Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver

Abstract

The cytosolic PEPCK gene is a model gene for assessing retinoid regulation of liver-specific genes encoding enzymes of carbohydrate metabolism. In vivo, we have demonstrated that the PEPCK gene is inhibited by vitamin A deficiency. Specifically, under conditions of food deprivation, induction of the PEPCK gene is inhibited in the vitamin A deficient mouse. Inhibition of the PEPCK gene by vitamin A deficiency is reversed by all- trans or 9- cis retinoic acid (RA) treatment. In a transgenic mouse model, a −460 and −355 bp PEPCK promoter fragment confers susceptibility to inhibition by vitamin A deficiency and responsiveness to all- trans RA treatment. However, there is a differential effect of 9- cis RA on the PEPCK promoter; the −460 fragment confers responsiveness to 9- cis RA, but the −355 fragment does not. Taken together, these results indicate that the PEPCK retinoic acid response element (RARE)1 is required for 9- cis RA induction—but not all- trans RA induction—of the PEPCK gene. In order to determine if vitamin A deficiency alters specific localized expression of the PEPCK gene in the periportal cells of the liver, the effect of vitamin A status on PEPCK localization in the liver was also measured. The PEPCK transgenes were expressed specifically in the periportal region of the liver acinus and although vitamin A deficiency caused a decrease in PEPCK transgene mRNA levels in periportal cells, it did not alter the periportal cell-specific pattern of expression. Retinoid treatment induced PEPCK transgene mRNA levels in the same population of cells, however, the −355 bp PEPCK promoter fragment did not respond to 9- cis RA treatment. In order to determine the nuclear transcription factor(s) responsible for retinoid regulation of the PEPCK gene in the liver, we investigated retinoic acid receptor (RAR)α and β and the retinoid X receptor (RXR)α—the major retinoid receptors in liver—in terms of expression and the ability of the receptors to bind the PEPCK RAREs. Vitamin A deficiency significantly decreased hepatic RARβ, but not RARα or RXRα mRNA levels. In situ hybridization showed that RARα, RARβ and RXRα mRNAs were localized in the periportal region, however, immunohistochemistry showed that RARα and RXRα were distributed evenly across the liver acinus, whereas only RARβ levels were higher in periportal cells. The binding of nuclear receptors to PEPCK RARE1, RARE2 and RARE3 indicates a complex pattern of retinoid receptor and orphan nuclear receptor binding.