Cyclooxygenase-2, the inducible isoform of COX, is constitutively expressed in epithelial and interstitial cells of the renal cortex and medulla. Earlier evidence from this laboratory has shown that hypertonicity, the physiological environment in the medulla, stimulates COX-2 expression and prostaglandin formation in the medulla and in a collecting duct-derived cell line through activation of MAP kinases. COX-2 products act to protect cells against osmotic stress by inhibition of apoptosis. Further studies of this pathway revealed that COX-2 induction and PGE formation was markedly reduced by antioxidants (NAC, tempol, or DPI) and that this was associated with a reduction in the phosphorylation of ERK kinases. Furthermore, hypertonicity in the presence of antioxidants induced apoptosis as assessed by caspase activity and laser scanning cytometry. These studies suggest a novel osmotic survival pathway along a ROS/Erk/COX-2 axis. To identify possible elements in the COX-2 5?UTR responsive to hyperosmotic stress, we cloned 3.2 kb of the mouse COX-2 promoter from C57BL6 mice and transiently transfected mouse collecting duct cells with variable length promoter-luciferase constructs. These studies suggest the presence of a hyperosmolarity responsive site between -1.63 kb and -1.27 kb upstream of the COX-2 transcription start site. Further studies showed that the ~100 bp sequence between -1361 bp and -1268 bp was required for the hyperosmolarity response. Electrophoretic mobility shift assays indicated distinct protein binding under hypertonic conditions that was not attributable to any of the known transcription factors found in this region (C/EBP?, OCT-1, HSF2, AP-1, HNF3, TCF11). Furthermore, the hyperosmotic response site does not contain the previously identified TONE consensus sequence.
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