Estrogen Stimulates Transcription of c-jun Protooncogene

Alessandro Weisz*, Luigi Cicatiello, Eliana Persicot, Marilina Scalonaf, and Francesco Bresciani Istituto di Patologia Generate e Oncologia Prima Facolta di Medicina e Chirurgia Universita di Napoli Napoli 1-80138, Italy

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 17/3-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. (Molecular Endocrinology 4: 1041-1050, 1990)

polypeptide growth factors. This led to the finding that cells respond to stimulation by growth factors with changes in the expression of specific genes, in particular the immediate early or competence genes that include the protooncogenes c-fos, c-myc, and c-myb (Refs. 1-3 and references therein). The induced gene products trigger a cascade of events that results in the progression through the cell cycle to the mitosis. Estrogen is a mitogen for the uterus of mammals, where it stimulates the growth of endometrial epithelia (4). Administration of estrogen to adult castrated rats increases cell proliferation in uterine epithelia by inducing the recruitment of quiescent (GO) cells into the cell cycle, while at the same time shortening the G1 and S phases (Ref. 5 and references therein, 6). Estrogen acts on endometrial cells as a growth-promoting factor; however, its mechanism of action differs greatly from that of polypeptide growth factors. The estrogen-receptor complex is a frans-acting transcription enhancer factor that binds to c/s-acting enhancer-like estrogen response elements (EREs) located within or near responsive genes to influence promoter activity (7-9). It is likely that estrogen, in growth-responsive cells, modulates the activity of genes whose products control the cell cycle. In line with this hypothesis, it was found that estrogen induces expression of c-fos and c-myc protooncogenes in both rat uterus and breast cancer cells in culture (10-15). The induction of c-fos gene expression by estrogen in rat uterus parallels the rate of formation of active estrogen-receptor complex and is not abolished by protein synthesis inhibitors, suggesting that transcriptional activation of c-fos is a primary direct response to the hormone (16, 17). FOS is a nuclear phosphoprotein that can associate in transcriptional complexes with JUN-C, the product of c-jun protooncogene (18, 19), to generate the composite transcription factor /t/n/AP-1 (20-22). The expression of cjun is generally regulated by the same growth-promoting factors that regulate the expression of c-fos (2327), suggesting that both products may be required to achieve the mitogenic effect. To test the effect of estrogen on c-jun gene expression, we measured its transcriptional activity and steady state mRNA concentration in the uterus of adult ovariectomized rats before and at different time intervals after ip injection of 17/3-

INTRODUCTION Molecular analysis of growth control in mammalian cells has largely focused on characterization of the cellular responses to growth-promoting factors, in particular 0888-8809/90/1041-1050S02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

1041

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Vol 4 No. 7

MOL ENDO-1990 1042

estradiol. We report that c-jun gene transcription is rapidly and transiently induced by estrogen. The effect of the hormone on this gene is resistant to the protein synthesis inhibitor cycloheximide, suggesting a direct transcriptional activation by the estrogen-receptor complex. The effect of estrogen was confined to proliferation-responsive cells of the uterus, since the c-jun mRNA concentration was unaffected in brain, heart, large intestine, kidney, liver, lung, muscle, and spleen of the same animals. Furthermore, estrogen activates transcription from a/tvn/AP-1 -responsive test gene containing four copies of the AP-1 motif from the a-domain of the polyoma virus enhancer (28, 29) cloned upstream of a globin promoter-chloramphenicol acetyl transferase (CAT) reporter gene. This test gene is responsive to the estrogen-receptor complex in estrogenresponsive MCF-7 human breast cancer cells, but not in estrogen-unresponsive HeLa cells.

-E,

0'

60'

90' 120' 240'

•••i. tt 1 *

c-jun c-fos

tubulin lamin C

_

30'

4,0

RESULTS Induction of c-jun mRNA by Estrogen in Rat Uterus The steady state concentration of c-jun mRNA was measured in uterine poly(A)+ RNA isolated from adult ovariectomized rats killed either before (0, -E 2 ) or at different times after ip injection of 17/3-estradiol (+E2). Analysis was performed by Northern blot hybridization using a 1.1-kilobase (kb) human c-jun cDNA clone containing the entire protein-coding sequence (24). Figure 1 (top) shows the results of a representative experiment as an actual autoradiograph of the nitrocellulose filter. For comparison, the results obtained hybridizing the same blot with v-fos (30), /3-tubulin (31), or lamin-C (32) DNA probes are also reported. Beta-tubulin and laminC gene expression does not change after estrogen treatment, and the levels of the corresponding mRNAs can be used to correct for changes in hybridization signal due to differences in the amount of RNA loaded in the gel. The c-jun radioactive probe detects in rat uterine RNA two bands, corresponding to mRNA species of 2.5 and 3.2 kb. The entity of both of these bands increases substantially after estrogen stimulation, starting 30 min after injection and lasting 120 min, before declining by 240 min. The data from quantitative densitometric scanning of the autoradiographic signals, corrected on the basis of /3-tubulin and lamin-C mRNA concentrations in the same blot, are also reported in Fig. 1 {bottom). Estrogen treatment induces an increase in c-jun mRNA concentration in the uterus (4- to 5-fold) within the first 30 min after injection that persists without significant variations for up to 120 min, before declining to the starting levels by 240 min. The 2.2-kb c-fos mRNA shows similar kinetics, but a different pattern of induction. Its basal concentration in nonstimulated uterus is lower than that of c-jun mRNA, it starts to increase (5- to 10-fold) within the first 30 min after injection and keeps increasing gradually, to peak after 120 min (20- to 40-fold induction) before declining by

• 0,8

60

120 minutes after E2 injection

180

^

240

Fig. 1. Effects of Estrogen on Steady State Concentrations of c-jun and c-fos mRNA in the Uterus of Ovariectomized Rats Kinetics of mRNA accumulation after injection of 17/3-estradiol were assessed by Northern blot analysis of poly(A)+ uterine RNA using c-jun, fos, /3-tubulin, or lamin-C DNA probes. Autoradiographs of Northern blots are presented at the top of the figure, and data from quantitative densitometric scanning of autoradiographic signals, corrected on the basis of /3-tubulin mRNA concentration in the same blot, are shown in graph form at the bottom.

240 min to a level 3- to 5-fold above that of unstimulated uterus. To study the tissue specificity of the c-jun mRNA response to estrogen, we examined expression of the protooncogene in different organs and tissues of the rat before and 2 h after injection of 17/3-estradiol. mRNAs of identical size to those detected in the uterus were observed in the different organs studied, although the level of expression varied greatly (Fig. 2, -E 2 ). When the data from quantitative densitometric scanning of the autoradiographic signals, corrected on the basis of lamin-C (32) mRNA concentration in the same blot, were compared, highest levels of expression were found in uterus, lung, brain, and large intestine, followed by liver and heart. In kidney, spleen, and skeletal muscle c-jun mRNA was detectable only after a longer autoradiographic exposure. Although the significance of these differences is unclear, it is interesting to note that a distribution of c-jun mRNA similar to what we observed in the adult female rat has been reported in adult mouse

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Estrogen Induction of c-jun Gene Transcription

Lu

1043

L

II

B

II

S

II

K

II

H

II

M

II

I

II

I

•%*** c-jun

lamin C

Fig. 2. Distribution of c-jun mRNA in Female Rat Organs and Tissues before and after Injection of 170-Estradiol Ten micrograms of poly(A)+ RNA from organs and skeletal muscle of ovariectomized rats, killed either before (-E2) or 120 min after ip injection of 17/3-estradiol (+E2), were fractionated by electrophoresis, transferred to nitrocellulose, and probed with 32Plabeled c-jun or lamin C cDNAs. Lu, Lung; L, liver; B, brain; S, spleen; K, kidney; H, heart; M, skeletal muscle; I, large intestine.

organs by Ryseck et al. (26). Higher levels of c-jun mRNA in mouse brain and intestine have been found also by Ryder and Nathans (25). When the animals were injected with 17/3-estradiol, no significant changes were observed in any of the organs and tissues tested (Fig. 2, +E2). Induction of a steady state c-jun mRNA level by estrogen is thus confined to the uterus in female rats, the only organ studied that responds to the hormone with increased cell proliferation. Induction of c-jun mRNA by Estrogen Is not Prevented by the Protein Synthesis Inhibitor Cycloheximide Modulation of the levels of specific RNAs by steroid hormones can occur in the absence of protein synthesis if this is a direct primary response to the hormone. We tested the effect of the protein synthesis inhibitor cycloheximide on the induction of c-jun mRNA by estrogen in rat uterus (Fig. 3). The animals were injected with cycloheximide 60 min before estrogen and killed 120 min after the second injection (E2, +CHX) or, alternatively, were injected with cycloheximide alone and killed 180 min later (0, +CHX). Under these conditions, protein synthesis was inhibited in rat organs by more than 95%, starting 20-40 min after the injection of cycloheximide (17, 33) (data not shown). Uterine poly(A)+ RNA was analyzed for c-jun mRNA as described above, and the results of a representative experiment are reported in Fig. 3. Cycloheximide treatment induced a substantial increase in the basal level of c-jun mRNA in the uterus (11-fold induction; compare the data reported in the lower part of Fig. 3 with the results reported in Fig. 1). This was true for c-fos mRNA also (Fig. 3) (17). Superinduction of immediate early gene mRNA by protein synthesis inhibitors is well known. It is primarily due to a prolongation of the half-life of the mRNA, with its consequent accumulation in the cell. Superinduction of c-jun mRNA by cycloheximide has been described in human, mouse, and rat fibroblasts in culture (24-27, 34). Estrogen increased the c-jun mRNA level in the

uterus to the same extent with or without cycloheximide (3.5- to 4-fold; compare data reported in the lower part of Fig. 3), suggesting that de novo protein synthesis was not required for this induction. Based on these results, we conclude that induction of c-jun mRNA is not mediated by the product of an estrogen-dependent gene, but is a primary response to the estrogen receptor in rat uterus. Induction of c-jun mRNA by Estrogen in Rat Uterus Is due to Increased Transcription To define the mechanism(s) underlying the effect of estrogen on c-jun mRNA concentration in rat uterus, we measured the transcription rate of this gene in isolated uterine nuclei by nuclear run-on transcription. This method allows assessment of changes in the transcription rate of the gene of interest [or density of RNA polymerase molecules on the gene, as described by Groudine et al. (35)]. Nuclei were isolated from the uterus of animals killed either before (-E2) or after injection of 17^-estradiol (+E2), and c-jun gene transcription was measured as described in Materials and Methods (Fig. 4). The expression of /3-tubulin and laminC genes is unaffected by estrogen in rat uterus (17) (data not shown). For comparison, the transcription rate of /3-tubulin and lamin-C genes was also measured in the same experiments. The result shows that c-jun gene transcription increases in the uterus by 30 min after injection of estrogen (2.5- to 3-fold increase; see Fig. 4, bottom). The increase in transcription lasts for up to 120 min before declining after 240 min toward the starting values. The kinetics of induction of c-jun mRNA accumulation and rate of transcription by estrogen are thus overlapping in rat uterus (compare results shown in Figs. 1 and 4). Moreover, the effects on mRNA concentration and gene transcription are comparable in magnitude (3- to 5-fold; compare again results shown in Figs. 1 and 4), suggesting that the action of the hormone on expression of this gene is primarily on its rate of transcription. This is different from the effects of

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MOL ENDO-1990 1044

Vol 4 No. 7

120'+ CHX

treatment with cycloheximide did not prevent the activation of c-jun transcription by estrogen, confirming that this is a primary response to the hormone. However, cycloheximide alone had a significant effect on the transcription of this gene, increasing it 3- to 5-fold (reproducible in two separate experiments). A possible explanation for the increase in c-jun transcription by cycloheximide is that blockade of protein synthesis decreases the cellular levels of labile inhibitor(s) of c-jun transcription, but it is not possible to exclude a different mechanism of action of cycloheximide, due to its pleiotropic effects on the cell and its toxicity in the animal.

I

0

E.

c-jun

c-fos tubulin

Effect of Estrogen on AP-1 Response Element (RE) Activity in MCF-7 and HeLa Cells

lamin C

0

1201 -CHX E2

JUN/TUBULIN

0.77(1 x)

2.98 (4 x)

FOS/TUBULIN

0.02

0.84

0

1201 +CHX E2

8.25(11 x) 11.14(14 x) 10.27

16.07

Fig. 3. Effect of Cycloheximide Treatment on Estrogen-Mediated Induction of c-jun mRNA in Rat Uterus Cycloheximide (50 mg/kg BW) in sterile saline was injected ip into ovariectomized rats. Animals were killed 180 min later (0), or 17/3-estradiol was injected 60 min after cycloheximide and the animals were killed 120 min after the second injection (E2). Ten micrograms of poly(A)+ uterine RNA were fractionated by electrophoresis, transferred to nitrocellulose, and probed with 32P-labeled c-jun, fos, /3-tubulin, or lamin-C DNA. Data are presented as actual autoradiographs of the nitrocellulose filters at the top of the figure and as data from quantitative densitometric scanning of autoradiographic signals, corrected on the basis of /3-tubulin mRNA concentration in the same blot, at the bottom (120' + CHX). For comparison, the c-jun and c-fos mRNA concentrations in the uterus before (0) and 120 min after injection of estrogen alone (E2) are reported (120' - CHX; see Fig. 1).

estrogen on c-fos and c-myc gene expression in the same experimental system, where the accumulation of the specific mRNA is 5-20 times more important than the increase in transcription of the corresponding gene (17). The specificity of the hybridization signals observed was confirmed by the fact that plasmid DNA alone, under the same conditions, did not show any significant hybridization signal (data not shown). The effect of cycloheximide on c-jun gene transcription was also tested with the run-on assay (Fig. 4, +CHX). Transcription rate was measured in vitro in uterine nuclei from animals treated with cycloheximide 60 min before estrogen and killed 120 min after the second injection (E2) or, alternatively, injected with cycloheximide alone and killed 180 min later (0). The

JUN C is a nuclear protein that can associate with FOS to generate the transcription factor AP-1. Since c-fos and c-jun genes are regulated by estrogen in proliferation-responsive cells, this must lead to changes in the activity of AP-1 REs in these cells. MCF-7 human breast cancer cells express the estrogen receptor and respond to estrogen with increased c-fos mRNA and cell growth (15). Moreover, they contain AP-1 and c-jun mRNA (Rosales, R., and A. Weisz, unpublished results). We studied in MCF-7 cells the effects of both estrogen and the estrogen receptor on the activity of a reporter plasmid containing four copies of the polyoma virus enhancer AP-1 RE (28, 36), cloned up-stream of a globin promoter-CAT gene. Figure 5 {left) shows the structure of the recombinant plasmids containing the wild-type (wt) sequence of the polyoma AP-1-binding site (underlined in Fig. 5; plasmid pGCAT PBx4) or a mutated sequence (plasmid pGCAT PABx4) that prevents binding of the factor and transcriptional activation (28, 37). MCF-7 cells were transfected using the calcium phosphate-DNA coprecipitation method before stimulation with 17/3-estradiol and assay of CAT activity in the extracts. An SV-40 enhancer and early promoterdriven /3-galactosidase (/8-gal) gene was always cotransfected, and the resulting levels of /3-gal activity were used to correct for changes in transfection efficiency, as described in Materials and Methods section. The results are reported in Fig. 5 (A) and in Table 1. The recombinant gene in plasmid pGCAT PBx4 was expressed at a relatively low level upon introduction in MCF-7 cells. The comparison of the activity of this plasmid with that of pGCAT PABx4, where the AP-1binding site is inactivated, shows a significant difference (pGCAT PBx4 was about 5-fold higher; see Table 1), suggesting that the polyoma AP-1-binding site affects transcription of this reporter in MCF-7 cells. Treatment with 17/3-estradiol increased CAT activity (~2-fold) only in cells transfected with pGCAT PBx4. The antiestrogens 4-hydroxytamoxyfen or ICI 164384 (ICI), which bind the estrogen receptor with high affinity without significantly affecting its activity on transcription (Refs. 38-39 and references therein), slightly decreased (~50% of the control value) the expression of pGCAT PBx4. It is possible that low amounts of estrogens, still

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Estrogen Induction of c-jun Gene Transcription

1045

12O'+CHX

-E, I 30'

60'

90'

120'

240*

I 0

E2

120' •CHX E2

lam jun tub

0

30'

60'

90'

120'

240'

0

JUN/LAMIN

0.16 (1x)

0.54 (3.0)

0.55 (3.1)

0.67 (3.7)

0.46 (2.5)

0.27 (1.5)

0.60 (3.3)

2.00 (11.1)

JUN/TUBULIN

0.16 (1x)

0.40 (2.5)

0.56 (3.5)

0.51 (3.2)

0.50 (3.1)

0.28 (1.8)

0.80 (5.0)

1.80 (11.3)

LAMIN/TUBULIN

0.92 (1x)

0.73 (0.8)

1.0 (1.1)

0.76 (0.8)

1.0 (1.1)

1.0 (1.1)

1.36 (1.5)

0.88 (0.9)

Fig. 4. Transcriptional Activation of c-jun Gene in Rat Uterus after Stimulation with Estrogen Analysis of relative transcription rate was performed using nuclear run-on assay. Plasmids containing c-jun (jun), nuclear envelope lamin (lam), or /3-tubulin (tub) cDNA bound to nylon were hybridized with 32P-labeled run-on transcripts from nuclei isolated from uteri of ovariectomized rats killed immediately before (0, -E 2 ) or at different times after ip injection of 170-estradiol (+E2). Transcription was also tested in nuclei isolated from uteri of animals injected with cycloheximide (120' + CHX) without (0) or with (+E2) estrogen, as described in Fig. 3. Results are presented as actual autoradiographs of the nylon filters (top) and as data from quantitative densitometric scanning of c-jun autoradiographic signals, corrected on the basis of lamin-C or /3-tubulin signals (bottom).

present in the charcoal-stripped serum, were antagonized by the antihormones, with a consequent reduction of estrogen receptor activity, since ICI effectively antagonized the stimulation by 17j8-estradiol (see lanes 6 in Fig. 5A and ICI+E2 in Table 1). However, a direct inhibitory effect of antiestrogens on putative activation by ligand-free receptors or the activation by these compounds of a regulatory pathway independent of the estrogen receptor cannot be excluded. One of the early effects of estrogen in MCF-7 cells is the rapid downregulation, or processing, of its receptor (40-42), with the consequent attenuation of the stimulatory effect of the hormone. For this reason, we cotransfected the plasmid HEO, containing a cDNA coding for the human estrogen receptor protein (43, 44) cloned in the eukaryotic expression vector pSG5 (45). In the presence of estrogen, HEO increased the expression of the plasmid pGCAT PBx4, but not pGCAT PABX4, to about 5-fold the control values (compare lanes 1 and 3 in Fig. 5A and see Table 1). Thus, estrogen and the estrogen receptor increase the transcriptional activity of this AP1 reporter gene in MCF-7 cells. Down-regulation of the estrogen receptor could be responsible for the low level of response observed. To study whether this response to the hormone is cell specific, we transfected the plasmids pGCAT PBx4 and pGCAT PABx4 in the estrogen-unresponsive HeLa cells, also cotransfecting HEO, since these cells do not contain estrogen receptor

(Fig. 5B). As a control, the expression vector pSG5 alone, without inserted cDNA, was alternatively cotransfected. The results show that in HeLa cells expression of the estrogen receptor does not confer estrogen responsivity to this reporter gene, suggesting that the estrogen receptor does not bind directly to the AP-1 RE. The reporter gene itself is active in Hela cells, since the basal activity of pGCAT PBx4 was 3- to 5-fold higher than that of pGCAT PABx4 (compare lanes 1 and 4 in Fig. 5B and see Table 1). The lack of response of pGCAT PBx4 to estrogen in HeLa cells is not due to inactivity of the estrogen-receptor complex under the conditions used for this study, since transcription of the reporter plasmid pVIT-TK-CAT, containing the ERE from the X. laevis vitellogenin A2 gene (46, 47) is activated in these cells by the estrogen receptor under comparable experimental conditions (Fig. 5B, lanes 7-10).

DISCUSSION The proliferative response of rat uterine cells has been extensively used as a model system to study in vivo the control of cell proliferation by estrogen. Hormone deprivation drastically reduces the proliferative activity of uterine cells, and administration of estrogen to im-

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Vol 4 No. 7

MOL ENDO-1990 1046

MCF-7

7 pGCATPABx4

pGCATPBx4

HELA

5 I0 » 8 13 0 QOAAQTOACTAACTQACCOCAQ

wt

CCTTCACTOATTQACTOOCQTC

5' 3-

53-

*p| C f*] C * °

TCQAOOAAOTOACTAACTO

C C T T C A C T a A T T O A C T C 0 TJO T C A 0 C T

PB

T C Q A Q O A A O T[A|A[O|T A A C T 0 AIG]C[AJC A Q C C T T C A(TJT|CJAT T O A C T[CJO|T|O T C A O C T

PAB

pGCATPBx4

pVlT-TK-CAT

pGCATPABx4

Fig. 5. Effect of Estrogen on AP-1 RE Activity in MCF-7 and HeLa Human Cancer Cells Left, Structure of recombinant plasmids containing wt (pGCAT PBx4) or mutated (pGCAT PABx4) AP-1 RE from the polyoma virus enhancer. The reporter recombinants contain four head to tail copies of enhancer oligonucleotides cloned up-stream of a rabbit /3-globin promoter-CAT gene. Boxed basepairs are mutations of the wt polyoma sequence (nucleotides 5109-5130) that prevent binding of PEA2 factor (28) (PB oligonucleotides) or PEA2 and AP1 factors (PAB oligonucleotides). Right, Effects of estrogen, antiestrogens, the estrogen receptor (HEO; 1 ^g). or the eukaryotic expression vector pSG5 (1 M9) on pGCAT PBx4 (1 fig), pGCAT PABx4 (1 ^g), and pVIT-TK-CAT (1 nQ) reporter plasmids in estrogen-responsive MCF-7 cells (A) or estrogenunresponsive HeLa cells (B). CAT activity was measured in whole cell extracts from cells transfected with the indicated plasmids and exposed for 24 h to 10~8 M 17j3-estradiol (E2), 10"7 M 4-hydroxytamoxyfen (OHT), 1O~7 M ICI, or 10~8 M E2 with 10~6 M ICI, corrected for changes in transfection efficiency, as described in Materials and Methods.

Table 1. Effect of Estrogen on AP-1 RE Activity in Estrogen-Responsive and Unresponsive Human Cancer Cells HeLa Reporter

Conditions

MCF-7 Cat activity (pmol/h-U(3-gal)

Fold induction

pGCAT PBx4

None HEO + E2 pSG5 + E2

3.2 3.1 3.8

(1.0) 0.9 1.2

pGCAT PABX4

None HEO + E2 pSG5 + E2

0.8 0.7 0.9

0.3 0.2 0.3

pVIT-TK-CAT

None HEO HEO + E2 pSG5 + E2

1.6 3.7 37.7 2.2

(1.0) 2.3 23.8 1.4

Reporter

pGCAT PBX4

pGCAT PABX4

Conditions

CAT activity (pmol/h-lip-gal)

Fold induction

None E2 HEO + E2 OHT

4.6 8.4 21.7 3.4

(1.0) 1.9 4.8 0.7

ICI ICI + E2

2.8 2.7

0.6 0.6

None E2 HEO + E2

1.0 1.2 1.0

0.2 0.3 0.2

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Estrogen Induction of c-jun Gene Transcription

mature or castrated animals induces a considerable mitogenic response in this organ, with the semisynchronous recruitment of cell in cycle, followed within 24-30 h by cell proliferation (for a review, see Ref. 5). It has been postulated that estrogen controls cell proliferation acting indirectly, via the local (paracrine, autocrine) or systemic (endocrine) production of growth factors (Refs. 48 and 49 and references therein), or direcly, acting as a mitogen for target cells (50, 51). There is a basic mechanism that controls cell division in eukaryotes, and the different growth-promoting factors act on the same set of genes or gene products to regulate cell proliferation (for reviews, see Refs. 5254). We postulated that estrogen, in proliferation-responsive cells, exerts its mitogenic potential by modulating the expression of these same genes. In line with this hypothesis, we have previously shown that ip injection of 17/3-estradiol to adult castrated female rats induces early transient expression of c-fos and c-myc protooncogenes in the uterus. Stimulation of c-fos gene transcription by estrogen is a direct primary response to the hormone, since it is not blocked by protein synthesis inhibitors, whereas activation of c-myc is probably a consequence of the changes induced by the hormone in the cell (17). Supporting this possibility, are the findings that the human c-fos, but not c-myc, gene promoter responds to the estrogen receptor in transfection experiments (Weisz, A., and R. Rosales, in preparation). We now present evidence that estrogen stimulates c-jun gene transcription in rat uterus. Also, in this case stimulation of transcription by the hormone does not require the synthesis of an intermediate protein, suggesting that c-jun gene promoter could be a target for regulation by the estrogen-receptor complex. The regulation of c-fos and c-jun gene transcription by estrogen in rat uterus differs from that of other estrogen-responsive genes in vertebrate tissues. In the presence of continous estrogenic stimulus, after a first short increase in transcription, these genes become refractory. This results in the transient accumulation of the specific mRNA, suggesting that a mechanism must exist to counterbalance the stimulatory effect of estrogen on the activities of these promoters in uterine cells. FOS and JUN-C have been postulated to bring about transcriptional repression of c-fos in a cooperation that involves the serum response element (dyad symmetry element) and/or the consensus AP-1-binding sites within the c-fos gene promoter (55-58). Estrogen induces expression of both c-jun and c-fos, with the consequent simultaneous increase in their products in the cell; this could interfere with activation of transcription of c-fos by the estrogen receptor, suggesting an antagonism between a positively acting transcription factor (the estrogen receptor) and a repressor of transcription. However, it is also possible that a yet unidentified factor, different from FOS or JUN-C, interferes with the activity of the estrogen receptor in the regulation of transcription of this gene. The same mechanism considered for c-fos could be acting on c-jun, with the exception that JUN-C has been shown to positively

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autoregulate its own promoter (59). The analysis in vitro of the regulation of c-jun gene promoter by the estrogen receptor will help to define the molecular bases of this response. The transcription factor AP-1 was first described as a DNA-binding activity in human HeLa cell extracts that specifically recognizes the enhancer elements of SV40 and the human metallothionein-IIA gene (60). AP-1 is a transcription factor that can be formed by a repertoire of protein complexes, including FOS and JUN-C (for reviews, see Ref. 30). AP-1-binding sites and AP-1 REs were identified in the control regions of cellular and viral genes that are stimulated by treatment of cells with phorbol esters and growth factors (61,62). In particular, the a-domain of the polyoma virus enhancer contains the binding site for a mouse cell factor called PEA1, that is a murine homolog of human AP-1, since PEA1 also interacts with the SV40 and c-fos enhancers (29); the DNA sequence recognized by PEA1 also binds JUNC and mediates the effects of phorbol esters on transcription in mouse cells (28, 36). We demonstrate that estrogen activates the transcription of a reporter gene containing a multimer of the polyoma AP-1-binding site. This response of the AP-1 RE to estrogenic stimulation is due to increased activity of AP-1 or a related factor, and not to the direct binding of the estrogen-receptor complex to the polyoma DNA sequence. This conclusion is supported by the following observations. 1) Mutations in the AP-1 RE that abolish binding of AP-1 also inhibit the response to the hormone (Fig. 5A). 2) The sequence tested does not contain estrogen receptor-binding sites, since the pentamer TGACC, which represents a possible ERE (38) and is present in the wt polyoma sequence (nucleotides 5122-5126 in Fig. 5), was mutated to an inactive TGAGC in the plasmids used for this study. 3) The response to estrogen is specific for MCF-7 cells and is not observed in HeLa cells transfected with the human estrogen receptor cDNA, where, instead, the transfected receptor can activate transcription from an ERE-containing reporter plasmid (Fig. 5B). A more likely explanation for this effect is that estrogen increases FOS, JUN-C, and/or related gene products in MCF-7 cells by increasing the expression of the corresponding genes in a cell-specific way. Supporting this possibility are the findings that estrogen increases c-fos mRNA levels in MCF-7 cells (15) and, as described in this report, c-fos and c-jun genes expression in responsive cells. The finding of prompt activation of c-jun and c-fos gene transcription by estrogen in proliferation-responsive cells suggests that this hormone is a mitogen in its own right, acting direcly to produce the mitogenic effect. However, as in the case of competence growth factors (63), induction of these immediate early genes is not sufficient to produce growth (64) (Weisz, A., unpublished data). Based on this assumption, one can predict that other target genes and gene products must be regulated by estrogen to achieve the full mitogenic effect. The identification of these genes and understanding of the mechanism of their response to the

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Vol 4 No. 7

MOL ENDO-1990 1048

hormone will help to define the way in which estrogen controls cell growth.

to Hyperfilm-MP (Amersham) x-ray film, with a DuPont Cronex screen (DuPont, Wilmington, DE) at - 8 0 C. Densitometric scanning of autoradiographs was performed with a UltroscanXL densitometer (LKB, Rockville, MD). Data are expressed as optical density units.

MATERIALS AND METHODS

Isolation of Nuclei and Nuclear Run-On Transcription Assay

Animals All animal experimentations were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Adult female Sprague-Dawley rats (225-250 g) from Charles River, Calco, Italia, ovariectomized under light ether anesthesia 10-14 days earlier, were injected ip with 1.5 Mg/100 g BW 17/3-estradiol in 0.15 ml 10% ethanol-90% PBS. At different times after injections, groups of 10-15 animals were killed by cervical dislocation, and uteri and other organs were dissected and immediately chilled in ice-cold buffer in preparation for isolation of nuclei or, alternatively, frozen in liquid nitrogen, pulverized, and freeze-dried for RNA extraction. Where indicated, animals were also injected ip with cycloheximide (50 mg/kg BW in sterile saline) and processed as described above. RNA Purification Total RNA was extracted with guanidinium thiocyanate (65) and purified by ultracentrifugation through a dense cushion of cesium chloride (66); the RNA pellet was then dissolved in 7.5 M guanidine hydrochloride (pH 7), ethanol precipitated, redissolved in TES buffer [10 HIM Tris-HCI (pH 7.5 at 25 C), 5 mM Na2EDTA, and 1 % sodium dodecyl sulfate (SDS)], extracted with chloroform-n-buthanol (4:1), and reprecipitated with ethanol. Polyadenylated [poly(A)+] and nonpolyadenylated [poly(A)~] RNA fractions were separated by affinity chromatography on oligo(dT)-cellulose (Pharmacia type 7, Piscataway, NJ). Purified poly(A)+ RNAs were ethanol precipitated and resuspended in H2O at 2 ng/n\ in preparation for electrophoretic analysis. Electrophoresis of RNA, Transfer to Nitrocellulose Membrane and Hybridization with 32P-Labeled DNA Probes Poly(A)+ RNA (10 /xg) was glyoxalated at 50 C for 1 h, fractionated in 1.5% agarose gels in 10 mM Na phosphate (pH 7), and transferred to nitrocellulose (BA 85, Schleicher and Schuell, Keene, NH) in 20 x SSC (1 x SSC = 0.15 M NaCI and 0.015 M Na citrate, pH 7) over a period of 24 h, as described by Thomas (67). Filters were then baked for 2 h at 80 C in a vacuum owen. Nitrocellulose filters were prehybridized for 2-12 h at 42 C with the following solution: 5 x SSC, 5 x Denhardt's (1 x Denhardt's mix = 0.2% each of BSA, polyvinylpyrrolidone, and Ficoll), 50 mM Na phosphate (pH 8), 0.25 mg/ml sonicated heat-denatured herring sperm DNA, and 50% (vol/vol) formamide. The hybridization was carried out for 16-24 h in 5 x SSC, 5 x Denhardt's, 20 mM Na phosphate (pH 6.5), 0.1 mg/ml sonicated heat-denaturated herring sperm DNA, 50% formamide, and 2 x 106 cpm/ml 32P-labeled DNA probes (SA, 1-2 x 109 cpm/^g DNA). Probes were prepared by digestion of plasmids with restriction endonucleases, purification of insert DNA by low melting point agarose gel electrophoresis, and 32P labeling of DNA by random primed DNA synthesis with random sequence hexanucleotide primers (Amersham, Arlington Heights, IL) and [«-32P]dCTP (3000 Ci/ mmol; Amersham), as described by Feinberg and Vogelstein (68). Plasmids used for preparation of the probes were the following: pUN 121 (human c-jun cDNA) (24), p-fos-1 (v-tos) (69), pT2 (chicken /3-tubulin cDNA) (31), and pcDNA7 (human lamin-C cDNA) (32). Filters were washed four times with 2 x SSC-0.1% SDS at room temperature for 5 min and twice with 0.5 x SSC-0.1% SDS at 42 C for 30 min, and then exposed

Nuclei were isolated from rat uterus, and elongation of nascent RNA chains in isolated nuclei was carried out as described by Weisz and Bresciani (17). Plasmid DNAs (10 M9 DNA/slot) immobilized on nylon membranes (Hybond-N, Amersham) were hybridized with 1-2 x 106 cpm/ml 32P-labeled RNA for 48-72 h at 37 C, as previously described (17). Filters were washed as follows: four times in 2 x SSC-0.1% SDS at 37 C for 20 min, twice in 2 x SSC at 37 C for 20 min, followed by incubation at 37 C for 30 min with 10 M9/ml RNase-A (Sigma, St. Louis, MO) in 2 x SSC, and once at 42 C in 2 x SSC0.1% SDS. Filters were then dryed and exposed to HyperfilmMP (Amersham) x-ray films, with a DuPont Cronex screen, for 24-48 h at - 8 0 C. Densitometric scanning of autoradiographs was performed with an Ultroscan-XL (LKB) densitometer. Data are expressed as optical density units. The DNA probes immobilized on the filters were the following: pUN 121 (c-jun), pT2 (/3-tubulin), and pcDNA7 (lamin-C). Transient Transfection and CAT Assay For transient transfection, MCF-7 cells were plated on day 1 at about 30% confluency in Dulbecco's Modified Eagle's Medium, without phenol red, containing 10% fetal calf serum, pretreated with dextran-coated charcoal to remove endogenous steroids (70), 0.6 Mg/ml insulin, and 100 U/ml penicillinstreptomycin. HeLa cells were plated at about 10% confluency in Dulbecco's Modified Eagle's Medium, without phenol red, containing 5% charcoal-stripped fetal calf serum and penicillinstreptomycin. On day 2, fresh medium was added, and cells were transfected, using the calcium phosphate-DNA coprecipitation method (71), with 1 M9 reporter plasmid, 3 ^g #galactosidase expression vector pCH110 (Pharmacia) as internal control for transfection efficiency, and carrier DNA (Bluescribe M13+), up to 20 ng total DNA/dish. Where described, 1 /xg plasmid HE0 (the eukaryotic expression vector pSG5 containing the cDNA for the human estrogen receptor) or 1 jug pSG5 was also added. After 4-12 h, cells were washed with medium for 30 min before the addition of fresh medium, with or without steroids as indicated, and incubated for 24 h. CAT enzyme assays were performed in whole cell extracts as described by Gorman et al. (72), after normalization for /3galactosidase activity [as described by Webster et al. (73)]. The acetylated and nonacetylated forms of [14C]chloramphenicol were separated by TLC, autoradiographed, and then excised and quantitated by liquid scintillation counting. CAT activity is reported as picomoles of [14C]chloramphenicol acetylated per h/unit /3-gal activity.

Acknowledgments We thank Richard Breathnach for the gift of human c-jun cDNA probe; Bohdan Wasylyk and Jose Perez-Mutul for the gift of plasmids pGCAT PBx4 and pGCAT PABx4; Pierre Chambon for the gift of plasmids HE0, pSG5, and pVIT-TK-CAT; Don Cleveland for the gift of chicken /3-tubulin probe; and Alan Wakeling (ICI Pharmaceuticals, United Kingdom) for providing 4-hydroxytamoxyfen and IC1164348. Moreover, we would like to thank R. Breathnach, B. Wasylyk, and Vijay Kumar for helpful suggestions; P. Chambon for critical comments and financial support; Ferdinando Auricchio, Maria Teresa Masucci, and Vincenzo Sica for critically reading the manuscript. Received February 15, 1990. Revision received April 16, 1990. Accepted April 19,1990.

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Estrogen Induction of c-jun Gene Transcription

Address requests for reprints to: Dr. Alessandro Weisz, Istituto di Patologia Generale e Oncologia, Prima Facolta di Medicina e Chirurgia, Universita di Napoli, Piazza S. Andrea delle Dame, 2, Napoli 1-80138, Italy. This work was supported by grants from the Italian Ministry for Public Education (MPI), the Italian National Council for Research (CNR; Contract 00547.44), and the AIRC. * EMBO Fellow in the Laboratoire de Genetique Moleculaire des Eukaryotes du CNRS, Unite 184 de Biologie Moleculaire et de Genie Genetique de I'lNSERM, Faculte de Medecine, Universite de Strasbourg, France, where part of the work was carried out. t Fellow of the Italian Association for Research on Cancer (AIRC).

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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 ...
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