Estrogen-modulated Estrogen Receptor·Pit-1 Protein Complex Formation and Prolactin Gene Activation Require Novel Protein Synthesis

Abstract

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER⅐Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER⅐Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER⅐Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER⅐Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER⅐Pit-1 protein complex formation in a dosedependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.The tissue-specific expression of the rat prolactin (PRL) 1 gene in the anterior pituitary gland is regulated by the synergistic action of two upstream regulatory elements: the distal enhancer and the proximal promoter (1, 2). Complex binding sites for trans-acting factors within these elements control tissue-specific expression and transcription efficiency (3, 4). A pituitary cell-specific transcription factor, Pit-1, a member of the POU domain family of transcription factors, binds to multiple sites in these elements and is required for the tissuespecific expression of rat PRL (5). Gene dysfunction analysis has shown not only that failure to express the PRL gene accounts for genetically dwarfed mice, but also that Pit-1 function is linked to transcriptional activation of the rat PRL gene in the anterior pituitary (6). Pit-1 is also involved in normal pituitary development and the proliferation of specific anterior pituitary cell types such as lactotrophs and somatotrophs (7).The Pit-1 protein by itself, however, is not sufficient for the tissue-specific expression of the rat PRL gene. The promoter activity of the rat PRL gene strongly depends on the synergistic interactions between Pit-1 and other promoter-specific transcription factors, including the thyroid hormone receptor, CAAT/enhancer-binding protein-␣, Ets-1, and c-Jun (8 -10). Moreover, rat PRL gene expression is regulated by the steroid hormone estrogen at the level of transcription (11). Evidence showed that estrogen receptor-␣ (ER-␣), which exhibits affinity for binding sites in the distal enhancer element of the rat PRL gene, synergizes with the Pit-1 protein to permit activation of the distal enhancer in a ligand-depen...

Transcriptional activation of heat shock protein 27 gene expression by 17beta-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists

“…ER interacts with several nuclear proteins that modulate ER action, and these include multiple coactivators that primarily interact with the AF2 domain of ER (Glass et al 1997, Zwijsen et al 1997, Lemon & Freedman 1999, McKenna et al 1999, Johansson et al 2000, Resnick et al 2000, Ying & Lin 2000. For example, both ER -AP1 and ER -Sp1 are ligand-activated transcription factor complexes in which ER does not directly bind DNA but interacts with another DNA-bound transcription factor.…”

Effects of Hormones Targeting Nuclear Receptors on Transcriptional Regulation of the Growth Hormone Gene in the MtT/S Rat Somatotrope Cell Line

“…Interestingly, two Pit-1-binding sites are located within the promoter (–123/–116 and –88/–81), and further deletion virtually abolished basal transcriptional activity in the MtT/S [5] as well as in the GC cell line [37]. Thus, ER might influence the GH gene transcription through protein-protein interaction between Pit-1 and ER, because the role of the ER/Pit-1 complex has recently been shown in the prolactin gene promoter [38, 39]. Alternatively, E 2 may increase expression of an undetermined protein, which in turn exerted a positive effect on GH gene expression, because the effect of estrogens on GH gene transcription had a relatively late onset (>24 h).…”

The effects of glutamate can be attenuated by estradiol via estrogen receptor dependent pathway in rat adrenal pheochromocytoma cells

“…It thus indicated that the possibility of ER-alpha involved in the estrogen-induced protection in PC12 cells. However, some studies found that higher levels of ER-alpha were needed to be introduced into PC12 cells by various means including transfection of ER-alpha cDNA in order to study the effects of estrogen [29,45]. To elucidate the potential involvement of ER-beta, the recently identified ER subtype, we measured the levels of ER-beta in PC12 cells via RT-PCR and found little expression.…”

“…Its mechanism of action appears to be complex and is mediated through either ER-dependent or ER-independent pathways. Via the ER-dependent pathway, estrogen binds to ERs and regulates the transcription of target genes, including genes involved in cell growth, survival and differentiation, such as the expression of anti-apoptotic genes Bcl-XL and Bcl-2 and pro-apoptotic genes BAD and c-jun [5,29,43]. Estrogen can also function via ER-independent pathway by interacting with MAPK and PI3-K signaling cascades, bind to NMDA receptors and act as an antioxidant without the involvement of transcriptional regulation [6,12,14].…”

“…The RT reaction was performed using 0.1-0.2 lg of total RNA in a 20-ll reaction mixture, and a portion of the RT product containing an equal amount of cDNA from each sample was then subjected to PCR. The PCR reaction mixture contained 2 ll of 10X PCR buffer, 1.6 ll of 50 lM MgCl 2 , 11.7 ll of diethylpyrocarbonate-treated water, 1.5 ll of RT-reaction mixture, 1 ll of 10 mM dNTPs, l unit of Taq DNA polymerase and 2 ll of test primers, including primers for Bcl-XL, BAD, BAX and c-jun [29] or ribosomal protein L19 (RPL19) primers (5¢-CTGAAGGTCAAAGGGAATGTG-3¢ and 5¢-GGACAGAGTCTTGAT-GATCTC-3¢; 125 lg/ ml). RPL19 is constitutively expressed and used as a control for the RT-PCR reaction [30].…”