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Endocrinology Vol. 138, No. 9 3836-3841
Copyright © 1997 by The Endocrine Society

ARTICLES

A Novel Estrogen-Enhanced Transcript Identified in the Rat Uterus by Differential Display1

Lynn M. Everett, Aixin Li, Gayatri Devaraju, Andrea Caperell-Grant and Robert M. Bigsby

Departments of Obstetrics and Gynecology and Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indianapolis 46202

Address all correspondence and requests for reprints to: Robert M. Bigsby, Ph.D., Indiana University School of Medicine, Department of Obstetrics and Gynecology, 1001 West Walnut Street (MF102), Indianapolis, Indiana 46202-5196.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen exerts its physiological effects in the uterus by inducing a cascade of transcriptional events; however, the number of genes known to be directly activated by estrogen in the uterus is small. In this study, immature ovariectomized rats were treated with estrogen or vehicle, and 3 h later the uterine horns were flushed to extract epithelial RNA. This RNA was used in the differential display technique to search for estrogen-responsive genes. Products of reverse transcriptase-PCR, made with pairs of arbitrary and oligo-deoxythymidine primers, were separated on denaturing polyacrylamide gels; candidate bands were excised and reamplified to produce probes for use in Northern blot analysis and screening of a {lambda}gt10 complementary DNA library made from rat uterus. A novel estrogen-enhanced transcript, designated EET-1, was identified from a differential display band, and the estrogen sensitivity of its expression was verified in Northern analysis. Characterization of EET-1 expression in the uterus showed that estrogen treatment resulted in a rapid and transient increase in EET-1 messenger RNA; steady state levels peaked between 2–3 h, returning to basal levels by 6 h. This increase was not abolished by pretreatment with cycloheximide, indicating that induction of EET-1 is a primary response to estrogen. Induction was specific to estrogen when extracts of whole uterus were examined; in the epithelium, there was also a slight response to progesterone. Expression of the gene was found in all organs surveyed; however, hormonal regulation was observed only in tissues of the reproductive tract and in the kidney.

Analysis of cloned EET-1 complementary DNA revealed a 2008-base sequence that showed 61% identity with a reported transcript that encodes a protein that plays a role in phorbol ester-induced regulation of the tumor necrosis factor-{alpha} gene. Potential casein kinase-2 and protein kinase C phosphorylation sites and a cysteine-rich region were identified in the amino acid sequence deduced from EET-1. Thus, it appears that EET-1 represents a primary estrogen response gene that may code for a phosphorylated protein involved in gene regulation through a protein kinase C-activated pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS, like all members of the steroid family of hormones, modify cellular physiology through the transactivational effects of their receptors (1, 2). Genes that are directly activated by steroid-receptor complexes, without the requirement for de novo protein synthesis, are referred to as primary response genes. Some of these encode other transcription factors, which then produce secondary and tertiary responses, and so on (2, 3, 4). It is believed that by stimulating primary response genes, estrogen activates a cascade of transcriptional events, products of which participate in physiological responses known to be estrogen dependent in the uterus, for example cell proliferation or embryo implantation.

To date, relatively few genes have been identified that are directly activated by estrogen (4, 5, 6, 7). The goal of this study was to identify estrogen-responsive genes in the uterine epithelium, determine whether they are primary response genes, and determine whether hormonal regulation is specific to the epithelium. The technique of differential display was used, and a novel transcript has been identified and designated EET-1. Characterization by Northern blot analysis indicates that estrogen stimulates accumulation of messenger RNA (mRNA) for EET-1 in the uterus, and that its induction is a primary response. A 2008-bp complementary DNA (cDNA) clone isolated from a {lambda}gt10 rat uterine library has been sequenced and reported to GenBank as accession no. U53184.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Use of and procedures performed on animals were approved by the local animal care and use committee. Immature (20–21d) female Sprague-Dawley rats (Harlan, Indianapolis, IN) were ovariectomized under general anesthesia. On the following 2 days, the animals were estrogen primed with 40 µg/kg BW 17ß-estradiol (E2; Sigma Chemical Co., St. Louis, MO) injected sc. After an additional 2 days without hormone treatment, animals were injected sc with vehicle or test substance. To yield starting material for the differential display assay, animals were injected with 4 µg/kg BW E2 3 h before harvest of epithelial RNA from the uterus. In separate experiments, animals were treated with test doses of E2, 16{alpha}-E2 (Steraloids, Pawling, NY), dexamethasone (DEX; Sigma), or dihydrotestosterone (DHT; Sigma). All of the above hormones were given as sc injections in 200 µl of a 1:20 solution of ethanol-0.9% NaCl. Progesterone (P4; Sigma) injections were 40 mg/kg BW in a 1:3 solution of ethanol-0.9% NaCl. Cycloheximide (Cx; Sigma) was administered by ip injection of 50 mg/kg BW in 800 µl Dulbecco’s PBS (Life Technologies, Grand Island, NY) 1 h before estrogen treatment. Animals were killed by cervical dislocation at the indicated times after estrogen injection.

RNA isolation
Uterine horns were removed and trimmed of fat and mesentery. One horn was homogenized in TriReagent (Molecular Research Center, Cincinnati, OH) using four 10-sec bursts of a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) at setting 9. Epithelial RNA of the other horn was isolated as described previously (4). Briefly, 0.8 ml solution D (guanidinium thiocyanate, sodium citrate, and Sarkosyl) was flushed through the horn over a 45-sec interval and added to 2.4 ml TriReagent-LS. For tissue specificity studies, whole organs were removed and immediately homogenized in TriReagent; RNA isolation was completed according to the protocol supplied by the manufacturer.

In some experiments total RNA was enriched for polyadenylated [poly(A)+] RNA before Northern analysis. Enrichment was carried out with the PolyATract mRNA Isolation System (Promega, Madison, WI) according to the manufacturer’s protocols.

Differential display and generation of probes
Candidates for estrogen-regulated genes were identified by differential display (8) using the RNAmap Kit (GenHunter Corp., Brookline, MA) according to the manufacturer’s instructions. Briefly, RNA isolates from uterine epithelium of vehicle-treated or estrogen-treated animals, as described above, were reverse transcribed using four separate oligo-deoxythymidine [oligo(dT)] primers degenerate at the second base from the 3'-end (i.e. dT12dMdA, dT12dMdC, dT12dMdG, and dT12dMdT, where M represents the degenerate base). Each primer directs synthesis of cDNA from about one fourth of the total RNA. The resulting subpopulations of cDNA were amplified by PCR in the presence of [35S]dATP using the original oligo(dT) downstream primer along with one of a set of five upstream arbitrary primers supplied with the kit (AP-1, 5'-AGCCAGCGAA-3'; AP-2, 5'-GACCGCTTGT-3'; AP-3, 5'-AGGTGACCGT-3'; AP-4, 5'-GGTACTCCAC-3'; AP-5, GTTGCGATCC-3'). Samples from each amplification reaction were loaded onto a 6% polyacrylamide-urea DNA sequencing gel and electrophoresed at 60 watts for 3 h. The gel was dried without fixing, and an autoradiograph was made to locate bands that appeared to be differentially expressed. Each PCR amplification reaction was performed four times, and only those differences that consistently appeared were considered relevant. The bands of interest were excised from the gel, and the cDNA was eluted and reamplified by PCR with the same primers as those used in the original PCR amplification. The cDNA thus produced was gel purified through low melting point agarose and used to probe Northern blots and a {lambda}gt10 library as described below.

Northern blot analysis
Total or poly(A)+-enriched RNA was quantified by spectrophotometry, and 12- to 20-µg samples of total RNA or 4-µg samples of poly(A)+-enriched RNA were run on 1.2% agarose-6% formaldehyde electrophoresis gels. The RNA was transferred to nylon membranes (MSI, Westboro, MA) by capillary blotting, baked at 65 C for 2 h, and prehybridized for 3 h at 42 C in 50% formamide, 5 x SSC (20 x SSC = 3 M sodium chloride-0.3 M sodium citrate, pH 7.0), 5 x Denhardt’s solution, 0.5% SDS, 100 µg/ml sheared salmon sperm DNA, and 1 µg/ml poly(A)+ DNA. Hybridization was carried out at 42 C for 16–18 h by the addition of [32P]deoxy-CTP-labeled probes (~25 ng cDNA; 50 µCi/reaction). cDNA probes were generated from differential display products as described above or from a cloned cDNA (see below). After hybridization, the membranes were washed for 15 min at room temperature in 200 ml of each of the following solutions: 2 x SSC-0.1% SDS, 1 x SSC-0.1% SDS, 0.2 x SSC-0.1% SDS, and 0.1 x SSC-0.1% SDS. The membranes were then exposed to autoradiographic film, and the optical density of the hybridization signals was quantified using a Bio-Rad model GS-670 imaging densitometer (Bio-Rad, Hercules, CA). To determine lane to lane loading variation, each blot was also probed with a control cDNA, CHO B, which corresponds to a transcript that is unaffected by hormone treatment (4). Final quantification normalized treatment effects against CHO-B expression.

cDNA library and sequence analysis
RNA was isolated from the uterus of ovariectomized immature rats treated with E2 for 3 h. RNA was enriched for poly(A)+ RNA as described above. The poly(A)+ RNA was reverse transcribed using an oligo(dT) primer. EcoRI linker arms were ligated to the cDNA. The library was ligated into {lambda}gt10 DNA and packaged into viral particles using the GigaPack II Gold system (Stratagene, La Jolla, CA). Bacterial plates were infected with virus, and plaque lifts were prepared. The lifts were probed with PCR-amplified cDNA representing individual bands on the differential display gel. Positive plaques were purified. The cDNA inserts were excised from the viral DNA and ligated into pGEM-7Zf+ (Promega). The pGEM clones were used as the source of cDNA for sequence analysis with the Sequenase version 2 DNA sequencing kit (U.S. Biochemical Corp., Cleveland, OH) according to the manufacturer’s recommendations. Two pGEM clones were sequence analyzed in both directions, such that nucleotide sequences reported were confirmed by at least four separate analyses.

Two procedures were used to ensure that the full 5'-end of the cDNA sequence was analyzed. A 5'-RACE (rapid amplification of 5'-cDNA ends) method was applied to uterine poly(A)+ RNA (isolated as described above) using a kit (5' RACE System, Life Technologies) according to the manufacturer’s protocols. Briefly, the first strand of cDNA was made using an oligonucleotide primer complementary to bases approximately 250 bases from the original 5'-end of the known sequence. This new cDNA was tailed with deoxy-ATP. The second strand of cDNA was made in a polymerase reaction using the supplied adapter primer, which contains a 3'-poly(dT) tail. The double stranded cDNA thus produced was ligated into pGEM-7Zf+, and this was grown as a clone in DH5{alpha} bacteria (Life Technologies). The inserts of two such clones were sequenced as described above. In addition, the original {lambda}gt10 cDNA library was PCR amplified using the internal antisense primer mentioned above and the forward or reverse primer sequences located in the {lambda}gt10 sequence flanking the inserted library. The {lambda}gt10 PCR reaction products were sequenced by a cycle-sequencing method using the SequiTherm EXCEL DNA sequencing kit (Epicenter Technologies, Madison, WI) according to the manufacturer’s protocols.

Cell culture
Cultures of uterine epithelial and stromal cells were prepared by enzymatic digestion as follows. Uterine horns were trimmed of fat and mesentery, slit open longitudinally, and cut into 3–4 pieces. The uterine pieces were incubated in 1% trypsin (Difco Laboratories, Detroit, MI) for 2 h at 4 C with gentle rocking. The digestion was stopped by the addition of 10% newborn calf serum (Life Technologies, Grand Island, NY) for 0.5 h at room temperature, and the supernatant containing the epithelial cells was removed. The remaining uterine pieces were then shaken in a solution of 0.05% trypsin and 0.05% collagenase A (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a 37 C water bath for 40 min to separate the stroma from underlying myometrium. Enzymatic activity was stopped by the addition of 15% newborn calf serum for 10 min at room temperature, and the solution was filtered through a no. 40-mesh Cellector tissue sieve (Bellco Glass, Vineland, NJ) to remove undigested pieces of uterine tissue. Separate cultures of epithelial and stromal cells were plated in Falcon six-well dishes (Becton Dickinson, Oxnard, CA) using the equivalent of two uteri per well. Cell types in individual cultures were verified by immunocytochemistry using antibodies against cytokeratin (Dako, Carpenteria, CA), a marker for epithelial cells, and desmin (Sigma), a marker for cultured stromal cells (not shown). For mixed cultures, the two cell solutions were combined and plated at approximately 850,000 cells/well. Plating medium consisted of DMEM-Ham’s F-12 nutrient mixture (1:1; Life Technologies) supplemented with 5% charcoal-stripped FBS (serum treated with 2 mg/ml dextran-coated charcoal), 10 µg/ml insulin (Sigma), 5 µg/ml transferrin (Sigma), and 0.5 µg/ml penicillin-streptomycin (Life Technologies). After 24 h of culture, cells were washed with PBS, and fresh medium was added to the wells. After an additional 24 h, mixed cultures were washed again with PBS and changed to either serum-free medium or medium containing 1% charcoal-stripped FBS. The cultures were given fresh medium again after 48 h. On the fifth day of culture, RNA was isolated from the cells using TriReagent according to the manufacturer’s protocol. Gels and Northern blots were prepared as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Northern blot analysis was used to verify the effect of E2 on expression of transcripts that were identified as being differentially displayed. Each probe for this initial Northern analysis was generated by PCR from the differential display product. One of these, designated EET-1, produced by the AP-5 and dT12dMdC primers, detected a mRNA band that was increased an average of 4-fold in extracts from the uterus of E2-treated animals (Fig. 1Go). There was no difference in the relative intensities of the EET-1 mRNA bands between the epithelial extract and the extract of whole uterus, indicating that it is not specific to the epithelium (Figs. 3Go and 4Go).



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  		Figure 1. Verification of estrogen responsiveness of the transcript identified by differential display. Animals were treated with vehicle (Veh and E) or 40 mg/kg BW progesterone (P and PE) at -0.5 h. At 0 h, either vehicle (Veh and P) or E2 (4 µg/kg BW, E and PE) was injected, and uteri were harvested at 3 h. RNA was pooled from uteri of four animals per treatment group, enriched for poly(A)+ RNA, and 4-µg samples from each treatment were applied to an agarose gel for electrophoresis. An adjacent lane was loaded with 10 µg total RNA to allow visualization of the ribosomal RNA, detected by ethidium bromide staining, as size standards. Blots were hybridized with a PCR-amplified cDNA derived from a differential display band, designated EET-1, that was the product of the primers AP-5 and dT12dMdC. This band was cut from the differential display gel, eluted, and reamplified. After gel purification, the EET-1 PCR product was used as a probe, priming its labeling reaction with dT12dMdC. The autoradiogram for EET-1 was developed after 3 days. The blot was stripped of probe and rehybridized with a probe for CHO-B; the newly probed blot was exposed to film for 16 h. The positions of the 28S and 18S ribosomal RNA bands seen in the adjacent, total RNA lane are indicated for size reference.

 


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  		Figure 3. Dose response and time course of EET-1 induction. Northern blots were prepared with epithelial RNA from uteri of animals treated with the indicated doses of E2 3 h before harvest of RNA, or (inset) RNA was collected from the epithelium and the whole uteri of animals killed at the indicated times after injection of 4 µg/kg BW E2. The blots were hybridized with a probe made from the 2008-bp cDNA sequenced in Fig. 2Go. To normalize for RNA loading variation, the amount of EET-1 mRNA was expressed as the ratio of the optical density of the EET-1 band to the optical density of the corresponding CHO-B band, and the ratio of each treatment was then related to the ratio of the control vehicle-treated animals.

 


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  		Figure 4. Hormone specificity of EET-1 induction. A, Animals were treated with various hormones for 2 h before tissue harvest. RNA extracts of whole uterus from four animals per treatment were pooled in each of three experiments; values represent the mean ± SEM. Treatments were as follows: Veh, vehicle; 17ß, E2 (4 µg/kg BW); 16{alpha}, 16{alpha}-estradiol (4 µg/kg BW); P, progesterone (40 µg/kg BW); DEX, 4 µg/kg BW; DHT, 4 µg/kg BW. B, Animals were pretreated with vehicle or P4 as described in Fig. 1Go, and tissues were collected 2 h after the test doses of E2 or vehicle. RNA was pooled from five animals for each treatment within an experiment; values represent the mean ± SEM of four experiments. Blots were hybridized, and results were calculated as described in Fig. 3Go. *, P < 0.05; +, P = 0.05 (vs. the vehicle control values).

 
A {lambda}gt10 cDNA library was screened with the PCR-amplified probe for EET-1. Several plaques were identified, and two were purified. The cDNA insert in these plaques was subcloned into a pGEM plasmid. Throughout the remainder of this report, the cloned cDNA was used to produce the probes in all Northern blot analyses.

Each strand of the cDNA in the two plasmid clones was sequenced. The analysis yielded a sequence of 2008 bases, shown in Fig. 2Go. The longest open reading frame that begins with an ATG sequence codes for a 161-amino acid protein. A search of GenBank databases showed that EET-1 exhibits a high degree of homology (61% identity) with a recently reported transcript (GenBank accession no. U77396) involved in phorbol ester-stimulated (PKC-activated) tumor necrosis factor-{alpha} (TNF-{alpha}) expression in human monocytes (Myokai, F., Boston University, personal communication).



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  		Figure 2. Nucleotide and deduced amino acid sequences of EET-1. The nucleotide sequence of the EET-1 cDNA was determined as described in the text. The longest open reading frame yields a deduced amino acid sequence of 161 residues before a stop signal (*) is encountered. Four potential phosphorylation sites were detected (bold T and S residues): a protein kinase C site at amino acid 157 and three Ck-2 sites at amino acids 22, 49, and 86. A cysteine-rich region with potential zinc fingers is seen in the carboxy-terminal portion (underlined amino acid sequence). Also, two potential ERE nucleotide sequences were detected in the 3'-untranslated region (double underline).

 
Further database analysis of the sequences using the ScanProsite program on the ExPASy WWW server (Geneva University Hospital and University of Geneva, Geneva, Switzerland) (9) revealed four potential phosphorylation sites (Fig. 2Go, bold T or S residues). There is a PKC phosphorylation site (TYK) at amino acid 157 and three residues identifiable as casein kinase-2 (CkII) sites (amino acids 22, 49, and 86). Furthermore, the CkII sites are conserved between the rat and human sequences even though there has been a change from serine to threonine residues at two of these positions (amino acids 22 and 86) between the two species. The EET-1 nucleotide sequence also showed high degrees of similarity with several mouse, rat, and human expressed sequence tags (EST), e.g. there was 94% nucleotide identity with a mouse embryo EST (GenBank AA032342) and 72% nucleotide identity with a human placental EST (GenBank C18902). The sequences corresponding to and surrounding the CkII phosphorylation sites were also found in these mouse and human ESTs. Also, there is a cysteine-rich domain (underlined) in the carboxy-terminal portion of the protein; although the sequence does not precisely match any of the known zinc finger motifs (10, 11), the spacing of the cysteine residues is such that it may allow for finger formation.

In the 3'-untranslated portion of the transcript, there are two 13-base sequences (underlined) that resemble known estrogen response elements (ERE). The first of these (GGTCAnnnTGGTC, nucleotides 1657–1669) is identical, except for a single nucleotide, to the ERE of the human pS2 gene (GGTCAnnnTGGCC) (12); the other is identical to the imperfectly palindromic Xenopus vitellogenin B1 ERE (AGTCAnnnTGACC, at nucleotides 1842–1854) (13). Whether these sequences function as response elements is currently under investigation. Neither of these potential ERE sequences is present in the reported sequence of the TNF{alpha}-inducible transcript; however, it does not appear that the complete cDNA sequence has been reported, as there is no polyadenylation signal present. A mouse EST derived from an embryo cDNA library (GenBank AA033070) is nearly identical to the 3'-untranslated region of EET-1, and it does contain the putative ERE sequences, suggesting that these sequences have been conserved at least between rat and mouse.

Several experiments were performed to characterize the estrogen responsiveness of EET-1 expression. Animals were treated with various doses of E2 for 3 h before tissue harvest. The minimum dose eliciting a response was 0.4 µg/kg BW. A dose of 0.8 or 4.0 µg/kg BW induced approximately a 2.7-fold increase in the level of EET-1 mRNA (Fig. 3Go).

To examine the time course of the estrogen response, animals were treated with vehicle or 4 µg/kg BW E2 and killed at indicated times thereafter. Epithelial and whole uterine RNA showed similar patterns (Fig. 3Go). There was a rapid increase in the mRNA level as early as 1 h after treatment. Levels peaked between 2–3 h and returned to basal levels by 6 h post treatment.

The effects of other steroid hormones and the interaction of E2 and P4 were analyzed. To test the hormonal specificity of EET-1 induction, animals were treated with E2, 16{alpha}-E2 (a short acting estrogen), P4, DEX, or DHT. Northern analyses of RNA extracts made from whole uterus showed that steady state levels of EET-1 mRNA were increased consistently by both estrogens, but not by the progestin, the glucocorticoid, or the androgen (Fig. 4AGo). Pretreatment with P4 did not modify the effect of E2 in either whole uterus or uterine epithelium (Fig. 4BGo); P4 given alone tended to increase the expression of EET-1 in the epithelium (P = 0.05).

The protein synthesis inhibitor Cx was administered to establish whether de novo protein synthesis was required for estrogen responsiveness. Treatment with Cx alone increased levels of the EET-1 transcript approximately 3-fold. Administration of E2 1 h after Cx resulted in a further increase in EET-1 mRNA, with levels averaging 1.6-fold those of Cx alone (Fig. 5Go).



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  		Figure 5. Effects of Cx Treatment. Animals were injected with vehicle (V) or 4 µg/kg BW E2 2 h before tissue harvest; others were treated with 50 mg/kg BW Cx 1 h before either vehicle (Cx) or E2 (CxE) and killed 2 h later. Total RNA from whole uterus was analyzed as described in Fig. 3Go. Data are expressed as mean ± SEM (n = 3). *, P < 0.05 compared with appropriate control, V or Cx.

 
One of the earliest events after estrogen stimulation in the uterus is a dramatic hyperemia (14). Therefore, primary cultures of uterine cells were used to confirm that EET-1 mRNA is actually of uterine origin and not the product of blood-borne cellular RNA. Northern blot analysis revealed the presence of the EET-1 transcript in both epithelial and stromal cultures (data not shown).

A number of organs in addition to uterus were tested for the presence of the EET-1 transcript. These included vagina, cervix, brain, heart, kidney, liver, and spleen. EET-1 mRNA was found in all organs tested; however, estrogen responsiveness was not universal. EET-1 mRNA increased with estrogen treatment in all three of the tissues of the reproductive tract (vagina, cervix, and uterus) as well as in the kidney (Fig. 6Go). The abundance of mRNA in heart, spleen (Fig. 6Go), brain, and liver (not shown) was unchanged with estrogen treatment.



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  		Figure 6. Tissue specificity of the estrogen response. Tissues from a variety of organs was collected from animals that had been treated with vehicle (V) or 4 µg/kg BW E2 (E) 2 h earlier. RNA (20 µg total RNA/lane) extracted from these organs was analyzed as described in Fig. 3Go. Blots shown are representative of three separate experiments. Autoradiograms for EET-1 were exposed for 3–7 days, whereas those for CHO-B required only 16-h exposure times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the initial results of differential display with the five arbitrary 5'-primers used in this study yielded several candidate genes, only one proved to be estrogen regulated and of uterine origin. This candidate, designated EET-1, displays a reproducible pattern of estrogen responsiveness and, in addition, was identified in cultures of rat uterine cells grown in serum-free medium, thus confirming its uterine tissue origin.

Because the increase in EET-1 mRNA levels is both rapid and transient, and because Cx fails to abolish the increase, it appears that EET-1 induction is a primary response to estrogen. The putative EREs found in the 3'-untranslated portion of the transcript may be responsible for the estrogen induction of EET-1. Although most enhancer elements identified in genes have been localized in a position 5' of the promoter region, a 3'-position is not unprecedented; for example, the progesterone receptor gene contains functional EREs in the transcribed sequence (7), c-fos is regulated by an ERE in a position 3' to the coding sequence (15), and c-jun contains an ERE in its coding sequence (16). Whether the putative EREs in the 3'-untranslated region of the EET-1 transcript are functional is the subject of continuing studies.

An investigation of the distribution of the EET-1 transcript revealed its expression in all organs surveyed; hormonal regulation, however, exhibited tissue specificity. No changes in mRNA levels were detected in brain, heart, liver, or spleen, but estrogen responsiveness was observed in all three tissues of the reproductive tract as well as in kidney. Although the kidney expresses estrogen receptor (ER) (17), and other estrogen-responsive genes have been identified in kidney (18, 19), this was an unexpected result, because the kidney is not considered to be a target of estrogen action in general. Furthermore, brain, heart, and liver also express low levels of ER and have estrogen-inducible genes (20), but estrogen failed to induce EET-1 in these organs. Therefore, the induction of EET-1 does not appear to be a generalized response to estrogen, but, rather, dependent upon the particular cellular context.

Although the induction of EET-1 in whole uterus appears to be specific to estrogen, this was not the case in the epithelium, where P4 treatment consistently resulted in an increase in EET-1 mRNA about half of that induced by estrogen. Other investigators have reported P4 induction of genes that are regulated primarily by estrogen. Das et al. found that both estrogen and P4 increased mRNA levels for the epidermal growth factor receptor in the uterus of adult mice; however, they could detect no bioactivity of the receptor in the absence of estrogen (21). In a study of immature mouse uterus by Gray et al., P4 induced a 4- to 5-fold increase in mRNA for the A chain of platelet-derived growth factor, whereas estrogen induction was about 20-fold (22). In the present study, the epithelial specificity of this response argues against any systemic increase in estrogen due to increased steroid biosynthesis. These preliminary findings on P4 action are the subject of continuing investigation.

Sequence analysis of EET-1 allows us to speculate that it codes for a protein that may be subject to posttranslational modification by kinases and that contains a structural motif that may allow for formation of novel zinc fingers. Such structural characteristics would be consistent with a regulatory role of EET-1. The similarity between the deduced amino acid sequences of EET-1 and a transcript involved in PKC regulation of TNF{alpha} expression suggests that EET-1 may serve as an intermediary factor linking estrogen receptor and growth factor receptor pathways. The transient character of its induction after estrogen treatment is also consistent with the pattern followed by the immediate early genes, c-fos, c-jun, and egr-1, after estrogen stimulation (3, 4, 23, 24, 25). Furthermore, like the immediate early genes, the levels of EET-1 mRNA increased during treatment with Cx; this, too, appears to be characteristic of regulatory genes (26, 27). Thus, EET-1 may represent a new immediate early gene with regulatory action. Testing of such speculation awaits further investigation.


   Footnotes
 
1 This work was supported by Grant HD-23244 from the NIH (to R.M.B.), a Biomedical Research Grant from Indiana University School of Medicine (to A.L.), and an Indiana University Graduate Student Fellowship (to L.M.E.). Back

Received February 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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