Effects of Calcium and Calcium lonophores on Prolactin Gene Expression in GH3 and 235-l Rat Pituitary Tumor Cells

Beverly

C. Delidow,

Melissa

Lail-Trecker,

and Bruce

A. White

Graduate Program in Developmental Biology Department of Anatomy University of Connecticut Health Center Farmington, Connecticut 06030

cultures of pituitary cells (1) and in the GH3 pituitary tumor cell line (2, 3). In GH3 cells cultured in a serumfree medium (SFM) containing low Ca2+ levels (about 10e6 M contaminating Ca2+), expression of both the PRL and GH genes declines to low levels over a l- to 2-day period (2). Addition of 0.5 mM CaCI, to GH3 cells in SFM specifically elevates PRL mRNA levels and PRL synthesis by 5 to 1 O-fold (2,3). The levels of GH mRNA and several other mRNAs [e.g. actin, histone 3, and glucose-regulated protein 78 (GRP78)] are not affected by Ca2+ (4, 5). Thus, a minimum level of extracellular Ca2+ appears to be required for the maintenance of high basal expression of the PRL gene in GH3 cells. The signal transduction pathway involved in this response is unknown, even to the extent that it is not clear whether extracellular Ca2+ acts at the cell surface or influences intracellular Ca*+ levels. Addition of 0.5 mM CaCI, to GH3 cell cultures appears to increase PRL mRNA levels primarily at a posttranscriptional step (4, 5). This conclusion was based on the finding that the apparent transcription rate of the PRL gene, measured by the nuclear run-on transcription assay, was increased by less than P-fold by Ca*‘, whereas the steady state cytoplasmic PRL mRNA levels were increased by 5 to IO-fold. Actinomycin D chase experiments indicated that cytoplasmic mRNA stability was not affected by Ca2+ (4) leading us to suspect that Ca 2+ increases PRL mRNA levels at a nuclear step after transcription. This is consistent with the observation that Ca2+ also increased the steady state level of PRL splicing intermediates in the nucleus

Previous observations that extracellular calcium (Ca”) enhanced PRL mRNA levels posttranscriptionally in GH3 rat pituitary tumor cells were made using double-stranded transcription probes. The effects of Ca*+ and the Ca*+ ionophore, ionomycin, on PRL gene expression in GH, and 235-l cells were investigated using site- and strand-specific probes. Treatment of GHB and 235-l ceils with 0.5 mM Ca*+ in serum-free medium specifically increased PRL mRNA levels by severalfold. In 235-l but not GH3 cells PRL gene transcription was comparably induced by Ca*+. Use of single-stranded 5’ and 3’ probes revealed no antisense transcription, nor any Ca*+ effect on transcriptional elongation. Treatment with Ca*+ plus ionomycin inhibited PRL mRNA levels and gene transcription in both cell lines. Although their PRL gene transcription rates are similar, several basic differences were noted between the cell lines. The 235-l cells exhibit a different profile of nuclear PRL pre-RNAs than GH3 cells. Also, mRNA levels for a Ca*+-regulated gene (GRP78) did not change in Ca*+-treated GHB cells but decreased in Ca*+-treated 235-l cells. lonomycin treatment increased GRP78 mRNA levels in both cell lines. Thus, addition of extracellular Ca*+ appears to affect [Ca*‘]i in 235-l but not GHI cells, while ionomycin affects [Ca’+]i in both cell lines. These data suggest that changing [Ca”J modulates PRL gene transcription. The comparative data suggest that posttranscriptional PRL regulation is Ca*+-regulated in GH3 cells, but is constitutive in 235-l cells. (Molecular Endocrinology 6: 1268-1276, 1992)

(6). In contrast to these reports, several studies have shown an induction of the expression of PRL-promoterreporter constructs in response to added Ca2+ (7, 8). Several of these studies used treatments which directly affect intracellular Ca *+ levels. Ca’+ channel agonists activate the expression of PRL promoter constructs in GH3 cells, while nimodipine, a Ca*+ channel blocker, inhibits both agonist-induced Ca*’ fluxes and reporter expression (7-9). Deletion studies indicate that both the distal and proximal regulatory regions of the PRL

INTRODUCTION

The expression of the rat PRL gene is specifically regulated by extracellular calcium (Ca”) in both primary 0666-6609/92/l 268-l 276$03.00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine

Society

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Transcriptional

and Posttranscriptional

Regulation

of PRL Gene

promoter contribute to Ca2+ regulation of PRL promoter constructs (7, 8). There is evidence that the effect of Ca2+ on the PRL promoter is mediated by one or more binding sites for the pituitary-specific transcription factor, Pit-l (10). Ca2+-responsive elements (CaREs) have also been discovered in other genes, including c-fos (11) and GRP78 (12). These CaREs were initially identified by Ca*’ ionophore treatment, which increases intracellularCa*+. The CaRE of the c-fos promoter has been shown to colocalize with the CAMP-responsive element, TGACGTTT (11). The regulatory region of GRP78 promoter conferring Ca*+ responsivenessalso contains this sequence(positions-153 to -146, relative to the cap site; 12, 13). Ca*+ionophoreshave alsobeen usedto examinethe mechanismof Ca*+regulationof PRL gene expression. Surprisingly, low doses (lo-’ M) of Ca*+ ionophores inhibit the induction of PRL mRNA by Ca*+in GH3cells (3) suggestingthat maintenanceof normalintracellular Ca*+ levels and/or gradients is required for PRL gene expression.Several inhibitory regulators of PRL act at a transcriptional level, including dopamine(14) dexamethasone(15, 16), and transforming growth factor-p (5). It is not known whether Ca’+ ionophoressimilarly depressPRL gene transcription. The presentstudy was undertakento morerigorously examine the effects of Ca2+on PRL gene transcription in GH3cells.Our previous resultswere obtained by runon transcription assays using a full-length PRL cDNA in the BluescriptSK vector as a probe. However, vector sequencesmay interfere with specificdetection of transcription (Murray, M. B., and H. C. Towle, personal communicationwithin Ref. 17). Additionally, apparent antisensetranscription has been detected across several genes (18-21) and our double-stranded probes could not distinguish between sense and antisense transcripts. Finally, hybridization to full-length cDNA probes alsocannot distinguishchangesin transcription initiationfrom changesin transcriptionalelongation.The importance of this distinction is underscored by the recent report that Ca*+ionophoresinduce murinec-fos gene expressionby removinga block to transcriptional elongation (22). Thus, in the present study, we have reexamined the effects of Ca*+ on endogenous PRL gene transcription using single-stranded(sense and antisense)probes as well as 5’-specific and 3’-specific probes. Additionally, we includeda second rat pituitary tumor cell line, the 235-l cells, which originated independently of GH cell lines(23) and in which the effects of Ca*+on PRL gene expressionhave not been studied. The 235-l cells are not well characterized, but differ from GHs cells in that they produce no GH (23, 24). Finally, we examinedwhether Ca*+ionophoresact at a transcriptionalor posttranscriptionallevel to inhibit PRL gene expression. RESULTS Effects of Ca*+ and Ca*+ lonophores Expression in GHJ and 235-l Cells

on PRL Gene

In the first seriesof experiments, GH3and 235-l cells were cultured in SFM + 0.5 mM CaC12for about 24 h.

Expression

1269

The cytoplasmicRNA was isolatedfrom these cellsand analyzed by Northern blot hybridization for PRL mRNA. As shown in Fig. 1 (left lanes), additionof 0.5 mMCaC12 to GH3 cellscultured in SFM induced a g-fold increase in cytoplasmic PRL mRNA levels (Fig. 1). The 235-l cells have considerably higher (13-fold) basal levels of PRL mRNA than GH3 cells. Ca*+ treatment of 235-l cellsincreasedthese levels by 2.5fold (Fig. 1). The presenceand/or length of the poly(A)+ tail has been shownto affect the stability of somemRNAs(2528), and serum induction of the mRNA for proliferin, a protein of the PRL-GH family, also inducesan increase in the extent of its polyadenylation (29). Furthermore, the decidual form of human PRL mRNA differs in size from the pituitary RNA due to the use of an upstream promoter and addition of another 5’noncoding exon (30, 31). Therefore, the size of the poly(A)+ tail of PRL mRNA in both cell lineswas determinedby hybridization of the samecytoplasmic RNAs to oligo-dT, followed by digestionwith RNaseH. The sizes of the deadenylated PRL RNAs were then determined by Northern blot hybridization and comparedto the undigestedsamples. The length of the PRL mRNA poly(A)+ tail in both cell types is approximately 250 base pairs(bp), and it is not affected by Ca*+ treatment (Fig. 1, right lanes). In addition, this analysisshows that there is no detectable difference in the length of the PRL mRNA itself in the two cell types. PRL gene transcription was measuredin the nuclei of the cellsfrom the experiment presentedin Fig. 1. As expected (4, 5) 0.5 mMCaC12had essentiallyno effect on PRL gene transcription in GH3 cells (Fig. 2) even though it increasedPRL mRNA levels by O-fold(Fig. 1). In contrast to GH3 cells, Ca*+ induced a 2- to 3-fold increase in both PRL gene transcription (Fig. 2) and PRL mRNA levels in 235-l cells (Fig. 1). In a separate experiment, Ca*+ again had no effect on PRL gene transcriptionin GH3cellsbut enhancedboth PRL mRNA GH3

1 Kb

I

GH3

235-I

+

ca**

OdT/RNH PRL InRWA lcvclr

23%

- C. * C.

f

-

l

-

-

-

-

1

l

-

-

l

-

l

+

+

+

+

0 75

Kb

12.8 89

32 0

Fig. 1. Effect of CaC12 Treatment on Cytoplasmic PRL mRNA Levels and Polyadenylation in GH3 and 235-l Cells GHB and 235-l cells were cultured in SFM + 0.5 mM CaCI, (Ca*+) for 24 h. Cytoplasmic RNA was isolated, and lo-rg aliquots were analyzed for PRL mRNA by Northern blot hybridization. These samples are presented in the leti lanes. The level of PRL mRNA in each sample is expressed below as the value relative to the GH:, SFM control. A second set of equal aliquots of RNA were deadenylated by incubation with oligo dT and RNase H (OdT/RNH), then precipitated and analyzed on the same Northern blot to compare with the intact RNAs. These samples are in the right lanes. The arrows indicate the positions of the 1 -kb mature PRL mRNA and the approximately 750-bp deadenylated PRL RNA.

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MOL ENDO. 1992 1270

Vol6 No. 8

235- 1

S”3

A

SFH

PRL

ctl’*

PRL

GH3

KS+

PRL

Ca/SFtt 1.2

KS+

ND 2.3

mRNA

PRL-ES 235-l Ca2+ Relative transcription

AT/A

R

+

-

-

+

1.2

2.9

PRL

1

0.8 0.1

1.2

Fig. 2. Effect of Ca*+ Treatment on PRL Gene Transcription in GH3 and 235-l Cells Nuclei were isolated in the 24-h Ca’+ induction experiment presented in Fig. 1 and used to perform run-on transcription in the presence of [32P]UTP. Labeled transcripts were hybridized to immobilized DNA probes containing a PRL cDNA clone (PRL), a PRL 3’-end genomic clone (PRL-E5), or plasmid DNA alone (KS+). Hybridized dots were analyzed using a Betascope blot analyzer. Images were obtained by autoradiography for 2 weeks at -70 C with an enhancing screen. Transcription levels were corrected for background hybridization to KS+ and are expressed relative to the GH3 SFM control. AT/AR represents the ratio of the Ca’+-induced change in PRL gene transcription to that in PRL mRNA levels (see Fig. 1). For GHB cells the ratio is significantly less than 1, suggesting that transcriptional effects cannot account for the increase in PRL mRNA levels.

and (Fig. gene (Fig.

gene transcription by 2- to 3-fold in 235-l cells 3). A comparison of the effects of Ca2+ on PRL transcription to its effects on PRL mRNA levels 2, AT/AR; Fig. 3A, Ca/SFM) shows very clearly

that these effects are on transcription in 235-l cells. However, in GHScells, PRL gene transcription rates in the presenceof Ca2+fail to account for the increasein PRL mRNA levels. In these two experiments,several additionalcontrols were introduced. In both GH3 and 235-l cells, no evidenceof a block to elongationwas observed, either by hybridizationto PRL-E5, a genomicclone containing the 3’-most 1.3 kilobases(kb) of the PRL gene (Fig. 2), or by hybridization to single-stranded5’- and 3’-PRL cDNA fragments(3 + 1 and 5 -+ 3, Fig. 38). Similarly, the use of the reverse single-strandedprobes (1 + 3 and 3 + 5) showed that transcription in the antisense direction does not occur in either cell line (Fig. 3B). WhileextracellularCaC12increasedPRL geneexpression in these experiments, Ca2+ionophoreslower PRL mRNA levels in GHBcells (3). As shown in Fig. 4, treatment of 235-l cells with the Ca2+ionophore ionomycin completely inhibited the 4.5-fold increase in PRL mRNA levels induced by Ca*+. This inhibition is not the result of toxicity, since, in the same cells,

PRL

2.2

KS+

ND 3.2

mRNA

GH3235- 1 - + - +

B cg7-+ transcription sense.

Antisense transcrlption

:I:

nn

nn

Fig. 3. Analysis of PRL Gene Transcription Using Full-Length or 3’ vs. 5’ and Sense vs. Antisense Probes Duplicate groups of GHB and 235-l cells were cultured in SFM + 0.5 mM CaC12 for 24 h. Cytoplasmic RNA was isolated for analysis of PRL mRNA by RNA dot blot hybridization, with results shown in the third row of each panel in A. Nuclei were used for run-on transcription assays. Labeled transcripts were hybridized to the PRL cDNA and plasmid control (A), as well as to PCR-generated single-stranded DNAs extending from PRL exons l-3 and 3-5 (B). Probes to detect sense (3 --, 1 and 5 + 3) and antisense (1 + 3 and 3 + 5) transcription were synthesized using the PRL cDNA as a template (see Materials and Methods) and were immobilized as for doublestranded probes. Hybridized dots were analyzed by autoradiography on x-ray film for 2 weeks at -70 C with an enhancing screen, followed by scanning densitometry of the films. Results from duplicate samples are shown for the full-length PRL probe in A. Representative results from the single-stranded sense and antisense probes are shown in B. The Ca/SFM column presents the PRL transcription or RNA level in Ca*+-treated cells, relative to those levels in SFM controls. ND, Not detectable.

ionomycin induced the levels of GRP78 mRNA by 4fold over the levels in Ca*‘-treated cells. In a separate experiment,ionomycin inhibitedPRL gene transcription by 50-60% in both cell lines (Fig. 5). lonomycin also comparablydecreasedPRL mRNA levelsand increased GRP78mRNA levelsinthese samples(data not shown). Again, the use of 5’ and 3’ single-strandedprobes demonstratedno block to elongationnor any antisense transcription (data not shown). The data presented above demonstrate that Ca*+ induces PRL gene expression in 235-l cells, as had

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Transcriptional and Posttranscriptional

Regulation of PRL Gene Expression

1271

Nuclear PRL Precursor RNAs in GHI and 235-l Cells

PRL

GRP78

1.0

0.6

2.2

Fig. 4. Ca*+ lonophores Inhibit PRL Gene Expression in 2351 Cells Duplicate groups of 235-i cells were cultured for 20 h in SFM alone, SFM plus 0.5 mM Ca*+, or SFM plus 0.5 mM Ca*+ plus 400 nM ionomycin (lono). Cytoplasmic RNA was isolated and 1 0-pg aliquots were analyzed for PRL and GRP78 mRNAs by Northern blot hybridization. Blots were analyzed by Betascope and autoradiography. The levels of PRL and GRP78 mRNA are expressed relative to the SFM controls and represent the averages of duplicate measurements.

In order to further examine basic differences in the processingof PRL gene transcripts in GH3and 235-l cells, we examined the effects of Ca” on the steady state levelsof nuclearPRL precursorRNAs by Northern blot hybridization. In GHBcells (Fig. 6, left side) there are three PRL pre-mRNA bands (or band clusters) of approximately 7, 5, and 3.5 kb. In severalexperiments, of which only one is shown, these bandswere induced in the presenceof Ca*+, as was the level of the 1-kb mature PRL mRNA. This suggests that the Ca*+-responsive posttranscriptional step in the production of PRL RNA occurs very early after transcription. In 235-l cells the PRL RNA precursor pattern consists of two bands only, at 7 and 4.5 kb (approximate; Fig. 6, right side). Unlike the GH3 cells, the levels of these precursors and the I-kb nuclear mRNA responded variably to Ca*+ treatment (in the experiment shown the levels were induced by Ca*+), although cytoplasmic PRL mRNA levels were consistently induced. Despite this variability in the responseto Ca*+, the two-band pattern was invariant. Thesedata provide further evidence for differences between GHBand 2351 cells in their processingof the PRL gene transcript. Effect of Ca*+ on GRP78 Expression

Caz+

Ion0

I/Ca

GH3

In the course of these experiments we also measured levels of GRP78 mRNA, which have been measured G”3

cyto nue,esr 235- I

235-I

nuc,e*r cyto

PRL KS+

Fig. 5. Ca*+ lonophore Inhibits PRL Gene Transcription in GH3 and 235-l Cells In the experiment presented in Fig. 3, additional groups of cells were treated for 20 h in SFM plus Ca*+ in the presence of 400 nM ionomycin (lono). After nuclear run-on transcription, labeled transcripts were hybridized to immobilized PRL cDNA, to single-stranded 3’ and 5’ sense and antisense probes (data not shown), and to a plasmid-only control. Hybridized dots were analyzed by autoradiography for 2 weeks at -70 C, followed by scanning densitometry of the films. l/Ca is the ratio of the PRL gene transcription rate in cells treated with Ca*+ plus lono to that of cells treated with Ca*+ alone. ND, Not detectable.

been previously shown for GH, cells. More importantly, these data indicate a fundamental difference in the mode of responseto Ca*+ between GH3 and 235-l cells. Extracellular CaCI, inducesPRL gene expression primarily at a posttranscriptionallevel in GH3 cells but acts at a transcriptionallevel in 235-l cells. Disruption of normal [Ca*+]i and intracellular [Ca*+] gradients by ionomycin treatment resulted in decreased PRL gene transcription in both cells.

-* Fold induction

31.5

5.6

- + 5.2

6.7

Fig. 6. The Pattern of Nuclear PRL RNA Precursors Differs in GH3 and 235-l Cells GHB and 235-l cells were cultured for 24 h in SFM or SFM plus 0.5 mM Cap+. Cells were collected and lysed in isotonic saline to isolate nuclear pellets. Both cytoplasmic and nuclear RNA were prepared, and lo-pg aliquots were analyzed by Northern blot hybridization, autoradiography, and scanning densitometry. The 18s and 28s ribosomal RNA bands were visualized by ethidium bromide staining of the gels, and their positions are given in each panel. The large arrows show the position of the 1-kb mature PRL mRNA. The open arrows indicate the positions and estimated sizes of the PRL RNA precursor bands. There are three such bands in GH3 cell RNAs (/eff panels) at 7.5,5, and 3.5 kb. There are two PRL precursor bands in 235-l RNA (right panels) at 7 and 4.5 kb. The smaller panels show lighter exposures of the 1-kb PRL mRNA bands, with their relative levels presented below.

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MOL 1272

ENDO.

Vol6

1992

previously as a control in GH3 cells. As expected (3, 5), addition of 0.5 mM CaCl* had little or no effect on the levels of GRP78 mRNA in GH3 cells (Fig. 7). This is consistent with the findings that, while the expression of the GRP78 gene is regulated by changes in intracellular Ca” concentrations ([Ca”],; 12, 32, 33), the [Ca*+], of GH3 cells is not appreciably altered by addition of 0.5 mM CaCI, (9; our unpublished data). In 235-l cells, on the other hand, treatment with Ca*+ induced a consistent 50% inhibition of GRP78 mRNA levels (Figs. 4 and 7). This unexpected effect has been observed in eight independent experiments. This suggests that addition of extracellular Ca*+ to 235-l cells causes an alteration of [Ca*+], that does not occur in GH3 cells, and may in part explain the transcriptional response of the PRL gene in 235-l cells to Ca*+.

DISCUSSION Transcriptional

Regulation of PRL Gene Expression

Earlier studies on 235-l cells indicate that Ca*+-active agents such as monensin and EGTA inhibit PRL secretion (23) and that maitotoxin, a Ca2+ channel activator, stimulates PRL secretion (34). The present study demonstrates that treatment of 235-l cells with 0.5 mM CaC12 induces PRL gene expression at a transcriptional level. Ca*+ did not affect the elongation of PRL transcripts in 235-l cells, suggesting that the effect of Ca*+ is on transcriptional initiation. Little is known about the signal transduction pathway by which addition of 0.5 mM CaCI, to 235-l cultures induces PRL gene transcription. Ca2+ treatment of 2351 cells reduced the levels of GRP78 mRNA, suggesting that addition of Ca*+ to the medium of 235-l cell cultures results in a significant repletion of [Ca’+]i (12,

SFM

Ca

32,33). Although further studies are needed to measure [Ca”]i in 235-l cells, we propose that addition of extracellular CaCI, to 235-l cells in SFM results in an increase in [Ca*‘]i, which, in turn, stimulates PRL gene transcription. This is consistent with studies showing that the expression of PRL-promoter-reporter constructs is stimulated in GH3 cells treated with Ca*+ channel agonists (8), which activate membrane Ca*+ channels and increase [Ca*+]i (9). TRH, a physiological regulator of PRL, has been shown to both require extracellular Ca*+ for its actions (6) and to induce an increase in [Ca”]i in GH3 and GH.& cells (35, 36). In contrast, treatment of cells with Ca*+ ionophores in the presence of extracellular Ca*+ induces a sustained supraphysiological rise in total cytoplasmic Ca*+ (3) and inhibits PRL gene transcription in both 235-l and GH3 cells. While indicating that optimal [Ca”], and/ or [Ca*+] gradients are required for maximal PRL gene transcription, these results are also consistent with the hypothesis that changes in [Ca’+]i produce changes in PRL gene transcription. An effect of Ca*+ and Ca*+ ionophores on transcriptional initiation would be expected to be mediated by sites within the PRL promoter region. Sequences capable of conferring Ca*+ inducibility on PRL promoterreporter constructs have been identified in both the distal enhancer (-1800 to -1500) and proximal enhancer/promoter (-200 to -1) regions (7, 8, 37). It has also been shown that the most proximal binding site for the pituitary-specific transcription factor, Pit-l, can confer Ca*+ inducibility on a reporter (10). It is interesting to note that in the region of the PRL promoter between the first (Pl) and second (P2) proximal Pit-l sites, the sequence TGACGAAA has been identified as a potential CAMP and phorbol ester response element (38). This sequence is similar to the region of the c-fos promoter believed to confer Ca*+-responsiveness (TGACGTTT; 11). The fos CaRE is identical to a sequence within the Ca’+-regulated region of the GRP78 promoter (12, 13). This element of the PRL promoter has not been directly tested for Ca2+-responsiveness, although 5’-promoter-deleted reporter constructs lacking this sequence show no less Ca2+ activation than those lacking the three immediately upstream Pit-l sites (10). Posttranscriptional Expression

Fig. 7. GRP78 mRNA Levels Are Responsive to Ca*+ Addition in 235-l but Not GHB Cells Cytoplasmic RNA from GH3 and 235-l cells treated for 24 h in SFM + Ca2+ was analyzed for GRP78 mRNA by Northern blot hybridization. A representative experiment from each cell type is shown for comparison. Such measurements have been previously published for GH:, cells (see text). The Ca’+-induced 50% decrease in GRP78 mRNA levels in 235-l cells has been observed in at least eight independent experiments.

No. 8

Regulation of PRL Gene

Addition of 0.5 mM CaCI, to GHB cell cultures in SFM increases PRL mRNA levels by severalfold. Nevertheless, we have been unable to detect a significant effect of this treatment on PRL gene transcription in the current study and in two previously published reports (4, 5). This finding prompts us to conclude that in GH3 cells, CaC12 increases PRL mRNA levels primarily by regulating some posttranscriptional step(s) in PRL RNA processing and/or transport. Although this conclusion is based on the inability to detect a difference in GH3 cell PRL gene transcription by the nuclear run-on assay,

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Transcriptional and Posttranscriptional

Regulation

of PRL Gene

the following findings attest to the fidelity of this assay in our hands: 1) identical results were obtained using 5’-specific, 3’-specific, and full-length cDNA probes; 2) no transcription was detected in the antisense direction; 3) in 235-l cells, Ca’+ induced comparable increases in PRL gene transcription and PRL mRNA levels; 4) Ca*+ ionophoreinduced decreases in PRL gene transcription were detected in both GH3 and 235-l cells; and 5) we have detected transcriptional induction of other genes (e.g. Ca*’ ionophore induction of GRP78 gene transcription) in GH3 cells (4). Given our hypothesis that an increase in [Ca”], induces an increase in PRL gene transcription, we propose that addition of 0.5 mM CaCI, to GH3 cells in SFM does not lead to a significant increase in [Ca’+]i. Hinkle et al. (9) reported a barely significant increase (P < 0.1) in [Ca’+]i in GH3 cells in response to 0.5 mM CaCI,. In several preliminary experiments using indomethacin lloaded GH3 cells, we have not observed a change in [Ca’+]i after the same treatment. Also, the treatment of GH3 cells with Ca*’ induces no decrease in GRP78 mRNA levels, which is consistent with unchanging [Ca”]i (12, 32, 33). Because Ca*+ has no effect on the cytoplasmic halflife of PRL mRNA (4) and we find that the levels of all three nuclear PRL precursor RNAs were increased by Ca*+, we suggest that the posttranscriptional step affected by Ca*’ occurs very early after completion of transcription. An early stabilization of liver/bone/kidney alkaline phosphatase gene transcripts has been postulated as the mechanism by which the levels of this RNA are increased in bone cells over those in other tissues in which the promoter is equally active (39). As we compared aspects of PRL gene expression between GH3 cells and 235-l cells, we uncovered other basic differences between the two cell types which further emphasize the presence of posttranscriptional controls. The two-band nuclear PRL pre-RNA pattern of 235-l cells, compared to the three precursor bands in the nuclear RNA of GH3 cells, indicates that 235-l cells process PRL transcripts in a qualitatively different manner. The absence of the smallest precursor band in 235-l cells may be due to more rapid processing of PRL gene transcripts during removal of the final intron. This difference in processing may contribute to the maintenance of high steady state levels of cytoplasmic PRL mRNA in 235-l cells, in the face of a low level of PRL gene transcription. Other studies have revealed that differences in RNA processing can lead to differences in amounts of specific mRNAs (39-41). Posttranscriptional Phenotypes

Regulation

and Acidophil

Since PRL production by normal pituitary cells is also regulated by Ca*+ (l), both posttranscriptional processing and transcriptional regulation may represent physiological mechanisms for controlling PRL levels. In this regard, the differences between GH, and 235-l cells are intriguing, because these two cell lines resemble

Expression

1273

different types of PRL-producing cells within the normal pituitary. GH3 cells produce both PRL and GH (42), like pituitary mammosomatotrophs (43), while 235-l cells produce PRL only (23, 24), as do pure mammotrophs. Because the primary transcriptional regulator of PRL, Pit-l (44, 45), also controls expression of the GH gene, posttranscriptional mechanisms may offer a greater range of specific control over both PRL and GH gene expression. It is possible that the differentiation of mammotrophs and mammosomatotrophs involves not only activation of PRL gene transcription by Pit-l, but also programing the capacities of different cell types to process PRL RNA. Thus, the constitutively active posttranscriptional processing of PRL transcripts in 235-l cells may be characteristic of pure mammotrophs, while the less efficient, Ca*‘-regulated processing of PRL RNA in GH3 cells may be representative of the dualsecreting mammosomatotroph. Such a mechanism requiring additional cellular differentiation for production of PRL RNA would help to explain the delay in accumulation of PRL RNA, relative to the appearances of both Pit-l and GH RNAs, in the pituitary of the developing mouse (46). We have recently studied PRL gene expression in PRL-deficient GC cells, which resemble pure somatotrophs. Surprisingly, GC cells transcribe the PRL gene at a rate similar to that observed in GH, and 235-l cells, even though these cells contain no detectable nuclear or cytoplasmic PRL mRNA (47). This is again suggestive of a role for posttranscriptional processes in regulating PRL RNA levels. Further studies will focus on the processing steps involved in regulation of PRL mRNA levels in GH, and 235-l cells, as well as on identification of the PRL RNA sequences responsible for mediating this response.

MATERIALS

AND

METHODS

Cell Culture and Ca2+ Treatments GH3 cells were obtained

from the American

Tissue Type

Collection (Rockville, MD) and were maintained in suspension culture as previously described (48). The 235-l cell line was generously donated by Dr. H. H. Samuels (New York University Medical Center, New York, NY) and was maintained on tissue culture plates in Joklik’s medium containing 10% iron-supplemented calf serum (HyClone Laboratories, Logan, UT) and

0.25 mM CaCl* to aid in attachment. For experiments, GH3 cells were collected by centrifugation, resuspended in SFM (48), and plated on culture dishes in the absence or presence of 0.5 mM CaC12. Because 235-l cells do not adhere well to fresh plates containing SFM, they were prepared for experiments by first feeding near-confluent (80-90%) plates with growth medium without CaCI, 1 to 2 days before beginning treatment. On the day of treatment, the culture medium was replaced with SFM, and the cells were incubated at 37 C for 60 min. The medium was then replaced with SFM with or without addition of 0.5 mM CaCI,. Cells were treated for 2024 h, as stated for individual experiments, in a humidified incubator at 37 C in 2% CO,.

Calcium lonophores lonomycin (Calbiochem, La Jolla, dimethylsulfoxide at a concentration

CA) was resuspended in of 7 mM. Aliquots of the

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MOL 1274

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1992

Vol6

diluted in dimethylsulfoxide at a final concentration

Transcription

to 400 of 400 nM.

PM

for use in

Assay

GH3 or 235-l cells (five IOO-mm dishes per group) were cultured for 20 h in SFM alone or in the presence of 0.5 mM CaCI, (Ca’+) or 0.5 mM Ca2+ plus 400 nM ionomycin. Cells were collected by scraping, pooled, washed once in ice-cold PBS, and then nuclei were isolated and used for run-on transcription as described (4, 5), using 300 &I [32P]UTP (650 Ci/mmol; ICN, Costa Mesa, CA) per reaction. Labeled run-on RNA (final vol 0.4 ml) was isolated essentially as described (5, 49). Total counts per min of 32P-IBbeled RNA in 5 ~1 of each sample was determined and equal counts per min (up to 4 x 1 06) in 1 ml TES buffer [lo mM N-Tris(hydroxymethyl) methyl2-aminoethanesulfonic acid (TES), pH 7.4, 10 mM EDTA, and 0.2% sodium dodecyl sulfate] were added to 1 ml TES buffer plus 0.6 M NaCI, then hybridized to DNA immobilized on nitrocellulose filters which were prehybridized as described (4). DNA dots were made with linearized and denatured plasmids containing either no insert (Bluescript KS+, Stratagene, La Jolla, CA), a PRL cDNA clone (PRL-SK+; 5), or PRL-ES, an EcoRI/BamHI fragment of the 3’-end of the rat PRL gene containing 73 bp intron D, exon 5 (the last exon), and about 1 kb 3’-flanking sequence. DNA dots were also made using four single-stranded ~NAs generated by performing asymmetric oolvmerase chain reaction (PCR) on the PRL cDNA. These bN& contain exons l-3 and 3-5 of the PRL cDNA and on both the antisense (probes, detect sense transcripts) and sense strands (controls, detect antisense transcripts). The generation of these probes is described below. Hybridization was carried out at 52 C for 2 days. Filters were washed twice in 2x SSC (0.3 M NaCI, 0.03 M sodium citrate, pH 7) for 1 h at 65 C, then treated with 1 fig/ml RNase A (Boehringer Mannheim, Indianapolis, IN) in 2x SSC for 30 min at 37 C. After a final wash in 2x SSC at 37 C for 1 h. filters were blotted dry and analyzed using a Betascope 603 blot analyzer (Betagen, Waltham, MA). The filters were then autoradiographed on Kodak X-Omat x-ray film (Eastman Kodak Co., Rochester, NY) for 2 weeks at -70 C with an enhancing screen. In some cases, films were analyzed by scanning densitometry using a Gilford Response spectrophotometer (CIBA-Corning Diagnostics, Medford, MA). RNA Isolation

and Northern

Blot Analysis

Cytoplasmic RNA was prepared from run-on cell lysates after removal of nuclei by centrifugation. Cytoplasmic lysates (l-2 ml) were combined-with an equal voI2x. protein&e K biffer (2x = 0.2 M Tris-Cl. DH 7.5. 0.44 M NaCI, 2% sodium dodecvl sulfate, and 25 mM i?DTA) plus 200 pg/ml proteinase K a& incubated for 30 min at 37 C. The samples were extracted once with phenol:chloroform (l:l), twice with chloroform, and then made 0.25 M in NaCl and ethanol precipitated. Five- to 1 O-microgram aliquots of RNA were electrophoresed on 1% agarose-formaldehyde gels and analyzed by Northern blot hybridization (50). For some experiments, RNA was analyzed by RNA dot & by cytoplasmic dot blot hybridization (48): Nuclear RNA was isolated from GHB and 235-l cells (five or six loo-mm dishes per group) that-were cultured in SFM with or without Ca2+ for 24 h. Cells were collected by scraping, pooled, washed once in ice-cold PBS, and then resuspended in 0.9 ml PBS. Cells were lysed by addition of 0.1 ml NP-40 (5% in PBS), incubated on ice for 3 min, then centrifuged at 1000 x g for 5 min at 4 C. Supernatants were used for preparation of cytoplasmic RNA as above. The nuclear RNA was isolated by guanidine extraction and centrifugation through CsCI, essentially as described (51). Ten-microgram aliquots of nuclear RNA were electrophoresed on 1% agaroseformaldehyde gels and analyzed by Northern blot hybridization (50).

Asymmetric Probes

PCR to Generate

Single-Stranded

No. 8

PRL DNA

Single-stranded probes encompassing the sense and antisense strands of PRL exons l-3 and 3-5 were made by performing asymmetric PCR on 500-ng aliquots of Pstl-cut PRL-SK+ (fstl releases the cDNA insert). The reactions contained 200 FM of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP) and 2.5 U Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) in 100 pl of the manufacturer’s recommended 1 x buffer. The reactions also contained 200 pmol and 4 pmol 5’- and 3’-oligonucleotide primers, respectively, for each fragment. For generation of the antisense probes, the ratios of the primers were reversed. To ensure production of sufficient probe DNA, five to eight reactions were run for each probe. The primers used, with their nucleotide positions within the PRL cDNA, were: PRL exon 1 5’: 9-“GTCAT CACCA TGAAC AGCCA?-28 PRL exon 3 3’: 274-5’GGGCA GTCAT TGATG GCCTT3’-255 PRL exon 3 5’: 255-“AAGGC CATCA ATGAC TGCCC3’-274 PRL exon 5 3’: 708-“TCTCA GATGT ACATG GAATGy-727 Amplification was carried out for 50 cycles as follows: 94 C, 1 min; 60 C, 2 min; and 72 C, 1 min. After the last cycle, the reactions were held at 72 C for an additional 5 min to finish any incomplete synthesis. The amplified single-stranded DNA was precipitated, quantified by measuring the ODpeO, and a small aliquot was run on a 1% agarose gel to verify the approximate correct size. The probes were then denatured as for plasmid DNAs, and 1.25-2.5 pg of each was dotted onto nitrocellulose for use in the run-on transcription assays. Acknowledgments We We and the

thank Dr. H. H. Samuels for providing the 235-l cell line. thank Puja Agarwal for her expert technical assistance John Lynch and Bill Billis for helpful suggestions during preparation of this manuscript.

Received May 4, 1992. Revision received June 9, 1992. Accepted June 10,1992. Address requests for reprints to: Dr. Bruce A. White, Department of Anatomy, University of Connecticut Health Center, 263 Farmington Ave., Farmington, Connecticut 06030. This work was supported by NIH Research Grant DK43064.

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MOL ENDO. 1992 1276

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Vol6 No. 8

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Recertification in Endocrinology, Diabetes, and Metabolism Endocrinology, Diabetes, and Metabolism Board of the American Board of Internal Medicine Drs. Martin I. Surks (Chairman), Glenn D. Braunstein, John D. Brunzell, Philip E. Cryer, Willa A. Hsueh, Robert A. Kreisberg, D. Lynn Loriaux, and Lawrence G. Raisz Since 1990, the duration of validity of certification by the American Board of Internal Medicine (ABIM) has been limited to 10 yr. This policy was adopted because medical information changes rapidly and the public needs assurance that certified internists have maintained their skills and kept their knowledge up to date. Time-limited certification and recertification are obligations of an accountable profession. The ABIM recently has completed and adopted plans for a comprehensive recertification program. Entry into Recertification: Diplomates may attempt recertification only in disciplines in which they were previously certified. Certified endocrinologists may seek recertification at any time after initial certification in endocrinology, diabetes, and metabolism only, in intermal medicine only, or in both. Diplomates can allow a time-limited certificate in internal medicine to expire without jeopardizing elrgrbrlrty for recertification in endocrinology, diabetes, and metabolism. However, expiration of a certificate in internal medicine or endocrinology means the ABIM no longer recognizes an individual as certified in that disipline. Certificates in internal medicine or endocrinology, diabetes, and metabolism issued before 1990 are not time-limited and are valid for life; individuals holding such certificates are eligible to seek recertification without placing existing certificates at risk. Please note that all successful candidates for recertification will be issued a certificate with diabetes in the title: Endocrinology, Diabetes, and Metabolism. The Recertification Process: The recertification process consists of three steps: documentation of clinical competence, completion of the self-evaluation process, and success on a proctored, written final examination. Dual Recertificaiton: The Board anticipates that most endocrinologists will seek recertification in internal medicine as well as endocrinology, diabetes, and metabolism and has, therefore, developed an efficient process for dual recertification that does not change the standards required for recertification in each discipline. For both the self-evaluation processes and the final examinations, substitution of required modules for self-selected modules can reduce the total number of modules required for recertification in internal medicine and endocrinology to six self-evaluation process modules (three in general internal medicine, three in endocrinology) and four final examination modules (two in general internal medicine, two in endocrinology). The score for each final examination will be determined independently. Thus, an individual can be successful in becoming recertified in endocrinology, diabetes, and metabolism but not in internal medicine, even when the processes are undertaken concurrently. Schedule of Availability: This comprehensive recertification program will become available in 1995. The self-evaluation process will be available continuously beginning in 1995. Final examinations will be available annually beginning in 1996. Diplomates who would like to become recertified before this time can take a regularly scheduled certificaiton examination for recertification credit (interim voluntary recertification).

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