Hu antigen R and tristetraprolin: Counter-regulators of rat apical sodium-dependent bile acid transporter by way of effects on messenger RNA stability

Abstract

The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) mediates intestinal, renal, and cholangiocyte bile acid reclamation. Transcriptional regulation of ASBT is well described, whereas information on posttranscriptional regulation is limited. Prior studies suggested that ontogeny of ASBT is controlled in part by changes in messenger RNA (mRNA) stability. We studied the role that Hu antigen R (HuR) and tristetraprolin (TTP) play in regulating the expression of mRNA that contains the 3' untranslated region (UTR) of rat ASBT. The 3'UTR was incorporated into an SV-40 driven luciferase reporter (rASBT3-luciferase) for rapid screening of regulatory effects. Silencing HuR reduced luciferase reporter activity, whereas silencing TTP enhanced luciferase activity. Conversely, overexpression of HuR enhanced rASBT3-luciferase reporter activity. The same 3'UTR fragments of rat ASBT were incorporated into a beta-globin coding mRNA construct for analysis of mRNA stability (rASBT3-βglobin). mRNA half-life was progressively shortened by the incorporation of increasing sized fragments of the 3'UTR. Silencing HuR shortened the half-life of rASBT3-βglobin containing 0.3 kb of the rat ASBT 3'UTR. Gel shift assays revealed binding of HuR and TTP to rat ASBT 3'UTR. Endogenously expressed human ASBT mRNA half-lives and steady-state protein levels in Caco-2 cells were repressed when HuR was silenced but was enhanced when TTP was silenced. Developmental changes in HuR and TTP protein abundance correlated with previously characterized ontogenic changes in rat ileal and renal ASBT expression. These studies not only show that ASBT expression is controlled at the level of mRNA stability by way of its 3'UTR, but also identify HuR and TTP as two key transacting factors that are involved in exerting counterregulatory effects on ASBT mRNA stability.