Involvement of Hepatocyte Nuclear Factor 1 in the Regulation of the UDP-Glucuronosyltransferase 1A7 (UGT1A7) Gene in Rat Hepatocytes1
- Department of Pharmacology and Toxicology, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia
Abstract
UDP-glucuronosyltransferase 1A7 (UGT1A7) is a major UGT contributing to the glucuronidation of xenobiotic phenols in rats. Its expression in rat liver is tightly regulated, with low constitutive and high inducible expression in response to aryl hydrocarbon receptor ligands and oltipraz. Previously, we reported the absence of 3-methylcholanthrene- or oltipraz-responsive elements in the 1.6-kbp region flanking the UGT1A7 promoter. However, potential binding sites were noted for several liver-enriched transcription factors. Here we show that deletion of the hepatic nuclear factor (HNF)3, HNF4, and CCAAT-enhancer binding protein-like binding sites had no effect on the expression of a UGT1A7 reporter plasmid, p(−965/+56)1A7-Luc, in primary rat hepatocytes. The full activity of the promoter was contained in the region between bases −157 and +76. Two sites of binding by rat liver nuclear proteins were detected in this region by DNase footprinting. PR-1 corresponded to the HNF1-like binding site between bases −52 and −38, whereas PR-2 was located between −30 to −6. Gel retardation studies supported the presence of HNF1α in the PR-1 DNA-liver nuclear protein complex. Mutation of PR-1 inhibited binding in the gel shift assay, prevented activation by overexpressed HNF1 in human embryonic kidney cells, and reduced by >80% the maximal luciferase activities expressed from basal and 3-methylcholanthrene-responsive UGT1A7 gene reporter constructs in primary rat hepatocytes. These data provide evidence for an important stimulatory role of HNF1 in promoting UGT1A7 gene expression in rat liver.
Footnotes
-
Send reprint requests to: Joseph Ritter, Ph.D., Department of Pharmacology and Toxicology, Virginia Commonwealth University, Box 980613, Richmond, VA 23298-0613. E-mail: jritter{at}hsc.vcu.edu
-
↵1 This work was supported by Grant R29-ES07762 from the National Institute of Environmental Health Sciences. R.M. and D.A. were supported by National Institute of Environmental Health Sciences Training Grant ES07087.
- Abbreviations:
- UGT
- UDP-glucuronosyltransferase
- HNF
- hepatic nuclear factor
- C/EBP
- CCAAT-enhancer binding protein
- HEK
- human embryonic kidney
- AHR
- aryl hydrocarbon receptor
- 3-MC
- 3-methylcholanthrene
- EMSA
- electrophoretic mobility shift assay
- PAS
- period/aryl hydrocarbon receptor nuclear translocator/aryl hydrocarbon receptor/single-minded
- kbp
- kilobase pair
- bp
- base pair(s)
-
- Received March 21, 2000.
- Accepted April 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
