The Roles of ATF3 in Liver Dysfunction and the Regulation of Phosphoenolpyruvate Carboxykinase Gene Expression

Amy E Allen-Jennings,Matthew G Hartman,Gary J Kociba,Tsonwin Hai

doi:10.1074/jbc.m200727200

Abstract

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element-binding protein family of transcription factors, is a transcriptional repressor, and the expression of its corresponding gene, ATF3, is induced by many stress signals. In this report, we demonstrate that transgenic mice expressing ATF3 in the liver had symptoms of liver dysfunction such as high levels of serum bilirubin, alkaline phosphatase, alanine transaminase, aspartate transaminase, and bile acids. In addition, these mice had physiological responses consistent with hypoglycemia including a low insulin:glucagon ratio in the serum and reduced adipose tissue mass. Electrophoretic mobility shift assays indicated that ATF3 bound to the ATF/cAMP-responsvie element site derived from the promoter of the gene encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Furthermore, transient transfection assays indicated that ATF3 repressed the activity of the PEPCK promoter. Taken together, our results are consistent with the model that the expression of ATF3 in the liver results in defects in glucose homeostasis by repressing gluconeogenesis. Because ATF3 is a stress-inducible gene, these mice may provide a model to investigate the molecular mechanisms of some stress-associated liver diseases.

Highlights

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element-binding protein family of transcription factors, is a transcriptional repressor, and the expression of its corresponding gene, ATF3, is induced by many stress signals
Electrophoretic mobility shift assays indicated that ATF3 bound to the ATF/cAMP-responsvie element site derived from the promoter of the gene encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK)
Because F1 mice derived from some TTR-ATF3 founders expressed ATF3 in the pancreas, we only describe phenotypes obtained from mice with hepatic but not pancreatic expression of ATF3 to exclude hepatic phenotypes resulting from indirect effects of pancreatic expression of ATF3

Summary

EXPERIMENTAL PROCEDURES

The TTR-ATF3 Transgenic Mice—The generation of these mice was described previously [21]. The phenotypes described in this report were observed in F1 hybrid mice derived from Ͼ8 founders. This reproducibility strongly suggests that these phenotypes were not because of the artifacts of integration sites. DNA-binding reactions and gel electrophoresis were carried out as described previously [11] using 32P-labeled double-stranded oligonucleotides containing the ATF/CRE site derived from the PEPCK promoter, 5Ј-AGGGCCCCTTACGTCAGGGCGAGA-3Ј (ATF/CRE site is underlined). The cell suspension with the DNA mixture was incubated at room temperature for 15 min and divided into three 6-cm plates containing 2 ml of medium. A mock transfection was carried out using 12 ␮g of pGEM3 to make the DNA mixture

RESULTS

TABLE I Comparison of serum biochemical parameters

DISCUSSION