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Endocrinology Vol. 138, No. 12 5256-5265
Copyright © 1997 by The Endocrine Society

ARTICLES

Estrogen Regulation of Peptidylglycine {alpha}-Amidating Monooxygenase Messenger Ribonucleic Acid Levels by a Nuclear Posttranscriptional Event1

Rajaâ El Meskini, Françoise Boudouresque and L’Houcine Ouafik

INSERM U297, Institut Federatif de Recherche Jean Roche, Faculté de Médecine Nord, 13916 Marseille Cedex 20, France

Address all correspondence and requests for reprints to: Dr. L’Houcine Ouafik, INSERM U297, Institut Federatif de Recherche Jean Roche, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France. E-mail: ouafik.h{at}jean-roche.univ-mrs.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptidylglycine {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyze the conversion of glycine-extended peptides into COOH-terminal amidated peptides. We have previously shown that PAM messenger RNA (mRNA) levels in the anterior pituitary of intact cycling adult female rats showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17ß-estradiol was accompanied by a 4.5 ± 0.5-fold decrease in total PAM mRNA and a 2-fold decrease in PAM activity in the anterior pituitary gland.

To investigate the cellular site at which 17ß-estradiol acts to affect the PAM mRNA, we made parallel measurements of the relative levels of PAM mRNA and nuclear precursor RNA and the relative rate of gene transcription after treatments designed to alter the estrogen status. The transcription rate experiments indicated that these 17ß-estradiol effects were not due to reduced PAM gene activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. Primary rat pituitary cell cultures from OVX and OVX-17ß-estradiol-treated rats in the presence of actinomycin D showed that 17ß-estradiol treatment decreased the half-life of PAM mRNA from 15–16 h to 8–9 h. There was no effect of 17ß-estradiol on PAM mRNA poly(A) tail length or site of polyadenylation. However, in this study the down-regulation of PAM was identified as a nuclear event. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that decreased PAM expression after 17ß-estradiol treatment was largely due to intranuclear destabilization of the primary transcript. The levels of nuclear precursor RNA were decreased roughly 5- to 6-fold in OVX+17ß-estradiol compared with OVX rats. The decrease in PAM mRNA is blocked by cycloheximide, indicating that its requires new protein synthesis. Mechanisms that would generate such an effect include altered stability of unprocessed message in the nucleus. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for PAM mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEPTIDYLGLYCINE {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3) is involved in the posttranslational processing of many prohormones and neuropeptides (1, 2). PAM catalyzes the formation of {alpha}-amidated peptides from peptide precursor molecules with a COOH-terminal glycine. Conversion of a peptidylglycine substrate into {alpha}-amidated product involves a two-step process resulting from the sequential actions of two enzymes activities (3, 4, 5). Both enzymes are derived from the bifunctional PAM protein (6, 7, 8). The first enzyme, peptidylglycine {alpha}-hydroxylating monooxygenase, produces an {alpha}-hydroxylated intermediate in the presence of copper, ascorbate, and molecular oxygen. The second enzyme, peptidyl-{alpha}-hydroxyglycine-{alpha}-amidating lyase, cleaves the peptidyl-{alpha}-hydroxyglycine intermediate to form the {alpha}-amidated peptide and glyoxylate. The two catalytic domains of the bifunctional PAM protein can be separated by endoproteolysis and can act independently (9). Alternative splicing of the primary transcript of a single copy gene located on human chromosome 5 (10, 11) generates numerous PAM messenger RNA (mRNA) transcripts (2). The PAM proteins in any tissue reflect both the forms of PAM mRNA present and the co- or posttranslational modifications that occur. PAM has broad substrate specificity, is found in a variety of tissues, and is regulated in response to endocrine manipulations (2, 12).

We recently demonstrated that 17ß-estradiol (E2) induces a decrease in the amount of PAM mRNA, thereby providing evidence for the pretranslational regulation of PAM synthesis by estrogen (13). There are many steps in the pathway of RNA metabolism at which steroid hormones might act to alter the levels of a given mRNA. The effects of steroid hormones are not limited to the modulation of transcriptional activity. Indeed, they have been found to regulate many of the subsequent steps from polyadenylation (14, 15), to posttranslational modification of the regulated proteins (16). In recent years, the regulation of cytoplasmic mRNA stability has emerged as an important control point in a variety of biological systems (17, 18). Glucocorticoids have been shown to enhance the stability of GH mRNA (14) and decrease the stability of ß-globin mRNA in differentiating erythroleukemia cells (19). In the chick oviduct, estrogen increases the stability of ovalbumin and conalbumin mRNA (20). Administration of E2 to male Xenopus laevis induces both the transcription of the vitellogenin genes and the stabilization of vitellogenin mRNA against cytoplasmic degradation (21).

The pretranslational control of PAM synthesis by estrogen could be exerted at transcriptional and/or posttranscriptional levels. To further investigate the molecular site of action of E2, we estimated the relative rates of PAM gene transcription after E2 treatment of ovariectomized (OVX) rats and compared these rates to changes in the relative levels of nuclear precursor and mature mRNA. In this report we show that E2 alters the rate of degradation of PAM primary transcripts and that this leads to the decrease of cytoplasmic PAM mRNA. This intranuclear mechanism occurs with an accompanying change in PAM mRNA cytoplasmic half-life and depends on de novo protein synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Ten-week-old female Sprague-Dawley rats (Dépré, Lyon, France) weighing 180–200 g were OVX, and rested for 1 week. Thereafter, OVX rats received for 1 week daily sc injections of E2 (4 µg diluted in 100 µl sesame oil) (Merck, Darmstadt, Germany) (OVX+E2). A control group (OVX rats) received injection of sesame oil only (13). At the end of each experiment, the animals were killed by decapitation, and trunk blood was collected for plasma LH measurements by RIA. Anterior pituitaries were rapidely removed and placed on ice, and the anterior pituitary lobe was separated from the neurointermediate lobe for nuclei isolation, RNA preparation, and primary cultures.

LH RIA
Plasma levels of LH were measured in duplicate in a single assay by a double-antibody RIA and expressed as nanograms per ml of NIAMDD rat LH-RP 2. Anti-rat LH CSU 120 was provided by Dr. G. D. Niswender (Colorado State University, Fort Collins, CO). The sensitivity of the assay was 0.25 ng/ml of plasma. The intra- and interassay coefficients of variation were below 10%.

Probe and DNA preparation
Rat PAM-1 complementary DNA (cDNA) probe (3.8 kb), encompassing nearly the entire PAM mRNA sequence, was prepared by digesting ZAP 6 clone containing the full-length 3.8-kb PAM cDNA with EcoRI (22). DNA restriction fragment, derived from the PAM genomic clone and localized to the 5'-region of the PAM gene, was subcloned at the appropriate restriction sites of pBS II SK-. Purified PAM intronic fragment G33 I-PAM (2.2 kb), verified to contain neither repetitive sequences nor PAM exonic sequences, was used to hybridize nuclear and cytosolic RNA (23).

To correct for the actual amount of RNA in each lane, blots were stripped and hybridized to cDNA probes derived from 18S frog ribosomal RNA (the 3.8- and 4.2-kb NcoI fragments derived from pX1r101a, kindly provided by Dr. Barbara Sollner-Webb, Johns Hopkins Medical School, Baltimore, MD). All DNA probes were random primed using DNA labeling beads [-deoxycytosine triphosphate] kit (Pharmacia Biotech, Gif sur Yvette, France) and [{alpha}-32P]deoxycytosine triphosphate to a specific activity of 1–2 108 cpm/µg.

A rat PRL (rPRL) cDNA-bearing plasmid, was generously provided by Dr. J. A. Martial (University of Liege, Liege, Belgium). The plasmid contains a 823-nucleotide sequence representing almost all of the bases complementary to mature rPRL mRNA (24). Cyclophilin cDNA-bearing plasmid (1B15) was kindly provided by Dr. B. A. Eipper (Johns Hopkins Medical School) (25). Cyclophilin cDNA was used in the run-on transcription assay as an internal reference not regulated by estrogen status (see Results).

RNA analysis
Total RNA was isolated from anterior pituitary as described previously (26). RNA samples (10 µg) were separated on a 1% agarose gel containing 2.2 M formaldehyde, transferred to Hybond-N membrane (Amersham Corp., Les Ulis, France), UV cross-linked, and hybridized to {alpha}-32P-labeled full-length PAM cDNA (3.8 kb) (22). Filters were prehybridized, hybridized, and washed as previously described (27). To correct for the actual amount of RNA in each lane, blots were stripped and hybridized to cDNA probes derived from 18S and 28S frog ribosomal RNA (27). The autoradiograms were analyzed by measurement of optical density by scanner-densitometer using NIH image 1.54 Software (National Institutes of Health, Bethesda, MD). The results are expressed as optical density (OD) of PAM mRNA/OD 18S ribosomal RNA (rRNA), with the panels representing the mean ± SEM for each group of rats.

Slot blot analysis of RNA from nuclei and cytoplasm
Nuclei were isolated exactly as described below. After Dounce homogenization and centrifugation, 2 volumes of solution GTC [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.1 M 2-mercaptoethanol] was added to the cytosol fraction; the nuclear pellet was washed and resuspended directly in the GTC solution, and the RNA was isolated as described above. RNA was denatured by heating at 65 C for 15 min in 2.2 M formaldehyde, 6xSSC (1xSSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and cooled on ice before applying on a Hybond-N membrane (Amersham) by using the Schleicher & Schuell (Keene, NH) Minifold II Slot-Blotter. After 1 x 106 cpm of the 32P-labeled probe/1 ml of hybridization solution was added, filters were hybridized and washed as described (27). Slot blots were scanned and quantified as above.

Preparation of rat anterior pituitary nuclei
Rat anterior pituitary nuclei were prepared for each treatment as described (23). The run-on assay was performed simultaneously with freshly prepared and frozen nuclei; the results showed that the freezing step has no effect on the transcriptional activity of PAM and PRL genes (data not shown).

Transcription run-on analysis
The nuclear transcription assay, isolation of 32P-labeled RNA, and hybridization conditions were described previously (23). Briefly, run-on transcription was carried out at 30 C for 40 min in reaction mixture containing 3 x 107 nuclei and 150 µCi of [{alpha}-32P]uridine triphosphate (3000 Ci/mmol) in a final volume of 0.3 ml. After the deoxyribonuclease I (75 U) and proteinase K (200 µg/ml) digestions, the labeled RNA was extracted with phenol and chloroform (vol/vol) and isopropanol-precipitated. The precipitation procedure was repeated two times.

Hybridization of run-on transcripts to filter-bound plasmid DNA
Ten micrograms each of plasmids rat (r) PAM-1 (22), rPRL (24), and cyclophilin (25) were denatured by 0.4 M NaOH treatment for 30 min at room temperature, neutralized with 2 M ammonium acetate, pH 7.5, and applied to Hybond-N membrane using a slot blot apparatus. Nonspecific binding was determined by hybridization to pBS II SK-. Prehybridization, hybridization, and washes were carried out as described by us (23). The blots were exposed to x-ray film (Kodak X-OMAT, Eastman-Kodak, Rochester, NY) at -70 C, and several different exposure times were chosen to obtain densitometric scans in the linear response range of the x-ray film. The autoradiograms were analyzed by measurement of OD by scanner-densitometer using NIH image 1.54 Software.

Tissue culture
Primary anterior pituitary cultures from OVX and OVX+E2 female Sprague Dawley rats were prepared essentially as described (23, 28). Cell yields were typically 1.0–1.5 x 106 cells per anterior pituitary lobe. The cells were cultured on polylysine-coated culture wells and maintained in DMEM (Life Technologies, Gaithersburg, MD) containing penicillin (50 U/ml) and streptomycin (50 µg/ml). OVX cells were cultured in 10% charcoal-stripped FCS (29) without phenol red whereas OVX+E2 cells were cultured in stripped FCS+E2. To limit further proliferation of fibroblasts, the cultures were treated with 10 µM cytosine-ß-D-arabinofuranoside (Sigma Chemical Co, St. Louis, MO) for 24 h. On the second day of culture, the cells were fed with DMEM containing 10% stripped FCS ± E2 and allowed to adhere to the plates by incubation for 3 days under a moist 5% CO2/95% air atmosphere. On day 5, cells were washed and incubated as above, with their respective media containing actinomycin D (5 µg/ml, Sigma) to block a new transcription, or cycloheximide (Sigma) to block protein synthesis. Preliminary experiments indicated that addition of cycloheximide (10 µg/ml) for 1 h reduced incorporation of [3H]leucine into trichloroacetic acid-precipitable material over that 1-h period by 90%. Following different periods of incubation, cells from individual wells were scraped in 0.5 ml guanidinium isothiocyanate for RNA analysis.

Polyadenylation (H-blot) analysis
A 25-mer oligonucleotide (5'-ATGAGCTAAACTTCCCTCGGGG-GTT-3') [H23] that was complementary to a region 262 nucleotides downstream of the translation termination codon and 354 nucleotides upstream of the polyadenylation site in PAM mRNA was synthesized (22). This PAM oligonucleotide was hybridized, in the absence or presence of oligo(dT)12–18 (Pharmacia Biotech) to total cellular RNAs, and the samples were digested with ribonuclease H (RNase H) as described by Carrazana et al. (30). RNAs were then phenol-chloroform extracted, fractionated through 1.5% agarose-2.2 M formaldehyde gels, and transferred to Hybond-N. Blots were hybridized to a SmaI-EcoRI PAM probe from the cDNA clone ZAP6 (22) that spans the 3'-untranslated RNA fragment generated by RNase H digestion. Samples hybridized to oligo(dT) and to the PAM oligonucleotide generated deadenylated fragments that were detected by the 3'-untranslated region probe, whereas samples hybridized to the PAM oligonucleotide alone generated fragments with poly (A) tails.

Statistical analysis
All results are expressed as the mean values ± SEM. Statistical analysis was performed by a one-way ANOVA followed by Fisher’s protected least significant difference test (Statview 512, Brain Power Inc., Calabasas, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of PAM expression by estrogen status
In OVX rats, the effectiveness of ovariectomy was demonstrated by a significant increase in the plasma LH levels. In E2-treated rats (OVX+E2), plasma LH levels were reduced significantly compared with those in OVX animals (1.55 ± 0.15 ng/ml vs. 3.41 ± 0.28 ng/ml, respectively; P < 0.0001) (Fig. 1BGo). Anterior pituitary PAM expression in OVX and OVX-E2-treated rats (OVX+E2) was assessed by Northern blot analysis. Total RNA prepared from the pituitaries of individual rats was subjected to Northern blot analysis, and PAM mRNA was visualized using a radiolabeled rPAM-1 cDNA capable of detecting all known forms of rPAM mRNA (Fig. 1AGo). Pituitary PAM mRNA size ranged from 3.6–3.8 kb, and the size distribution was unaltered by estrogen status (13). The amount of rRNA present in each sample was determined by hybridization to a cDNA probe for rRNA. The illustration of uncorrected PAM mRNA levels showed a significant difference between OVX and OVX+E2 animals (Fig. 1CGo). To ensure that 18S rRNA can be used for comparative purposes, the uncorrected 18S rRNA levels were illustrated (Fig. 1DGo). These data demonstrated that 18S rRNA levels in OVX and OVX+E2 rats are not significantly different. The amount of PAM mRNA in each sample was then normalized to the amount of rRNA (Fig. 1BGo).



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  		Figure 1. Effect of estrogen status on expression of PAM mRNA in anterior pituitary and plasma LH levels. A, An aliquot of total RNA (10 µg) from pituitaries of individual female OVX and OVX+E2 rats was fractionated on a denaturing 1% agarose gel and transferred to Hybond-N membrane. The blot was hybridized with a full-length rPAM-1 cDNA probe and exposed to x-ray film at -70 C with an intensifying screen. The blot was subsequently stripped and reprobed with a cDNA probe corresponding to 18S rRNA to permit correction for the amount of sample actually transferred to Hybond-N membrane. B, The autoradiograms were densitometrically analyzed, and the levels of PAM mRNA were normalized to levels of rRNA on the same blot (solid bar). Plasma LH levels (hatched bar) were assayed for each experimental animal, as described in Materials and Methods. Each bar represents the mean ± SEM of four independent experiments (three rats in each experiment). The asterisk indicates that the value for OVX+E2 rats is significantly different from OVX rats (*, P < 0.0001). C, The uncorrected PAM mRNA levels are illustrated, and the asterisk indicates that the value is significantly different between both groups (*, P < 0.04). D, The levels of 18S rRNA were not significantly different between OVX+E2 and OVX animals.

 
Northern blot analysis demonstrated that there was a 4.8 ± 0.5-fold decrease in PAM mRNA levels in the OVX+E2 rats compared with OVX rats (mean ± SEM of four experiments with three rats each) (Fig. 1BGo). These levels of PAM mRNA expression found in both groups were similar to those reported in previous experiments (13). Accordingly, the following studies were directed toward understanding the precise molecular mechanisms regulating PAM gene expression. To investigate the mechanism whereby E2 decreases PAM mRNA concentration, we first sought the cellular PAM mRNA stability in primary anterior pituitary culture; second we measured the rate of PAM gene transcription and the nuclear PAM RNA level.

Role of PAM mRNA transcripts stability
To further characterize the effect of E2 on PAM mRNA stability, PAM mRNA decay was evaluated in the presence of actinomycin D (5 µg/ml) to block new transcription. Dispersed anterior pituitary cells prepared from OVX and OVX-E2 treated animals were maintained in DMEM containing actinomycin D with 10% stripped FCS ± E2. Northern blot analysis was performed with total RNA prepared 0, 5, 10, 15, and 24 h after the actinomycin D was added (Fig. 2AGo). The half-life of PAM mRNA was estimated by regression analysis of the decline in mRNA levels with time by scanning laser densitometer. The amount of PAM mRNA was normalized to the amount of 18S rRNA (Fig. 2BGo). The apparent half-life of PAM mRNA in cells from OVX rats was 15 ± 1 h whereas it decreased approximately to 8 ± 1 h in cells from OVX+E2 rats (n = 4, mean ± SEM; P < 0.002). This result suggests that E2 acts to destabilize PAM mRNA in anterior pituitary cells from OVX+E2 animals.



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  		Figure 2. Estimation of PAM mRNA apparent half-life. Rat primary anterior pituitary cultures prepared from OVX and OVX+E2 animals were maintained for 3 days in DMEM plus 10% charcoal stripped serum ± E2. On day 5, cells were washed twice and incubated with medium containing actinomycin D (5 µg/ml). At the indicated intervals after the addition of actinomycin D, duplicate plates of cells were harvested for isolation of RNA. A, Total RNA was subjected to Northern blot analysis. The blot was hybridized with the PAM cDNA probe. PAM mRNA levels were corrected for hybridization to 18S rRNA. Quantitative analysis of the blots was performed as described in Fig. 1BGo. B, The y-axis is expressed as the percentage of maximum PAM mRNA remaining and is plotted on a logarithms scale. Symbols represent the mean and SEM of triplicate samples.The asterisk indicates that the value is significantly different between both groups (*, P < 0.002). Similar data were obtained from four independent experiments. The point without an error bar had a SEM of 1% or less.

 
PAM gene transcription is not decreased by E2
To determine the mechanism(s) responsible for the 4- to 5-fold decrease in PAM mRNA, nuclear run-on transcription assays were performed (23). This assay measures the density of RNA polymerase II molecules on the PAM transcription unit and thus reflects the transcriptional activity of the gene.

Before use cyclophilin gene expression as an internal reference to normalize hybridization conditions in the run-on transcription assays, we investigated the effect of estrogen status on cyclophilin mRNA levels in anterior pituitary gland. Anterior pituitary cyclophilin expression was assessed by Northern blot analysis. Total RNA prepared from individual rat anterior pituitaries was visualized using a radiolabeled cyclophilin cDNA. The blot was hybridized to a cDNA probe for rRNA (Fig. 3AGo). The amount of cyclophilin mRNA (1 kb) in each sample was then normalized to the amount of rRNA (Fig. 3BGo). There was no significant difference in cyclophilin mRNA levels between OVX and OVX+E2 animals. These data demonstrate that cyclophilin expression is not regulated by estrogen status in anterior pituitary gland and consequently it can be used to normalize hybridization conditions in our experiments.



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  		Figure 3. Effect of estrogen status on the accumulation of cyclophilin mRNA in anterior pituitary. A, Total RNA (10 µg) isolated from anterior pituitaries of individual OVX and OVX+E2 rats was subjected to Northern blot analysis. The blot was hybridized with rat cyclophilin cDNA probe. The membrane was subsequently stripped and hybridized with 18S ribosomal cDNA. B, The autoradiograms were densitometrically analyzed, and the amount of cyclophilin mRNA was normalized to the amount of rRNA. Data are presented as mean ± SEM of four independent experiments. No significant difference in cyclophilin mRNA levels was observed between both groups.

 
The cytosolic fraction of the nuclei isolation procedure was prepared for Northern blot analysis as an independent means of examining the influence of E2 on PAM mRNA levels. Consistent with the data shown in Fig. 1Go, E2 reproducibly caused a decrease in PAM mRNA levels (data not shown).

The relative rate of PAM gene transcription was investigated in the nuclear fraction from the same anterior pituitaries. When the amount of 32P-labeled nascent RNA complementary to PAM cDNA was compared with that hybridizable with cyclophilin cDNA (Fig. 4AGo), no alteration in PAM gene transcription was observed in OVX and OVX+E2 rats (Fig. 4BGo). Similar results were obtained when the same experiment was performed using a genomic clones covering almost the entire PAM gene (data not shown). To ensure that the nuclear preparations had been active, rPRL cDNA was introduced as a gene known to be transcriptionally activated by estrogen (31). As shown in Fig. 4Go, the rPRL gene is regulated by estrogen. The transcription Slot blot and its quantification showed that E2 treatment caused a 5- to 6-fold increase in specific PRL gene transcription compared with ovariectomy levels (Fig. 4BGo); these data are consistent with the report that estrogen acts to transcriptionally activate the PRL gene in rat anterior pituitary gland (31). Thus, the conditions used for nuclear isolation and assay appear to be appropriate to detect bona fide transcriptional changes. Nonspecific hybridization measured with the immobilized pBS II SK- plasmid DNA was negligible (Fig. 4AGo). Transcription was inhibited by greater than 95% by the addition of 2 µg {alpha}-amanitin per ml, demonstrating that these transcripts were synthesized by RNA polymerase II, the enzyme responsible for the transcription of pre-mRNA (Fig. 4CGo) (32). These results suggested that a posttranscriptional mechanism was responsible for the E2-induced decrease in PAM mRNA levels in anterior pituitary OVX rats.



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  		Figure 4. Run-on transcription analysis of PAM and PRL genes transcription after estrogen treatment in OVX anterior pituitary rat. A, Transcriptional activity of the PAM gene measured with rPAM-1 cDNA (3.8 kb). Nuclei were isolated from pituitaries of OVX and OVX+E2 female rats and allowed to continue RNA synthesis in vitro in the presence of [{alpha}-32P]uridine triphosphate. 32P-labeled RNA transcripts newly synthesized were hybridized with filters containing plasmid cDNA sequences for rPAM-1, rPRL, and cyclophilin. rPRL cDNA was used as the gene known to be responsive to changes in estrogen status and to ensure that the nuclei preparations had been active. Cyclophilin was used as an internal reference not regulated under estrogen status. A plasmid without insert (pBS SK-) was used as a nonspecific hybridization control. B, Quantification of the signals shown in panel A was performed by densitometry analysis, and the rPAM-1 cDNA or rPRL cDNA was normalized to the signal obtained with cyclophilin cDNA. The ratios of the relative density values were used to express relative PAM and PRL gene transcription. Data are presented as mean ± SEM of three independent experiments. The asterisk indicates that the PRL gene transcriptional activity in OVX+E2 rats is significantly different than OVX rats (*, P < 0.0001). C, Effect of {alpha}-amanitin on PAM gene transcription. 32P-labeled nuclear RNAs obtained from an in vitro transcription in the presence of 2 µg/ml {alpha}-amanitin (a concentration that specifically inhibited RNA polymerase II) were hybridized to the membranes as described in Materials and Methods.

 
The abundance of nuclear PAM mRNA is reduced by E2
Regulation of cytoplasmic message stability is the most frequently documented means of posttranscriptional regulation. To determine whether the posttranscriptional down-regulation of PAM mRNA by E2 was a cytoplasmic or nuclear event, cellular RNAs were fractionated and analyzed by slot blotting. Anterior pituitary nuclear RNAs prepared from OVX and OVX+E2 rats were initially hybridized to PAM cDNA spanning the full length of PAM mRNA (Fig. 5AGo) (22). A 5- to 7-fold decreased expression of PAM mRNA was detected as expected in the cytosolic fraction and intact anterior pituitary gland tissue preparation of E2-treated animals (Fig. 5BGo). Nuclear PAM RNA levels were down-regulated 4- to 5-fold of control (OVX) levels following E2 treatment (Fig. 5BGo). The magnitude of this effect on nuclear PAM RNA levels was similar to the {approx} 5-fold decrease observed when total PAM RNA was examined (Fig. 5BGo). An E2-induced mechanism involving inhibition of nuclear export of PAM mRNA was ruled out since there was no build-up of PAM mRNA in the nuclei of OVX+E2 rats.



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  		Figure 5. Effect of E2 on PAM RNA levels in nuclear and cytosolic fractions. Nuclear, cytosolic, and total RNAs were isolated from anterior pituitaries of OVX and OVX+E2 rats. Slot blots containing 10 µg of each nuclear, cytosolic, and total RNA samples were hybridized to probes corresponding to rPAM-1 cDNA (A), and the intron localized to the 5'-region of PAM gene (I-PAM) (C), respectively (see Materials and Methods). The blot was reprobed with 32P-labeled ribosomal cDNA to normalize data for quantification. Quantitative analysis of the blots shown in panels A and C was performed as described in Fig. 1BGo [B (rPAM-1 cDNA) and D (I-PAM)]. Each bar represents the mean ± SEM; the asterisk indicates that the value is significantly different between OVX and OVX+E2 rats (*, P < 0.001; * *, P < 0.02). Similar data were obtained from four independent experiments.

 
Several recent investigations have described nuclear posttranscriptional regulation involving changes in the stability and accumulation of primary (unspliced) transcripts (33, 34, 35). To investigate this possibility, nuclear RNA slot blots were rehybridized with a PAM intronic fragment I-PAM, derived from PAM genomic clone G33 (23). We have shown previously that I-PAM contains no repetitive sequences or PAM exon sequences (23). With the I-PAM, a 5- to 6-fold decrease in the levels of unspliced PAM RNA in OVX+E2 rats was obtained (Fig. 5Go, C and D). The observed effect on the intron-containing RNA may reflect altered stability of the unprocessed message. Furthermore, a mechanism involving inhibition of pre-RNA splicing could be ruled out since there was no buildup of unspliced PAM RNA in the nuclei of OVX+E2 rats. Taken together, the data shown in Fig. 5Go indicated that E2 caused down-regulation of unprocessed (unspliced) nuclear PAM RNA and consequently leads to the decrease observed in the amount of processed nuclear PAM mRNA levels. The same results were obtained using other intronic fragments derived from different PAM genomic clones (data not shown).

Since E2 caused a down-regulation for PAM mRNA at the nuclear level, it was of interest to assess the rPRL RNA levels in the nuclear preparations for comparaison.

Anterior pituitary nuclear RNAs prepared from OVX and OVX+E2 rats were hybridized to rPRL cDNA (Fig. 6AGo). As expected, a 4- to 7-fold increased expression of rPRL mRNA was detected in the cytosolic fraction and intact anterior pituitary gland tissue preparation of E2-treated animals (Fig. 6BGo). Nuclear rPRL RNA levels increased 10- to 11-fold in OVX+E2 animals (Fig. 6BGo). The finding that E2 increases the levels of rPRL mRNA is consistent with an effect of E2 to stimulate PRL gene transcription.



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  		Figure 6. Effect of estrogen on levels of nuclear and cytosolic PRL mRNA. A, The same blot illustrated in Fig. 5Go was reprobed with rPRL cDNA probe. B, The amount of PRL mRNA was normalized to the amount of rRNA (18S) rRNA signals for quantification. Each bar represents the mean ± SEM; similar data were obtained from three independent experiments. The asterisk indicates that the values are significantly different between OVX and OVX+E2 animals (*, P < 0.0001; **, P < 0.002).

 
PAM mRNA polyadenylation is unaffected by E2
The results presented above suggested that the E2 exerts a posttranscriptional effect on PAM expression in the nucleus at the level of stability of the unprocessed message. Several studies have demonstrated posttranscriptional regulation involving changes in poly (A) tail length on fully spliced nuclear RNAs (36, 37). We therefore investigated whether changes in the site or extent of polyadenylation correlated with the E2-induced decrease in nuclear PAM mRNA levels. To investigate the site of polyadenylation, samples of anterior pituitary total RNA from OVX and OVX+E2 rats were hybridized simultaneously to oligo(dT) and to an antisense PAM oligonucleotide (25-mer) complementary to the 3'-untranslated region of PAM mRNA (See Materials and Methods). Following digestion with RNase H, the 3'-deadenylated fragments were detected by Northern blotting and hybridization to a probe spanning the 3'-untranslated region of PAM mRNA. The data revealed that the size of the deadenylated fragment was unaffected by E2 treatment (Fig. 7Go). Therefore the site of polyadenylation of PAM mRNA was not altered by E2 treatment. To investigate the effects of E2 on the length of PAM mRNA poly (A) tail, total RNAs were hybridized to the PAM oligonucleotide alone before digestion with RNase H. The size distribution of the resulting 3'-polyadenylated fragments was also unaffected by E2 treatment (Fig. 7Go). The data showed that PAM mRNA has a discrete 25-residue poly (A) tail. Therefore, there were no detectable changes in the site of polyadenylation or in the length of the poly (A) tail of PAM mRNA that correlated with E2-induced changes in PAM mRNA levels.



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  		Figure 7. PAM mRNA polyadenylation in anterior pituitary gland. Total RNAs from OVX and OVX+E2 rats were subjected to RNase H digestion following hybridization to a mixture of oligo (dT) and an antisense oligonucleotide complementary to the 3'-untranslated region of PAM mRNA [H23+oligod(T)]. RNAs were similarly digested with RNase H following hybridization to the PAM oligonucleotide alone (H23). The resulting 3'- deadenylated and 3'-polyadenylated PAM mRNA fragments were detected by Northern blot hybridization to a probe specific for the 3'-end of PAM mRNA.

 
E2 decreased PAM expression is dependent on protein synthesis
Whether newly made protein is required for the decrease of PAM mRNA was explored by the use of the protein synthesis inhibitor cycloheximide. Anterior pituitary cells prepared from OVX and OVX+E2 animals were cultured as described in Materials and Methods. Cells were incubated with 10 µg/ml of cycloheximide. PAM expression was assessed by Northern blot analysis. Treatment with the protein synthesis inhibitor for 2 h or 4 h prevented E2 decrease in PAM mRNA levels (Fig. 8Go). Cycloheximide did not significantly affect the level of 18S rRNA (data not shown). These results demonstrate that E2-caused down-regulation of PAM mRNA is sensitive to protein synthesis inhibitors.



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  		Figure 8. E2 decrease of PAM mRNA depends upon protein synthesis. Rat primary anterior pituitary cultures prepared from OVX and OVX+E2 animals were maintained for 4 days in DMEM plus 10% stripped FCS ± E2. On day 5, cells were washed twice and incubated with medium containing cycloheximide (10 µg/ml). At the indicated intervals after the addition of cycloheximide, triplicate plates of cells were harvested for isolation of RNA. PAM mRNA levels were corrected for hybridization to 18S rRNA for quantification as described in previous figures. Each bar represents the mean ± SEM of three independent experiments. The asterisk indicates that the differences in values between both groups are statistically significant (*, P < 0.02).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have attempted to determine the primary site at which E2 acts to control the expression of the PAM gene in anterior pituitary gland. For this purpose, parallel measurements were made of relative PAM mRNA levels and nuclear precursor RNA levels and of the rate of gene transcription in OVX and OVX+E2 rats. To investigate whether differential changes at transcriptional rates might account for the differences in mRNA levels, we performed in vitro run-on transcription assays on nuclei prepared from anterior pituitary glands of OVX and OVX+E2-treated animals. Our results clearly demonstrate no change in the transcriptional activity of PAM gene in OVX and OVX+E2 rats, indicating that the transcription rate does not account for the great relative changes observed for the PAM mRNA levels. The data demonstrate that E2 exerts its effect on PAM biosynthesis at the posttranscriptional level.

The most commonly observed mechanism of posttranscriptional regulation of mRNA accumulation affects mRNA stability in the cytoplasm (38). Actinomycin D showed a 50% decrease in the half-life of PAM mRNA in primary anterior pituitary cultures prepared from OVX+E2 compared with OVX rats. These data demonstrated that anterior pituitary PAM mRNA is destabilized in E2-treated animals. There is a growing body of data to indicate that the 3'-untranslated regions of a number of mRNAs contain sequences that serve as determinants of RNA stability (39, 40). We therefore examined whether estrogen might alter the 3'-processing and polyadenylation of PAM mRNA. The data in Fig. 7Go indicate that estrogen treatment produces no alterations in the 3'-untranslated region of the message. One interesting observation came out of these experiments: PAM mRNA has a remarkably short poly (A) tail, the length of which is unaffected by estrogen.

However, the magnitude of the E2 effect observed when total RNA was analyzed could be accounted for by the effect seen when nuclear RNA was analyzed (Fig. 5Go). The analysis of nuclear RNAs clearly demonstrated a decrease in levels of intron-containing RNA in the E2-treated rats. This phenomenon could be explained by altered stability of the unprocessed PAM message since the transcription of PAM gene was not altered. As the levels of the nuclear precursor were altered proportionally to cellular mRNA levels, the posttranscriptional regulation would have to occur at some nuclear step preceding the accumulation of the precursor for PAM mRNA. This finding thus implies that the stability of the nuclear precursor is altered in response to estrogen treatment. In OVX+E2 animals, the PAM gene is transcribed at the same rate as in OVX animals, but a large proportion of the precursor is degraded before processing and transport to the cytoplasm take place.

Posttranscriptional regulation of PAM expression has been demonstrated in other experimental systems. For example, in rat anterior pituitary gland we have shown that hypothyroidism stimulates PAM mRNA levels by increasing the RNA stability in the cytoplasm (23). Therefore, although the precise mechanism remains to be identified, the present report provides the first evidence for nuclear posttranscriptional regulation of PAM expression. Nuclear posttranscriptional regulation is emerging as an important means of controlling gene expression. Regulation of specific cytoplasmic mRNA levels by altering the stability of nuclear transcripts has been shown in a variety of biological systems. For example, it has been demonstrated that the regulation of malic enzyme (35), class I major histocompatibility complex (41), myeloperoxidase (42), {alpha}1-acid glycoprotein (43), dihydrofolate reductase (44), eIF-2{alpha} (33), and the hepatic spot 14 (45) expression occurs at the level of nuclear RNA stability. In the case of Xenopus ribosomal protein genes, a specific posttranscriptional regulation mediated through regulation of processing efficiency and stability of intranuclear transcripts has been proposed (46). Nuclear mechanisms affecting the splicing patterns (47) and polyadenylation (36, 37) have also been demonstrated.

In conclusion, the present study demonstrates that estrogen causes down-regulation of nuclear PAM RNA levels in anterior pituitary gland. However, the precise mechanisms by which the stability of nuclear RNA is controlled have not been established. Based on cycloheximide data, E2 appears to destabilize PAM RNA through a mechanism involving a new protein synthesis. The putative destabilizing protein(s) may recognize either a specific sequence or a secondary structure in PAM mRNA transcripts or, alternatively, another regulatory factor(s) specifically bound to the PAM transcripts. Experiments are in progress for identification of such a factor(s). The PAM gene expression is a potentially interesting system for studying the complexity of hormonal regulation of gene expression at the posttranscriptional level.


   Acknowledgments
 
Reagents for LH RIA were provided by the NIDDK Hormone Distribution Program. We wish to thank Drs. Charles Oliver and Anne Dutour Meyer for their critical reading of the manuscript. We thank F. Youssouf and J. C. Orsoni for their technical help.


   Footnotes
 
1 This work was supported by INSERM. Back

Received June 4, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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