrated, there was nonormalization for transfection efficiency. In transient transfectionexperiments performed in parallel, the degree of induction by hypertonicity was less pronounced (data not shown), consistent with the nature of the COX-2minimal promoter ( 5 ). Inductionwas more apparent after normalization for cotransfected -galactosidasereporter gene; however, normalization of studies of this sort viacotransfection 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 andare susceptible to the nonphysiological superinduction( 15 ) that accompanies atonicity-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 ofimmunoblot data wherein a representative figure is shown. Statisticalsignificance was ascribed to P
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Fig. 1. Hypertonic NaCl increases cyclooxygenase (COX)-2 mRNA expression. Effect ofthe indicated concentration of supplemental NaCl on COX-2 mRNA abundance at 6and 24 h of treatment, as assessed via RNase protection assay in renalmedullary mIMCD3 cells. Open arrowhead, COX-2-protected antisense riboprobe;filled arrowhead, actin control; P, undigested probe-only lane. Figure isrepresentative of 2-3 experiments (depending on condition).8 w' r2 {: M' Y- ^2 C" [( W

Fig. 2. Pharmacological inhibitors of the EGF receptor (EGFR) kinase blocktonicity-dependent COX-2 expression at the mRNA and protein levels. Effect ofcontrol treatment (C) or supplemental NaCl (N; 100 mM x 6 h) on COX-2mRNA ( A ) or protein ( B ) abundance in the presence or absenceof pretreatment (100 nM x 30 min) with the EGFR kinase inhibitorsPD-153035 ( PD) or AG-1478 ( AG), as assessed via RNase protection assay( A ) or anti-COX-2 immunoblot ( B ) in renal medullary mIMCD3cells. Open arrowhead, COX-2-protected antisense riboprobe ( A ) orimmunoreactive COX-2; filled arrowhead, actin control ( A ). P,undigested probe-only lane. C : effect of EGF (10 nM) or hypertonicstress (NaCl; 100 mM) for the indicated interval (in min) on tyrosinephosphorylation (on residue Y1068) of the EGFR, as assessed throughanti-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 suchexperiments, respectively (depending on condition).  q0 H% a: g8 |9 M
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Fig. 3. Hypertonic stress increases reporter gene activity driven by the COX-2promoter. Reporter gene assay of mIMCD3 cells stably transfected with aconstruct encoding the luciferase (Luc) reporter gene driven by 0.7 kb of thehuman COX-2 promoter, and then subjected to the indicated solute stress forthe indicated interval, in the presence or absence of the EGFR kinaseinhibitor PD-153035. Data are means ± SE of 3-4 separateexperiments (depending on condition), with determinations performed intriplicate for each experiment. Data for each experiment were normalized tocontrol treatment in the absence of PD-153035. Effect of PD-153035 was nottested under conditions represented by urea (200 mM) and NaCl (50 and 150 mM). P
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Fig. 4. Inhibition of the EGFR kinase blocks tonicity-dependent PGE 2 generation. Effect of supplemental NaCl (100 mM x 6 or 16 h) onPGE 2 release in cell culture medium in the presence or absence ofpretreatment (100 nM x 30 min) with PD-153035, as quantitated viaanti-PGE 2 RIA in renal medullary mIMCD3 cells. Data are means± SE of 3 separate experiments, with determinations performed intriplicate for each experiment. P

Fig. 5. Inhibition of the EGFR kinase blocks tonicity-dependent phosphorylation ofERK, 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 absenceof pretreatment (100 nM x 30 min) with PD-153035, as quantitated viaanti-P-MAPK immunoblotting; open arrowheads, migration of relevant MAPK;molecular mass markers are indicated on left. JNK migrated as twobands at 46 and 54 kDa, consistent with published reports. The bandmigrating at 42 kDa on the anti-P-JNK most likely represented nonspecificbinding to activated ERK. Figure is representative of 2-3 experiments(depending on condition).5 q7 [) x/ M1 J9 i! y. O# F

Fig. 6. Hypertonicity-inducible COX-2 expression is sensitive to inhibition ofMEK1/2. Anti-COX-2 immunoblot depicting effect of control treatment (C) ortreatment 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 (50µM) and U-0126 (10 µM). Figure is representative of 2 experiments.5 P! }) z* n7 @& s9 q7 r0 ~0 H( n' X
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Fig. 7. Hypertonicity-inducible COX-2 expression is sensitive to inhibition ofmetalloproteinases. Anti-COX-2 immunoblot depicting effect of controltreatment (C) or hypertonic NaCl [100 mM NaCl (N)] for 6 h and in the presenceof the metalloproteinase inhibitors N -{ DL -[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl}- L -3-(2'-naphthyl)-alanyl- L -alanine2-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 oncondition).

Fig. 8. Hypertonicity-dependent COX-2 regulation is HSP60 independent. Effect ofcontrol treatment or hypertonic stress (100 mM NaCl for the indicatedinterval) on HSP60 abundance, as measured by anti-HSP60 immunoblotting ofmIMCD3 whole cell detergent lysates. Figure is representative of 2experiments.

Fig. 9. Tonicity-dependent COX-2 upregulation is absent from an EGFR kinase-nullcell line. A : effect of EGF treatment (10 nM x 5 or 30 min) onERK phosphorylation in the EGFR-positive cell lines 3T3 and MDCK and in theEGFR-null Chinese hamster ovary (CHO) cell line. B : effect ofhypertonic 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 andin the EGFR-null CHO cell line. Figure is representative of 2-3experiments (depending on condition).
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RESULTS
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Consistent with the data of others, hypertonic stress (50-150 mMNaCl) increased COX-2 expression at the mRNA level at 6 and 24 h of treatment,as assessed via RNase protection assay ( Fig1 ). There was no effect on control (actin) mRNA expression inresponse to these stimuli.
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Because we have observed other potentially tonicity-dependent signalingevents to be mediated via transactivation of the EGFR, we determined theeffect of pharmacological inhibition of the EGFR on tonicity-dependent COX-2expression. Two highly specific and potent EGFR kinase inhibitors, PD-153035and AG-1478, both dramatically inhibited tonicity-dependent COX-2 mRNAexpression ( Fig.2 A ).# @7 I" W8 Q$ y0 v, r6 B. Q" K

Because upregulation at the mRNA level in response to hypertonic stress isfrequently 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), wesought to determine the effect on COX-2 expression at the protein level.Again, consistent with the data of others, hypertonic stress dramaticallyincreased COX-2 expression at the protein level( Fig. 2 B ), suggesting physiological relevance. Because the degree of cell confluence may influencesignaling events, experiments were performed in parallel using subconfluentcells with comparable results (data not shown). As in the mRNA studies, bothof the EGFR inhibitors markedly decreased the tonicity-dependent COX-2 upregulation ( Fig.2 B ).3 Z; ^6 u+ X1 X5 s
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We sought to confirm that the degree of hypertonicity used in this modelwas sufficient to activate the EGFR kinase. Hypertonic NaCl (100 mM) inducedtyrosine phosphorylation of this receptor (on residue Tyr 1068 ) to adegree comparable to that induced by the bona fide EGFR ligand EGF( Fig. 2 C ).& K; t6 u1 g: O" T: g
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To address the upregulation of COX-2 expression in a more mechanistic fashion, we examined the effect of hypertonicity on reporter gene activity ofan expression plasmid encoding luciferase and under control of the proximal 0.7 kb (-724 to  7, relative to the transcriptional start site) ofthe human COX-2 promoter ( 43 ).When stably transfected in mIMCD3 cells, the reporter gene exhibitedapproximately fivefold greater activity after 6 h of hypertonic stress (100 mMNaCl; Fig 3 ). This effect wasalso dose dependent within the range of 50-150 mM NaCl. The effect ofurea was much more modest (27% increase) but achieved statisticalsignificance. The tonicity-dependent upregulation was partially blocked whencells were pretreated with the EGFR kinase inhibitor PD-153035( Fig. 3 ). The effect of theimpermeant solute mannitol was equivalent to that of NaCl in this assay, aswas its sensitivity to PD-153035 (data not shown). Because the most widelyrecognized tonicity-responsive enhancer element is TonE, we sought thiselement in the COX-2 promoter. Using the reported canonical sequence for TonE(YGGAANNNYNY; see Ref. 31 ), wewere unable to identify this cis -acting element in the proximal 0.7kb of the COX-2 5'-flanking sequence (data not shown).. R; ]8 ]/ f: Y: K
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We assessed PGE 2 generation as an index of COX-2 function. Hypertonic NaCl increased PGE 2 release in cell supernatants in atime-dependent fashion, and this effect was blocked by EGFR kinase inhibition( Fig. 4 ). Of note, the largeerror 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 ordinalrelationship was identical.

ERK and JNK have been implicated in COX-2 regulation, particularly inmedullary epithelial cells( 47 ) where they may function downstream of EGFR in a signaling cascade. We therefore assessed the effect ofEGFR kinase inhibition on activation of all three of the principal MAPKfamilies by hypertonic stress. As anticipated and as previously shown in thismodel, hypertonicity increased ERK activation as assessed via anti-P-ERKimmunoblotting ( Fig. 5 ). Consistent with our earlier observation( 52 ), EGFR kinase inhibitionmarkedly blunted the effect of tonicity on ERK activation. In contrast, therewas virtually no effect of EGFR kinase inhibition on tonicity-dependent JNK orp38 phosphorylation.
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Because ERK appeared to be a principal effector of EGFR in this and othercontexts, we tested the effect of pharmacological inhibition of ERK activationon tonicity-inducible COX-2 expression. We used two differentmitogen/extracellular signal-regulated kinase (MEK) inhibitors (and inhibitorsof ERK activation), PD-98059 and U-0126, in part to permit discriminationbetween the anticipated ERK1/2-dependent phenomenon and a potential ERK5-dependent phenomenon ( 23 )recently reported in another model( 17 ). Both MEK inhibitorssubstantially blocked the effect of NaCl on this end point, consistent with arole for MEK (and hence ERK) activation in tonicity-responsive COX-2 expression. Of note, the inhibitors were equally effective when applied at 50µM for PD-98059 and 10 µM for U-0126, strongly suggesting that ERK5 wasnot 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 wasequivalent to that of equiosmolar NaCl, and this effect was equally sensitiveto MEK inhibition ( Fig. 6 ).

EGFR activation generally occurs via metalloproteinase-dependent cleavageof an EGF-like ectodomain that then acts in an autocrine or juxtacrine fashion(reviewed in Ref. 16 ). To testthis possibility in preliminary fashion in the present context, we investigated the ability of two metalloproteinase inhibitors to abrogate theeffect 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, whereasvehicle 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 wereunable to use the specific hydroxamate-based metalloproteinase inhibitors(e.g., ilomastat) because the vehicle concentration required by theserelatively insoluble compounds (i.e., DMSO at 0.5-2%) was sufficient tointerfere with COX-2 expression (data not shown). In support of ouralternative approach, a recent report documented functional equivalencebetween TAPI and a hydroxamate-based inhibitor in at least one context( 42 ).4 l: {( c; ~  O# c0 z

HSP60 expression induces COX-2 expression( 4 ), and hypertonic stress hasbeen associated with increased expression of HSPs in renal epithelial cells( 10 ). We investigated thepossibility that HSP60 may mediate the effect of hypertonicity on COX-2 expression. Abundant HSP60 expression was detected in mIMCD3 cells( Fig. 8 ); however, this levelwas unaffected by hypertonic stress or by the presence of EGFR kinaseinhibitors./ V& n+ T( u2 U' N& G
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Last, we sought an EGFR-null model in which to test for the presence oftonicity-dependent signaling. Although the EGFR is nearly ubiquitous amongcultured cell lines, CHO cells reportedly lack this tyrosine kinase( 29 ). We compared CHO cellswith two additional cell lines known to express the EGFR: 3T3 cells and therenal epithelial MDCK cell line. We first assessed the ability of EGF toactivate ERK in each cell line. As anticipated, EGF induced a marked increasein ERK phosphorylation in both the 3T3 and MDCK cells at 5 and 30 min oftreatment but produced no effect in the CHO cells( Fig. 9 A ). Thisconfirmed the EGF nonresponsiveness of the CHO line. With this validated model in hand, we next assessed the ability of hypertonic stress to upregulate COX-2expression in each cell line. Similar to the effect in mIMCD3 cells,hypertonic stress dramatically increased COX-2 expression in both 3T3 and MDCKcells ( Fig. 9 B ); in marked contrast, however, there was essentially no effect in the EGFRkinase-null CHO cells. For comparison, we also determined the effect of themedullary solute, urea, on COX-2 expression in each of these models. LikeNaCl, urea produced no effect in CHO cells. The urea effect was also much lessprominent than that of hypertonic NaCl in both 3T3 and MDCK cells. These datawere consistent with the relatively modest effect of urea vis-à-vis theCOX-2 promoter ( Fig. 3 ). Ofnote, introduction of an expression vector encoding EGFR in CHO cells failedto recapitulate tonicity-inducible COX-2 expression. We infer that EGFRexpression is necessary but not sufficient in this context; CHO cells may lackeither the EGFR ligand (i.e., HB-EGF) or the relevant ligand-directedmetalloproteinase, both of which are required for an intact signalingmodule.8 Z: U3 t# p6 A& o
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DISCUSSION% S  R8 m/ }) F/ i! Z. A/ O

We extend the observations of Yang et al.( 47 ), who reported MAPKdependence of renal medullary epithelial cell COX-2 expression in response tohypertonic stress. We found that two potent and highly specific inhibitors ofthe EGFR kinase blocked tonicity-dependent upregulation of COX-2 mRNAexpression, protein expression, transcription, and PGE 2 synthesis.This signaling event was independent of HSP60 expression, which induces COX-2in at least one model ( 4 ). Theinhibitors of EGFR kinase partially blocked tonicity-dependent ERK activation,as would be expected from the data of Yang et al.( 47 ), but failed tosignificantly 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 toexhibit the tonicity-dependent COX-2 expression evident in the EGF-responsive3T3, MDCK, and mIMCD3 cell lines. In aggregate, these data strongly suggest that the EGFR kinase mediates the effect of hypertonicity on COX-2 expressionin the renal medullary cell model and that ectodomain cleavage of an EGFRligand may be required.
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Although medullary COX-2 expression and action likely play a pivotal rolein renal physiology, particularly in response to dehydration, much of thiseffect has been attributed to actions of the medullary interstitial cells andadjacent 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 otherrenal epithelial cells in vitro( 13, 47, 48 ), particularly in thecontext of hypertonic stress( 47, 48 )." ]% G) l1 C* f( G; h  n0 e! Y) y

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 aninhibitory effect of the general tyrosine kinase inhibitor genistein( 47 ). We, in contrast, found aprofound effect in the mIMCD3 cell line at the mRNA and protein level and atthe level of PGE 2 production. The study of Yang et al.( 47 ) was performed using themIMCD-K2 cell line ( 25 ),whereas the present study employed the mIMCD3 line( 34 ). Tyrphostin-A23 andtyrphostin-A51 exhibit IC 50 for EGFR kinase of 40 µM and 800 nM, respectively, and were applied at 10 µM( 47 ); it is conceivable thatinsufficient cellular levels were achieved, particularly for tyrphostin-A23. While the present studies were being completed, Guo et al. ( 17 ) reported the EGFRdependence of COX-2 expression in a rat intestinal epithelial cell model inresponse to the peptide hormone gastrin, in further support of this mode ofregulation.

Importantly, these are not the first data implicating a role for the EGFRin 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. Incontrast, King et al. ( 24 )reported activation of the EGFR by osmotic shock in the absence of receptordimerization. Sheikh-Hamad and co-workers( 38 ) describedtonicity-dependent association of the following three proteins: theosmotically inducible CD9( 36 ), 1 -integrin( 37 ), and the EGFR ligand HB-EGF. A related phenomenon was observed recently in normal human renaltissue ( 32 ). Moreover,expression of HB-EGF appears to be itself osmotically regulated( 1, 27 ). Interestingly, HB-EGF andCD9 also interact with A disintegrin and metalloprotease domain 10 (also knownas Kuzbanian), and this metalloproteinase may mediate the ectodomain sheddingand 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 hasnot been observed in the context of hypertonic stress.$ t0 T8 p  \' |. V/ Y% k, G

The EGFR ligand conferring transactivation in the present context isunknown. Potential ligands, including EGF, amphiregulin, epiregulin,transforming growth factor-, and HB-EGF, all exist as precursors with asingle membrane-spanning domain and exhibit autocrine or juxtacrine actionafter cleavage by one or more metalloproteinases (reviewed in Ref. 33 ). Our data using the metalloproteinase inhibitors TAPI and doxycycline support a role for thismechanism in tonicity-dependent COX-2 regulation, but they do not permitdiscrimination among potential EGFR agonists. Neutralizing antibodies specificfor 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 ahuman renal epithelial cell line suitable for studying this phenomenon.

In our studies, the proximal 0.7 kb of the murine COX-2 promoter wassufficient to confer tonicity responsiveness to a heterologous reporter gene.Although lacking a canonical tonicity-responsive TonE element, this generegion contains several cis -acting elements validated in othercontexts. For example, a cAMP-response element (CRE) is instrumental in theCOX-2 response to endotoxin, as are tandem CCAAT/enhancer-binding proteinsites ( 22, 30, 43 ); the CRE site, however, isalso responsive to platelet-derived growth factor( 44 ), in a JNK-dependentfashion. An NF- B binding site was implicated in the COX-2 response totumor necrosis factor- ( 45 ). Although most of thesedata were acquired in heterologous expression systems, studies have emergedusing more native models. For example, both in cultured medullary interstitialcells exposed to hypertonicity( 19 ) and in cultured thickascending limb cells exposed to low-salt or low-chloride conditions( 7 ), the NF- B sitemediated COX-2 transcriptional induction. Our data clearly support a role forEGFR kinase signaling in tonicity-dependent upregulation of COX-2 expression in the well-studied mIMCD3 model; whether this effect requires the CRE [asmight be suggested by earlier data with another agonist of a receptor tyrosinekinase, PDGF ( 44 )], theNF- B element [consistent with the interstitial cell and thick ascending limb models ( 19 )], or stillanother element, it clearly occurs in a TonE-independent fashion. Consistentwith these data, EGFR kinase inhibition failed to influence TonE- andTonEBP-dependent transcription( 52 )., l& U# ^: }; D' m

In summary, we have shown that the well-described ERK-dependent upregulation in COX-2 expression accompanying hypertonic stress in medullaryepithelial cells is mediated via transactivation of the EGFR. The EGFR agonistmediating this effect and the additional upstream activating events remainobscure.# g1 p. a0 h4 T0 U

DISCLOSURES
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This work was supported by National Institute of Diabetes and Digestive andKidney Diseases Grant DK-52494, the American Heart Association, and theDepartment of Veterans Affairs.
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ACKNOWLEDGMENTS
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We thank Dr. H. Herschman for the kind gift of the COX-2 promoter inpXP2.! ~& V( w0 x1 V" w
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