Cell type-specific induction of cyclin D and cyclin-dependent kinase inhibitor p27(kip1) expression by estrogen in rat endometrium.

Cyclins, cyclin-dependent kinases (CDKs) and the CDK inhibitor p27(kip1) are known to be involved in the regulation of G(1)/S phase transition by estrogen in the rodent endometrium. Little is known, however, of the cell-specific location and regulation of these proteins during this process, or the way they mediate the differential effect of estrogen in the epithelium and stroma of the endometrium. Here we studied the cell-specific regulation of D-type cyclin (D(1-3)), of cyclin A and E, of CDK(2) and p27(kip1) by 17beta-estradiol in the endometrium of ovariectomized rats. Time-course changes in these proteins in the endometrium of ovariectomized rats were examined by immunohistochemistry at 2, 4, 8, 12, 20, 28 and 32 h after estrogen stimulation. The expression of proliferation cell nuclear antigen (PCNA) was also studied as a marker of proliferating cells. As expected from previous studies, all the proteins investigated were up-regulated by estrogen, with peak times from 8 to 32 h. The induction of cyclin D(1) is predominant in the glandular epithelium, whereas cyclin D(3) increases mainly in the luminal epithelium. The up-regulation of p27(kip1) is restricted to stromal cells with a 'gradient-like' expression pattern, in which the sub-epithelial (functional) layer showed stronger staining than the basal layer. The differential regulation of cyclins and p27(kip1) in the epithelium and stroma of the endometrium appear indicative of distinct actions of estrogen in different cell types in the uterus, as D-type cyclins mediate the proliferative effect of estrogen in epithelial cells while p27(kip1) might help prevent the same effect in the stroma.

Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1.

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G1 phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.

Estrogens do not modify MAP kinase-dependent nuclear signaling during stimulation of early G(1) progression in human breast cancer cells.

Estrogens are direct mitogens for hormone-responsive human breast cancercells, where they promote cell cycle progression and induce transcriptional activation of "immediate early" and cyclin genes. Nongenomic signaling by estrogens, including rapid changes of mitogen-activated protein(MAP) kinase and other signal-transduction-cascades activity, has been proposed to be essential for the mitogenic actions of these hormones and their nuclear receptors. Because regulation of gene transcription is considered a key step in cell cycle control by mitogenic protein kinase cascades, here we investigated the possibility that estrogen might induce the activation of extracellular signal-regulated kinase (Erk) 1/2-, c-Jun NH(2)-terminal kinase-, p38- or protein kinase A-responsive transcription factors in the cell nucleus during stimulation of early G(1) progression, a timing coincident with the maximum effects of these hormones on such enzyme activity. No significant changes in protein kinase-mediated transcription factor activity could be detected here after estrogen stimulation of either MCF-7 or ZR-75.1 cells. Furthermore, these steroids were able to induce activation of the human CCND1 gene promoter, accumulation of cyclin D1 and pRb phosphorylation, all key events in cell cycle stimulation by mitogens, even in the presence of Erk1/2 activation blockade by a MAP kinase-activating kinase (Mek)1/2 inhibitor. Thus, estrogens do not appear to convey significant protein kinase-dependent signaling to the cell nucleus during the early phases of human breast cancer cell stimulation. Furthermore, hormonal regulation of G(1) gene transcription can occur even without additional activation of the Mek-Erk1/2 pathway by estrogen receptors.

The antiestrogen ICI 182,780 inhibits proliferation of human breast cancer cells by interfering with multiple, sequential estrogen-regulated processes required for cell cycle completion.

Antiestrogens are widely used for breast cancer treatment, where they act primarily by inhibiting the mitogenic action of estrogens on tumor cells. The effects of the pure antiestrogen ICI 182,780 on estrogen-regulated cell cycle phase-specific events were investigated here in synchronously cycling human breast cancer (HBC) cells. In early G(1)-arrested MCF-7 or ZR-75.1 cells, 17beta-estradiol (E2) induces rapid activation of the cyclin/Cdk/pRb pathway, as demonstrated by D-type G(1) cyclins accumulation during the first few hours of hormonal stimulation, followed by sequential accumulation of E, A and B1 cyclins and progressive pRb phosphorylation, as cells progress through the cell cycle. When added to quiescent cells together with E2, ICI 182,780 prevents all of the above hormonal effects. Interestingly, in mid-G(1) cells (2-8 h into estrogen stimulation) the antiestrogen causes rapid reversal of hormone-induced D-type cyclins accumulation and pRb phosphorylation, and still fully inhibits G(1)-S transition rate, while in late-G(1) cells it does not prevent S phase entry but still inhibits significantly DNA synthesis rate, S-phase cyclins accumulation and pRb hyperphosphorylation. These results indicate that pure antiestrogens prevent multiple estrogen-induced cell cycle-regulatory events, each timed to allow efficient G(1) completion, G(1)-S transition, DNA synthesis and cell cycle completion.

Ketoconazole inhibits lipopolysaccharide-induced activation of the nitric oxide synthase gene in the murine macrophage cell line J774.

The aim of this study was to determine whether ketoconazole can affect the expression of the nitric oxide (NO) synthase gene in the murine macrophage cell line J774. The inducible enzyme (i-NOS) is activated in murine macrophages by LPS and cytokines. Exposure of the J774 cell line to ketoconazole for 24 h did not induce any NO release. Cells preincubated with ketoconazole and treated with LPS showed a significant decrease in nitrite levels in the culture medium, compared with controls (cells treated with LPS alone). The addition of 1 mM N-monomethyl-L-arginine (L-NMMA), a structural analogue of arginine, reduced nitrite levels by about 88+/-9.2% in cells treated with LPS alone, whereas in those treated with ketoconazole + LPS, the levels were comparable to the baseline values detected in control cells. Northern blotting, used to assess i-NOS mRNA expression in the J774 cells, showed that ketoconazole reduced the LPS-induced increase in i-NOS mRNA activation by about 50%. These results support another mechanism for the antiinflammatory effect of ketoconazole (i.e. reduction in i-NOS gene expression and consequently inhibition of reactive radical NO production), that may explain the antierythema and antiedema action of this compound, besides its antimycotic effects.

Constitutive overexpression of cyclin D1 does not prevent inhibition of hormone-responsive human breast cancer cell growth by antiestrogens.

Cyclin D1 is a target for positive regulation by estrogens in growth-responsive cells, in which it mediates their mitogenic effects. Amplification and overexpression of the cyclin D1 gene (CCND1) might thus represent a genetic lesion inducing hormone-independent growth of transformed cells. Indeed, cyclin D1 overexpression has been found in up to 50% of primary breast cancers, and in about one-third of these cases, this is linked to amplification of the 11q13 chromosomal region, which also includes the CCND1 gene. These tumors are predominantly estrogen receptor-positive, and for this reason, these patients are often selected for adjuvant antiestrogen therapy. No information is available, however, as to whether cyclin D1 overexpression due to gene amplification might interfere with and reduce antiestrogen efficacy. This was investigated here by taking advantage of an experimental model that reproduces cyclin D1 overexpression resulting from increased CCND1 gene dosage in hormone-responsive human breast cancer cells. For this, MCF-7 cells stably transfected with a tet-inducible cyclin D1 expression vector were tested for their in vitro response to steroidal (ICI 182,780) and nonsteroidal (trans-4-hydroxytamoxifen) antiestrogens under condition of low (endogenous only) or high (exogenous) cyclin D1 levels. Results show that although cyclin D1 overexpression seems to interfere with the early cell cycle effects of antiestrogens, it does not prevent their cytostatic actions, so that growth of cyclin-overexpressing MCF-7 cells is still efficiently inhibited in vitro by these drugs.

Estrogen induces early and timed activation of cyclin-dependent kinases 4, 5, and 6 and increases cyclin messenger ribonucleic acid expression in rat uterus.

Cyclin-dependent kinases (cdks) are serine-threonine protein kinases that play a key role in the regulation of the mitotic cycle, in transcription initiation, and in the control of specific metabolic pathways in eukaryotic cells. cdk activity is controlled via phosphode-phosphorylation of the catalytic subunits of these enzymes and their physical association with cyclins and cdk inhibitors. In adult rats, estrogen stimulation results in massive proliferation of endometrial epithelial cells, accompanied by functional and structural modifications in all other tissue components of the uterus. We report here that administration of 17 beta-estradiol (E2) to adult ovariectomized rats induces within the first 25 h significant activation of cdk 4, 5, and 6, but not cdk 2, in the uterus, accompanied by increased expression of D-type (D1-3), A and E cyclin messenger RNAs (mRNAs). Furthermore, expression of the cdk inhibitor p27Kip1, a key regulator of uterine functions, is induced by E2 in this organ. Analysis of RNA extracted from E2-stimulated rat endometria shows early accumulation of D1 and D3, but not D2, cyclin mRNA, preceded by transient accumulation of c-fos mRNA. These results indicate an involvement of cdks and cyclins in estrogen actions in adult rat uterus and suggest that cyclins D1 and D3 are part of the molecular pathway that allows hormonal regulation of G1 progression in endometrial cells.

Inhibition of inducible nitric oxide synthase gene expression by glucocorticoid-induced protein(s) in lipopolysaccharide-stimulated J774 cells.

Glucocorticoids inhibit inducible-type NO synthase activity in a variety of cell types. We report here that proteins recovered from the medium of dexamethasone-treated J774 macrophages (1, 10, 100 microg/ml) inhibited lipopolysaccharide-stimulated nitrite generation by 10.0 +/- 3.0%, 32.3 +/- 5.3% and 55.0 +/- 6.0%, respectively, and inducible NO synthase mRNA expression in these cells. Immunoblotting analysis of crude and partially purified glucocorticoid-induced proteins with an anti-lipocortin-1 polyclonal antiserum revealed the presence of lipocortin-1-like immunoreactive species with a molecular mass of 35-37 kDa. Furthermore, inhibition of lipopolysaccharide-induced nitrite production by glucocorticoid-induced proteins in J774 cells was reversed by addition of anti-lipocortin-1 neutralizing polyclonal antibody (1:60 dilution; 4 h before lipopolysaccharide). Comparison of glucocorticoid-induced proteins inhibition of both nitrite production and inducible NO synthase mRNA expression suggests that these effects result mainly from inhibition of lipopolysaccharide-mediated inducible NO synthase gene expression. These results indicate that negative regulation of inducible NO synthase by glucocorticoids is, at least in part, mediated by glucocorticoid-induced proteins that involve also members of the lipocortin-like superfamily.

17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells.

MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.

Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-gamma, bacterial lipopolysaccharide and NG-monomethyl-L-arginine.

Cytokines and bacterial lipopolysaccharides (LPSs) stimulate nitric oxide production in macrophages by inducing transcription of the gene coding for the inducible isoform of nitric oxide synthase (iNOS). We have cloned the mouse iNOS gene promoter and analysed its structural features and its response to interferon-gamma (IFN-gamma) and Escherichia coli LPS in RAW 264.7 mouse macrophage-like cells. Transcription of a recombinant reporter gene including the promoter and 4 kb of its 5'-flanking DNA, linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, is stimulated by IFN-gamma and, more efficiently, by LPS upon transient transfection in RAW 264.7 cells. Two upstream DNA regions are required for maximal promoter activation of LPS: the first maps between positions -1541 and -775 and the other between -420 and -47, with respect to the major transcriptional start site of the iNOS gene. The upstream-most region also mediates promoter trans-activation by IFN-gamma. As reported earlier for transcription of the endogenous iNOS gene, combined stimulation of RAW 264.7 cells with IFN-gamma and LPS results in lower activation of the transfected promoter, when compared with LPS alone. NG-Monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase activity, enhances iNOS gene mRNA induction and promoter activation by IFN-gamma and LPS, indicating that nitric oxide can influence negatively the reponsiveness of this gene to inducers. These results suggest the possibility of a negative regulatory feedback exerted by iNOS on the transcriptional activation of its own gene.

Stimulation of human breast cancer MCF-7 cells with estrogen prevents cell cycle arrest by HMG-CoA reductase inhibitors.

Inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, such as Simvastatin and Lovastatin, reduce the rate of DNA synthesis and proliferation of a wide variety of cell types in vitro, by inducing a cell cycle arrest in G1. In estrogen-free medium, DNA synthesis is reduced by more that 90% following exposure of normal and transformed human breast epithelia] cells to 20 microM Simvastatin or Lovastatin for 24 to 42 hrs. We show here that stimulation of estrogen responsive MCF-7 cells with nanomolar concentrations of 17beta-estradiol (E2) prevents inhibition of DNA synthesis by these compounds. The effect of the hormone is antagonized by both steroidal and non steroidal antiestrogens, and it is not detectable in estrogen receptor-negative MCF-10a cells. Cell cycle analysis demonstrates that HMG-CoA reductase inhibitors are unable to induce G1 arrest of MCF-7 cells in the presence of E2.

17 beta-Estradiol overcomes a G1 block induced by HMG-CoA reductase inhibitors and fosters cell cycle progression without inducing ERK-1 and -2 MAP kinases activation.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

In vivo functional analysis of the mouse estrogen receptor gene promoter: a transgenic mouse model to study tissue-specific and developmental regulation of estrogen receptor gene transcription.

Understanding the molecular and morphological basis of estrogen responsiveness in the various tissues and organs that make up an adult organism and its onset during ontogenesis requires identification of the genetic controls that determine timed expression of the estrogen receptor (ER) gene in multiple cell types. With this goal in mind, we describe here the results of the functional analysis of the mouse (m) ER gene promoter, carried out in vivo in transgenic mice. The mER gene promoter was cloned and spliced to the coding sequence of the bacterial lacZ gene (fused to the nuclear localization signal of SV40 large T: nls-beta-GAL) and then stably reintegrated into the genome of mice. Analysis of beta-GAL mRNA and protein expression in multiple organs of both female and male transgenic animals was then performed. Results show that the transgenic mER promoter, much like the endogenous one, is active in several organs and tissues of adult female and male mice. The first 0.4 kilobases of 5'-flanking DNA (up to -364) are sufficient to direct widespread expression of the transgene in mouse organs. This indicates that genetic elements functional in various cell types are included in this segment. Furthermore, the first exon and intron of the mER gene are necessary to achieve sexually dimorphic expression of the transgene in neurons located at specific sites within the central nervous system. These mER promoter transgenic mice will be useful in mapping estrogen- responsive cell types under different physiological and pathological conditions in vivo, in defining ontogenesis of estrogen action in the mouse, and in studying the mechanisms that regulate ER gene transcription.

Dual mechanism for the control of inducible-type NO synthase gene expression in macrophages during activation by interferon-gamma and bacterial lipopolysaccharide. Transcriptional and post-transcriptional regulation.

Production of nitric oxide (NO) by macrophages is enhanced upon activation by bacterial endotoxins and cytokines mainly via an increase of the intracellular content of the inducible isoform of nitric oxide synthase (i-NOS). We have studied in detail the effect of several modulators of macrophage activity on steady state levels of i-NOS mRNA in the mouse macrophage-like cell line RAW 264.7. Bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) were found to be effective inducers of i-NOS mRNA, in accordance with their known ability to stimulate both i-NOS activity and NO production in macrophages from different sources, while TNF-alpha, IL-1, or IL-6 was ineffective in this regard. Accumulation of i-NOS mRNA in response to either LPS or IFN-gamma stimulation was accompanied by increased i-NOS gene transcription, as detected both by using a nuclear "run-on" transcription assay and by transient transfection of the cloned gene promoter in RAW 264.7 cells. Co-stimulation of the cells with both inducers resulted in higher steady state levels of i-NOS mRNA in the absence, however, of a corresponding potentiation of the rate of gene transcription. This was due primarily to a considerable effect of LPS on i-NOS mRNA stability, with prolongation of its half-life from 1-1.5 h, in the presence of IFN-gamma alone, to 4-6 h in the presence of both LPS and IFN-gamma.

Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription.

Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.

Identification of a specific pattern of "immediate-early" gene activation induced by estrogen during mitogenic stimulation of rat uterine cells.

Estrogen hormones are potent mitogens for certain target tissues, where they stimulate cell growth by inducing recruitment of quiescent cells in cycle and by fostering cell cycle progression. To define the molecular bases of the mitogenic action of these steroid hormones, the pattern of "immediate-early" gene expression was monitored during the early phases of estrogen stimulation of rat uterine cells in vivo. Nuclear run-on transcription and/or Northern blot RNA analysis indicate that c-jun, junB, jun-D, c-fos, TIS 1 (also called NGFI-B or nur/77) and TIS 8 (zif-268, krox24, egr-1, or NGFI-A) genes are all transiently activated in the uterus (up to 20-fold) within 30-120 min after treatment of adult ovariectomized rats with a mitogenic dose of 17b-estradiol. Conversely, JE gene mRNA accumulates progressively in estrogen-stimulated uterine cells, whereas TIS 11 and 21 genes are only slightly responsive to the hormone (less than twofold induction) and fos B,fra-1,fra-2,krox20 (egr-2), TIS 7 and 10, KC, and c-rel mRNAs are undetectable in rat uterus either before or after estrogen treatment. Stimulation in the presence of cycloheximide shows that only c-jun, jun-D, c-fos, and JE gene activations are primary responses to the hormone in rat uterine cells. These findings establish the direct mitogenic role of estrogen and identify for the first time a specific genetic program activated by these steroid hormones during stimulation of target cell proliferation. Furthermore, since most of the activated genes encode for transcription factors, these results enable us to envision how the mitogenic signal transmitted by the hormone can be elaborated and amplified within target cells by the products of estrogen-responsive genes, leading to a cascade of growth-dependent gene regulation, cell cycle progression, and, ultimately, cell division.

Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells.

Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus.

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.

Estrogen stimulates transcription of c-jun protooncogene.

Estrogen is a mitogen for the rat uterus, where it induces transient activation of c-fos and c-myc protooncogene expression, followed by increases in DNA synthesis and cell proliferation. JUN-C, the product of the c-jun protooncogene, is a nuclear protein that can interact with FOS to modulate the activity of AP-1-responsive promoters. To test whether c-jun is a target for estrogen regulation, we measured the effects of 17 beta-estradiol on the expression of this gene in rat uterus. A human c-jun cDNA probe detects in rat uterus two mRNA species of 2.5 and 3.2 kilobases. Treatment of the animals with estrogen results in a rapid transient increase in the concentrations of these mRNAs; a 4- to 5-fold increase over the prestimulation level was detected starting 30 min after estrogen injection and lasting for 2 h, with a return to the prestimulation level after 4 h. In accordance with the results obtained by analysis of the mRNA, we found that estrogen increases 3- to 4-fold c-jun gene transcription in the uterus, at the same time it induces its mRNA accumulation. The ability of estrogen to induce c-jun gene expression was not abolished by the protein synthesis inhibitor cycloheximide, suggesting that transcriptional activation of this protooncogene is a primary response to the hormone. Furthermore, we found that in the estrogen-responsive MCF-7 human mammary carcinoma cells, estrogen stimulates transcription of a reporter gene containing four copies of a jun/AP-1 response element. These data demonstrate that c-jun gene expression is regulated by estrogen and suggest that JUN-C could play a role in the activation of cell proliferation by estrogen.