Hormonal regulation and expression of the jun-D protooncogene in specific cell types of the rat uterus

J. SteroidBiochem.Molec.Biol.Vol.46, No. 3, pp. 281-287, 1993 Printe.d

0960-0760/93 $6.00+ 0.00 PergamonPressLtd

in Great Britain

H O R M O N A L R E G U L A T I O N AND EXPRESSION OF THE jun-D P R O T O O N C O G E N E IN SPECIFIC CELL TYPES OF THE RAT UTERUS KENNETHP. NEPHEW,1 DAVID K. WEBB, 1. KAMIL CAN AKCALI, l BRUCEC. MOULTON2 and SOHAIBA. KHANI~ Departments of IAnatomy and Cell Biology and 2Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521, U.S.A.

(Received 19 February 1993;accepted 7 May 1993) Summary--Steroid hormone regulation and cell-type specific expression of the jun-D protooncogene in rat uterus was examined. Adult, ovarieetomized rats were injected with progesterone, testosterone, 17fl-estradiol (E2-17fl), 16ct-estradiol (E2-16ct), dexamethasone or cycloheximide. Uteri were collected between 0 and 6 h post-treatment. Northern blot analysis of uterine RNA revealed that induction of jun-D was specific for estrogenic steroids, as progesterone and testosterone had no effect on expression of this member of the jun gene family. Treatment with E2-17fl increased jun-D mRNA levels by approx. 5-fold, with expression reaching peak levels at 3 h after treatment and declining thereafter. Administration of E2-16~t, a short-acting estrogen that does not cause uterine cell proliferation, increased expression of jun-D but with different kinetics compared to the long-acting E2-17fl. The mRNA levels ofjun-D increased by 3-fold 1 h after administration of E2-16~ but declined soon after. Slight induction ofjun-D mRNA by dexamethasone was apparent, but to a much lesser extent compared to estrogen. The protein synthesis inhibitor, cycloheximide, did not block jun-D induction, indicating that this is an "immediate early" response. Expression of Jun-D protein was examined by immunohistochemical methods. E2-17fl treatment activated jun-D primarily in the nuclei of luminal and glandular epithelial cells of the endometrium. These results demonstrate that hormonal induction ofjun-D is specific for estrogens and that uterine expression of this protooncogene occurs in a cell-type specific manner.

amino acid sequence, with Jun-D more closely related to c-Jun than to Jun-B [1, 2]; however, The protooncogene jun-D is a cellular homolog several observations indicate that the different of the transforming gene of avian sarcoma virus jun genes are not regulated coordinately. Com17 [1, 2]. It was the third member of the m a m - pared to c-jun and jun-B, jun-D m R N A is more malian jun family identified, after c-jun and abundant in nongrowing 3T3 cells, is expressed jun-B. The jun genes are members of a larger to a lesser extent by serum growth factors and gene family encoding nuclear proteins that can is more stable after serum stimulation [1]. In associate with products of thefos gene family to differentiated myocytes [10], the jun-D gene is generate an AP-1 transcription factor com- expressed constitutively at high levels and not plex [3-9]. Although Jun/Fos heterodimers bind regulated during myoblast proliferation or more efficiently, the Jun proteins alone can bind differentiation, in contrast to c-jun and jun-B. to an AP-1 site and modify gene transcrip- Peptide growth factors rapidly induce extion [6, 9]. These oncoprotein dimers may play a pression of c-jun andjun-B in human m a m m a r y key role(s) in the regulation of normal cell carcinoma cells, but neither m R N A level nor growth, perhaps as initiators of a cascade of transcription rate ofjun-D are modulated [11]. events involved in cell proliferation and differ- Thus, it appears that each of the jun genes is entiation. likely to have unique controlling elements that The three Jun proteins are closely related in govern responses to specific intra- or extracellular signals, perhaps in a tissue specific manner. *Present Address: Laboratory of Molecular Genetics, NaUterine function is controlled primarily by tional Institute of Aging, 4940 Eastern Avenue, Baltithe interaction of estrogen and progesterone. more, MD 21224. tTo whom correspondence should be addressed. Estrogen stimulates proliferation of uterine INTRODUCTION

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endometrial epithelial cells, but is not mitogenic to stromal and myometrial cells[12-15]. The growth stimulatory effect of estrogen presumably involves regulation of the expression of genes whose products control the cell cycle, such as jun and fos protooncogenes. We and others [16-21] have shown that administration of estradiol-17fl (E2-17/3) to ovariectomized (OVX) rats induces rapid increases in expression ofjun gene family members and c-fos, supporting the hypothesis that these protooncogenes play a role in estrogen-stimulated growth. E 2-17/3 induces c-fos expression specifically in the epithelial cells of the adult rat uterus [22]; however, it is presently not known which uterine cell types express Jun oncoproteins in response to estrogen. In this report, hormonal regulation and cell-type specific expression of jun-D in uteri of OVX rats were investigated. Of the steroid hormones examined, both 17/3- and 16~-estradiol (a short-acting estrogen that does not cause proliferation of uterine cells) elevated in vivo levels ofjun-D transcripts. Immunohistochemical analysis revealed that E2-17/3 activated jun-D specifically in luminal and glandular epithelial cells of the endometrium.

EXPERIMENTAL

Animals Mature Sprague-Dawley rats (150-200g, Zivic Miller) were ovariectomized 10 days before use. Animals were housed in facilities illuminated between 0500 and 1900h. Animals treated with steroid hormones received a 0.1 ml s.c. injection in the periscapular region of 1/zg of E2-17fl (Sigma Chemical Co., St Louis, MO) in sesame oil, or 2rag of medroxyprogesterone acetate (Upjohn, Kalamazoo, MI) in an aqueous suspension, or 20/~g of dexamethasone (Sigma) in an aqueous solution or 2rag of testosterone propionate (Sigma) in sesame oil. Controls received sesame oil (Sigma) injections only. Estrogen-16~ (E216~; kindly provided by Marion Merrell Dow Research Institute, Marion Merrell Dow Inc., Cincinnati, OH) was administered by intraperitoneal injection of 3 p g in 1 ml of buffered saline containing 5% ethanol. Animals treated with cycloheximide received 5 mg in 1 ml of sterile saline via an intraperitoneal injection.

Total RNA analysis

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Northern

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Animals were killed by cervical dislocation. Uteri were quickly excised, trimmed of extraneous connective tissue and frozen on dry ice. Total RNA was isolated from whole uteri after homogenization in "Total RNA Isolation Reagent" (Molecular Genetics Group, Inc., Cincinnati, OH) using a Polytron homogenizer (Brinkman, Westbury, NY). Uteri from 5 animals were pooled for the preparation of a single sample of RNA. Pelleted RNA was resuspended in formamide (Formazol; Molecular Genetics Group, Inc.). The concentration of total RNA was determined by measuring the optical density at A260. The concentrations and the quality of the RNA preparations were confirmed by comparing 28 and 18 S ribosomal RNA bands stained with ethidium bromide on an agarose-formaldehyde gel. The Northern blot procedure utilized in these experiments has been described previously [19]. Each blot analysis was repeated three times. Briefly, 50/~g samples of total RNA were electrophoresed on agarose-formaldehyde gels and then transferred to Duralon (Stratagene, La Jolla, CA) by capillary blot. Molecular weight marker RNA was run on each gel to measure transcript size. Filters were pre-hybridized for 15min at 65°C with Rapid Hybridization Buffer solution (Amersham Life Science, Arlington Heights, IL) and hybridized for 2 h in the same solution with 106cpm/ml 32p-labeled jun-D cDNA probe or a constitutively expressed cDNA probe designated as 1A [23] (obtained from C. R. Lyttle, University of Pennsylvania). Probes were labeled by the random primer labeling method (Stratagene). The specific activity of the probes ranged from 1-2× 109cpm//zg DNA. The jun-D cDNA (ATCC No. 63025) is a 1.9 kb mouse cDNA cloned into the pGEM2 plasmid and excised with EeoRI. Filters were washed twice with 2 × SSPE, 0.1% SDS at room temperature for 15min, twice with 1 × SSPE, 0.1% SDS at 65°C for 10min, twice with 0.7 × SSPE, 0.1% SDS at 65°C for 15 min and then exposed to Kodak XAR X-ray film at - 7 0 ° C for 1 week. The relative intensity of the RNA bands was measured using a Hoefer Scientific Instruments GS300 scanning densitometer, jun-D expression in each lane was corrected for RNA content using the 1 A m R N A hybridization signal.

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jun-D regulation and expression in rat uterus

Immunohistochernistry Frozen uterine tissues were cut in 15-20/~m sections on a Leitz Kryostat model 1720 and placed on gelatin-coated glass sides. Sections were fixed in 4% paraformaldehyde for 0.5 h at 4°C and then incubated overnight in blocking buffer. The sections were then incubated for 48 h at 4°C with primary antibody. The primary antibody used in this experiment was kindly provided by Dr R. Bravo (Bristol-Myers Squibb). The anti-Jun-D (783/4) antibody is a polyclonal antibody generated in rabbits against a Jun-D MS2 polymerase fusion protein containing amino acids 1 to 102. Biotinylated goat anti-rabbit IgG was used as a secondary antibody. Sections were then incubated with avidin and biotinylated horse radish peroxidase complexes and stained by peroxidase reaction with nickel-enhanced 3,3' diaminobenzidine as a chromogen. The Jun fusion protein that was used to affinity absorb the anti-Jun-D (783/4) antibody was the same fusion protein used to generate the antibody. The cDNA encoding this protein was cloned into the heat inducible plasmid pEX-34 provided by R. Bravo. The fusion protein was purified by gel electrophoresis, electroeluted and dialyzed against 10 mM ammonium carbonate, as described previously by Kovary and Bravo [25].

s c a n n i n g of the resultant autoradiographs. Data were normalized with the 1 A hybridization signal. The 1 A m R N A is constitutively expressed in rat uterus and not stimulated by treatment with E2-17fl [23]. Figure 2(C) illustrates that levels ofjun-D m R N A were elevated 2- to 5-fold at 0.5 to 3 h after estrogen treatment. Results of our steroid-specificity experiments, at least at the doses tested in the present study, agree with observations for c-jun and jun-B, two other known members of this gene family, ([18-20] and our observations in press). Furthermore, the time course we observed is similar to that reported by Chiapetta et al. [20] for E2-17fl induction in immature rat uteri and by CicatieUo et al. [21] who used a nuclear run-on assay to examine jun-D induction in adult rat uteri.

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RESULTS AND DISCUSSION The experiments described in this communication were directed toward elucidating factors that regulate expression ofjun-D m R N A levels in rat uterus in vivo, as well as determining which uterine cell types express this oncoprotein. Induction of jun-D expression by E 217fl has been shown [20, 21], but it was not clear if jun-D activation is specific for this steroid. Low but detectable levels of jun-D transcripts were present in uterine tissues of OVX rats. Treatment with medroxyprogesterone acetate, testosterone propionate or dexamethasone did not markedly induce uterine expression ofjunD m R N A (Fig. 1). However, the levels of the 1.9 kbjun-D m R N A were rapidly elevated after a single injection of E2-17fl, reaching a maximal level by 3 h post-injection (Fig. 2). By 6 h after E2-17fl treatment, levels of jun-D m R N A had dropped toward baseline values. Quantitative increases in jun-D transcripts following EE-17fl administration were estimated by densitometric SBMB 46/3~B

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inhibitor, cycloheximide, on induction ofjun-D mRNA (A). Rats (5 per sample) were treated with progesterone (1 mg/100 g BW medroxyprogesterone acetate), MPA, testosterone propionate (1 mg/100g BW), dexamethasone (10#g/100g BW), DEX, or cycloheximide (1.5#g/100g BW), CHX, 3 h before sacrifice. Total RNA was prepared from 5 pooleduteri per treatment and subjectedto Northern blot analysis as described in the Experimentalsection. The l A mRNA hybridizationsignal (B) was used to correct for RNA content in each lane.

KENNETH P. NEPHEW et al.

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In uterus, it is apparent that estrogen induces expression of the jun genes in a synchronous manner. In other experimental systems, however, differences in levels of expression among the members of the jun gene family are apparent. Compared to c-jun and jun-B mRNA, expression of jun-D is constitutively high in quiescent 3T3 cells [1]. jun-D expression is insensitive to serum stimulation, in contrast to the rapid increase in levels of c-jun and jun-B mRNAs after serum addition [1, 2, 26]. Insulin, epidermal growth factor and transforming growth

factor-~ increase levels of c-jun and jun-B m R N A in MCF-7 human breast cancer cells, but expression of jun-D m R N A remains unchanged[ll]. There are also apparent differences in magnitude of uterine expression of the jun genes, but the significance of this observation is not clear. It has been shown in other experimental systems that Jun-B functions as a negative regulator of c-Jun [27-29]. Thus, an abundance ofjun-B m R N A may serve to limit uterine cell signalling by c-Jun. In addition, an increase in jun-B could potentially favor homodimerization of Jun-B with itself or with other jun gene family members and/or aid in heterodimerization of Jun-B with members of the fos gene. A single injection of E2-16~t is insufficient to induce uterine epithelial cell proliferation; repeated injections at 3 h intervals are required to induce the proliferative response [24]. However, the fact that one injection of E2-17 fl will stimulate D N A synthesis in rat uterus has led to the supposition that immediate early gene activation is linked to the uterine proliferative response to estrogen. For this reason, we examined the linkage between induction of jun-D expression and induction of the proliferative response of uterine epithelial cells to estrogen. The results are illustrated in Fig. 3. A single injection of E2-16~ induced expression ofjun-D m R N A in uteri of adult, OVX rats by approx. 3-fold over expression in untreated rats by 1 h after administration. This induction was transient and had begun to decline by 3 h after injection. These results demonstrate for the first time that jun-D expression can be induced without inducing the proliferative response of uterine epithelial cells to estrogen. It has also been reported that administration of this estrogen induces c-jun, jun-B and c-los in rat uterus ([24] and our observations in press). Collectively, these results support the hypothesis that induction of jun and c-los gene expression per se is not suffÉcient to mediate the mitogenic response to estrogen. It is possible that E2-17fl, although mitogenic in its own right, acts in concert with other growth factors to achieve a full growth response. It is also likely that estrogen activates still unidentified genes required for uterine cell proliferation and requisite to attain the full growth response. To investigate the possibility that induction of jun-D m R N A is a primary response to estrogen, we studied the effect of the protein synthesis inhibitor, cycloheximide (CHX), on E2-17 fl

jun-D regulation and expression in rat uterus

induction of jun-D mRNA expression. Pretreatment of rats with CHX for 1 h prior to E2-17//administration failed to block induction ofjun-D mRNA expression (data not shown). Moreover, CHX pre-treatment alone or in combination with E2-17//induced a remarkable increase in jun-D mRNA. The results are shown in Fig. 1. Superinduction ofjun protooncogenes by protein synthesis inhibitors is well recognized ([18,22,30] and our observations in press), suggesting that in the unstimulated uterus,

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285

mechanisms are already present to amplify the hormone signal. It is interesting, however, that CHX treatment superinduces jun expression, but puromycin, another protein synthesis inhibitor, does not [20]. In contrast to the report by Cicatiello et al. [21] of two jun-D transcripts (sizes 1.6-1.8 and 2.0-2.1 kb) in rat uterus after CHX treatment, we observed only one jun-D mRNA species in uteri from control rats or those treated with CHX or steroid hormones. Only a single jun-D mRNA species has been reported in other experimental systems [1, 2, 11, 20, 31]. In OVX mature rats, ¢stradiol stimulates the proliferation of endometrial epithelial cells, but is not mitogenic in the stroma and myometrium [12-15]. To identify the uterine tissue responding to estradiol with increased Jun-D expression, an anti-Jun-D antibody was used in immunohistochemical studies. To confirm the specificity of the antibody preparation, all immunohistochemical procedures were performed on tissue sections in the absence of the primary antibody. In other control experiments, the primary antibody was neutralized with antigen incubation overnight with excess Jun-D fusion protein before use of the supernatant for immunohistochemistry. In both control experiments, no positive staining was evident[Fig. 4(A and B)], thus confirming the specificity of the Jun-D antibody. Sections were incubated with secondary antibodies alone as a control for background staining. In sections from untreated control animals reacted with anti-Jun-D antibody, some nuclear staining in luminal and glandular epithelial cells was evident [Fig. 4(C)]. After 3 h of treatment with E2-17fl, the intensity of nuclear staining appeared to increase, as did the ability to distinguish individual epithelial cell nuclei [Fig. 4(D)]. Nuclear staining of epithelial cells remained very strong 6 h after E2-17fl administration [Fig. 4(E and F)], thus implying a nuclear function for jun-D. Even after 24h post-E2-17fl injection, luminal and glandular epithelial cells displayed strong nuclear staining (data not shown), and staining was still evident at 48 h, albeit to a lesser degree. This was not surprising, as Jun-D has been identified as the most stable member of the jun gene family [25]. Anti-Jun-D staining was apparent only in a few stromal and myometrial cells, supporting the concept that Jun-D proteins participate in estrogen-induced proliferation and growth of uterine epithelial cells. The simultaneous presence of

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Fig. 4. Immunohistocbemical localization of Jun-D in uterine sections from OVX rats. Specificity of the Jun-D polyclonal antibody was confirmed by (A) pre-absorbing the primary Jun-D antibody with antigen prior to use; and (B) performing immunohistochemical procedures in the absence of primary antibody.

Note the lack of staining in both A and B (200 × magnification).(C) Control (no treatment) section after incubation with anti-Jun-D (200 x ). Only faint staining is discernable. (D) 3 h after E 2-17fl administration (200 x ). Nuclei of the uterine luminal epithelial cells are stained positivelyfor Jun-D. (E and F) 6 h after E2-17fl administration (200 x and 400 x, respectively).Intense nuclear staining in luminal and glandular epithelial cells. Jun-D (present study), c-Jun and Jun-B (our observations in press) and c-Fos [24] in uterine epithelial cells in response to estrogen is significant. These oncoproteins can associate and form functional dimers [2, 4-9] with different D N A binding affinities, modulatory actions or stability. Jun proteins appear to differ in their ability to bind AP-1 [8]. Furthermore, Jun-D can bind AP-1 in the absence of c-Fos, but Jun proteins bind D N A more efficiently in the presence o f c - F o s [2, 6, 7]. Induction of various combinations of Jun/Fos homo- and heterodimers

by estrogen would provide a uterine cell multiple pathways to alter its response to various stimuli and ultimately control expression of target genes. In conclusion, regulation of uterine growth and differentiation by estrogen is thought to occur through a cascade of events. As proposed by Spelsberg et al. [32] and DeAngelo and Gorski [33], a class of "early response genes," those genes important in regulating the expression of later classes of genes, respond quickly to estrogen. The "early genes" are then

jun-D regulation and expression in rat uterus

followed by expression of a second class of"late genes," which would include general structural genes important for overall uterine metabolism. The data presented here, together with that of other investigators discussed above, clearly lend credence to the hypothesis thatjun-D and other members of the jun gene family are immediate early genes which play a key role(s) in amplifying the uterine response to estrogenic stimulation. Acknowledgements--This work was supported by a grant from NIH (HD22918) to S.A.K. The generous gift of Jun-D antibody from Dr Rodrigo Bravo (Bristol-Myers Squibb) is greatly appreciated. The authors thank Merrell Dow Research Institute for providing the 16~t- estradiol.

15. 16. 17. 18. 19.

20.

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