Developmental and tissue-specific regulation of hepatocyte nuclear factor 4-alpha (HNF4-alpha) isoforms in rodents

Abstract

Hepatocyte nuclear factor 4-alpha (HNF4-alpha) regulates expression of a number of genes in several metabolic organs. The HNF4-alpha gene has two promoters and encodes at least nine isoforms through differential splicing. In mouse liver, transcription initiates at promoter 2 (P2) during fetal life, but switches to P1 at birth. Developmental and tissue-specific expression of HNF4-alpha in other organs is largely unknown. Here, we examined expression of P1- and P2-derived transcripts in a number of mouse and rat tissues. Both P1 and P2 were active in mouse fetal liver, but P2-derived isoforms were detected 50% more abundantly than P1 transcripts. Conversely, the adult mouse liver expressed significantly higher levels of P1- than P2-derived mRNA. In contrast, in the rat, P1 was used more predominantly in both fetal and adult liver. Exposure of fetal rats to the synthetic glucocorticoid dexamethasone caused suppression of P2 while enhancing hepatic expression of transcripts from P1. This was associated with increased expression of erythropoietin and phosphoenolpyruvate carboxykinase, which are key HNF4-alpha targets in the liver. Unlike liver, the kidney and stomach used promoters more selectively, so that only P1-derived isoforms were detected in fetal and adult kidneys in mice or rats, whereas the stomach in both species expressed P2-derived transcripts exclusively. No significant HNF4-alpha mRNA was detected in the spleen. These data reveal striking developmental and tissue-specific variation in expression of HNF4-alpha, and indicate that this can be influenced by environmental factors (such as exposure to glucocorticoid excess), with potential pathophysiological consequences.

Figure 1

Systematic representation of the HNF4-α gene structure. Exons are shown as boxes. The arrows represent the two alternative promoters and the lines linking exons indicate splicing events.

Figure 2

Expression of P1 and P2 HNF4-α isoforms in mouse liver. HNF4-α mRNA from mouse fetal (E15 and E19) and adult liver was measured by real-time RT-PCR using P1- or P2-specific primers. GAPDH was used as control housekeeping gene. (A) RT-PCR products separated on agarose gel and (B) relative mRNA content (mean ± SEM) in the liver at different ages. *p < 0.05 versus P1; $p < 0.05 versus fetal (E15, E19).

Figure 3

Expression of P1 and P2 HNF4-α isoforms in rat liver. HNF4-α mRNA from rat fetal (E15 and E21) and adult liver was measured by real-time RT-PCR using P1- or P2-specific primers. GAPDH was used as control housekeeping gene. (A) PCR products separated on agarose gel and (B) relative mRNA content (mean ± SEM) in the liver at different ages. *p < 0.05 versus P2; $p < 0.05 versus E15.

Figure 4

Expression of P1 and P2 HNF4-α isoforms in kidney, stomach, and spleen. HNF4-α mRNA isolated from adult mouse (A) and rat (B) kidney, stomach, or spleen was analyzed by real-time RT-PCR using P1- or P2-specific primers. GAPDH was used as control housekeeping gene. *p < 0.05 P1 versus P2.

Figure 5

Effect of dexamethasone on HNF4-α and its target genes. Hepatic expression of HNF4-α, PEPCK, EPO, and glucokinase (GK) mRNA was measured in E21 fetuses of dams that received dexamethasone (DEX) or vehicle (CON) during the last week of pregnancy. Results (mean ± SEM) represent mRNA levels relative to control animals (n = 8 per group). *p < 0.05 versus control.