Vol. 10, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1990, p. 442-448
0270-7306/90/020442-07$02.00/0 Copyright © 1990, American Society for Microbiology
Transcriptional and Posttranscriptional Regulation of the Rat Prolactin Gene by Calcium GREGORY M. PRESTON, WILLIAM M. BILLIS, AND BRUCE A. WHITE* Graduate Program in Developmental Biology, Department of Anatomy, University of Connecticut Health Center, Farmington, Connecticut 06032 Received 25 August 1989/Accepted 25 October 1989
The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2", serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2" addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2+-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2' to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2' has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca2+ appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.
expression through posttranscriptional mechanisms has not been examined. Interestingly, thyrotropin-releasing hormone increased prolactin mRNA by both transcriptional stimulation and specific stabilization of prolactin mRNA (19). In light of this finding, and of an increasing number of reports of posttranscriptional control of gene expression (5, 9, 25, 27, 30, 33, 36), we compared the effects of Ca2+ on the transcriptional rate of the endogenous prolactin gene with its effects on steady-state prolactin mRNA levels. We report here that a modest (less than twofold) increase in prolactin gene transcription was accompanied by a significant (greater than fivefold) increase in prolactin mRNA. These data indicate that Ca2+ stimulates prolactin gene expression primarily at a posttranscriptional level. Furthermore, Ca2+ appears to act on prolactin mRNA through a mechanism that is independent of ongoing protein synthesis.
The prolactin gene is expressed at a high basal level in the adult rat pituitary lactotroph and in pituitary tumor GH3 cells (3). This fact suggests the existence of a lactotroph-specific mechanism that promotes prolactin expression in the absence of external hormonal factors. We and others have demonstrated that the basal-level enhancement of the rat prolactin gene is maintained by a Ca2"-dependent mechanism (13, 35, 37, 38, 42). For example, when GH3 cells are cultured in a low-Ca2 serum-free medium (SFM), prolactin mRNA declines to a barely detectable level. Subsequent addition of CaCl2 results in a large and sustained increase in prolactin mRNA levels (39, 42). This Ca2+-dependent enhancement of prolactin gene expression is highly specific, since Ca2' has little or no effect on the mRNA levels for growth hormone, metallothionein, histone 3, or glucoseregulated protein 78 (GRP78) (13, 42, 43). In addition, manipulation of the culture medium Ca2+ concentration had no discernible effect on the synthesis of numerous proteins other than prolactin, as detected by two-dimensional gel electrophoresis (35). At least part of the Ca2'-dependent enhancement of prolactin gene expression in GH3 cells appears to be due to transcriptional stimulation (18). In a study using transfected hybrid constructs containing the prolactin promoter linked to the chloramphenicol acetyltransferase (CAT) gene, Jackson and Bancroft (18) obtained a four- to eightfold stimulation of CAT activity by addition of 0.5 mM CaCl2 to GH3 cells cultured in SFM. Analysis of 5'-deleted prolactin-CAT constructs demonstrated that a Ca2+-responsive element resides within the first 174 base pairs of upstream flanking DNA sequence (18). The possibility that Ca2+ may also increase prolactin gene ,
*
MATERIALS AND METHODS
DNA clones. The prolactin cDNA clone (pPRL-1) was provided by R. Maurer (University of Iowa College of Medicine, Iowa City). The hamster GRP78 (p3C5) and histone 3 (pAAD3.7) cDNA clones were provided by A. Lee (University of Southern California School of Medicine, Los Angeles). Clones p(C2AT)19 and pML(C2AT)19 were provided by R. Roeder (Rockefeller University, New York, N.Y.). Cell culture. GH3 cells (American Type Culture Collection) were cultured in suspension as previously described (41). At the time of an experiment, cells were centrifuged out of growth medium and resuspended in SFM (41). The exact treatment for each experiment is described in the legend to the corresponding figure. Ionomycin (Calbiochem-Behring) was diluted in dimethyl sulfoxide and used at a final concentration of 600 nM. Cycloheximide (Sigma Chemical Co.) was
Corresponding author. 442
VOL. 10, 1990
CALCIUM REGULATION OF PROLACTIN GENE EXPRESSION
dissolved in phosphate-buffered saline and used at a final concentration of 2.5 p.M. Dactinomycin (Calbiochem-Behring) was dissolved fresh in phosphate-buffered saline and used at a final concentration of 2 pug/ml. Nuclear run-on assays. Nuclear run-on assays were performed as previously described (14, 15), with the following modifications. GH3 cells (5 x 107 to 20 x 107) were pelleted at 400 x g at 40C for 5 min. The cell pellet was washed in 5 ml of phosphate-buffered saline and centrifuged as described above. The cells were suspended in 4.5 ml of lysis buffer (14, 15) without Nonidet P-40, 500 p.l of 5% Nonidet P-40 was added, and the cells were incubated on ice for 3.5 min. Nuclei were pelleted by centrifugation at 1,000 x g at 40C for 5 min. The supernatant was saved and used for the analysis of cytoplasmic mRNA. The cDNAs were linearized, denatured, and immobilized on either nitrocellulose (Duralose-UV; Stratagene) or nylon (Duralon-UV; Stratagene) filters. The filters were prehybridized in 50% formamide-5 x SSPE (23)-S5 x Denhardt solution (23)-0.1% sodium dodecyl sulfate (SDS)-100 pug of denatured, sheared, sonicated salmon sperm DNA per ml at 520C for up to 24 h. Hybridizations were performed at 520C for 48 to 72 h in 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 7.4)-0.2% SDS-10 mM EDTA600 mM NaCl as described previously (14). Negative controls (i.e., hybridization of [32P]RNA to pBR322 DNA) were either nondetectable or at least twofold below the weakest signal from an experimental sample. Isolation and measurement of cytoplasmic mRNA. Cytoplasmic RNA was prepared for cytoplasmic dot hybridization as described previously (38). For analysis of mRNAs by either RNA dot or Northern (RNA) blot hybridization, cytoplasmic RNA was prepared by the method of Greenberg and Ziff (14) except that the spin step with vanadyl-ribonucleoside complexes (VRC) was excluded. Samples were blotted or dotted onto Duralose-UV (Stratagene) or DuralonUV (Stratagene). Membranes were prehybridized as described above for 6 to 16 h at 42°C. DNAs were labeled with [32P]dCTP by nick translation (Amersham Corp.) and purified by Sephadex G-50 chromatography. The radiolabeled DNA (2 x 107 cpm) was boiled for 5 min, cooled on ice, and added directly to the prehybridization solution. Samples were hybridized for 2 days, washed once in 0.1 X SSC (SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS at room temperature for 10 min and then twice in the same solution at 50°C for 30 min, and exposed to Kodak XAR5 film. Nuclear extract preparation and in vitro transcription. We obtained two unique clones, p(C2AT) and pML(C2AT), that allow rapid and direct detection of promoter-dependent in vitro transcription products by RNA polymerase 11 (32). Plasmid pPRL1957(C2AT) was constructed by inserting the prolactin gene promoter sequence from -1957 to -14 in front of the guanosine-free cassette ofp(C2AT). Using this in vitro transcription system and GH3 cell nuclear extracts, we have observed that (i) little or no transcription is detected from the promoterless plasmid, pC2AT; (ii) transcription is not detected from a construct containing the prolactin gene promoter in the reverse orientation; and (iii) transcription from pPRL1957(C2AT) is inhibited by a-amanitin (G. M. Preston and B. A. White, submitted for publication). Nuclear extracts were prepared as described by Dignam et al. (11) except that 0.5 mM spermidine was added to all buffers and 0.2 mM phenylmethylsulfonyl fluoride was added to buffers A and D. In vitro transcription reactions (20 p.l) contained 300 ng of DNA template and 3 mg of nuclear
443
extract per ml in a buffer containing 20 mM N-2-hydroxy-
ethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.9),
8 mM MgCl2, 60 mM KCl, 0.5 mM dithiothreitol, 0.1 mM EDTA, 0.25 mM spermidine, 12% glycerol, 2% polyethylene glycol 8000, 0.6 mM ATP, 0.6 mM CTP, 0.1 mM 3'O-methyl-GTP (Pharmacia, Inc.), 30 p.M UTP, 20 p.Ci of [k-32P]UTP (650 Ci/mmol; ICN Pharmaceuticals Inc.), 25 U of human placental RNase inhibitor (Amersham), and 20 U of RNase T1 (Boehringer Mannheim Biochemicals). After 60 min at 30'C, the reactions were stopped by adding 60 p.l of 14 mM Tris (pH 7.5)-7 mM EDTA-1% SDS and digested with 20 pug of proteinase K at 50'C for 60 min. The samples were then extracted twice with 80 p.l of phenol-chloroform (1:1, vol/vol), and the RNA was precipitated by adding 9 p.l of 2.5 M sodium acetate (pH 5.5) and 220 ,ul of 95% ethanol. The RNA pellets were dissolved in S to 10 ,ul of gel loading buffer (98% formamide, 1% xylene cyanol, 1% bromophenyl blue), resolved on a 4.5% acrylamide gel containing 7 M urea in 1 x TBE (90 mM Tris, 90 mM boric acid, 0.2 mM EDTA [pH 8.0]), and autoradiographed at -70°C with an intensifying screen. Quantification of autoradiograms. Autoradiograms were quantified by scanning densitometry using a Gilford Response spectrophotometer and by scanning filters with a Betascope 603 blot analyzer (Betagen). Several film exposures were obtained to ensure that intensities of the images were within the linear range of the film. RESULTS Transcriptional and posttranscriptional regulation by Ca2+. We have previously demonstrated that prolactin mRNA levels can be reversibly diminished by culturing GH3 cells in SFM in the absence of added CaCl2. Under these conditions, the cells become mitotically quiescent and remain viable for about 1 week. Addition of 0.4 mM CaCl2 to GH3 cells in SFM results in a specific increase in prolactin mRNA levels (39; see Fig. 3). The results from many time course experiments (see, for example, Fig. 3) have shown that prolactin mRNA levels increase slowly over a period of several hours. Prolactin mRNA levels remain stably elevated as long as CaCl2 is present and cells remain viable. To examine the potential contributions of transcriptional and posttranscriptional control in the Ca2'-induced stimulation of prolactin gene expression, we compared the effects of Ca2+ on the transcriptional rate of the endogenous prolactin gene (as measured by a nuclear run-on transcription assay) with effects on prolactin mRNA levels (as measured by Northern blot hybridization). Data from one experiment that is representative of five replicate experiments are shown in Fig. 1. Ca2+ increased prolactin gene transcription less than twofold. By contrast, the steady-state levels of prolactin RNA were increased by 6- to 10-fold. Essentially the same fold increase was obtained with varying amounts of RNA input (Fig. 1, top two rows). Decreasing the amount of filter-immobilized prolactin cDNA plasmid from 20 to 10 pug had no effect on the signal obtained (data not shown). Therefore, the inability to detect a large increase in prolactin gene transcription was not due to saturating amounts of prolactin RNA transcripts. These data demonstrate that Ca2+ induces a small increase in the transcriptional rate of the endogenous prolactin gene and that Ca2+ primarily acts at a posttranscriptional level to increase prolactin mRNA levels. In a separate experiment, we included a positive control to test the fidelity of the nuclear run-on transcription assay.
444
PRESTON ET AL.
A
SFM
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Transcription
PRL (3x 106cPm)[ PRL (6x IO6cpm)
[
pBR322 (6x 06cPm)[
B Cytoplasmic mRNA
PRL[
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MOL. CELL. BIOL.
Ca
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FIG. 1. Effects of 0.4 mM CaC12 on pirolactin (PRL) gene transcription and prolactin mRNA levels. Duplicate cultures of GH3 for 48 cells were treated in SFM or SFM plus 0.~4 mM CaCl2 h. Prolactin gene transcription was mea by nuclear assay (A), and prolactin mRNA was me hybridization (B) as described in Materia ls and Methods. Panel A shows autoradiograms after hybridization of filter-immobilized prolactin cDNA to 3 x 106 and to 6 x 106 c,pm of nuclear RNA or of filter-immobilized pBR322 DNA to 6 x 1(o6 cpm of nuclear RNA.
(Ca2+)
asured
rbn-on
The Ca2" ionophore ionomycin increases GRP78 mRNA levels in several cell types through trainscriptional activation of the GRP78 gene (31). Ca2" inc reased prolactin gene transcription 1.4-fold but increased F)rolactin mRNA levels about 5-fold (Fig. 2). In contrast, ion omycin increased both GRP78 gene transcription and GRP78 mRNA levels by about fivefold. Neither treatment significaLntly altered histone 3 gene transcription or histone 3 mRNjA levels. These results show that the conditions used for thie run-on transcription assay can detect a large increase in transcription and further support the existence of a significant and specific posttranscriptional effect of Ca2+ on prolactiin gene expression.
uz
a 4) L u
LL.
Run-On
mRNA
Prolactin
Run-On
Some investigators have reported rapid and transient hormonally induced increases in prolactin gene transcription (20, 24). Although the slow and sustained Ca2--induced increase in prolactin mRNA levels cannot be due solely to a rapid and transient transcriptional response, such a response could lead to the initial increase in prolactin mRNA levels. Therefore, we examined the effects of Ca2+ on prolactin gene transcription at several earlier time points. As expected (38), prolactin mRNA levels increased progressively during the 24-h period following CaCl2 addition (Fig. 3). In contrast, no significant change in prolactin gene transcription was detected for the first 12 h after CaCl2 addition (Fig. 3). At 24 h, prolactin gene transcription had increased by 1.4-fold over values for SFM controls, whereas prolactin mRNA levels were elevated 5.2-fold over values for SFM controls. Ca2" does not increase the activity of nuclear tissue-specific transcription factors. Tissue-specific transcription of the rat
prolactin gene has been obtained in vitro (6, 16, 21, 26), and
several cell- and tissue-specific nuclear proteins have been purified and cloned from GH cells (4, 7, 8, 17, 22). One possible mechanism by which prolactin gene transcription might be reduced in SFM and increased in SFM plus CaCl2 is by the alteration of the activity of nuclear cell-specific factors. This is a particularly attractive hypothesis because Ca2' regulates basal prolactin gene transcription in the absence of serum and hormones. This hypothesis was tested by using an in vitro transcription assay with nuclear extracts isolated from cells cultured in SFM and SFM plus 0.4 mM CaCl2 for 24 h. In this experiment, analysis of the cytoplasmic fractions by cytoplasmic dot hybridization indicated a 4.8-fold increase in prolactin mRNA transcripts in Ca 2induced cells relative to levels in SFM controls (data not shown). Nuclear extracts from SFM control and Ca2 induced cells transcribed approximately equally well from both the prolactin gene promoter and the adenovirus major late promoter (Fig. 4). This finding was not altered by
mRNA
GRP78
_______I___I___ __ I_ Run-On
mRNA
Histone 3
FIG. 2. Effects of CaCl2 and ionomycin on the transcriptional rates and steady-state mRNA levels of the prolactin gene, the GRP78 gene, and the histone 3 gene. GH3 cells were maintained for 24 h in SFM (O), in SFM plus 0.4 mM CaCl2 (U), or in SFM plus 0.4 mM CaCl2, with 600 nM ionomycin present during the last 4 h of culture (5). Transcription was measured by a nuclear run-on assay (using 6 x 106 cpm of nuclear RNA), and mRNA levels were measured by cytoplasmic dot hybridization as described in Materials and Methods. The means and ranges of values from duplicate cultures are shown. Values are presented as the fold increase over values for the SFM control.
CALCIUM REGULATION OF PROLACTIN GENE EXPRESSION
VOL. 10, 1990
Promoter: Extract:
51
445
AdML
PRL
.m -
I -
+
I
+
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4 z 0 -J
3
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FIG. 3. Time course of Ca2"-induced increase in prolactin mRNA levels and prolactin gene transcription. GH3 cells were cultured overnight in SFM, followed by addition of 0.4 mM CaCl2. Prolactin mRNA levels (0) and prolactin gene transcription (0) were measured at the indicated times after CaCl2 addition. Values represent the means and ranges of determinations from duplicate cultures. Values are presented as the fold increase over values for the time zero SFM control.
addition of CaCl2, ethylene-glycol-bis(P-aminoethyl ether)N,N,N',N-tetraacetic acid (EGTA), or calmodulin directly to the extracts or by varying the amount of nuclear extract or DNA template in the in vitro transcription reactions (data not shown). Also, addition of phosphatase inhibitors (e.g., sodium fluoride and molybdate) during preparation of nuclear extracts from Ca2+-induced cells did not enhance the transcriptional activity of these extracts (data not shown). Thus, the small Ca2+-induced increase in prolactin gene transcription is not accompanied by a similar increase in the activity of cell- or tissue-specific transcription factors. Ca2" increases prolactin mRNA in the absence of ongoing protein synthesis. The relatively slow Ca2+-induced increase in prolactin mRNA levels raises the possibility that this increase is a secondary response to a change in some other gene product. Therefore, we tested for the requirement of ongoing protein synthesis by examining the effects of 2.5 p.M cycloheximide on the ability of Ca2? both to increase and to maintain high levels of prolactin mRNA. This amount of cycloheximide inhibited protein synthesis in GH3 cells cultured in SFM by about 90% (data not shown). Cycloheximide had no effect on the ability of Ca2+ to increase prolactin mRNA levels during an 8-h incubation (Fig. 5A). In a separate experiment, GH3 cells were first cultured overnight in SFM plus 0.4 mM CaCl2 and then treated for 8 h with cycloheximide. Cycloheximide had no effect on the ability of Ca2+ to maintain high levels of prolactin mRNA (Fig. SB). Also, cycloheximide had little or no effect on prolactin mRNA levels in GH3 cells cultured in SFM without CaCl2 (data not shown). As a positive control for cycloheximide, histone 3 mRNA levels were also measured, since histone mRNA levels are known to increase in the presence of protein synthesis inhibitors (28). In all samples, cycloheximide treatment significantly increased histone 3 mRNA levels (Fig. 5). These data indicate that Ca2+ does not
FIG. 4. Effects of Ca2+
on
the activity of GH3 cell nuclear
extracts in an in vitro transcription assay. GH3 cells were cultured in
SFM (-) or SFM plus 0.4 mM CaCl2 (+) for 48 h and then processed for nuclear extract isolation and cytoplasmic dot hybridization as described in Materials and Methods. Equivalent amounts of extract were assayed in duplicate in an in vitro transcription assay as described in Materials and Methods. Symbols: -_, prolactin (PRL) gene promoter-C2AT transcript; cam adenovirus major late (AdML) promoter-C2AT transcript.
require ongoing protein synthesis in order to increase prolactin mRNA levels. Ca2" has no effect on prolactin mRNA clearance in the presence of dactinomycin. One way in which mRNAs are regulated posttranscriptionally is through preferential mRNA stabilization. Several studies have successfully utilized a dactinomycin RNA clearance approach to detect
A
13
w
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1990, p. 442-448
0270-7306/90/020442-07$02.00/0 Copyright © 1990, American Society for Microbiology
Transcriptional and Posttranscriptional Regulation of the Rat Prolactin Gene by Calcium GREGORY M. PRESTON, WILLIAM M. BILLIS, AND BRUCE A. WHITE* Graduate Program in Developmental Biology, Department of Anatomy, University of Connecticut Health Center, Farmington, Connecticut 06032 Received 25 August 1989/Accepted 25 October 1989
The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2", serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2" addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2+-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2' to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2' has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca2+ appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.
expression through posttranscriptional mechanisms has not been examined. Interestingly, thyrotropin-releasing hormone increased prolactin mRNA by both transcriptional stimulation and specific stabilization of prolactin mRNA (19). In light of this finding, and of an increasing number of reports of posttranscriptional control of gene expression (5, 9, 25, 27, 30, 33, 36), we compared the effects of Ca2+ on the transcriptional rate of the endogenous prolactin gene with its effects on steady-state prolactin mRNA levels. We report here that a modest (less than twofold) increase in prolactin gene transcription was accompanied by a significant (greater than fivefold) increase in prolactin mRNA. These data indicate that Ca2+ stimulates prolactin gene expression primarily at a posttranscriptional level. Furthermore, Ca2+ appears to act on prolactin mRNA through a mechanism that is independent of ongoing protein synthesis.
The prolactin gene is expressed at a high basal level in the adult rat pituitary lactotroph and in pituitary tumor GH3 cells (3). This fact suggests the existence of a lactotroph-specific mechanism that promotes prolactin expression in the absence of external hormonal factors. We and others have demonstrated that the basal-level enhancement of the rat prolactin gene is maintained by a Ca2"-dependent mechanism (13, 35, 37, 38, 42). For example, when GH3 cells are cultured in a low-Ca2 serum-free medium (SFM), prolactin mRNA declines to a barely detectable level. Subsequent addition of CaCl2 results in a large and sustained increase in prolactin mRNA levels (39, 42). This Ca2+-dependent enhancement of prolactin gene expression is highly specific, since Ca2' has little or no effect on the mRNA levels for growth hormone, metallothionein, histone 3, or glucoseregulated protein 78 (GRP78) (13, 42, 43). In addition, manipulation of the culture medium Ca2+ concentration had no discernible effect on the synthesis of numerous proteins other than prolactin, as detected by two-dimensional gel electrophoresis (35). At least part of the Ca2'-dependent enhancement of prolactin gene expression in GH3 cells appears to be due to transcriptional stimulation (18). In a study using transfected hybrid constructs containing the prolactin promoter linked to the chloramphenicol acetyltransferase (CAT) gene, Jackson and Bancroft (18) obtained a four- to eightfold stimulation of CAT activity by addition of 0.5 mM CaCl2 to GH3 cells cultured in SFM. Analysis of 5'-deleted prolactin-CAT constructs demonstrated that a Ca2+-responsive element resides within the first 174 base pairs of upstream flanking DNA sequence (18). The possibility that Ca2+ may also increase prolactin gene ,
*
MATERIALS AND METHODS
DNA clones. The prolactin cDNA clone (pPRL-1) was provided by R. Maurer (University of Iowa College of Medicine, Iowa City). The hamster GRP78 (p3C5) and histone 3 (pAAD3.7) cDNA clones were provided by A. Lee (University of Southern California School of Medicine, Los Angeles). Clones p(C2AT)19 and pML(C2AT)19 were provided by R. Roeder (Rockefeller University, New York, N.Y.). Cell culture. GH3 cells (American Type Culture Collection) were cultured in suspension as previously described (41). At the time of an experiment, cells were centrifuged out of growth medium and resuspended in SFM (41). The exact treatment for each experiment is described in the legend to the corresponding figure. Ionomycin (Calbiochem-Behring) was diluted in dimethyl sulfoxide and used at a final concentration of 600 nM. Cycloheximide (Sigma Chemical Co.) was
Corresponding author. 442
VOL. 10, 1990
CALCIUM REGULATION OF PROLACTIN GENE EXPRESSION
dissolved in phosphate-buffered saline and used at a final concentration of 2.5 p.M. Dactinomycin (Calbiochem-Behring) was dissolved fresh in phosphate-buffered saline and used at a final concentration of 2 pug/ml. Nuclear run-on assays. Nuclear run-on assays were performed as previously described (14, 15), with the following modifications. GH3 cells (5 x 107 to 20 x 107) were pelleted at 400 x g at 40C for 5 min. The cell pellet was washed in 5 ml of phosphate-buffered saline and centrifuged as described above. The cells were suspended in 4.5 ml of lysis buffer (14, 15) without Nonidet P-40, 500 p.l of 5% Nonidet P-40 was added, and the cells were incubated on ice for 3.5 min. Nuclei were pelleted by centrifugation at 1,000 x g at 40C for 5 min. The supernatant was saved and used for the analysis of cytoplasmic mRNA. The cDNAs were linearized, denatured, and immobilized on either nitrocellulose (Duralose-UV; Stratagene) or nylon (Duralon-UV; Stratagene) filters. The filters were prehybridized in 50% formamide-5 x SSPE (23)-S5 x Denhardt solution (23)-0.1% sodium dodecyl sulfate (SDS)-100 pug of denatured, sheared, sonicated salmon sperm DNA per ml at 520C for up to 24 h. Hybridizations were performed at 520C for 48 to 72 h in 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 7.4)-0.2% SDS-10 mM EDTA600 mM NaCl as described previously (14). Negative controls (i.e., hybridization of [32P]RNA to pBR322 DNA) were either nondetectable or at least twofold below the weakest signal from an experimental sample. Isolation and measurement of cytoplasmic mRNA. Cytoplasmic RNA was prepared for cytoplasmic dot hybridization as described previously (38). For analysis of mRNAs by either RNA dot or Northern (RNA) blot hybridization, cytoplasmic RNA was prepared by the method of Greenberg and Ziff (14) except that the spin step with vanadyl-ribonucleoside complexes (VRC) was excluded. Samples were blotted or dotted onto Duralose-UV (Stratagene) or DuralonUV (Stratagene). Membranes were prehybridized as described above for 6 to 16 h at 42°C. DNAs were labeled with [32P]dCTP by nick translation (Amersham Corp.) and purified by Sephadex G-50 chromatography. The radiolabeled DNA (2 x 107 cpm) was boiled for 5 min, cooled on ice, and added directly to the prehybridization solution. Samples were hybridized for 2 days, washed once in 0.1 X SSC (SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS at room temperature for 10 min and then twice in the same solution at 50°C for 30 min, and exposed to Kodak XAR5 film. Nuclear extract preparation and in vitro transcription. We obtained two unique clones, p(C2AT) and pML(C2AT), that allow rapid and direct detection of promoter-dependent in vitro transcription products by RNA polymerase 11 (32). Plasmid pPRL1957(C2AT) was constructed by inserting the prolactin gene promoter sequence from -1957 to -14 in front of the guanosine-free cassette ofp(C2AT). Using this in vitro transcription system and GH3 cell nuclear extracts, we have observed that (i) little or no transcription is detected from the promoterless plasmid, pC2AT; (ii) transcription is not detected from a construct containing the prolactin gene promoter in the reverse orientation; and (iii) transcription from pPRL1957(C2AT) is inhibited by a-amanitin (G. M. Preston and B. A. White, submitted for publication). Nuclear extracts were prepared as described by Dignam et al. (11) except that 0.5 mM spermidine was added to all buffers and 0.2 mM phenylmethylsulfonyl fluoride was added to buffers A and D. In vitro transcription reactions (20 p.l) contained 300 ng of DNA template and 3 mg of nuclear
443
extract per ml in a buffer containing 20 mM N-2-hydroxy-
ethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.9),
8 mM MgCl2, 60 mM KCl, 0.5 mM dithiothreitol, 0.1 mM EDTA, 0.25 mM spermidine, 12% glycerol, 2% polyethylene glycol 8000, 0.6 mM ATP, 0.6 mM CTP, 0.1 mM 3'O-methyl-GTP (Pharmacia, Inc.), 30 p.M UTP, 20 p.Ci of [k-32P]UTP (650 Ci/mmol; ICN Pharmaceuticals Inc.), 25 U of human placental RNase inhibitor (Amersham), and 20 U of RNase T1 (Boehringer Mannheim Biochemicals). After 60 min at 30'C, the reactions were stopped by adding 60 p.l of 14 mM Tris (pH 7.5)-7 mM EDTA-1% SDS and digested with 20 pug of proteinase K at 50'C for 60 min. The samples were then extracted twice with 80 p.l of phenol-chloroform (1:1, vol/vol), and the RNA was precipitated by adding 9 p.l of 2.5 M sodium acetate (pH 5.5) and 220 ,ul of 95% ethanol. The RNA pellets were dissolved in S to 10 ,ul of gel loading buffer (98% formamide, 1% xylene cyanol, 1% bromophenyl blue), resolved on a 4.5% acrylamide gel containing 7 M urea in 1 x TBE (90 mM Tris, 90 mM boric acid, 0.2 mM EDTA [pH 8.0]), and autoradiographed at -70°C with an intensifying screen. Quantification of autoradiograms. Autoradiograms were quantified by scanning densitometry using a Gilford Response spectrophotometer and by scanning filters with a Betascope 603 blot analyzer (Betagen). Several film exposures were obtained to ensure that intensities of the images were within the linear range of the film. RESULTS Transcriptional and posttranscriptional regulation by Ca2+. We have previously demonstrated that prolactin mRNA levels can be reversibly diminished by culturing GH3 cells in SFM in the absence of added CaCl2. Under these conditions, the cells become mitotically quiescent and remain viable for about 1 week. Addition of 0.4 mM CaCl2 to GH3 cells in SFM results in a specific increase in prolactin mRNA levels (39; see Fig. 3). The results from many time course experiments (see, for example, Fig. 3) have shown that prolactin mRNA levels increase slowly over a period of several hours. Prolactin mRNA levels remain stably elevated as long as CaCl2 is present and cells remain viable. To examine the potential contributions of transcriptional and posttranscriptional control in the Ca2'-induced stimulation of prolactin gene expression, we compared the effects of Ca2+ on the transcriptional rate of the endogenous prolactin gene (as measured by a nuclear run-on transcription assay) with effects on prolactin mRNA levels (as measured by Northern blot hybridization). Data from one experiment that is representative of five replicate experiments are shown in Fig. 1. Ca2+ increased prolactin gene transcription less than twofold. By contrast, the steady-state levels of prolactin RNA were increased by 6- to 10-fold. Essentially the same fold increase was obtained with varying amounts of RNA input (Fig. 1, top two rows). Decreasing the amount of filter-immobilized prolactin cDNA plasmid from 20 to 10 pug had no effect on the signal obtained (data not shown). Therefore, the inability to detect a large increase in prolactin gene transcription was not due to saturating amounts of prolactin RNA transcripts. These data demonstrate that Ca2+ induces a small increase in the transcriptional rate of the endogenous prolactin gene and that Ca2+ primarily acts at a posttranscriptional level to increase prolactin mRNA levels. In a separate experiment, we included a positive control to test the fidelity of the nuclear run-on transcription assay.
444
PRESTON ET AL.
A
SFM
I-1-I
Transcription
PRL (3x 106cPm)[ PRL (6x IO6cpm)
[
pBR322 (6x 06cPm)[
B Cytoplasmic mRNA
PRL[
~I
MOL. CELL. BIOL.
Ca
r
2.
24
Ca41;
M '1.6
*- ;-@
2.0
8.9
FIG. 1. Effects of 0.4 mM CaC12 on pirolactin (PRL) gene transcription and prolactin mRNA levels. Duplicate cultures of GH3 for 48 cells were treated in SFM or SFM plus 0.~4 mM CaCl2 h. Prolactin gene transcription was mea by nuclear assay (A), and prolactin mRNA was me hybridization (B) as described in Materia ls and Methods. Panel A shows autoradiograms after hybridization of filter-immobilized prolactin cDNA to 3 x 106 and to 6 x 106 c,pm of nuclear RNA or of filter-immobilized pBR322 DNA to 6 x 1(o6 cpm of nuclear RNA.
(Ca2+)
asured
rbn-on
The Ca2" ionophore ionomycin increases GRP78 mRNA levels in several cell types through trainscriptional activation of the GRP78 gene (31). Ca2" inc reased prolactin gene transcription 1.4-fold but increased F)rolactin mRNA levels about 5-fold (Fig. 2). In contrast, ion omycin increased both GRP78 gene transcription and GRP78 mRNA levels by about fivefold. Neither treatment significaLntly altered histone 3 gene transcription or histone 3 mRNjA levels. These results show that the conditions used for thie run-on transcription assay can detect a large increase in transcription and further support the existence of a significant and specific posttranscriptional effect of Ca2+ on prolactiin gene expression.
uz
a 4) L u
LL.
Run-On
mRNA
Prolactin
Run-On
Some investigators have reported rapid and transient hormonally induced increases in prolactin gene transcription (20, 24). Although the slow and sustained Ca2--induced increase in prolactin mRNA levels cannot be due solely to a rapid and transient transcriptional response, such a response could lead to the initial increase in prolactin mRNA levels. Therefore, we examined the effects of Ca2+ on prolactin gene transcription at several earlier time points. As expected (38), prolactin mRNA levels increased progressively during the 24-h period following CaCl2 addition (Fig. 3). In contrast, no significant change in prolactin gene transcription was detected for the first 12 h after CaCl2 addition (Fig. 3). At 24 h, prolactin gene transcription had increased by 1.4-fold over values for SFM controls, whereas prolactin mRNA levels were elevated 5.2-fold over values for SFM controls. Ca2" does not increase the activity of nuclear tissue-specific transcription factors. Tissue-specific transcription of the rat
prolactin gene has been obtained in vitro (6, 16, 21, 26), and
several cell- and tissue-specific nuclear proteins have been purified and cloned from GH cells (4, 7, 8, 17, 22). One possible mechanism by which prolactin gene transcription might be reduced in SFM and increased in SFM plus CaCl2 is by the alteration of the activity of nuclear cell-specific factors. This is a particularly attractive hypothesis because Ca2' regulates basal prolactin gene transcription in the absence of serum and hormones. This hypothesis was tested by using an in vitro transcription assay with nuclear extracts isolated from cells cultured in SFM and SFM plus 0.4 mM CaCl2 for 24 h. In this experiment, analysis of the cytoplasmic fractions by cytoplasmic dot hybridization indicated a 4.8-fold increase in prolactin mRNA transcripts in Ca 2induced cells relative to levels in SFM controls (data not shown). Nuclear extracts from SFM control and Ca2 induced cells transcribed approximately equally well from both the prolactin gene promoter and the adenovirus major late promoter (Fig. 4). This finding was not altered by
mRNA
GRP78
_______I___I___ __ I_ Run-On
mRNA
Histone 3
FIG. 2. Effects of CaCl2 and ionomycin on the transcriptional rates and steady-state mRNA levels of the prolactin gene, the GRP78 gene, and the histone 3 gene. GH3 cells were maintained for 24 h in SFM (O), in SFM plus 0.4 mM CaCl2 (U), or in SFM plus 0.4 mM CaCl2, with 600 nM ionomycin present during the last 4 h of culture (5). Transcription was measured by a nuclear run-on assay (using 6 x 106 cpm of nuclear RNA), and mRNA levels were measured by cytoplasmic dot hybridization as described in Materials and Methods. The means and ranges of values from duplicate cultures are shown. Values are presented as the fold increase over values for the SFM control.
CALCIUM REGULATION OF PROLACTIN GENE EXPRESSION
VOL. 10, 1990
Promoter: Extract:
51
445
AdML
PRL
.m -
I -
+
I
+
W LAI
C,)
4 z 0 -J
3
0
-*
LiL
04
so
s
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e
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6
12 TIME (hr)
24
FIG. 3. Time course of Ca2"-induced increase in prolactin mRNA levels and prolactin gene transcription. GH3 cells were cultured overnight in SFM, followed by addition of 0.4 mM CaCl2. Prolactin mRNA levels (0) and prolactin gene transcription (0) were measured at the indicated times after CaCl2 addition. Values represent the means and ranges of determinations from duplicate cultures. Values are presented as the fold increase over values for the time zero SFM control.
addition of CaCl2, ethylene-glycol-bis(P-aminoethyl ether)N,N,N',N-tetraacetic acid (EGTA), or calmodulin directly to the extracts or by varying the amount of nuclear extract or DNA template in the in vitro transcription reactions (data not shown). Also, addition of phosphatase inhibitors (e.g., sodium fluoride and molybdate) during preparation of nuclear extracts from Ca2+-induced cells did not enhance the transcriptional activity of these extracts (data not shown). Thus, the small Ca2+-induced increase in prolactin gene transcription is not accompanied by a similar increase in the activity of cell- or tissue-specific transcription factors. Ca2" increases prolactin mRNA in the absence of ongoing protein synthesis. The relatively slow Ca2+-induced increase in prolactin mRNA levels raises the possibility that this increase is a secondary response to a change in some other gene product. Therefore, we tested for the requirement of ongoing protein synthesis by examining the effects of 2.5 p.M cycloheximide on the ability of Ca2? both to increase and to maintain high levels of prolactin mRNA. This amount of cycloheximide inhibited protein synthesis in GH3 cells cultured in SFM by about 90% (data not shown). Cycloheximide had no effect on the ability of Ca2+ to increase prolactin mRNA levels during an 8-h incubation (Fig. 5A). In a separate experiment, GH3 cells were first cultured overnight in SFM plus 0.4 mM CaCl2 and then treated for 8 h with cycloheximide. Cycloheximide had no effect on the ability of Ca2+ to maintain high levels of prolactin mRNA (Fig. SB). Also, cycloheximide had little or no effect on prolactin mRNA levels in GH3 cells cultured in SFM without CaCl2 (data not shown). As a positive control for cycloheximide, histone 3 mRNA levels were also measured, since histone mRNA levels are known to increase in the presence of protein synthesis inhibitors (28). In all samples, cycloheximide treatment significantly increased histone 3 mRNA levels (Fig. 5). These data indicate that Ca2+ does not
FIG. 4. Effects of Ca2+
on
the activity of GH3 cell nuclear
extracts in an in vitro transcription assay. GH3 cells were cultured in
SFM (-) or SFM plus 0.4 mM CaCl2 (+) for 48 h and then processed for nuclear extract isolation and cytoplasmic dot hybridization as described in Materials and Methods. Equivalent amounts of extract were assayed in duplicate in an in vitro transcription assay as described in Materials and Methods. Symbols: -_, prolactin (PRL) gene promoter-C2AT transcript; cam adenovirus major late (AdML) promoter-C2AT transcript.
require ongoing protein synthesis in order to increase prolactin mRNA levels. Ca2" has no effect on prolactin mRNA clearance in the presence of dactinomycin. One way in which mRNAs are regulated posttranscriptionally is through preferential mRNA stabilization. Several studies have successfully utilized a dactinomycin RNA clearance approach to detect
A
13
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