The stability of transferrin receptor (TfR) mRNA is regulated by iron availability. When a human plasma‐cytoma cell line (ARH‐77) is treated with an iron source (hemin), the TfR mRNA is destabilized and a shorter TfR RNA appears. A similar phenomenon is also observed in mouse fibroblasts expressing a previously characterized iron‐regulated human TfR mRNA (TRS‐1). In contrast, mouse cells expressing a constitutively unstable human TfR mRNA (TRS‐4) display the shorter RNA irrespective of iron treatment. These shorter RNAs found in both the hemin‐treated ARH‐77 cells and in the mouse fibroblasts are shown to be the result of a truncation within the 3′ untranslated regions of the mRNAs. The truncated RNA is generated by an endonuclease, as most clearly evidenced by the detection of the matching 3′ endonuclease product. The cleavage site of the human TfR mRNA in the mouse fibroblasts has been mapped to single nucleotide resolution to a single‐stranded region near one of the iron‐responsive elements contained in the 3′ UTR. Site‐directed mutagenesis demonstrates that the sequence surrounding the mapped endonuclease cleavage site is required for both iron‐regulated mRNA turnover and generation of the truncated degradation intermediate. The TfR mRNA does not undergo poly(A) tail shortening prior to rapid degradation since the length of the poly(A) tail does not decrease during iron‐induced destabilization. Moreover, the 3′ endonuclease cleavage product is apparently polyadenylated to the same extent as the full‐length mRNA.

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A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)

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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