Abstract
Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.
Highlights
Inorganic sulfate is one of the most abundant anions in mammalian blood and is essential for numerous physiological functions [1]
In the original studies by Forman et al [10], rodent Fxr␣ was shown to be expressed in the liver, intestine, adrenal cortex, and renal tubules
Vates hepatic and intestinal target genes involved in the maintenance of bile acid, lipid, and glucose homeostasis
Summary
Materials—Expression constructs for the human FXR␣1 and FXR␣2 (pcDNA-hFXR␣1 and pcDNA-hFXR␣2) and human RXR␣ (pCMX-hRXR␣) have been described previously [20]. 3-(2,6-Dichlorophenyl)-4-(3Ј-carboxy-2-chloro-stilben-4-yl)oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic ligand for FXR␣, was a gift from Drs Tim Willson and Patrick Malloney (GlaxoSmithKline). ␣-Naphthylisothiocyanate (ANIT) was purchased from Sigma. RNA Isolation and Real Time Quantitative PCR—RNA from tissues was isolated using TRIzol reagent (Invitrogen). Real time PCR was performed essentially as described [21]. RNA samples were subjected to real time quantitative PCR analysis in duplicate by Xenoport Inc. For the ANIT experiment, 8 –10-week-old male and female Fxr␣Ϫ/Ϫ mice and their wild-type C57BL/6J littermates were gavaged with a single dose of vehicle (olive oil) or ANIT dissolved in vehicle (75 mg/kg). Animals were euthanized at 48 h after the ANIT dose, and the indicated tissues were excised and homogenized in TRIzol reagent (Invitrogen) for RNA extraction. Inorganic sulfate levels in urine or serum were assayed by a turbidimetric method as described [33]. Statistical Analysis—Mean values and S.E. were determined by the analysis of multiple independent samples, each assayed in duplicate or triplicate, as indicated in the figure legends. A two-tailed Student’s t test was used to calculate p values
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