Urea inducibility of egr-1 in murine inner medullary collecting duct cells is mediated by the serum response element and adjacent Ets motifs

The renal medullary solute urea increases transcription and protein expression of the zinc finger-containing transcription factor Egr-1 in a renal epithelial cell-specific fashion. Transient transfection of mIMCD3 cells with a luciferase reporter gene driven by 1.2 kilobases of the murine egr-1 5'-flanking sequence showed 4-fold increase in reporter gene activity with 200 mM urea treatment. The effect of impermeant solutes such as NaCl was much less pronounced, whereas the permeant solute glycerol had no effect. In addition, this same sequence, minus the egr-1 minimal promoter, conferred urea responsiveness to a heterologous (thymidine kinase) promoter. Whereas deletion of two putative AP-1 sites from the sequence had no effect upon urea inducibility, elimination of the five putative serum response elements (SREs) abolished the urea effect. Progressive deletion of the SREs caused a corresponding diminution in urea effect. Two key tandem SREs (SRE-3 and SRE- 4), in conjunction with their two adjacent clusters of Ets motifs, were sufficient to confer urea responsiveness to a reporter gene. This response was markedly attenuated in the absence of either cluster of Ets motifs and was abolished if both clusters were deleted. By electrophoretic mobility shift assay, formation of the ternary complex was constitutive and was demonstrable in vitro despite the presence of 200 mosm urea or NaCl. Therefore urea-inducible egr-1 transcription in renal medullary cells is mediated through the SRE and adjacent Ets motifs; ternary complex formation is not inhibited even in the presence of physiological hyperosmolality.