.-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 PGE 2 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 hypertonicitydependent 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/kgH 2 O. 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 PGH 2 , 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, 2...