In Vivo Regulation of Zif268 Messenger RNA Expression by 17/3-Estradiol in the Rat Uterus
Larry J. Suva, Sandra C. Harm, Russell M. Gardner, and Mark A. Thiede Department of Bone Biology and Osteoporosis Research Merck, Sharp, and Dohme Research Laboratories (L.J.S., S.C.H. M.A.T.) West Point, Pennsylvania 19486 Department of Biology Villanova University (R.M.G.) Villanova, Pennsylvania 19085-1699
Administration of 17/3-estradiol (E2) induces a mitogenic response in the rat uterus. Previous studies have shown that this effect involves the transient activation of c-fos and c-myc expression, followed by significant increases in both DNA synthesis and cell proliferation. Z/7268 is a zinc finger-containing, DNA-binding transcription factor that has been implicated in the regulation of cell growth and development and has been shown to be coregulated with c-fos in a number of systems. To determine whether Zif268 is also a target for estrogen regulation, we measured the effects of E2 on Z/7268 mRNA expression in the uterus of the ovariectomized rat. In this report we demonstrate that although low levels of Z/7268 mRNA expression are detectable in the uteri from ovariectomized control rats, treatment with E2 (4, 40, or 400 M9/kg BW) induces a rapid and transient 45- to 50-fold increase in the level of Z/7268 mRNA 2 h after E2 treatment. The elevated levels of Z/7268 mRNA returned to basal 6 h after hormone treatment. Lower doses of E2 (0.004, 0.04, and 0.4 fig/kg) had little or no effect on Zif268 mRNA expression, while higher doses of E2 (4-400 Mg/kg) resulted in maximal increases in Z/7268 expression. Dexamethasone, 5a-dihydrotestosterone, and progesterone had no effect on uterine Zif268 mRNA expression, and the induction of Zif268 by E2 was abolished by pretreating the animals with the RNA synthesis inhibitor actinomycin-D. In addition, stimulation of Z/7268 mRNA expression was observed with the short-acting estrogen estriol, suggesting that the response may be specific for estrogenic steroids. These data demonstrate that one of the initial responses induced by estrogen in the rat uterus involves increased Z/7268 gene expression and suggest that Z/7268 (as well as c-fos and c-myc) may play a transcriptional role in the estrogen regulation
of cell growth. (Molecular Endocrinology 5: 829-835, 1991)
INTRODUCTION A major effect of the acute administration of 1 "7/3estradiol (E2) to ovariectomized female rats is the stimulation of DNA synthesis and cell proliferation in the uterus (1). The uterine response initially involves hyperemia, followed by hypertrophy and hyperplasia within 24 h of hormone treatment (2, 3). In addition to these gross changes, E2 induces the stimulation of many classes of RNA (4-6), including a number of protooncogenes, including c-fos, c-myc, N-myc, c-jun, c-ras and c-erb-B (7-13). The response of the uterus to E2 is, therefore, a useful model in which to elucidate the molecular mechanisms involved in regulating the massive effects of hormone-induced cell proliferation. Among the molecular events that control the initial phase of transcriptional activation are several genes encoding known or presumed transcription factors, such as c-jun, c-fos, and c-myc, which may regulate the ensuing genetic program (14-16). One of these putative transcription factor genes, Zif268 (also referred to as NGFI-A, Egr-I, and Krox-24) (19-22), is rapidly upregulated in various cultured cell lines by growth factors (19-22), retinoic acid (23), and cellular depolarization (16) and is expressed in a number of different tissues and organs (16, 18-20, 24). The Zif268 gene product contains three tandemly repeated zinc finger sequences, a common feature of many DNA-binding transcription factors, and is homologous to the Xenopus transcription factor TFIIIA (25, 32). Numerous studies have associated Zif268 expression with proliferation (16-22) and differentiation (26, 27) both in vitro and in vivo. In addition, Zif268 mRNA expression in rat brain is increased after seizure (28,29). Although the precise function of the Zif268 protein is unknown, Zif268 binds
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to a specific sequence in genomic DNA and activates transcription (30, 31), perhaps by a similar mechanism as a frans-acting regulatory factor. Since the immature rat uterus is commonly used to study the molecular mechanisms of steroid hormone action (9, 13, 33-37), we sought to determine whether increased Zif268 expression was controlled by E2. This report demonstrates that Zif268 mRNA expression is rapidly increased (45- to 50-fold by 2 h) after a single ip injection of E2. In addition, this response is dose dependent, specific for estrogenic steroids, and apparently transcriptional in nature.
accumulation of Zif268 mRNA 2 h after treatment (Fig. 1A). Levels of Zif268 mRNA fall precipitously over the next 2 h and return to basal levels 12 h after hormone addition (Fig. 1A). We consistently observed that E2 stimulated the accumulation of 0-actin mRNA during this time course. Consistent with previous findings (13, 17), the levels of 0-actin mRNA peaked later (4-6 h) than those of Zif268 mRNA and remained elevated even at times when Zif268 mRNA levels were declining. As expected, over the same time course as E2 treatment, the injection of saline had no effect on the expression of Zif268 mRNA in the uterus (Fig. 1B).
RESULTS
Dose-Related Increase in Zif268 mRNA Expression by Estrogen in Rat Uterus
Increased Expression of Z/7268 mRNA by Estrogen in Rat Uterus The level of Zif268 mRNA was measured in uterine RNA isolated from ovariectomized immature rats at various times after ip injection of E2. Although Zif268 mRNA is detectable in the uteri of untreated ovariectomized animals (Fig. 1A, 0 h), a single dose of E2 (40 Mg/kg) stimulates a rapid 45- to 50-fold increase in the
E2 (40 ug/kg) 0 0.5h 1h 2h 4h 6h 12h Zif 268
We next examined the increase in uterine Zif268 mRNA expression as a function of the dose of E2. Groups of rats were injected ip with increasing doses of E2, and uteri were removed for RNA analysis 2 h after injection (Fig. 2). At doses of 0.004, 0.04, and 0.4 Mg/kg, E2 had little or no effect on the level oi Zif268 mRNA expression in the rat uterus; however, doses of 4.0, 40, and 400 Mg/kg produced 45-fold elevations in Zif268 expression 2 h after hormone treatment (Fig. 2). In the above study we examined the regulation of Zif268 mRNA expression in the uterus 2 h after treatment with E2. Since the increase in Zif268 mRNA expression was apparent only after the injection of 4.0, 40, or 400 Mg/kg E2) we next tested whether the highest dose of E2 used (400 Mg/kg) would alter the magnitude or duration of Zif268 mRNA expression. The injection of 400 Mg/kg E2 had no significant effect on either the peak level of expression or the time course of Zif268 mRNA accumulation in the uterus (Fig. 3).
(3-actin
0)
Saline
B o
o
C\J
.c
.c
Tt
CO
9
Zif 268 -
Zif 268 •
(3-actin *•
(3-actin •
Fig. 1. Time Course of the Induction of Zif268 mRNA by E2 in Rat Uterus Total RNA (30 Mg/'ane) was prepared from uteri removed from ovariectomized rats 0-12 h after treatment with E2 (40 Mg/kg; A) or saline (B). Each lane contains RNA from five animals. The position of the 3.7-kilobase Zif268 mRNA is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter. Results are representative of two additional experiments that yielded similar results.
o o
c — ft
Tfr
CO
Fig. 2. Dose-Related Effects of E2 on the Expression of Zif268 mRNA in Rat Uterus Total RNA (30 fig/\ane) was prepared from uteri removed from ovariectomized rats (five per sample) 2 h after treatment with E2 at the indicated doses or a vehicle (saline) control. Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
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831
Estrogen Regulation of Zif268 mRNA
E2 (400 |ag/kg) o
0 2h 4h 6h 8h
S
J
O
Zif 268 P-actin Fig. 3. Time Course of Zif268 mRNA Expression after Treatment with 400 Mg/kg E2 in Rat Uterus Animals were given E2 (400 Mg/kg), and uteri (five per sample) were removed 0, 2, 4, 6, and 8 h after treatment. Total RNA (30 ^g/lane) was hybridized with a Zif268 cDNA probe. Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
Steroid Specificity of Increased Zif268 mRNA Expression To investigate the specificity of the Zif268 mRNA response to E2, we examined whether Zif268 mRNA expression in the rat uterus was regulated by nonestrogenic steroid hormones. Levels of Zif268 mRNA were measured 2 h after a single ip injection of dexamethasone (600 Mg/kg), 5a-dihydrotestosterone (400 Mg/kg), or progesterone (40 mg/kg). Of the steroids tested in this experiment, only E2 (40 ^g/kg) stimulated the expression of Zif268 mRNA (50-fold) in the uterus (Fig. 4). With this protocol, the increase in Zif268 mRNA expression in the uterus is specific for estrogen; however, these data do not exclude the possibility that other nonestrogenic steroids may regulate Zif268 expression under different experimental conditions. To examine the specificity of the estrogenic regulation of Zif268 mRNA in this tissue, the level of Zif268 mRNA was measured in uterine RNA at various times after ip injection of estriol (E3), a short-acting estrogen which is equipotent with E2 in inducing early hypertropic growth responses in the uterus (41). We found that, like E2, a single dose of E3 (40 Mg/kg) stimulates a modest 2- to 3-fold increase in Zif268 mRNA expression 1 h after treatment, which increases to maximal levels (50-fold increase) within 2 h after E3 treatment (Fig. 5). The elevated level of Zif268 mRNA expression falls sharply over the next 2 h and returns to basal levels 4 h after hormone administration (Fig. 5). These data confirm the estrogen specificity of the Zif268 response with respect to both time course and peak effect after estrogen treatment.
Zif 268
(V
UJ
--**«^m^^^m .
p-actin Fig. 4. Steroid Specificity of the Induction of Zif268 mRNA in Rat Uterus Ovariectomized animals were injected ip with vehicle (saline), 600 Mg/kg dexamethasone (DEX), 400 Mg/kg 5«-dihydrotestosterone (DHT), 40 Mg/kg E2, or 40 mg/kg progesterone (PRG). Uteri (five per sample) were removed 2 h after hormone treatment, and total RNA (30 iiq/\ar\e) was hybridized with a Zif268 cDNA probe. Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
E3 (40 |ig/kg) 0 0.5h 1h 2h 4h 6h
Zif 268 • P-actin • Fig. 5. Time Course of Induction of Zif268 mRNA by E3 in Rat Uterus Total RNA (30 Mg/'ane) was prepared from uteri (five per sample) removed from ovariectomized rats 0-6 h after treatment with E3 (40 Mg/kg). Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /?actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
Effect of Actinomycin-D on Estrogen-Stimulated Z/7268 mRNA Expression in the Uterus To investigate whether the increased expression of Zif268 mRNA represents a transcriptional effect of estrogen resulting from new RNA synthesis, we studied
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MOL ENDO-1991 832
the effect of the transcription inhibitor actinomycin-D on this increase. Animals were pretreated with actinomycin-D (8 mg/kg) or vehicle for 2 h and then challenged with a single ip injection of either vehicle or E2 (40 ng/ kg). As shown in Fig. 6, actinomycin-D abolished the increase in Zif268 mRNA that followed E2 administration.
DISCUSSION
The proliferative activity of rat uterine tissue in response to estrogen treatment in the immature rat has been widely used as an in vivo model to study the molecular mechanism(s) of steroid hormone action. E2 has been demonstrated to induce a biphasic uterine growth response (2, 3). The initial phase of the mitogenic response involves a series of early hypertropic responses, 4-6 h after hormone treatment, and a series of late responses, which develop 24-30 h after hormone administration (42). The early responses induced by E2 in the immature rat uterus are characterized by altered expression of genes encoding protooncogenes (7-13), growth factors (35, 36), and their receptors (11). As these responses are associated with the regulation of cell proliferation and differentiation, it has been suggested that they may mediate the E2-stimulated growth response in the uterus (33, 43). In this study we have demonstrated that E2 produces a rapid stimulation of the transcription factor Zif268 in the immature rat uterus. After a single ip injection of E2,
Saline
Act D
S E2
S E2
Zif 268 •
p-actin Fig. 6. Effect of Actinomycin-D on E2 Induction of Zif268 mRNA Total RNA was prepared from uteri removed from different groups of animals (five per sample). One group received vehicle for 2 h, followed by a second ip injection of either vehicle (S) or 40 M9/kg E2. A second group received 8 mg/kg actinomycinD (Act D), followed 2 h later by an ip injection of either vehicle (S) or 40 /xg/kg E2. Total uterine RNA (30 ixg/\ane) was hybridized with a Zif268 cDNA probe. Hybridization to the 3.7kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of one additional experiment that yielded similar results.
Zif268 mRNA is rapidly and transiently increased, reaching a peak level (45- to 50-fold increase) within 2 h and declining to basal levels over the next 10 h (Fig. 1A). The time course of E2-stimulated expression of Zif268 mRNA develops after the time course of occupancy of nuclear estrogen receptors with E2, which has been demonstrated to peak 30-60 min after E2 treatment (33, 38, 44), and with increased uterine DNA synthesis (39, 40) after E2 administration. The pattern of Zif268 mRNA expression in the uterus after E2 treatment is very similar to the pattern observed for mRNA encoding the epidermal growth factor receptor (12, 45), c-fos (7, 8), N-myc (13), c-myc (8,13, 34), and c-jun (9, 10) and coincides with the established time courses of many early uterine growth responses. The doses of E2 used that produced pronounced changes in Zif268 mRNA levels in the immature rat uterus were within the physiological range of doses used to initiate other uterine growth responses (46) and suggest a potential role for Zif268 in mediating the growth and development of this tissue. In addition, we examined whether this response could be reproduced in ovariectomized adult rats and mice, and have observed identical results (data not shown). These data together suggest that the increase in Zif268 mRNA expression is physiologically relevant and neither age nor species specific. Given the wide array of agents known to induce Zif268 expression (16, 19-23), it is interesting that the rapid induction of Zif268 mRNA in the uterus is specific for estrogens. This is particularly intriguing since uterine tissue possesses receptors for glucocorticoids (47), androgens (48, 48), and progesterone (50, 51) as well as functional estrogen receptors. It is also possible that nonestrogenic steroids may regulate Zif268 mRNA expression in the uterus or other tissues under different experimental conditions or with different kinetics. Interestingly, the 5'-regulatory region of the rat Zif268 gene for which sequence data are available (52) contains no consensus pallindromic estrogen response elements. However, a number of sequence elements homologous to regions of the c-fos gene promoter that have been identified as binding sites for both the estrogen receptor and transcription factor AP-1 (53) are located in the Zif268 gene promoter region. While this similarity is interesting, as yet it is not known if any of these sequence elements are involved in the E2 regulation of Zif268 in this tissue. E3, a short-acting estrogen with altered receptorbinding affinity, has been shown to elicit early uterine hypertropic growth responses of equal magnitude to those caused by E2 (40); however, unlike E2, E3 does not stimulate cell division. These growth effects have been confirmed using other short-acting estrogens (54) and suggest that the uterine responses are not a simple cascade of events, with the early hypertropic responses leading directly to the hyperplastic growth response. Indeed, Anderson et al. (44) have suggested a sustained input theory for steroid action in which occupied estrogen receptors must remain in the nucleus of target cells
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833
Estrogen Regulation of Zif268 mRNA
for defined periods in order to induce the molecular events that eventually culminate in the hyperplastic growth response of the tissue. In this study the rapid and specific induction of Zif268 mRNA by both E2 and E3 in the uterus suggests that Zif268 mRNA expression may play a role in the early stages of the estrogeninduced responsiveness of this tissue. The complete blockade of the rapid E2 stimulation of Zif268 mRNA expression by actinomycin-D suggests that this response is mediated by an effect of E2 on the rate of Zif268 transcription. The dose of actinomycin-D used in these studies has been shown previously to be effective in inhibiting the synthesis of other E2-stimulated RNAs in the uterus (55). Although these pharmacological studies only suggest a transcriptional effect of estrogen on Zif268 expression, previous studies have demonstrated that the regulation of Zif268 is primarily transcriptional (16, 21-23). However, nuclear run-on experiments will be necessary to unequivocally define the precise mechanism(s) of E2 stimulation of Zif268 expression in the uterus. To date, the precise function of the Zif268 gene product in any system is unknown, but given the broad spectrum of stimuli that induce Zif268 expression (16, 18-23), it is likely that it has an important function in many fundamental biological processes. Recent reports identifying specific Zif268 DNA-binding sites (31, 32) and the homology with known transcription factors (14, 21, 22, 24, 33) imply that Zif268 is a frans-acting regulatory molecule. It is likely that many specific interactions occur between E2-inducible genes, such as Zif268, in the rat uterus. Presumably, it is this interrelationship that is responsible for the control of E2induced growth and/or differentiation in this tissue. By analogy with competence growth factors, the transient induction of Zif268 and other immediate early genes by E2 may render the cell competent to progress through the cell cycle (56). Given the dramatic changes in the levels of gene expression that occur in the rat uterus after E2 treatment, identifying the potential function(s) of Zif268 in mediating various cellular responses requires identification of the genes, or gene products, regulated by Zif268 and localization of the sites of synthesis and action of Zif268 in vivo.
Louis, MO). Before death, animals were injected ip with treatment compounds in a vehicle consisting of 0.9% NaCI-0.4% ethanol. Control animals received only vehicle. At different times after injection, animals were killed, and uteri were removed, immediately frozen in liquid nitrogen, and pooled in groups of five. RNA Purification Total RNA was prepared using a modification of the guanidinium isothiocyanate method (57). Pools of frozen uteri were homogenized in 9 ml of a solution containing 4 M guanidinium isothiocyanate (Bethesda Research Laboratories, Gaithersberg, MD), 0.03 M sodium acetate, 0.1 M /3-mercaptoethanol, and 0.4 g/ml cesium chloride using a Brinkmann Polytron (Westbury, NY). The homogenates were clarified by a prespin at 12,000 x g, and 7 ml clarified supernatant were layered over 3 ml of a solution containing 5.7 M cesium chloride and 0.03 M sodium acetate. Gradients were centrifuged at 35,000 rpm for 14-16 h in a Beckman 70.1 Ti rotor. Pelleted RNA was washed with 70% ethanol, resuspended in water, and precipitated with ethanol. Yields of total RNA were determined by measuring absorbance at 260 nm. Northern Blot Analysis Total RNA (30 ^g) was separated by electrophoresis through a 1 % agarose (Bethesda Research Laboratories) gel containing 0.66 M formaldehyde (J. T. Baker, Phillipsburg, PA) and transferred to nitrocellulose (Schleicher and Schuell, Keene, NH). After transfer, filters were baked at 80 C for 2 h under vacuum. The filters were prehybridized in a buffer containing 50% deionized formamide (Bethesda Research Laboratories), 5 x SSC (1 x SSC = 0.15 M NaCI-0.015 M Na3.citrate), 50 rriM sodium phosphate (pH 7.0), 5 x Denhardt's solution, 1 % skim milk powder, 100 Mg/ml sonicated salmon sperm DNA, and 0.1% sodium dodecyl sulfate at 42 C. Filters were hybridized in the same buffer to a 1.6-kilobase BglH fragment of Zif268 cDNA (22) (106 cpm/ml) labeled with [
Larry J. Suva, Sandra C. Harm, Russell M. Gardner, and Mark A. Thiede Department of Bone Biology and Osteoporosis Research Merck, Sharp, and Dohme Research Laboratories (L.J.S., S.C.H. M.A.T.) West Point, Pennsylvania 19486 Department of Biology Villanova University (R.M.G.) Villanova, Pennsylvania 19085-1699
Administration of 17/3-estradiol (E2) induces a mitogenic response in the rat uterus. Previous studies have shown that this effect involves the transient activation of c-fos and c-myc expression, followed by significant increases in both DNA synthesis and cell proliferation. Z/7268 is a zinc finger-containing, DNA-binding transcription factor that has been implicated in the regulation of cell growth and development and has been shown to be coregulated with c-fos in a number of systems. To determine whether Zif268 is also a target for estrogen regulation, we measured the effects of E2 on Z/7268 mRNA expression in the uterus of the ovariectomized rat. In this report we demonstrate that although low levels of Z/7268 mRNA expression are detectable in the uteri from ovariectomized control rats, treatment with E2 (4, 40, or 400 M9/kg BW) induces a rapid and transient 45- to 50-fold increase in the level of Z/7268 mRNA 2 h after E2 treatment. The elevated levels of Z/7268 mRNA returned to basal 6 h after hormone treatment. Lower doses of E2 (0.004, 0.04, and 0.4 fig/kg) had little or no effect on Zif268 mRNA expression, while higher doses of E2 (4-400 Mg/kg) resulted in maximal increases in Z/7268 expression. Dexamethasone, 5a-dihydrotestosterone, and progesterone had no effect on uterine Zif268 mRNA expression, and the induction of Zif268 by E2 was abolished by pretreating the animals with the RNA synthesis inhibitor actinomycin-D. In addition, stimulation of Z/7268 mRNA expression was observed with the short-acting estrogen estriol, suggesting that the response may be specific for estrogenic steroids. These data demonstrate that one of the initial responses induced by estrogen in the rat uterus involves increased Z/7268 gene expression and suggest that Z/7268 (as well as c-fos and c-myc) may play a transcriptional role in the estrogen regulation
of cell growth. (Molecular Endocrinology 5: 829-835, 1991)
INTRODUCTION A major effect of the acute administration of 1 "7/3estradiol (E2) to ovariectomized female rats is the stimulation of DNA synthesis and cell proliferation in the uterus (1). The uterine response initially involves hyperemia, followed by hypertrophy and hyperplasia within 24 h of hormone treatment (2, 3). In addition to these gross changes, E2 induces the stimulation of many classes of RNA (4-6), including a number of protooncogenes, including c-fos, c-myc, N-myc, c-jun, c-ras and c-erb-B (7-13). The response of the uterus to E2 is, therefore, a useful model in which to elucidate the molecular mechanisms involved in regulating the massive effects of hormone-induced cell proliferation. Among the molecular events that control the initial phase of transcriptional activation are several genes encoding known or presumed transcription factors, such as c-jun, c-fos, and c-myc, which may regulate the ensuing genetic program (14-16). One of these putative transcription factor genes, Zif268 (also referred to as NGFI-A, Egr-I, and Krox-24) (19-22), is rapidly upregulated in various cultured cell lines by growth factors (19-22), retinoic acid (23), and cellular depolarization (16) and is expressed in a number of different tissues and organs (16, 18-20, 24). The Zif268 gene product contains three tandemly repeated zinc finger sequences, a common feature of many DNA-binding transcription factors, and is homologous to the Xenopus transcription factor TFIIIA (25, 32). Numerous studies have associated Zif268 expression with proliferation (16-22) and differentiation (26, 27) both in vitro and in vivo. In addition, Zif268 mRNA expression in rat brain is increased after seizure (28,29). Although the precise function of the Zif268 protein is unknown, Zif268 binds
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to a specific sequence in genomic DNA and activates transcription (30, 31), perhaps by a similar mechanism as a frans-acting regulatory factor. Since the immature rat uterus is commonly used to study the molecular mechanisms of steroid hormone action (9, 13, 33-37), we sought to determine whether increased Zif268 expression was controlled by E2. This report demonstrates that Zif268 mRNA expression is rapidly increased (45- to 50-fold by 2 h) after a single ip injection of E2. In addition, this response is dose dependent, specific for estrogenic steroids, and apparently transcriptional in nature.
accumulation of Zif268 mRNA 2 h after treatment (Fig. 1A). Levels of Zif268 mRNA fall precipitously over the next 2 h and return to basal levels 12 h after hormone addition (Fig. 1A). We consistently observed that E2 stimulated the accumulation of 0-actin mRNA during this time course. Consistent with previous findings (13, 17), the levels of 0-actin mRNA peaked later (4-6 h) than those of Zif268 mRNA and remained elevated even at times when Zif268 mRNA levels were declining. As expected, over the same time course as E2 treatment, the injection of saline had no effect on the expression of Zif268 mRNA in the uterus (Fig. 1B).
RESULTS
Dose-Related Increase in Zif268 mRNA Expression by Estrogen in Rat Uterus
Increased Expression of Z/7268 mRNA by Estrogen in Rat Uterus The level of Zif268 mRNA was measured in uterine RNA isolated from ovariectomized immature rats at various times after ip injection of E2. Although Zif268 mRNA is detectable in the uteri of untreated ovariectomized animals (Fig. 1A, 0 h), a single dose of E2 (40 Mg/kg) stimulates a rapid 45- to 50-fold increase in the
E2 (40 ug/kg) 0 0.5h 1h 2h 4h 6h 12h Zif 268
We next examined the increase in uterine Zif268 mRNA expression as a function of the dose of E2. Groups of rats were injected ip with increasing doses of E2, and uteri were removed for RNA analysis 2 h after injection (Fig. 2). At doses of 0.004, 0.04, and 0.4 Mg/kg, E2 had little or no effect on the level oi Zif268 mRNA expression in the rat uterus; however, doses of 4.0, 40, and 400 Mg/kg produced 45-fold elevations in Zif268 expression 2 h after hormone treatment (Fig. 2). In the above study we examined the regulation of Zif268 mRNA expression in the uterus 2 h after treatment with E2. Since the increase in Zif268 mRNA expression was apparent only after the injection of 4.0, 40, or 400 Mg/kg E2) we next tested whether the highest dose of E2 used (400 Mg/kg) would alter the magnitude or duration of Zif268 mRNA expression. The injection of 400 Mg/kg E2 had no significant effect on either the peak level of expression or the time course of Zif268 mRNA accumulation in the uterus (Fig. 3).
(3-actin
0)
Saline
B o
o
C\J
.c
.c
Tt
CO
9
Zif 268 -
Zif 268 •
(3-actin *•
(3-actin •
Fig. 1. Time Course of the Induction of Zif268 mRNA by E2 in Rat Uterus Total RNA (30 Mg/'ane) was prepared from uteri removed from ovariectomized rats 0-12 h after treatment with E2 (40 Mg/kg; A) or saline (B). Each lane contains RNA from five animals. The position of the 3.7-kilobase Zif268 mRNA is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter. Results are representative of two additional experiments that yielded similar results.
o o
c — ft
Tfr
CO
Fig. 2. Dose-Related Effects of E2 on the Expression of Zif268 mRNA in Rat Uterus Total RNA (30 fig/\ane) was prepared from uteri removed from ovariectomized rats (five per sample) 2 h after treatment with E2 at the indicated doses or a vehicle (saline) control. Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
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831
Estrogen Regulation of Zif268 mRNA
E2 (400 |ag/kg) o
0 2h 4h 6h 8h
S
J
O
Zif 268 P-actin Fig. 3. Time Course of Zif268 mRNA Expression after Treatment with 400 Mg/kg E2 in Rat Uterus Animals were given E2 (400 Mg/kg), and uteri (five per sample) were removed 0, 2, 4, 6, and 8 h after treatment. Total RNA (30 ^g/lane) was hybridized with a Zif268 cDNA probe. Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
Steroid Specificity of Increased Zif268 mRNA Expression To investigate the specificity of the Zif268 mRNA response to E2, we examined whether Zif268 mRNA expression in the rat uterus was regulated by nonestrogenic steroid hormones. Levels of Zif268 mRNA were measured 2 h after a single ip injection of dexamethasone (600 Mg/kg), 5a-dihydrotestosterone (400 Mg/kg), or progesterone (40 mg/kg). Of the steroids tested in this experiment, only E2 (40 ^g/kg) stimulated the expression of Zif268 mRNA (50-fold) in the uterus (Fig. 4). With this protocol, the increase in Zif268 mRNA expression in the uterus is specific for estrogen; however, these data do not exclude the possibility that other nonestrogenic steroids may regulate Zif268 expression under different experimental conditions. To examine the specificity of the estrogenic regulation of Zif268 mRNA in this tissue, the level of Zif268 mRNA was measured in uterine RNA at various times after ip injection of estriol (E3), a short-acting estrogen which is equipotent with E2 in inducing early hypertropic growth responses in the uterus (41). We found that, like E2, a single dose of E3 (40 Mg/kg) stimulates a modest 2- to 3-fold increase in Zif268 mRNA expression 1 h after treatment, which increases to maximal levels (50-fold increase) within 2 h after E3 treatment (Fig. 5). The elevated level of Zif268 mRNA expression falls sharply over the next 2 h and returns to basal levels 4 h after hormone administration (Fig. 5). These data confirm the estrogen specificity of the Zif268 response with respect to both time course and peak effect after estrogen treatment.
Zif 268
(V
UJ
--**«^m^^^m .
p-actin Fig. 4. Steroid Specificity of the Induction of Zif268 mRNA in Rat Uterus Ovariectomized animals were injected ip with vehicle (saline), 600 Mg/kg dexamethasone (DEX), 400 Mg/kg 5«-dihydrotestosterone (DHT), 40 Mg/kg E2, or 40 mg/kg progesterone (PRG). Uteri (five per sample) were removed 2 h after hormone treatment, and total RNA (30 iiq/\ar\e) was hybridized with a Zif268 cDNA probe. Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
E3 (40 |ig/kg) 0 0.5h 1h 2h 4h 6h
Zif 268 • P-actin • Fig. 5. Time Course of Induction of Zif268 mRNA by E3 in Rat Uterus Total RNA (30 Mg/'ane) was prepared from uteri (five per sample) removed from ovariectomized rats 0-6 h after treatment with E3 (40 Mg/kg). Hybridization to the 3.7-kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /?actin to the same filter (lower panel). Results are representative of two additional experiments that yielded similar results.
Effect of Actinomycin-D on Estrogen-Stimulated Z/7268 mRNA Expression in the Uterus To investigate whether the increased expression of Zif268 mRNA represents a transcriptional effect of estrogen resulting from new RNA synthesis, we studied
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Vol 5 No. 6
MOL ENDO-1991 832
the effect of the transcription inhibitor actinomycin-D on this increase. Animals were pretreated with actinomycin-D (8 mg/kg) or vehicle for 2 h and then challenged with a single ip injection of either vehicle or E2 (40 ng/ kg). As shown in Fig. 6, actinomycin-D abolished the increase in Zif268 mRNA that followed E2 administration.
DISCUSSION
The proliferative activity of rat uterine tissue in response to estrogen treatment in the immature rat has been widely used as an in vivo model to study the molecular mechanism(s) of steroid hormone action. E2 has been demonstrated to induce a biphasic uterine growth response (2, 3). The initial phase of the mitogenic response involves a series of early hypertropic responses, 4-6 h after hormone treatment, and a series of late responses, which develop 24-30 h after hormone administration (42). The early responses induced by E2 in the immature rat uterus are characterized by altered expression of genes encoding protooncogenes (7-13), growth factors (35, 36), and their receptors (11). As these responses are associated with the regulation of cell proliferation and differentiation, it has been suggested that they may mediate the E2-stimulated growth response in the uterus (33, 43). In this study we have demonstrated that E2 produces a rapid stimulation of the transcription factor Zif268 in the immature rat uterus. After a single ip injection of E2,
Saline
Act D
S E2
S E2
Zif 268 •
p-actin Fig. 6. Effect of Actinomycin-D on E2 Induction of Zif268 mRNA Total RNA was prepared from uteri removed from different groups of animals (five per sample). One group received vehicle for 2 h, followed by a second ip injection of either vehicle (S) or 40 M9/kg E2. A second group received 8 mg/kg actinomycinD (Act D), followed 2 h later by an ip injection of either vehicle (S) or 40 /xg/kg E2. Total uterine RNA (30 ixg/\ane) was hybridized with a Zif268 cDNA probe. Hybridization to the 3.7kilobase Zif268 mRNA (upper panel) is indicated by the arrow. Comparison of RNA loading is shown by the hybridization of /3-actin to the same filter (lower panel). Results are representative of one additional experiment that yielded similar results.
Zif268 mRNA is rapidly and transiently increased, reaching a peak level (45- to 50-fold increase) within 2 h and declining to basal levels over the next 10 h (Fig. 1A). The time course of E2-stimulated expression of Zif268 mRNA develops after the time course of occupancy of nuclear estrogen receptors with E2, which has been demonstrated to peak 30-60 min after E2 treatment (33, 38, 44), and with increased uterine DNA synthesis (39, 40) after E2 administration. The pattern of Zif268 mRNA expression in the uterus after E2 treatment is very similar to the pattern observed for mRNA encoding the epidermal growth factor receptor (12, 45), c-fos (7, 8), N-myc (13), c-myc (8,13, 34), and c-jun (9, 10) and coincides with the established time courses of many early uterine growth responses. The doses of E2 used that produced pronounced changes in Zif268 mRNA levels in the immature rat uterus were within the physiological range of doses used to initiate other uterine growth responses (46) and suggest a potential role for Zif268 in mediating the growth and development of this tissue. In addition, we examined whether this response could be reproduced in ovariectomized adult rats and mice, and have observed identical results (data not shown). These data together suggest that the increase in Zif268 mRNA expression is physiologically relevant and neither age nor species specific. Given the wide array of agents known to induce Zif268 expression (16, 19-23), it is interesting that the rapid induction of Zif268 mRNA in the uterus is specific for estrogens. This is particularly intriguing since uterine tissue possesses receptors for glucocorticoids (47), androgens (48, 48), and progesterone (50, 51) as well as functional estrogen receptors. It is also possible that nonestrogenic steroids may regulate Zif268 mRNA expression in the uterus or other tissues under different experimental conditions or with different kinetics. Interestingly, the 5'-regulatory region of the rat Zif268 gene for which sequence data are available (52) contains no consensus pallindromic estrogen response elements. However, a number of sequence elements homologous to regions of the c-fos gene promoter that have been identified as binding sites for both the estrogen receptor and transcription factor AP-1 (53) are located in the Zif268 gene promoter region. While this similarity is interesting, as yet it is not known if any of these sequence elements are involved in the E2 regulation of Zif268 in this tissue. E3, a short-acting estrogen with altered receptorbinding affinity, has been shown to elicit early uterine hypertropic growth responses of equal magnitude to those caused by E2 (40); however, unlike E2, E3 does not stimulate cell division. These growth effects have been confirmed using other short-acting estrogens (54) and suggest that the uterine responses are not a simple cascade of events, with the early hypertropic responses leading directly to the hyperplastic growth response. Indeed, Anderson et al. (44) have suggested a sustained input theory for steroid action in which occupied estrogen receptors must remain in the nucleus of target cells
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833
Estrogen Regulation of Zif268 mRNA
for defined periods in order to induce the molecular events that eventually culminate in the hyperplastic growth response of the tissue. In this study the rapid and specific induction of Zif268 mRNA by both E2 and E3 in the uterus suggests that Zif268 mRNA expression may play a role in the early stages of the estrogeninduced responsiveness of this tissue. The complete blockade of the rapid E2 stimulation of Zif268 mRNA expression by actinomycin-D suggests that this response is mediated by an effect of E2 on the rate of Zif268 transcription. The dose of actinomycin-D used in these studies has been shown previously to be effective in inhibiting the synthesis of other E2-stimulated RNAs in the uterus (55). Although these pharmacological studies only suggest a transcriptional effect of estrogen on Zif268 expression, previous studies have demonstrated that the regulation of Zif268 is primarily transcriptional (16, 21-23). However, nuclear run-on experiments will be necessary to unequivocally define the precise mechanism(s) of E2 stimulation of Zif268 expression in the uterus. To date, the precise function of the Zif268 gene product in any system is unknown, but given the broad spectrum of stimuli that induce Zif268 expression (16, 18-23), it is likely that it has an important function in many fundamental biological processes. Recent reports identifying specific Zif268 DNA-binding sites (31, 32) and the homology with known transcription factors (14, 21, 22, 24, 33) imply that Zif268 is a frans-acting regulatory molecule. It is likely that many specific interactions occur between E2-inducible genes, such as Zif268, in the rat uterus. Presumably, it is this interrelationship that is responsible for the control of E2induced growth and/or differentiation in this tissue. By analogy with competence growth factors, the transient induction of Zif268 and other immediate early genes by E2 may render the cell competent to progress through the cell cycle (56). Given the dramatic changes in the levels of gene expression that occur in the rat uterus after E2 treatment, identifying the potential function(s) of Zif268 in mediating various cellular responses requires identification of the genes, or gene products, regulated by Zif268 and localization of the sites of synthesis and action of Zif268 in vivo.
Louis, MO). Before death, animals were injected ip with treatment compounds in a vehicle consisting of 0.9% NaCI-0.4% ethanol. Control animals received only vehicle. At different times after injection, animals were killed, and uteri were removed, immediately frozen in liquid nitrogen, and pooled in groups of five. RNA Purification Total RNA was prepared using a modification of the guanidinium isothiocyanate method (57). Pools of frozen uteri were homogenized in 9 ml of a solution containing 4 M guanidinium isothiocyanate (Bethesda Research Laboratories, Gaithersberg, MD), 0.03 M sodium acetate, 0.1 M /3-mercaptoethanol, and 0.4 g/ml cesium chloride using a Brinkmann Polytron (Westbury, NY). The homogenates were clarified by a prespin at 12,000 x g, and 7 ml clarified supernatant were layered over 3 ml of a solution containing 5.7 M cesium chloride and 0.03 M sodium acetate. Gradients were centrifuged at 35,000 rpm for 14-16 h in a Beckman 70.1 Ti rotor. Pelleted RNA was washed with 70% ethanol, resuspended in water, and precipitated with ethanol. Yields of total RNA were determined by measuring absorbance at 260 nm. Northern Blot Analysis Total RNA (30 ^g) was separated by electrophoresis through a 1 % agarose (Bethesda Research Laboratories) gel containing 0.66 M formaldehyde (J. T. Baker, Phillipsburg, PA) and transferred to nitrocellulose (Schleicher and Schuell, Keene, NH). After transfer, filters were baked at 80 C for 2 h under vacuum. The filters were prehybridized in a buffer containing 50% deionized formamide (Bethesda Research Laboratories), 5 x SSC (1 x SSC = 0.15 M NaCI-0.015 M Na3.citrate), 50 rriM sodium phosphate (pH 7.0), 5 x Denhardt's solution, 1 % skim milk powder, 100 Mg/ml sonicated salmon sperm DNA, and 0.1% sodium dodecyl sulfate at 42 C. Filters were hybridized in the same buffer to a 1.6-kilobase BglH fragment of Zif268 cDNA (22) (106 cpm/ml) labeled with [
