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Endocrinology, doi:10.1210/en.2003-0260
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Endocrinology Vol. 144, No. 11 4790-4798
Copyright © 2003 by The Endocrine Society

Differential Regulation of Two 3' End Variants of P450 Aromatase Transcripts and of a New Truncated Aromatase Protein in Rabbit Preovulatory Granulosa Cells

Vincent Hanoux, Hélène Bouraïma, Hervé Mittre, Colette Féral and Annie Benhaïm

Laboratoire de Biochimie, Équipe d’Accueil 2608, Unité Sous Contrat de l’Institut National de la Recherche Agronomique, Centre Hospitalier Universitaire Côte de Nacre, 14032 Caen Cedex, France

Address all correspondence and requests for reprints to: Dr. Vincent Hanoux, Laboratoire de Biochimie, Équipe d’Accueil 2608, Unité Sous Contrat de l’Institut National de la Recherche Agronomique, Centre Hospitalier Universitaire Côte de Nacre, 14032 Caen Cedex, France. E-mail: benhaim-a{at}chu-caen.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In rabbit granulosa cells, two cytochrome P450 aromatase (P450 arom) mRNAs issued from promoter II were described: a full-length and a truncated transcript. Western blot analysis showed two P450 arom proteins with apparent molecular masses of 53 and 46 kDa, which are consistent with the predicted theoretical sizes of proteins encoded by these two transcripts. To examine the involvement of the truncated transcript in the regulation of P450 arom gene expression, the level of each transcript was specifically quantified in cultured granulosa cells by competitive quantitative RT-PCR. FSH induced a dose-dependent increase in both estradiol production and P450 arom mRNAs levels with a much more enhancement in the full-length mRNA. The half-life of the transcripts could not explain this differential regulation. Upon dibutyryl cAMP stimulation, the full-length mRNA was less abundant than the truncated one. In contrast, Western blot analysis revealed a stimulation of the 53-kDa protein content, whereas the 46-kDa protein amount was apparently unaffected. TGFß in FSH-stimulated conditions decreased both estradiol production and P450 arom transcripts levels. TGFß did not modify estradiol production and aromatase protein amounts induced by dibutyryl cAMP, whereas the two P450 arom mRNAs levels were increased. In conclusion, we report for the first time that a protein encoded by a truncated P450 arom mRNA could be involved in the regulation of estrogen production. Moreover, we show that the two P450 arom mRNAs are regulated in a differential manner, probably through hormonal control of the alternative splicing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BIOSYNTHESIS OF ESTROGENS from androgens is catalyzed by a microsomal enzymatic complex named aromatase, which is composed of a specific cytochrome P450 aromatase (P450 arom) and an ubiquitous nonspecific flavoprotein, the NADPH cytochrome P450 reductase (1). In several species, such as rabbit (2, 3), cow (4), sheep (5), and human (1, 6, 7), P450 arom is encoded by the cyp 19 gene, which is composed of nine coding exons (exons II–X) and several tissue-specific promoters associated with the specific 5'-untranslated exons I.

In gonads, the P450 arom gene expression is mainly driven by the promoter II, and alternative splicing in the coding region of P450 arom RNA is responsible for distinct mRNA variants. Thus, in rat ovary, three P450 arom mRNAs have been detected (8). The largest species contains the nine coding exons, whereas the two smaller lack exon X, which codes for the heme-binding domain (9). In male rat germ cells two unusual isoforms of P450 arom mRNAs are expressed, and their sequences analysis demonstrated that the last coding exon was lacking (10). In ovarian follicle of the fish Medaka, one of the two cDNAs described also lacks the sequence coding for the heme-binding domain as the result of an additional nucleotide that causes a frameshift in the open reading frame (11). In rabbit preovulatory granulosa cells, two P450 arom mRNAs issued from promoter II are expressed. Sequence analysis of these two cDNAs revealed that the largest species (2.9 kb) is composed of nine coding exons, whereas the smallest one (1.5 kb) lacks exon X (12). However, it must be mentioned that proteins encoded by these 3' end variants of P450 arom mRNAs have never been detected, and the role of these truncated transcripts remains unknown.

The aim of this study was to analyze the involvement of the truncated P450 arom mRNA in the regulation of P450 arom gene expression in cultured granulosa cells from rabbit preovulatory follicles. Studies of the regulation of P450 arom gene expression are usually focused on the total level of specific transcripts, but alternative splicing events could be involved in the control of P450 arom gene expression.

Here we report that the truncated P450 arom mRNA is more abundant than the full-length P450 arom mRNA in basal conditions. Moreover, the expression of these transcripts is differentially regulated by FSH and dibutyryl cyclic AMP (db cAMP), probably through a modulation of the alternative splicing. Furthermore, TGFß a paracrine/autocrine regulator that has been detected in ovarian tissue of several species (13), exerts an inhibitory effect on FSH-stimulated P450 arom gene expression in rabbit granulosa cells. We also show for the first time that a truncated P450 arom mRNA is translated in a protein, which may suggest a possible involvement of this inactive aromatase in the regulation of estrogen production in rabbit ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
MEM with Earle’s salts and Eurobio Taq II polymerase were obtained from Eurobio (Les Ulis, France). Purified ovine FSH (o-FSH.20, 4453 IU/mg) was provided by the National Hormone and Pituitary Program, NIDDK, and Dr. A. F. Parlow. Fetal calf serum (FCS) was obtained from BioWhittaker (Emerainville, France). Actinomycin D, db cAMP, ethidium bromide, Tris base, Triton X-100, sodium dodecyl sulfate (SDS), NaCl, EDTA, phenylmethanesulfonylfluoride, sodium fluoride, sodium orthovanadate (NaO), leupeptin, aprotinin, pepstatin, and ß-glycerophosphate were obtained from Sigma-Chimie (L’Isle d’Abeau, France). Equine chorionic gonadotropin was obtained from Intervet (Angers, France), and embutramide T61 was purchased from Distrivet (Paris, France). RNasin, deoxy-NTPs (dNTPs), and Moloney murine leukemia virus reverse transcriptase were purchased from Promega (Lyon, France). Recombinant human TGFß1 was obtained from R&D Systems (Abingdon, UK). Oligo(deoxythymidine)12–18 was obtained from Amersham Pharmacia Biotech (Orsay, France), and primers were purchased from Invitrogen (Paisley, Scotland, UK).

Animals
Animals used in this study were immature female HY albinos rabbits, 12–14 wk of age (Elevage Gastebled, Hottot Les Bagues, France), and were housed individually in a controlled photoperiod of 14 h of light and 10 h of darkness. They were given a supply of standard rabbit food and water and allowed to feed ad libitum. To obtain preovulatory follicles, female rabbits received 200 IU equine chorionic gonadotropin daily for 2 d. Animals were killed 4 d later by intracardiac injection of 3 ml of the narcotic T61. Animals were bred under standard conditions according to the instructions of Ministère de l’Agriculture et de la Pêche-Service Santé Animale (France).

Granulosa cells isolation and culture
Granulosa cells of preovulatory follicles were obtained as previously described by Féral et al. (14) and were cultured in MEM supplemented with 5% FCS in an humidified atmosphere of 5% CO2 in air at 37 C for 24 h to allow cell attachment. Then, supernatants were removed, and cells were incubated for an additional 24 h in MEM supplemented with 2.5% FCS with androstenedione (10-7 M) with or without FSH (0.5–5 ng/ml), or db cAMP (2.5 x 10-3 M) and in the presence or absence of recombinant human TGFß1 (1 ng/ml). Then cell-free supernatants were collected and frozen until estradiol measurement. Cell layers were harvested for total protein extraction or were lysed for RNA extraction.

Protein extraction and Western blotting
Granulosa cells were harvested with the trypsin/EDTA method. Briefly, adherent granulosa cells were washed twice with PBS without Ca2+ or Mg2+ and were treated for 5 min at 37 C with PBS containing 0.5 g/liter trypsin and 0.2 g/liter EDTA. Trypsin activity was stopped by the addition of MEM supplemented with 5% FCS (vol/vol). Granulosa cells pellets were frozen at -80 C until protein extraction.

For total protein extraction, granulosa cells pellets were resuspended in homogenization buffer [20 mM Tris base (pH 8.0), containing 150 mM NaCl, 1% Triton X-100, 0.5 mM EDTA, 1 µg/ml phenylmethanesulfonylfluoride, 10 mM sodium fluoride, 1 mM NaO, 10 µg/ml leupeptin, 50 µg/ml aprotinin, 1 µM pepstatin, and 10 mM ß-glycerophosphate]. Samples were placed on ice for 30 min and homogenized every 5 min. Then, samples were centrifuged at 4 C for 15 min at 11,000 x g to remove cellular debris, and the protein concentration in the supernatant was measured with the Bradford micro-method. Finally, samples were frozen at -80 C until Western blotting.

Proteins were separated on a 12% polyacrylamide denaturing gel at 40 mA/gel for 4.5 h using Tris/glycine/SDS running buffer [25 mM Tris base, 200 mM glycine (pH 8.3), and 0.1% SDS]. Then proteins were blotted onto nitrocellulose membranes (Amersham Pharmacia Biotech, Orsay, France) for 1 h at 15 V using a semidry transfer system (OWL, Illkirch, France). Membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS) (pH 7.4) supplemented with 5% nonfat dried milk. Then they were incubated overnight at 4 C with a monoclonal anti-P450 arom antibody (Serotec, Oxford, UK) diluted 1:500 in TBS supplemented with 1% nonfat dried milk. The mouse monoclonal anti-P450 arom antibody used for this experiment has been raised against a conserved peptide corresponding to amino acids 376–390 within human aromatase (15). Membranes were then incubated for 1 h at room temperature with a sheep antimouse immunoglobulin antibody (Amersham Pharmacia Biotech) diluted 1:2000 in TBS supplemented with 1% nonfat dried milk, and revelation of antibody-protein complexes was carried out using the enhanced chemiluminescence visualization system (ECL, Amersham Pharmacia Biotech).

Competitive quantitative RT-PCR
Total RNA was isolated according to the method described by Chomczynski and Sacchi (16), and its integrity was estimated by gel electrophoresis. The totality of total RNA was reverse transcribed using 0.4 µg oligo(deoxythymidine)12–18, 800 µM of each dNTP, 40 U RNasin, and 400 U Moloney murine leukemia virus reverse transcriptase in the reaction buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol] in an 80-µl final volume. Elongation was performed for 1 h at 37 C on a RoboCycler (Stratagene Europe, Amsterdam, The Netherlands), and the enzyme was inactivated for 5 min at 95 C.

Three transcripts were quantified in this study: the full-length and truncated P450 arom mRNAs and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA for normalization of data. Quantification of the full-length P450 arom mRNA and GAPDH mRNA was performed by coamplifying a sample of cDNA with a known quantity of a heterologous internal standard. The truncated P450 arom mRNA was quantified using the same strategy except for the internal standard used, which was a homologous standard (17). Competitive PCRs were performed using 4 µl cDNA, 30 pmol of each primer (primer 137, 5'-TAACTCTGGCAAAGTGGATGTTGTCG-3'; primer, 481 5'-TCGTGGTTCACGGCCCATCACA-3' for specific quantification of the GAPDH mRNA; primer 1367, 5'-AAACTTCGCAAAGAATGTTCCTTACAGG-3'; primer 1813, 5'-TGGGACAAGAGCACATGACTGATAGC-3' for specific quantification of the full-length P450 arom mRNA; primer INAC5, 5'-GTGTGTGCTGGAGATGCTGATTGCG-3'; primer INAC3, 5'-CGGGAAGGAATTTTGACTTGCACAGTGAGA-3' for specific quantification of the truncated P450 arom mRNA], 200 µM of each dNTP, 1.5 mM MgCl2, 1.5 U Eurobio Taq II polymerase in the reaction buffer [67 mM Tris-HCl (pH 8.8), 75 mM KCl, 16 mM (NH4)2SO4, and 0.01% Tween 20], and a known quantity of internal standard in a 50-µl final volume. For the full-length P450 arom mRNA and the GAPDH mRNA, amplification was performed with an initial step (2 min at 95 C); 35 cycles of 45 sec at 95 C, 45 sec at 61 C, and 45 sec at 72 C; and a final step of elongation for 10 min at 72 C. For the truncated P450 arom mRNA, PCR conditions are 2 min at 95 C, 35 cycles of 1 min at 95 C, 1 min at 68 C, and 1 min at 72 C; and a final step of elongation for 10 min at 72 C. All PCRs were performed on a RoboCycler (Stratagene Europe). Amplification products were separated on a 3% agarose gel stained with ethidium bromide (1 µg/ml) for 1.25 h at 12 V/cm. The intensity of staining was measured by densitometry using Bioprofil software (Vilbert Lourmat, Marne La Vallée, France).

Half-life of P450 arom mRNAs
To study the stability of P450 arom transcripts, granulosa cells (6 x 105 cells/treatment) were incubated with or without FSH (5 ng/ml) for 3 h, and then actinomycin D (2.5 µg/ml) was added to the culture medium. RNA was extracted at 0, 2, 3, 4, 5, and 6 h, and P450 arom mRNAs decay was quantified by competitive quantitative RT-PCR. The level of each P450 arom mRNA is expressed as a percentage of the level determined at 0 h (referred to as 100%). Four separate experiments were carried out. The half-life was calculated assuming that the decrease in the mRNA followed a stochastic process, according to the equation: C = Coe-kdt (18). In the equation, kd represents the mRNA decay, and for C = Co/2, we obtained the half-life.

Sequencing of the 5' end of intron IX
Genomic DNA was isolated by high salt extraction according to the method described by Miller et al. (19). The amplification products obtained using the Universal Genome Walker kit (Clontech, Saint Quentin, France) were sequenced on a 377 ABI PRISM DNA sequencer (PerkinElmer, Courtaboeuf, France) with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Courtaboeuf, France).

Estradiol production
The level of estradiol in culture medium was measured by RIA using a specific antibody (Sigma-Chimie). The sensitivity was 3 pg/tube. The intra- and interassay coefficients of variation were 5% and 9%, respectively.

Data analysis
The levels of P450 arom mRNAs were normalized to the level of the GAPDH mRNA used as an internal control. The level of total P450 arom mRNAs was obtained by adding the amounts of the two P450 arom mRNAs. Each data point represents the mean ± SEM of at least three experiments. The dose-dependent effect of FSH and half-life data were analyzed by t test. The Wilcoxon test was used for the other data. Differences were considered significant at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the truncated P450 arom mRNA and detection of P450 arom mRNAs and proteins in granulosa cell culture
Sequence analysis of the truncated P450 arom cDNA previously described (12) showed that this transcript included a short specific sequence instead of exon X. Therefore, the 5' end of intron IX was sequenced to determine the genomic sequence involved in the production of this transcript. Sequence analysis showed that a polyadenylation signal was embedded in the 5' end of intron IX, and the sequence upstream of this polyadenylation signal was similar to the specific sequence of the truncated P450 arom cDNA (Fig. 1AGo). Thus, the truncated P450 arom mRNA was deduced to be the result of alternative use of this polyadenylation signal (Fig. 1BGo). When granulosa cells were cultured under basal conditions (control), the two variants of P450 arom mRNAs were detected. The truncated P450 arom mRNA was 7-fold greater than the full-length P450 arom mRNA (P <= 0.05; Fig. 2AGo).



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  		FIG. 1. Alternative splicing mechanism generating the full-length and truncated P450 arom mRNAs. A, Partial sequence of the 3' end of exon IX and of the 5' end of intron IX (in bold). The stop codon and the polyadenylation signal (in italics) are underlined. This sequence is reported in EMBL Bank under accession number AY254893. B, Diagrammatic representation of genomic DNA fragments involved in the generation of full-length and truncated P450 arom mRNAs. Intron and exon sequences are denoted by bold uppercase letters and boxed uppercase letters, respectively. Stop codons and polyadenylation signals (in italics) are underlined. Resulting amino acid sequences are shown above and below.

 


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  		FIG. 2. Detection of P450 arom transcripts (A) and P450 arom proteins (B) in cultured granulosa cells. Granulosa cells (3 x 105 cells for quantification of P450 arom mRNAs and 7 x 106 cells for detection of P450 arom proteins) were incubated in MEM-2.5% FCS with androstenedione (10-7 M). After 24 h in culture, P450 arom mRNAs were specifically quantified by competitive quantitative RT-PCR, and P450 arom proteins were analyzed by Western blot (180 µg total protein). The blot shows a representative experiment. Results are the mean ± SEM of six different experiments. *, P <= 0.05 compared with the 2.9-kb P450 arom mRNA.

 
Total proteins were extracted from granulosa cells cultured under these conditions and were analyzed by Western blot to detect the aromatase proteins encoded by the two P450 arom mRNAs. Two bands with apparent molecular masses of 53 and 46 kDa were visualized (Fig. 2BGo). The sizes of these proteins are consistent with the predicted theoretical sizes of proteins encoded by these two P450 arom mRNAs. Under these conditions, the 46-kDa protein was approximately 2-fold more abundant than the 53-kDa protein (Fig. 2BGo).

Effect of FSH on P450 arom mRNAs and estradiol production
Culture of granulosa cells in the presence of increasing concentrations of FSH (0.5–5 ng/ml) resulted in a dose-dependent increase in both estradiol production and total P450 arom mRNAs level (Fig. 3Go, A and B). Specific quantification of each P450 arom transcript demonstrated a dose-dependent enhancement of the two mRNAs (Fig. 3CGo). Under basal conditions, the truncated P450 arom mRNA was 7-fold greater than the full-length P450 arom mRNA (P <= 0.05), whereas in the presence of FSH, there was no statistical difference between the levels of the two P450 arom transcripts (Fig. 3CGo).



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  		FIG. 3. Dose-dependent effects of FSH on estradiol production (A), total P450 arom mRNAs (B), and the two P450 arom mRNAs (C) in cultured granulosa cells. Granulosa cells (3 x 105 cells/treatment) were incubated in MEM-2.5% FCS with androstenedione (10-7 M) in the presence or absence of increasing doses of FSH. After 24 h in culture, estradiol production was measured by RIA, and P450 arom mRNAs were quantified by competitive quantitative RT-PCR. Results are the mean ± SEM of three different experiments. *, P <= 0.05; **, P <= 0.01; ***, P <= 0.001 (compared with each respective control).

 
Effect of TGFß on P450 arom mRNAs and estradiol production induced by FSH
With 5 ng/ml FSH, TGFß (1 ng/ml) decreased both estradiol production and total P450 arom mRNAs (28% and 46%, respectively; P <= 0.05) compared with FSH alone (Fig. 4Go, A and B). Moreover, TGFß decreased to the same extent (~50%; P <= 0.05) the levels of the two P450 arom mRNAs induced by FSH (Fig. 4CGo). Basal P450 arom mRNAs levels and estradiol production were not significantly modified by TGFß (data not shown).



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  		FIG. 4. Effects of TGFß on estradiol production (A), total P450 arom mRNAs (B), and the two P450 arom mRNAs (C) induced by FSH in cultured granulosa cells. Granulosa cells (3 x 105 cells/treatment) were incubated in MEM-2.5% FCS with androstenedione (10-7 M) and FSH in the presence or absence of TGFß. After 24 h in culture, estradiol production was measured by RIA, and P450 arom mRNAs were quantified by competitive quantitative RT-PCR. Results are the mean ± SEM of six different experiments. *, P <= 0.05 (compared with FSH alone).

 
Effect of db cAMP on P450 arom mRNAs, estradiol production, and P450 arom proteins
When granulosa cells were exposed to 2.5 x 10-3 M db cAMP, estradiol production and total P450 arom mRNAs were increased 6- and 25-fold, respectively, above control levels (P <= 0.01; Fig. 5Go, B and A). However, the two P450 arom mRNAs were not enhanced in the same ratio. Indeed, the full-length and truncated transcripts were increased 55- and 21-fold, respectively, by db cAMP (P <= 0.01; Fig. 5CGo). Thus, the ratio between the two mRNAs (truncated mRNA/full-length mRNA) was reduced in db cAMP-treated cultures compared with basal conditions (2.5 vs. 7, respectively; P <= 0.01; Fig. 5CGo).



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  		FIG. 5. Effects of db cAMP on estradiol production (A), total P450 arom mRNAs (B), the two P450 arom mRNAs (C), and P450 arom proteins (D) in cultured granulosa cells. Granulosa cells (3 x 105 cells/treatment for quantification of P450 arom mRNAs and 7 x 106 cells/treatment for detection of P450 arom proteins) were incubated in MEM-2.5% FCS with androstenedione (10-7 M) in the presence or absence of db cAMP. After 24 h in culture, estradiol production was measured by RIA, P450 arom mRNAs were quantified by competitive quantitative RT-PCR and P450 arom proteins were analyzed by Western blot (180 µg total protein/lane). The blot shows a representative experiment. Results are the mean ± SEM of seven different experiments. **, P <= 0.01 (compared with each respective control).

 
P450 arom protein expression was studied in the presence or absence of 2.5 x 10-3 M db cAMP. Under basal conditions, the level of the 46-kDa protein was greater than that of the 53-kDa protein, as already observed (Fig. 5DGo). The db cAMP treatment enhanced 53-kDa protein content approximately 2-fold without affecting the level of the 46-kDa protein (Fig. 5DGo).

Effect of TGFß on P450 arom mRNAs, estradiol production, and P450 arom proteins induced by db cAMP
The effect of TGFß on P450 arom mRNAs, estradiol production, and P450 arom proteins in db cAMP-treated granulosa cells was investigated. TGFß (1 ng/ml) in the presence of 2.5 x 10-3 M db cAMP increased the level of total P450 arom mRNAs (4-fold; P <= 0.01), whereas estradiol production was not modified (Fig. 6Go, B and A). Moreover, TGFß increased 2- and 4-fold, respectively, the levels of full-length and truncated P450 arom mRNAs induced by db cAMP (Fig. 6CGo). Thus, TGFß added to db cAMP increased the ratio between the two transcripts (truncated mRNA/full-length mRNA) compared with db cAMP alone (6 vs. 2.5, respectively; P <= 0.01; Fig. 6CGo). TGFß did not significantly affect the levels of 53- and 46-kDa aromatase proteins induced by db cAMP (Fig. 6DGo).



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  		FIG. 6. Effects of TGFß on estradiol production (A), total P450 arom mRNAs (B), the two P450 arom mRNAs (C), and P450 arom proteins induced by db cAMP in cultured granulosa cells. Granulosa cells (3 x 105 cells/treatment for quantification of P450 arom mRNAs and 7 x 106 cells/treatment for detection of P450 arom proteins) were incubated in MEM-2.5% FCS with androstenedione (10-7 M) and db cAMP in the presence or absence of TGFß. After 24 h in culture, estradiol production was measured by RIA, P450 arom mRNAs were quantified by competitive quantitative RT-PCR, and P450 arom proteins were analyzed by Western blot (180 µg total protein/lane). The blot shows a representative experiment. Results are the mean ± SEM of seven different experiments. *, P <= 0.05; **, P <= 0.01 (compared with db cAMP alone).

 
Effect of FSH on the P450 arom mRNAs stability
We investigated whether the differential regulation of P450 arom transcripts induced by FSH was associated with modulation of mRNA stability. Actinomycin D was used to examine the effect of FSH on P450 arom mRNA decay (Fig. 7Go, A and B). A 50% decrease in the initial quantity of P450 arom mRNAs was observed after 4 ± 0.5 h for the full-length P450 arom mRNA and after 2.7 ± 1 h for the truncated mRNA under basal conditions (Fig. 7CGo). The addition of FSH (5 ng/ml) did not modify the profiles of the curves (Fig. 7Go, A and B), and the half-life values were similar (3.9 ± 1.3 h for the full-length P450 arom mRNA and 2.7 ± 0.5 h for the truncated P450 arom mRNA; Fig. 7CGo).



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  		FIG. 7. Determination of the half-life of each P450 arom mRNAs in control and FSH-stimulated conditions after the addition of actinomycin D (2.5 µg/ml) to granulosa cells culture medium. Granulosa cells were incubated with androstenedione (10-7 M). A and B, Amounts of full-length and truncated P450 arom transcripts, respectively (as a percentage of the amount determined at 0 h); C, half-life values of the two P450 arom mRNAs. Results are the mean ± SEM of four different experiments. NS, Not significant compared with 2.9 kb P450 arom mRNA levels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report describing the presence of a truncated P450 arom protein in primary culture of rabbit preovulatory granulosa cells. We also show a differential regulation of the two PII-derived P450 arom transcripts. Moreover, the truncated P450 arom mRNA is thought to arise from the alternative use of a polyadenylation signal embedded in the 5' end of cyp 19 intron IX.

We report the presence of two P450 arom proteins in cultured granulosa cells: one with a molecular mass of 53 kDa, which could be encoded by the full-length P450 arom mRNA, and the other with a molecular mass of 46 kDa, which could be encoded by the truncated P450 arom mRNA. We can speculate about the role of this truncated protein in the regulation of P450 arom gene expression, because that 46-kDa protein contains the substrate-binding domain, but not the heme-binding domain. If the structure of the substrate-binding domain is similar for the two proteins, we could suggest that in particular physiological conditions, the truncated protein could bind the androgen substrate and thus could modulate the aromatase activity as a steroid-binding protein.

Our results show that the levels of P450 arom proteins are not in agreement with the levels of P450 arom mRNAs in the presence of db cAMP. Indeed, in that condition only the 53-kDa protein is increased, whereas the full-length P450 arom transcript is less abundant. This suggests the occurrence of a regulation at the translation level that could result in a more efficient translation rate for the full-length P450 arom mRNA and/or in a longer half-life for the 53-kDa protein induced by db cAMP.

The quantification of the two P450 arom mRNAs clearly shows a differential hormonal regulation of the expression of these transcripts. A differential regulation of GnRH receptor mRNAs, which occurs through a direct effect of estradiol, has also been demonstrated in the ovine pituitary gland (20). However, the mechanism of this differential regulation has not been specified. As our data demonstrate that FSH does not modulate the P450 arom transcript stability, an alternative splicing process could be involved in the observed differential regulation.

Alternative splicing is an important molecular mechanism that increases the protein diversity derived from a single gene through selective inclusion or exclusion of RNA sequence during posttranscriptional processing. The protein isoforms produced have distinct and sometimes opposite functions, underscoring the importance of this process. However, several types of alternative splicing exist (21). Our data show that the truncated P450 arom mRNA is issued from the alternative use of a polyadenylation signal embedded in the 5' end of cyp 19 intron IX. This type of RNA processing has been well characterized for the calcitonin/calcitonin gene-related peptide gene. Indeed, a polyadenylation enhancer element located in an intron downstream of exon 4 has been identified within the calcitonin/calcitonin gene-related peptide gene, and binding of a complex of several splicing factors on this site enhances polyadenylation at this exon (21).

Hormonal control of alternative splicing events in the coding region of P450 arom has been suggested in immature rat Sertoli cells by Pezzi et al. (22). They have described the occurrence of two altered aromatase transcripts under T3 treatment. However, these researchers did not examine the ratios of mRNA splice variants and did not detect aromatase protein encoded by truncated transcripts. Moreover, they did not look for the splicing mechanism. Another example concerns the human glucocorticoid receptor (hGR) gene, where alternative splicing generates a nonhormone-binding splice variant (hGRß) that differs from the wild-type receptor (hGR{alpha}) only at the carboxyl terminus (23). Furthermore, TNF{alpha} regulates hGR expression in a differential manner (24). Indeed, activation of a TNF-responsive nuclear factor-{kappa}B DNA-binding site 5' to the hGR promoter leads to a much greater increase in hGRß mRNA than in hGR{alpha} mRNA. These results suggest a coordinate regulation of transcription and splicing by nuclear receptors and are in agreement with Auboeuf et al. (25), who proposed that activated steroid receptors bind to target DNA response elements and promote the recruitment of coregulators that are involved in both transcriptional and splicing regulation. On the other hand, in ewe thecal cells, RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression (26). The changes from the ovulatory to the anovulatory state, which are accompanied by a decrease in the ovarian response to LH, may be correlated with changes in the regulation of LH receptor expression by alternative splicing of its mRNAs. The origin of the variety of LH receptor transcripts has been attributed to alternative transcription start sites, alternative RNA splicing, or the use of alternative polyadenylation site heterogeneity within their 3'-untranslated regions.

Our data demonstrate an inhibitory effect of TGFß on FSH-stimulated P450 arom gene expression in cultured granulosa cells. TGFß modulates aromatase activity in granulosa cells in a species-specific manner. For instance, this growth factor increases estradiol production and aromatase activity in rat granulosa cells (27) and decreases aromatase activity in porcine granulosa cells (28), but is not efficient in bovine granulosa cells (29). However, in these studies the effect of TGFß on P450 arom mRNAs levels has not been investigated.

In rabbit granulosa cells we observed that the inhibitory effect of TGFß on FSH-stimulated P450 arom gene expression occurs at a pre-cAMP step. Such a pre-cAMP inhibitory effect of TGFß has been described in MATLyLu rat prostate cancer cells (30), 3T3 fibroblasts (31), and it has been shown in porcine Sertoli cells (32) that TGFß reduces FSH-induced aromatase activity through an enhancement of cAMP phosphodiesterase activity, which leads to a decrease in the FSH-induced cAMP level. On the other hand, a down-regulatory effect of TGFß on FSH receptor (FSHR) expression could explain the pre-cAMP inhibitory effect induced by TGFß. Thus, it has been demonstrated in porcine and rat granulosa cells that TGFß modulates FSHR expression differently. Indeed, in rat granulosa cells TGFß increases FSHR binding by attenuating the down-regulatory action of FSH on FSHR expression, whereas in porcine granulosa cells, it reduces FSHR binding at FSH concentrations that enhance FSHR expression (33).

In our work, TGFß at the post-cAMP step had no effect on either estradiol production or P450 arom protein contents induced by db cAMP, whereas a 4-fold increase in the level of the db cAMP-stimulated P450 arom mRNAs was observed. This increase in the transcripts levels does not seem sufficient to induce a variation in both P450 arom proteins content and estradiol production. Indeed, we observed that the strong increase in the level of the full-length P450 arom mRNA induced by db cAMP (55-fold) led to a slight increase in the 53-kDa protein content (2-fold).

In conclusion, our study demonstrates a differential regulation of P450 arom mRNAs derived from the same promoter and favors studies of the expression of each transcript. Moreover, it emerges from our results that a truncated P450 arom mRNA and the inactive protein encoded by this transcript could be involved in the regulation of estrogen production by granulosa cells. Further studies will be necessary to clarify the mechanism controlling alternative polyadenylation, which is responsible for the differential regulation, and to characterize the involvement of this new protein in the regulation of estrogen production.


   Footnotes
 
Abbreviations: db cAMP, Dibutyryl cAMP; dNTP, deoxy-NTP; FCS, fetal calf serum; FSHR, FSH receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hGR, human glucocorticoid receptor; P450 arom, cytochrome P450 aromatase; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline.

Received February 28, 2003.

Accepted for publication July 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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