Regulation of the cytosolic aspartate aminotransferase housekeeping gene promoter by glucocorticoids, cAMP, and insulin

Abstract

The cytosolic aspartate aminotransferase (cAspAT) is a ubiquitous enzyme that displays liver-specific hormonal regulation. In the hepatoma cell line Fao, both the activity and the mRNA level of cAspAT are increased by glucocorticoids. This effect is potentiated by cAMP and inhibited by insulin. Using in vivo run-on experiments, we showed that these effectors act at the transcriptional level. A cAspAT gene fragment containing 2405 bp of the promoter was sequenced. Deletion fragments of this promoter were inserted upstream of the CAT gene, and the regulation of their activity was assayed following transfection in Fao cells. Stable transfection experiments established that the construct including the entire 2.405-kb fragment undergoes positive regulation by glucocorticoids and cAMP and negative regulation by insulin similar to the regulation of the endogenous gene. A physical separation of the positive and negative control elements is suggested by the fact that cAMP acted on the -682/-26-bp fragment (a 2-fold increase of the stimulation by dexamethasone), whereas the negative regulation by insulin (50% of the stimulation by dexamethasone) required the -1983/-1718-bp fragment. Both regions were required for maximal glucocorticoid activity (6-9-fold increase of CAT activity). We conclude that at least two regulatory regions, a proximal and a distal one, are required for full hormonal regulation of the cAspAT gene.

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

“…We have previously shown that a 2.4-kb fragment of the cAspAT gene promoter displays a hormonal regulation similar to that of the endogenous gene in Fao rat hepatoma cells (1). In Fao cells stably transfected by this promoter fragment preceding the CAT gene, glucocorticoids induced the promoter activity whereas insulin inhibited and cyclic AMP potentiated the glucocorticoid effect.…”

“…Alternatively, this band could be due to the binding of another rat liver cytosolic protein, an unlikely explanation in view of the supershift experiments (see below). In contrast, the GR (1,3, and 7 p.l) of a purified rat liver GR preparation were incubated with labeled GRE A and GRE pal. GR monomer-and GR dimer-containing complexes are indicated to the left.…”

Effects of fenofibrate on plasma and hepatic transaminase activities and hepatic transaminase gene expression in rats

“…Taking the functions of transaminases into account, transaminase synthesis must be altered as well as for other enzymes involved in the amino acid/ glucose metabolism pathways if hormonal or nutritional fluctuation, which leads to acceleration or deceleration of gluconeogenesis, occurs. In fact, the expression of the rat cytosolic AST gene in the hepatoma cell line Fao is modified by glucocorticoids, insulin and cAMP (Aggerbeck et al, 1993;Barouki et al, 1989) and by a protein-rich diet or during prolonged fasting (Horio et al, 1988). Further, ALT and AST levels in the serum and organs are increased by these hormonal and nutrition- et al, 1966;Ramesh and Pugalendi, 2005;Rosen et al, 1959; Hoffman et al, 1989).…”

The repression of nuclear factor I/CCAAT transcription factor (NFI/CTF) transactivating domain by oxidative stress is mediated by a critical cysteine (Cys-427)

“…They were derived from the pGal(399-499), pGalOct2 and pGalSp1 plasmids (described in [9]) in which the simian virus 40 (SV40) promoters have been replaced by Rous sarcoma virus (RSV) promoters because the SV40 promoter is very sensitive to oxidative stress whereas that of RSV is not [4]. Amino acid point mutations in the TAD of NFI\CTF were produced using a double PCR technique with mutated oligonucleotides [13].…”