Hypotonicity increases transcription, expression, and action of Egr-1 in murine renal medullary mIMCD3 cells.
In cells of the murine renal inner medullary collecting duct (mIMCD3) cell line, acute hypotonic shock (50% dilution of medium with sterile water but not with sterile 150 mM NaCl) increased Egr-1 mRNA abundance 2.5-fold at 6 h, as determined by Northern analysis. This increase was accompanied by increased Egr-1 transcription, as quantitated by luciferase reporter gene assay. Increased transcription was dose dependent, additive with other Egr-1 transcriptional activators, and occurred in the absence of overt cytotoxicity, as quantitated via a fluorometric viability assay. In addition, hypotonic stress increased Egr-1 protein abundance, which was accompanied by augmented Egr-1-specific DNA binding ability, as measured via electrophoretic mobility shift assay. Increased DNA binding was further associated with increased transactivation by Egr-1, demonstrated through transient transfection of mIMCD3 cells with a luciferase reporter gene driven by tandem repeats of the Egr-1 DNA consensus sequence. Taken together, these data indicate that hypotonic stress activates Egr-1 transcription, translation, DNA binding, and transactivation in renal medullary cells. This phenomenon might play a role in the acquisition of the adaptive phenotype in response to hypotonic stress in cells of the renal medulla in vivo.
Zhang
Z
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Division of Nephrology, Hypertension, and Clinical Pharmacology, Oregon Health Sciences University, Portland, USA.
Cohen
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D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D051766:Early Growth Response Protein 1; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007038:Hypotonic Solutions; D017874:Immediate-Early Proteins; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D013312:Stress, Physiological; D014157:Transcription Factors / Q000096:biosynthesis* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014508:Urea / Q000494:pharmacology; D016335:Zinc Fingers
D003470:Culture Media; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D007038:Hypotonic Solutions; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D014508:Urea