Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription.

Abstract

In this report, we describe apolipoprotein II (apoll) gene expression in cell lines derived by stable expression of the chicken estrogen receptor in LMH chicken hepatoma cells. In cell lines expressing high levels of receptor (LMH/2A), apoll gene expression is increased by estrogen 300-fold compared with levels in the receptordeficient parent LMH line. LMH/2A cells show apoll mRNA induction and turnover kinetics similar to those in chicken liver. Inhibition of protein synthesis with cycloheximide (CHX) or puromycin following estrogen withdrawal superinduces apoll mRNA without affecting apoll mRNA stability. Superinduction is due to an estrogen-independent reactivation of apoll gene transcription. The apoll gene can be reactivated by CHX for up to 24 h following hormone withdrawal, suggesting that the gene is in a repressed yet transcriptionally competent state. These results reveal two distinct events necessary for termination of estrogen receptormediated transcription. The first event, removal of hormone, is sufficient to stop transcription when translation is ongoing. The second event is revealed by the CHX-induced superinduction of apoll mRNA following hormone withdrawal. This superinduction suggests that deactivation of estrogen receptor-mediated transcription requires a labile protein. Furthermore, reactivation of apoll gene expression by CHX and estrogen is additive, suggesting that estrogen is unable to overcome repression completely. Thus, a labile protein may act to repress estrogen receptor-mediated transcription of the apoll gene.In avian liver, estrogens regulate the expression of a number of egg yolk precursor proteins required for the transport of nutrients to the developing oocyte (5). Among the proteins regulated by estrogen are vitellogenin I (VTGI), VTGII, and VTGIII (63, 64), apolipoprotein B (apoB) (17) and apoll (18) of very low density lipoprotein, and riboflavin-and biotinbinding proteins (16,66). The primary action of estrogen is to increase transcription of the genes encoding these proteins via binding to the estrogen receptor. In addition to regulating transcription, estrogen also alters the cytoplasmic stability of apoll and VTGII mRNAs (30,71). Analysis of the mechanisms of transcriptional regulation of these genes as well as the study of mRNA turnover in avian liver cells has been hampered by the absence of a suitable homologous cell line. Studies of gene activation and estrogen-dependent alterations in chromatin structure and DNA methylation patterns have been restricted to animal studies or tissue homogenates (2,3,14,15,38,42,67,68

Selective estrogen receptor α activation disrupts sex organ differentiation and induces expression of vitellogenin II and very low-density apolipoprotein II in Japanese quail embryos

“…Induction of apoVLDL II and VTG II through ERa agrees with in vitro studies showing that ERa is a more potent activator of transcription from the consensus Xenopus VTG A2 estrogen response element (GGTCAnnnTGACC; identical in quail) than ERb (Loven et al 2001). Furthermore, apoVLDL II gene expression is increased 300-fold in chicken hepatoma cells transfected with chicken ERa compared with receptor-deficient cells (Sensel et al 1994). By contrast, a recent study suggests that VTG expression in rainbow trout is induced through ERb and not ERa.…”

Interaction of proteins with transcriptionally active estrogen receptors.

“…Nevertheless, while the RIPs we detected bind to the ER with similar characteristics, they may not all serve the same function. It is possible, for example, that some play a role in receptor deactivation, a process that appears to require labile proteins (31). The regulation of RIP140 and RIP80 expression by estrogens in ZR75-1 cells would be consistent with such a role in repression of the activated receptor, as a negative feedback control mechanism.…”

Positive and Negative Regulation of Chicken Anemia Virus Transcription

“…All cell types had detectable ER mRNA, although there was very little in DF-1 cells. The LMH/2A cell line was derived from LMH cells stably transfected to overexpress the estrogen receptor (58), and this overexpression was confirmed by both RT-PCR and immunofluorescence (data not shown).…”

“…The chicken hepatocyte cell lines LMH (4) and LMH/2A were also obtained from the American Type Culture Collection. The LMH/2A cell line overexpresses ER 150 times compared with the parent LMH cell line (58). These cells were maintained in Waymouth's MB752/1 medium (Invitrogen) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 g/ml) and grown on 0.1% gelatin-coated flasks.…”

“…The following hormones were used (all from Sigma-Aldrich Corp.): 17␤-estradiol (E2, 10 nM to 1 M), dexamethasone (1 M), 9-cis-retinoic acid (10 nM), all-trans-retinoic acid (10 nM), 3,3Ј,5-triiodo-L-thyronine sodium salt (T3, 100 nM), progesterone (10 nM), and testosterone (10 nM). For LMH and LMH/2A cells, serum supplementation included 2% chicken serum as has been reported previously (4,31,58). Twelve-well plates were used for EGFP protein measurement by fluorometer, with each treatment run in triplicate wells and each assay repeated three times or as indicated in the figure legends.…”