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Endocrinology Vol. 139, No. 6 2831-2837
Copyright © 1998 by The Endocrine Society

ARTICLES

Vasopressin Messenger Ribonucleic Acid Regulation via the Protein Kinase A Pathway1

Rodica L. Emanuel, Yasumasa Iwasaki, Zoya K. Arbiser, Elena M. Velez, Charles H. Emerson and Joseph A. Majzoub

Division of Endocrinology (R.L.E., Z.K.A., E.M.V., J.A.M.), Department of Medicine, Children Hospital, Harvard Medical School, Boston, Massachusetts 02115; University of Massachusetts Medical Center (C.H.E.), Worcester, Massachusetts 01655; and Nagoya University School of Medicine (Y.I.), Nagoya 466, Japan

Address all correspondence and requests for reprints to: Rodica L. Emanuel, Division of Endocrinology, Department of Medicine, Children Hospital, Harvard Medical School, Boston, Massachusetts 02115.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of vasopressin (VP) gene expression in vivo by osmotic stimuli results in an increase in both messenger RNA (mRNA) content and polyadenylate [poly(A)] tail length. VP gene transcription in vitro is stimulated by protein kinase A (PKA) activation. To examine the role of PKA in the regulation of VP mRNA poly(A) metabolism, constructs of the rat VP gene were permanently transfected into the mouse anterior pituitary cell line, AtT-20. Treatment with forskolin of cells expressing the intact VP gene resulted in increased VP gene transcription, an increase in the content of VP mRNA, and a shift toward VP mRNA species with longer poly(A) tails accompanied by the loss of VP mRNA species with shorter poly(A) tails. We uncoupled the PKA-stimulated appearance of long-tailed species from the disappearance of short-tailed species, suggesting that the size shift was caused by a coincident, but uncoupled net increase in VP mRNA species with elongated poly(A) tails and net loss of mRNA species with short poly(A) tails. These data indicate that activation of the PKA second-messenger pathway both enhances transcription of the VP gene and causes an increase in the average length of VP mRNA poly(A) tails. This latter effect, by shifting upwards the average poly(A) tail size, could result in increased translational efficiency or stability of VP mRNA, thereby providing an additional mechanism by which PKA may enhance gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RAT vasopressin (VP) messenger RNA (mRNA) content increases in vivo after osmotic and circadian stimuli (1, 2, 3), which is, at least in part, caused by the stimulation of transcription (4, 5). These changes have also been observed in vitro, where forskolin, an activator of adenylate cyclase and protein kinase A (PKA), stimulates VP mRNA content in primary cultures of fetal rat hypothalamic cells (6) and in continuous cultures of a human cell line expressing the endogenous VP gene (7). Two putative cAMP response elements (CREs) have been identified in the VP promoter (8, 9); several investigators (10, 11, 12), using heterologous transient transfection systems, have shown that PKA activators stimulate the transcription of VP gene constructs containing these putative CREs.

Along with an increase in VP mRNA content, activation of VP gene expression in vivo by osmotic (1, 13) and circadian (14) stimuli, and in vitro in primary cultures of rat fetal hypothalamic cells by forskolin (6), is associated with a shift in the size of VP mRNA toward species containing longer polyadenylate [poly(A)] tails. In hypothalamus, poly(A) tail length of VP mRNA increases from a basal length of 225–275 (mean, 250) nucleotides to 375–425 (mean, 400) nucleotides after osmotic stimulation (1). This size shift could result from the appearance of newly transcribed species bearing longer poly(A) tails, coupled with enhanced degradation of preexisting mRNAs bearing short poly(A) tails, or cytoplasmic polyadenylation of preexisting mRNA. Because poly(A) tail length is important for the regulation of both mRNA translatability and stability (15), we have studied the regulation of VP mRNA polyadenylation by the PKA second-messenger pathway using the mouse corticotroph cell line AtT-20. These cells have a robust response to cAMP stimulation, and they properly express and process other transfected genes (16). We stably transfected these cells with several constructs of the rat VP gene and have examined the effects of PKA activation on VP mRNA content and poly(A) tail length, as well as the role of transcription and translation processes in mediating these effects. Our findings indicate that PKA stimulates both the transcription of the VP gene, the appearance of VP mRNA with long poly(A) tails, as well as the disappearance of VP mRNA species bearing shorter poly(A) tails.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture reagents, ribonuclease (RNase) H, and the 0.16–1.7 RNA ladder were purchased from Gibco-BRL (Gaithersburg, MD). FBS and horse serum were obtained from Hyclone (Logan, UT). 35S-methionine and GeneScreen Plus membrane were obtained from NEN-Dupont (Boston, MA) and {alpha}-32P-uridine 5'-triphosphate from Amersham Corp. (Arlington Heights, IL). Oligo(dT)12–18 was purchased from Collaborative Research Inc. (Waltham, MA) and T3 RNA polymerase from Stratagene (La Jolla, CA). Additional chemicals and reagents of molecular biology grade were purchased from Sigma (St. Louis, MO).

Plasmid construction and DNA preparation
A schematic diagram of plasmid constructs is shown in Fig. 1Go. Construct arginine vasopressin (AVP)-1 contains 8.2 kb of the rat VP gene, including 3.0 kb of the promoter, 3.2 kb of the 3'-flanking region, and the entire coding region. Construct AVP-2 contains 377 bp of the rat VP promoter region, the entire coding region, and 187 nucleotides of the 3'-flanking region. Construct AVP-3 is identical to AVP-2 in the coding and the 3'-flanking regions, but it differs in the 5'-flanking region, where the 377 bp of VP promoter was replaced by 550 bp of the rous sarcoma virus (RSV) promoter. The nontranslatable constructs {Delta}AVP-2 and {Delta} AVP-3 were created by mutating all ATG codons of AVP-2 to either AGG (T->G at positions 33 and 42); ATC (G->C at position 46); and CTG (A->C at positions 140 and 1532) [for nucleotide position numbering, we followed the scheme of Schmale et al. (17), with the position of transcription start site labeled as +1]. All mutations were created by recombinant PCR (18) using two PCR fragments (PCR1 and PCR2), which were generated using oligonucleotides containing the desired mutations: PCR-1s (sense strand nucleotides 10–55), PCR-1r (antisense strand nucleotides 171–131), PCR-2s (sense strand nucleotides 1519–1544), and PCR-2r (antisense strand nucleotides 1945–1922). PCR-1 and PCR-2 were ligated into AVP-2 and AVP-3 at DraIII/KpnI and PstI/NarI sites, respectively. All mutations created unique restriction sites, which were confirmed to be present by restriction digest analysis. Plasmid RSV-Luc, used for the evaluation of the transient transfection efficiency, contains 0.53 kb of RSV-LTR in the polylinker site of pXp1 luciferase vector.



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  		Figure 1. Structure of VP probes and gene constructs used to create specific stable AtT-20 corticotroph cell lines. AVP-1, AVP-2, and AVP-3 contain the entire coding region of the VP gene (from nucleotide +1 to 1985). AVP-1, containing 3.0 and 3.2 kb of the 5'- and 3'-flanking regions of the VP gene, respectively, was used to create cell line 3019. AVP-2, containing 377 nucleotides of the 5'- and 187 nucleotides of the 3'-flanking regions of the VP gene, was used to create cell line 182. In construct AVP-3, 550 nucleotides of the RSV promoter were inserted 38 bp upstream of the transcription initiation site of the VP gene. The 3'-flanking region contains 187 nucleotides past the poly(A) addition site of the VP gene. AVP-3 was used to create cell line 23. A putative CRE, present only in AVP-1 and AVP-2 constructs, is shown as a hatched square. The polyadenylation site, indicated by an arrow at nucleotide 1985, is present in all three constructs. Location of the cRNA probes rVP1, rVP3, and rVP4 and oligonucleotide rVP2 is shown at the bottom of the figure (see Materials and Methods).

 
Cell culture, transfection, and drug treatments
AtT-20/D16V (AtT-20), a mouse pituitary cell line, was grown in Ham’s F10 media supplemented with 10% FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were grown at 36.5 C in a humidified atmosphere of 10% CO2/90% air and were subcultured every 7–10 days after treatment with trypsin-EDTA. Stable cell lines were created using the polybrene technique (19) with 10–30 µg plasmid DNA and 2 µg of the neomycin selectable marker RSVneo (for constructs AVP-1, AVP-2, and {Delta}AVP-2) or SV2neo (for constructs AVP-3 and {Delta}AVP-3). Individual VP-expressing colonies were identified by the presence of VP peptide in the media (measured by RIA). The results reported herein for each cell line are representative of independent clones and have been confirmed in at least three experiments.

Transient transfections were performed using 0.6 µg DNA from AVP-3 or {Delta}AVP-3, 0.15 µg RSV-Luc (as an internal control), and 2.25 µg carrier DNA (pBluescribe) using the calcium phosphate method (16). Cells were grown for 48 h, washed with PBS, and incubated in serum-free Ham’s F10 media for 6 h. Media and cells were then collected for measurement of VP peptide content and luciferase activity, respectively.

Sixty to 70% confluent cells grown in culture for 10 days were washed once and fed with prewarmed Ham’s F10 media containing either vehicle alone or vehicle and drug. Forskolin was dissolved in 95% ethanol and used at a final concentration of 10 µM; cycloheximide was dissolved in methanol and used at a concentration of 100 µg/ml. The rate of 35S-methionine incorporation into tricholoracetic acid-precipitable protein, in the presence or absence of cycloheximide, was measured using the procedure described by Gay (20). RNA transcription was inhibited using 65 µM of the adenosine analog 5,6-dichloro-ribofuranosyl benzimidazole (DRB) (21).

RNA isolation and filter (Northern) hybridization
Total RNA was extracted from cells lysed directly in guanidinium thiocyanate/phenol solution (22). For a given experiment, control and forskolin-treated samples were analyzed on the same RNA blot. Total RNA (1–10 µg/lane) was size-fractionated in 1.4% agarose-formaldehyde gels and transferred to GeneScreen-plus membranes. Blots were hybridized to 32P-labeled antisense complementary RNA (cRNA) probes encoding rat VP (see below) at 65 C for 24 h, washed at 65 C in several changes of 0.1% SDS/15 mM NaCl/1.5 mM sodium citrate, and exposed for 1–4 days to Kodak XAR-5 film (VWR, Boston, MA) with an intensifying screen (23, 24). Autoradiograms were scanned with a laser ultrascan XL densitometer (Pharmacia Biotechnology, Piscataway, NJ). The amount of mRNA in each lane was corrected for the total amount of RNA loaded by scanning the 18s rRNA band from the ethidium bromide-stained gel. A 0.16- to 1.7-kb RNA ladder (Gibco-BRL) was used as a size indicator. The distance of VP mRNA migration (measured from the top of the gel to the center of the VP mRNA band) was read from the autoradiogram and transformed into kilobase pairs using a plot of the RNA ladder bands (0.16, 0.28., 0.4, 0.53, 0.78, 1.28, 1.52, and 1.77 kb) vs. the distance of migration (from the photograph of the ethidium bromide-stained gel).

3' mRNA structural analysis using RNase H
RNase H digestions were performed using the method described by Carrazana and colleagues (1). Ten micrograms of total RNA was hybridized at 22 C for 30 min with 500 pmol of either oligo(dT)12–18 or the 23 nucleotide-long probe rVP2, complementary to bases 1917–1939, located in the 3'-untranslated region of VP mRNA, 50 nucleotides upstream from the poly(A) addition site (Fig. 1Go). RNase H digestion of VP mRNA, in the presence of oligo(dT)12–18, results in the deadenylation of mRNA, whereas digestion in the presence of rVP2 yields two fragments: one consisting of the body of VP mRNA up to the site of hybridization of rVP2, and the second containing the poly(A) tail attached to the terminal 50 nucleotides of the 3' untranslated region. Products of RNase H digestion were analyzed by Northern blot, using 32P-labeled cRNA probes rVP3 and rVP4 (see below).

cRNA probes
32P-labeled cRNA probes were synthesized from DNA templates using {alpha}-32P uridine 5'-triphosphate (25). cRNA probe rVP3, used to identify intact or deadenylated VP mRNA, is complementary to nucleotides 1790–1985 of the VP gene; probe rVP4, used to identify poly(A) tails after their cleavage from VP mRNA bodies, is complementary to nucleotides 1977–1990 (Fig. 1Go). The probes were synthesized using T3 RNA polymerase, as reported previously (1). The rVP1 template was obtained by PCR amplification of intron I of rat VP gene using as a template clone AVP-2 and intronic primers complementary to nucleotides 164–185 (sense) and 1313–1293 (antisense). The downstream primer contains, at the 5' end, the T3 RNA polymerase sequence. Probe rVP1 detects a 1.7-kb precursor of VP mRNA containing intron I. The mouse ß-actin cRNA probe was obtained from a complementary DNA, kindly provided by B. Spiegelman (26).

VP RIA
VP peptide, secreted into culture media, was measured using a specific RIA, as described previously (6).

Statistical analysis
Each experiment contained at least three independent observations per group, and each complete experiment was repeated at least twice. All values are expressed as the mean ± SEM. To evaluate potential differences in VP mRNA size, under two different conditions (with and without forskolin stimulation), the two mRNA species were run in two adjacent gel lanes, and their distances of migration were measured as described above (see RNA and filter [Northern] hybridization). Statistical analysis of differences between two groups was performed using the two-tailed, unpaired Student’s t test, with significance defined as P < 0.05. To determine whether a significant change in VP mRNA content or poly (A) tail length occurred over time, one-way ANOVA was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of forskolin on VP gene expression in cell lines transfected with VP-promoter containing constructs
To evaluate the effect of forskolin on VP gene transcription, cells from line 3019 (expressing construct AVP-1, Fig. 1Go) were exposed to forskolin for 0, 1, 3, 5, and 24 h. Total RNA was subjected to RNA blot analysis and probed with cRNA probe rVP1, specific for intron I of the rat VP gene (Fig. 1Go). As shown in Fig. 2AGo (upper panel), the 1.7-kb VP mRNA precursor was maximally stimulated 5-fold after 1 h of forskolin treatment and was returned to control levels after 5 h. Rehybridization of the same RNA blot with probe rVP3 (Fig. 1Go), which detects mature VP mRNA, revealed that treatment with forskolin significantly increased VP mRNA content 2-fold after 1 h, maximally 4-fold after 3 h (from 0.97 ± 1.2 arbitrary densitometric units to 5.15 ± 1.75, n = 4, P < 0.05) and returned toward control level after 24 h (Fig. 2AGo, middle panel). The increase in VP mRNA content was accompanied by an upward shift in the average size of VP mRNA, by approximately 92 nucleotides, between 0 and 3 h after forskolin addition (from 674 ± 4.0 to 763 ± 26, n = 3, P < 0.01, Fig. 2AGo, middle panel). This shift in the average size of VP mRNA was most evident in autoradiograms obtained after very short exposure, indicating that it was not simply caused by an increase in the width of the mRNA band caused by the increase in the content of VP mRNA (data not shown). Treatment with 1 mM 8Bromo cAMP (8Br cAMP) also resulted in a similar time-dependent increase in VP mRNA content and length (data not shown). ß-actin mRNA content and size were unchanged after forskolin treatment (data not shown). The effects of forskolin on VP mRNA content and size were identical in cell line 182, which contains construct AVP-2 (Fig. 1Go), lacking all but 377 bp of 5'-flanking region and 187 bp of 3'-flanking region (data not shown). Removal of VP mRNA poly(A) tails by digestion with RNase H resulted in deadenylated VP mRNA bodies of identical sizes (~610 bp, P = 0.519) in both control and forskolin-treated cells, indicating that the increase in mRNA size is caused by enhanced polyadenylation (Fig. 2BGo), as is the case in vivo, after osmotic and circadian stimulation (1, 14). To determine whether the forskolin-induced increase in VP mRNA content and polyadenylation are dependent upon transcription, 3019 cells were treated with the generalized transcriptional inhibitor DRB. At the optimal dose of 65 µM, DRB alone did not have any effect on the content or size of the VP mRNA (data not shown). When forskolin treatment of 3019 cells was preceded by a 1-h incubation with DRB, the forskolin-induced increase in VP mRNA content and polyadenylation did not occur (Fig. 2CGo, P > 0.1, using one way ANOVA)



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  		Figure 2. Forskolin-stimulated transcription of the VP gene is associated with an increase in content and poly(A) tail length of VP mRNA. AtT-20 cells (line 3019), expressing VP construct AVP-1, were exposed to 10 µM forskolin for 1, 3, 5, and 24 h. A, Total cellular RNA was gel fractionated (10 µg/lane) and subjected to RNA blot analysis using the intron-specific cRNA probe rVP1 (upper panel) or exon C-specific cRNA probe rVP3 (middle panel). B, Analysis of VP mRNA after digestion with RNase H. Total RNA (5 µg), obtained from control (-) and forskolin-stimulated (+) VP-expressing cell line 3019 was digested with RNase H in the presence (+) or absence (-) of oligo(dT)12–18, gel-fractionated and hybridized with probe rVP3. Rat hypothalamic RNA (HT, 2 µg) was included as a control. The migration of RNA size markers (in nucleotides) is indicated on the right. C, Active transcription is required for forskolin-induced increase in VP mRNA content and polyadenylation. VP-expressing cell line 3019 was treated with the transcription inhibitor DRB, 1 h before the addition of forskolin, for the periods of time indicated. Total RNA was analyzed as described in B. In A–C, 18s rRNA content was measured (bottom panel of A–C) and used to correct for the amount of total RNA loaded in each lane.

 
Effect of forskolin on VP gene expression in cells transfected with a construct driven by the forskolin-unresponsive RSV promoter
The DRB-induced block in forskolin stimulation of VP mRNA content and poly(A) tail length suggested that these two effects require specific transcription of either the VP gene or other gene(s). To test the former possibility, forskolin treatment of cells containing the VP structural gene under the control of the RSV promoter (construct AVP-3, Fig. 1Go), which is unresponsive to cAMP (27), was performed. RNA blots of total RNA from cell line 23, treated with forskolin, were probed with rVP1, complementary to intron I of the VP gene. No change in the content of the 1.7-kb VP mRNA precursor was observed for up to 7.5 h after the addition of forskolin (Fig. 3AGo), confirming that the RSV promoter is transcriptionally unresponsive to forskolin (compare with Fig. 2CGo). Forskolin treatment, for 6 h, of cell line 23 resulted in a decrease in the content of mature VP mRNA (from 2.6 ± 0.18 densitometric units to 1.2 ± 0.22, n = 3, P = 0.06), which was associated with the preferential loss of shorter VP mRNA species (Fig. 3BGo, left panel). RNase H digestion was performed on total cellular RNA from cell line 23 in the presence of either oligo(dT)12–18 (to assess the size of deadenylated VP mRNAs), or the 23-mer oligonucleotide rVP2 (to assess the size of VP mRNA poly(A) tails). In the presence of oligo(dT)12–18, deadenylated VP mRNAs from control and forskolin-stimulated cells were of identical sizes of approximately 610 nucleotides (Fig. 3BGo, middle panel). Digestion with RNase H, in the presence of oligonucleotide rVP2, revealed that in the presence of forskolin, short poly(A) tails (containing 80–120 nucleotides) were lost, whereas longer poly(A) tails (containing 160–180 nucleotides) were preserved (Fig. 3BGo, right panel).



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  		Figure 3. Forskolin induces a decrease in the VP mRNA content in cell line 23, expressing the VP gene driven by the forskolin-unresponsive RSV promoter (construct AVP-3). A, Line 23 was treated with forskolin for 0–7.5 h, total cellular RNA was gel fractionated (10 µg/lane), and RNA blot analysis was performed using the intron-specific cRNA probe rVP1. B, VP mRNA analysis after digestion with RNase H. Line 23 cells were treated for 6 h, without (-) or with (+) forskolin, and total RNA was extracted and digested with RNase H in the presence of oligo(dT)12–18 or oligonucleotide rVP2. VP mRNA and poly(A) tails were identified using cRNA probe rVP4. The migration of RNA size markers (in nucleotides) is indicated on the right. In A and B, 18s rRNA content was measured (bottom panel of A and B) and used to correct for the amount of total RNA loaded in each lane. Similar results were obtained from two independent experiments.

 
Effect of protein synthesis inhibition on forskolin-induced changes in VP mRNA
To establish whether the forskolin-evoked changes in VP mRNA content and polyadenylation require protein synthesis, cell line 3019, expressing the VP gene driven by the VP promoter (construct AVP-1, Fig. 1Go), was exposed to forskolin for 6 h in the presence or absence of the generalized protein synthesis inhibitor, cycloheximide. The optimal dose of cycloheximide (required for inhibition of protein synthesis, as determined by measurement of 35S-methionine incorporation into total protein) was found to be 100 µg/ml (data not shown). Inhibition of protein synthesis did not prevent the forskolin-induced appearance of VP mRNA species with longer poly(A) tails (Fig. 4Go). However, cycloheximide treatment prevented the forskolin-induced disappearance of VP mRNA species bearing shorter poly(A) tails (Fig. 4Go, compare second and fourth lanes).



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  		Figure 4. Forskolin regulation of VP mRNA content and polyadenylation in the absence of protein synthesis. Cell line 3019, containing construct AVP-1, was cultured for 6 h, in the presence (+) or absence (-) of cycloheximide and forskolin, and total RNA was extracted and subjected to RNA blot analysis (10 µg/lane) and hybridized using probe rVP3. 18s rRNA content of each lane is shown below the autoradiogram.

 
To determine whether the above-described effects of cycloheximide on VP mRNA are caused by a specific block in the translation of VP mRNA, AtT-20 cells were permanently transfected with a mutant of constructs AVP-2 and AVP-3 (Fig. 1Go), {Delta}AVP-2, and {Delta}AVP-3, respectively. {Delta}AVP-2 and {Delta}AVP-3 lack all ATG codons, so that VP mRNA expressed from these constructs should not be translatable. To confirm that VP mRNA expressed from this type of construct cannot be translated, VP secretion was measured in media in which cells transiently cotransfected with either AVP-3 or the nontranslatable mutant, {Delta}AVP-3 and RSV-Luc were cultured for 48 h. AtT-20 cells, transfected with construct AVP-3, secreted 29 ± 6 pg VP peptide/ml·6 h (n = 3); no VP peptide was detected in the media collected from cells transfected with construct {Delta}AVP-3.

The effect of forskolin on VP mRNA was compared in translation-competent cell line 182 (which expresses the construct AVP-2) and the translation-incompetent cell line 182-{Delta} AUG (which expresses construct {Delta}AVP-2). Cells were treated for 6 h with forskolin, after which, total RNA and poly(A) tails (obtained by RNase H digestion in the presence of rVP2) were analyzed by RNA blot and probed with cRNA probe rVP4 (which hybridizes to both intact VP mRNA and cleaved VP poly(A) tails. Forskolin induced an increase in VP mRNA content with longer poly(A) tail species and the loss of VP mRNA species with short poly(A) tails from the cells containing the intact VP structural gene (Fig. 5Go, lanes 1 and 2, upper and lower panels). In cells expressing the nontranslatable construct {Delta}AVP-2 (line 182-{Delta}AUG), forskolin induced an increase in VP mRNA content and a more heterogeneous increase in poly(A) tail length, but it failed to induce the loss of VP mRNA species with short poly(A) tails (Fig. 5Go, lanes 3 and 4, upper and lower panels).



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  		Figure 5. Effect of forskolin on VP mRNA in cell lines expressing translatable or untranslatable VP mRNA. Cell lines 182 and 182-{Delta}AUG were cultured for 6 h, in the presence (+) or absence (-) of forskolin, and total cellular RNA was analyzed by RNA blot (10 µg/lane). Intact VP mRNA (shown in the upper panels) and poly(A) tails obtained after digestion of intact RNA with RNase H in the presence of oligonucleotide rVP2 (shown in the lower panels) were identified using probe rVP4. The migration of RNA size markers (in nucleotides) is indicated on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that cAMP stimulation of the VP gene, stably transfected into AtT-20 cells, results in increased VP gene transcription, mRNA accumulation, and elongation of the average length of VP mRNA poly(A) tails by approximately 90 nucleotides. This PKA-induced shift in poly(A) tail length could be caused by cytoplasmic addition of poly(A) to preexisting mRNA species or, alternatively, to a coincident, but uncoupled, net increase in VP mRNA species with elongated poly(A) tails and net loss of mRNA species with short poly(A) tails. We found evidence that favors the latter hypothesis, for we could uncouple the PKA-stimulated appearance of long-tailed species from the disappearance of short-tailed species: the PKA-stimulated increase in mRNA species with long poly(A) tails was transcription- (but not translation-) dependent, whereas the PKA-stimulated decrease in mRNA species with short poly(A) tails was translation- (but not transcription-) dependent. These data indicate that activation of the PKA second-messenger pathway, in addition to its known effect to enhance transcription of the VP gene, also causes an increase in the average length of VP mRNA poly(A) tails. This is likely to be a more general phenomenon, because concomitant increases in mRNA content and a shift toward longer poly(A) tail length have been reported after stimulation of several genes (including those encoding GH, insulin, TSH, GnRH, LH, and CRH) (28, 29, 30, 31, 32, 33).

The dependence of the increase in VP mRNA poly(A) tail length upon transcription was shown by a block of the forskolin-induced increase in poly(A) tail length, both after treatment of cells with DRB, a generalized inhibitor of transcription (21) and after the use of the RSV promoter, transcription of which is unresponsive to cAMP (27). Although the kinetics of mRNA poly(A) metabolism have not been well studied in mammalian cells, the availability of several yeast mutants has shed light on this area (34). Yeast mRNAs are initially transcribed with maximally long poly(A) tails, which undergo progressive cytoplasmic shortening (35, 36). It is therefore reasonable to posit that enhanced transcription of the VP gene by cAMP would result in the synthesis of increased amounts of VP mRNAs bearing long poly(A) tails, consistent with our findings. That the cAMP-induced loss in VP mRNAs with short poly(A) tails persisted (even when mRNA was transcribed from the cAMP-unresponsive RSV promoter) indicates that VP mRNA transcription is not required for this process.

Although the cAMP-induced decrease in VP mRNA species with short poly(A) tails is not dependent on VP gene transcription, it is dependent upon translation. This was seen both when translation was globally inhibited (using cycloheximide) and when VP mRNA translation was specifically inhibited by the use of a translation-incompetent VP mRNA. Our findings could be explained by the stimulation of VP mRNA translation by cAMP, and the coupling between translation with poly(A) tail shortening and mRNA degradation. Several recent reports, using a variety of mammalian systems, have shown that cAMP enhances the translation of specific mRNAs (37, 38, 39, 40). Moreover, reports from several investigators indicate an obligatory association between the translation and degradation of mRNA, and that, below a minimal poly(A) tail length, the degradation of a mRNA is greatly accelerated (15, 35, 36, 41, 42, 43, 44). Thus, it is possible that cAMP causes a loss of VP mRNAs bearing short poly(A) tails, by stimulating translation of VP mRNA in general, which is coupled to poly(A) tail shortening and degradation of mRNAs, particularly those with the shortest poly(A) tails. This, coupled with the cAMP-stimulated transcription of VP mRNAs with long poly(A) tails (as discussed above), could account for the net upward shift in VP mRNA poly(A) tail length caused by cAMP treatment (Fig 6Go).



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  		Figure 6. Schematic model of VP gene regulation by cAMP, under basal and stimulated conditions, including the transcription-dependent increase in VP gene transcription and mRNA content and the translation-dependent elimination of mRNA with short poly(A) tails. See Discussion for details.

 
There is increasing evidence that the translational efficiency of mRNA depends on the presence of poly(A) tails with greater than a minimum length (45). Because the translational efficiency decreases only when the poly(A) tail length of a mRNA is shortened to less than 12–15 residues (34), the increase in VP mRNA poly(A) tail length by approximately 90 nucleotides, after forskolin treatment, might allow mRNA molecules with longer poly(A) tails to participate in more translation cycles. Thus, by effecting an upward shift in the average poly(A) tail size, cAMP could cause an increase in translational efficiency of VP mRNA, thereby synergizing with cAMP stimulation of VP gene transcription to provide an additional mechanism by which PKA may enhance gene expression.


   Acknowledgments
 
We thank James Bonner for the rat genomic library, I. Richardson for AtT-20/D16V cells, and R. Pratt for RSVneo plasmid. We acknowledge the excellent technical assistance of Ms. O. Cortez.


   Footnotes
 
1 This work was supported by NIH Grants R01-DK-4017001, 5-P50-HL-36568, and 5-R01-NS-29384. Back

Received November 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Carrazana EJ, Pasieka KB, Majzoub JA 1988 The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo. Mol Cell Biol 8:2267–2274[Abstract/Free Full Text]
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  4. Herman JP, Schafer MK, Watson SJ, Sherman TG 1991 In situ hybridization analysis of arginine vasopressin gene transcription using intron-specific probes. Mol Endocrinol 5:1447–1456[CrossRef][Medline]
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