Hydroxytamoxifen induces a rapid and irreversible inactivation of an estrogenic response in an MCF-7-derived cell line.

The MVLN cell line was established in our laboratory from MCF-7 cells by stable transfection with the luciferase gene under the control of an estrogen-responsive element from the Xenopus vitellogenin A2 gene. This cell line allowed us to visualize the induction by hydroxytamoxifen of a heterogeneity in the cell population with regard to the expression of the luciferase gene. Treated cells lost their estradiol-inducible luciferase activity, progressively and irreversibly; the luciferase expression of 80% of the cells was irreversibly inactivated by a 12-day hydroxytamoxifen treatment. We showed that this inactivation process was specific for an estrogenic response and was mediated by the estrogen receptor. Tamoxifen itself gave rise to such an inactivation, whereas other compounds belonging to the triphenylethylenic family but differently substituted on the ethylenic carbon and the ICI 164,384 compound were not as efficient. This irreversible inactivation was accompanied by a sharp decrease in the luciferase mRNA level; however, the estrogen receptor function and the cellular transcriptional machinery were not affected by the treatment. Although this antiestrogen treatment neither affected the estrogen-dependent cell growth nor irreversibly inhibited the expression of the natural pS2 gene, these results highly suggest that long-term antiestrogen therapy may lead to some heterogeneity in tumor cells throughout the course of patient treatment.

A novel growth hormone response element unrelated to STAT (signal transducer and activator of transcription)-binding sites is a bifunctional enhancer.

Regulation of gene expression by GH has so far been shown to be mediated by a few cis-acting elements, most of which are signal transducer and activator of transcription (STAT)-binding sites. Here we have characterized a novel GH-response element present in the promoter of rat serine protease inhibitor (spi) genes. It consists of a 13 nucleotide-long GAGA box containing two GAGGAG repeats separated by a G, structurally unrelated to STAT-binding sites. In hepatocytes, the spi GAGA box behaves as a position-dependent bifunctional enhancer controlling basal and GH-dependent transcription. In addition, spi GAGA box oligonucleotides inhibit cell-free transcription driven by GAGA box-containing as well as GAGA box-less promoters, suggesting that the spi GAGA box interacts directly or indirectly with component(s) of the basic transcriptional machinery. Mobility shift assays showed that this GAGA box is specifically recognized by nuclear factors that are unrelated to previously characterized proteins binding to purine-rich elements or to GH-activated STATs. Finally, experiments performed with cells expressing wild type, truncated, or mutated forms of the GH receptor indicate that protein kinase Janus kinase 2 is involved in the GH-dependent activation of the spi GAGA box. These studies reveal the existence of an as yet unidentified Janus kinase-2-dependent, STAT-independent pathway in GH activation of gene expression.

Transcription of the rat serine protease inhibitor 2.1 gene in vivo: correlation with GAGA box promoter occupancy and mechanism of cytokine-mediated down-regulation.

Two GH-response elements (GHREs) and a single glucocorticoid (GC)-response element were found to regulate activity of the rat serine protease inhibitor 2.1 gene (spi 2.1) promoter in vitro. To assess the physiological relevance of these observations, we have investigated the relationship existing between the level of spi 2.1 gene transcription, structural modifications of the chromatin, and in vivo nuclear protein-promoter interactions monitored by genomic footprinting, in control, hypophysectomized, and inflamed rats. We also addressed the mechanism of inflammation-mediated gene down-regulation. We found that a high level of spi 2.1 gene transcription correlates with hypersensitivity of the promoter to deoxyribonuclease I (DNase I) and maximal occupancy of the GAGA box (GHRE-I). The failure of GAGA-box binding proteins (GAGA-BPs) to interact with the GAGA box appears to result from an impairment in GH action due to its absence (i.e. hypophysectomized animals) or to the appearance of a cytokine-mediated GH-resistant state (i.e. inflamed rats) in liver. Unlike the GAGA box, signal transducer and activator of transcription (STAT) factor-binding sites included in the GHRE-II were never found to be protected against DNase I attack but displayed a differential DNase I reactivity depending on the level of gene transcription. Alterations in DNase I reactivity of the GC-response element region suggest that GC receptor-GC complexes may associate, in a transient manner, with the promoter in the actively transcribing control state. Taken together, our studies suggest a mechanism of spi 2.1 gene activation in vivo whereby the GH-dependent chromatin remodeling caused by or concomitant to the recruitment of GAGA-box binding proteins is the first compulsory and presumably predominant step.

Characterization of three transcriptional repressor sites within the 3' untranslated region of the rat serine protease inhibitor 2.3 gene.

The activity of the rat serine protease inhibitor 2.3 gene (spi 2.3) is controlled by several positive promoter elements [Simar-Blanchet, A.-E., Paul, C., Mercier, L. & Le Cam, A. (1996) Eur. J. Biochem. 236, 638-648] and a negative element located in the 3' untranslated gene region (3' UTR) [Le Cam, A. & Legraverend, C. (1996) Eur. J. Biochem. 231, 620-627]. In the present studies, we dissected the 348-bp spi 2.3 3' UTR silencer to precisely define repressor sites and look for specifically interacting proteins. Three short elements referred to as A (nucleotides 1751-1776 in the cDNA), B (nucleotides 1812-1827) and C (nucleotides 1958-1974) sites repressed transcription from the homologous spi 2.3 promoter as well as from a heterologous minimal promoter containing the spi GAGA box enhancer. All three sites harbor a (TTTC) motif whose mutation affected silencer activity that was also dependent on flanking sequences. Those sites share the (TTTC) motif and a CCAAT/enhancer-binding-protein(C/EBP)-binding site with a fatty-acid-binding-protein gene promoter element shown to interact specifically with a transcriptional repressor [He, G. P., Muise, A., Wu Li, A. & Ro, H.-S. (1995) Nature 378, 92-96]. This repressor is however unlikely to mediate spi 2.3 3' UTR silencer action since it was not detected in rat hepatocytes. In vitro footprinting of the spi 2.3 3' UTR silencer region revealed a strong interaction with liver nuclear proteins. Among the six identified footprints, three of them (F-II, FIII and F-IV) bound C/EBPs and mapped in regions harboring the repressor function. Binding of C/EBPs to all three spi 2.3 3' UTR repressor sites, although rather weak, was confirmed by electrophoretic mobility shift assays that otherwise failed to reveal specific interactions with other liver nuclear proteins in vitro. However, none of the most largely liver expressed C/EBP species (i.e. alpha, beta and delta) activated the spi 2.3 3' UTR silencer function in NIH 3T3 cells, suggesting that binding of those transcription factors did not mediate the transcriptional repression.

Regulation of expression of the rat serine protease inhibitor 2.3 gene by glucocorticoids and interleukin-6. A complex and unusual interplay between positive and negative cis-acting elements.

The rat serine protease inhibitor 2.3 gene (spi 2.3) is almost completely silent in normal animals and is transiently expressed during acute inflammation. It encodes a potential anti-elastase which is likely to play a major physiological role for the host defense. Two well-known inflammatory mediators, glucocorticoids and interleukin-6 (IL-6) activate the spi 2.3 promoter and increase steady-state levels of mRNA in cultured hepatocytes. GC activation is mediated by a single glucocorticoid-response element which seems to act autonomously. A unique array of four functional IL-6-response sites was identified in the spi 2.3 promoter. Three of them (C-II--IV) bear structural identity to the CCAAT/enhancer-binding-protein-binding site consensus sequence, whereas the fourth closely resembles the consensus kappa B nuclear factor recognition motif. The C-IV element, which is the most active, contains the motif 5'-CTGGGA and binds the IL-6-inducible acute-phase response factor present in liver nuclear extracts from inflamed rats. Both basal and IL-6-dependent activities of each individual cytokine-response element tested separately are strongly down regulated by a recently identified regulatory sequence, located in the 3' untranslated region of the spi 2.3 gene. However, this repressor element does not significantly affect overall IL-6-dependent spi 2.3 promoter activity. This suggests that, in the context of the active gene in vivo, all four IL-6-response sites, which are largely redundant, cooperate to overcome the strong repressive effect of the 3' untranslated region silencer and are needed to bring about a maximal IL-6 response. These data reveal a novel type of regulation of an acute-phase gene involving different classes of IL-6-response elements controlled by a repressor and acting in conjunction with a glucocorticoid-response element.