Transcriptional Regulation of Metabolic Genes by Glucocorticoid Receptor

Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements (GRE) to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing (ChIPseq) and microarray, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR binding regions (GBR) in or near these genes and the genes' expression was regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 (IGF-1) pathways. We found that overexpression of p85 alpha, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85 alpha expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of IRS-1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85 alpha expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a GRE was identified in the p85 alpha GBR. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85 alpha plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes./nInterestingly, glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, in mouse C2C12 myotubes and gastrocnemius muscle. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene we identified three glucocorticoid receptor (GR) binding regions (GBRs): one in upstream of the transcription start site, -17kbGBR; and two in introns, +45kbGBR and +71kbGBR Together, these three GBRs contain 4 glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay showed that 30 min Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR, but not in +71kbGBR. Upon 60 min Dex treatment; however, the sensitivity to MNase was elevated in +71kbGBR, but not in +45kbGBR. Dex treatment for 30 and 60 min did not affect the chromatin structure of the -17kbGBR whose GRE was located in a linker region, whereas the GREs of +45kbGBR and +71kbGBR were located on nucleosomes. Dex treatment for 30 min also increased the levels of acetylated histone H3 and H4 in all three GBRs. Finally, using chromatin conformation capture assay, we showed that Dex treatment increased the interaction between the -17kbGBR and two genomic regions: one located around +500 bp and the other located around +73 kb. Overall, our results indicate that glucocorticoids activated FoxO3 gene transcription through multiple GREs. Each GRE was regulated by distinct mechanisms, and DNA looping is likely involved in this transcriptional activation process./nMoreover, glucocorticoids are important regulators of lipid homeostasis; however, chronically elevated glucocorticoid levels can induce hypertriglyceridemia and hepatic steatosis. The occupied glucocorticoid receptor (GR) functions as a transcription factor, although genes regulated by GR and involved in lipid metabolism are not fully understood. Angiopoietin-like 4 (ANGPTL4) is a secreted protein inhibiting extracellular lipoprotein lipase. It is synthesized and secreted during fasting and under elevated glucocorticoid level conditions to promote adipocyte intracellular lipolysis. Here, I show that dexamethasone (a synthetic glucocorticoid) treatment increased Angptl4 mRNA levels in mouse primary hepatocytes and rat H4IIE hepatoma cells, and elevated the transcriptional rate of Angptl4 in H4IIE cells. Using bioinformatics and chromatin immunoprecipitation (ChIP), a glucocorticoid receptor (GR) binding site was identified within the rat Angptl4 gene. I further confirmed that this glucocorticoid response element (GRE) with mutagenesis and reporter assay. Dex treatment also increased histone H4 acetylation and DNase I accessibility around the GRE genomic regions. In Angptl4-/- total knockout mice, glucocorticoid-induced hypertriglyceridemia and hepatic steatosis were significantly reduced; suggesting glucocorticoid-promoted flux of triglyceride from white adipose tissue to liver requires Angptl4. Notably, hypertriglyceridemia is frequently associated with insulin resistance. I found that insulin suppressed Dex-induced Angptl4 gene expression in mouse primary hepatocytes and H4IIE cells. Using compounds inhibiting distinct molecules in the insulin signlaling pathway, PI3K, Akt, and GSK-3 are involved in the suppression effect of insulin. Moreover, mutating a FOX transcription factor binding site, located ~40 base pair downstream from the Angptl4 GRE, compromised glucocorticoid-induced luciferase activity. In addition to GR, both FoxO1 and FoxA2 proteins are recruited to the Angptl4 GR binding region in ChIP assay. When treating the cells with insulin, GR recruitment was abolished. Finally, reducing FoxO1 expression by RNA interference in H4IIE cells decreased Dex-induced Angptl4 gene expression, mimicking the insulin response. These data suggest that FoxO1 is involved in insulin suppression of glucocorticoid-induced Angptl4 gene expression. In summary, my study established that 1) Angptl4 is a direct GR target and participates in glucocorticoid-regulated triglyceride metabolism 2) the transcription of Angptl4 is under the control of both insulin and glucocorticoids, where insulin can suppress glucocorticoid-induced Angptl4.

Advisor: Wang, Jen-chywan