High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA.
Hypertonicity increases mRNA and protein abundance of the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), contributing to increased transcription of downstream osmoprotective genes. Previously, this was attributed to increased transcription of TonEBP/OREBP because no change was found in its mRNA stability. However, there is no direct evidence for increased transcription, and the 3'-untranslated region (UTR) of TonEBP/OREBP contains numerous adenylate/uridylate-rich elements, which can modulate RNA stability. Therefore, we have reinvestigated the effect of hypertonicity on TonEBP/OREBP mRNA stability. We find that, in mouse inner medullary collecting duct cells, raising osmolality from 300 to 500 mosmol/kgH(2)O by adding NaCl increases TonEBP/OREBP mRNA to a peak of 2.3-fold after 4 h, followed by a decline. TonEBP/OREBP protein increases to a sustained peak of 3.0-fold at 8 h. To determine the stability of TonEBP/OREBP mRNA, we measured the rate of its decrease after inhibiting transcription with actinomycin D, finding that it is stabilized for 6 h after addition of NaCl. This stabilization is sufficient to explain the increase in mRNA without any change in transcription. To investigate how hypertonicity stabilizes TonEBP/OREBP mRNA, we tested luciferase reporters containing parts of the TonEBP/OREBP mRNA UTR. Inclusion of both the 5'- and 3'-UTR increases reporter activity, consistent with mRNA stabilization. Surprisingly, however, it is the 5'-UTR that stabilizes; the 3'-UTR, by itself, decreases reporter activity. We concluded that 1) hypertonicity stabilizes TonEBP/OREBP mRNA, contributing to its increase, and 2) stabilization depends on the presence of the 5'-UTR.
Cai
Qi
Q
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-1603, USA.
Ferraris
Joan D
JD
Burg
Maurice B
MB
D020413:3' Untranslated Regions; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012462:Saline Solution, Hypertonic; D017673:Sodium Chloride, Dietary / Q000494:pharmacology*; D015534:Trans-Activators / Q000096:biosynthesis*; D014157:Transcription Factors; D014542:Uridine Monophosphate / Q000378:metabolism
D020413:3' Untranslated Regions; C492200:Nfat5 protein, mouse; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012462:Saline Solution, Hypertonic; D017673:Sodium Chloride, Dietary; D015534:Trans-Activators; D014157:Transcription Factors; D003609:Dactinomycin; D000249:Adenosine Monophosphate; D014542:Uridine Monophosphate; D008156:Luciferases