©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Post-transcriptional Regulation of the Stanniocalcin Gene by Calcium (*)

(Received for publication, October 18, 1994)

Tannis J. Ellis Graham F. Wagner (§)

From the Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stanniocalcin (STC) is a Ca-regulating hormone produced by the corpuscles of Stannius in bony fish. Calcium has been shown to stimulate STC synthesis at multiple levels including the level of gene expression. The purpose of this study was to determine the effects of Ca on STC mRNA stability. The half-life of STC mRNA was measured in primary cultured trout corpuscles of Stannius cells maintained in either normal (1.2 mM) or high (1.9 mM) levels of extracellular calcium and treated with the transcriptional inhibitor alpha-amanitin. In cells maintained in 1.2 mM Ca, STC mRNA levels decreased progressively over time with an estimated half-life of 71 h. However, message levels remained unchanged for up to 4 days in cells maintained in 1.9 mM Ca, indicating that the transcript had been stabilized in response to Ca stimulation. Blocking transcription prior to exposing cells to high Ca did not alter the stabilizing effects of the cation, indicating that synthesis and processing of the mRNA transcript were not involved in message stabilization. Inhibiting protein synthesis with cycloheximide also had no influence on the stabilizing effects of high calcium. The experiments involving cycloheximide further suggested that the mechanism of mRNA stabilization involved protein-nucleic acid interactions in the cytoplasm, whereby the polysomal complex protected the mRNA from degradation. These data demonstrate that the stimulatory effect of Ca on STC gene expression is due, in part, to mRNA stabilization.


INTRODUCTION

Stanniocalcin (STC) (^1)is an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) in bony fishes. The CS glands were first identified in fishes by Stannius (1) and have subsequently been shown to arise embryologically from kidney tubule cells(2, 3) . A role for the CS glands in Ca homeostasis was demonstrated in a classic study, whereby surgically removing them in eels caused a form of hypercalcemia that could be alleviated by administering CS extracts to the fish(4) . The hypercalcemia was later discovered to be caused by accelerated Ca uptake from the aquatic environment by the gills, suggesting that the CS glands contained an inhibitor of gill Ca transport(5, 6, 7) . The active principal was subsequently shown to be a homodimeric glycoprotein now known as stanniocalcin(8) .

Studies in salmonid fishes have shown that the secretion of STC is positively regulated by extracellular levels of ionic Ca such that there is a time- and concentration-dependent relationship between Ca levels and measurable hormone release(9) . Upon release, STC reduces gill Ca transport from the aquatic environment into the blood stream(5, 6, 7, 8, 10, 11) and stimulates phosphate reabsorption by the kidneys(12) , thereby bringing about a restoration of normocalcemia. Consequently, STC working in concert with the kidneys and gills is responsible for Ca homeostasis in bony fish.

High Ca also affects the biosynthesis of STC in CS cells, a regulatory response that would be required to sustain a higher output of STC during prolonged exposures to elevated Ca levels in either the extracellular or aquatic environment. This occurs on at least two levels. Calcium stimulates STC synthesis from pre-existing mRNA and can have profound effects on steady-state levels of STC mRNA (13, 14) . Moreover, this response is specific to the STC gene as Ca has absolutely no effects on the housekeeping genes, beta-actin and ribosomal RNA, in STC cells. The ability of Ca to increase STC mRNA levels could occur via transcriptional as well as post-transcriptional mechanisms, in other words, at the level of mRNA synthesis and/or mRNA degradation. Therefore, to gain further insight as to the mechanisms involved in Ca-regulated STC gene expression, we have characterized the effects of Ca at the post-transcriptional level. The data presented here demonstrate that the stimulatory effect of Ca on steady-state STC mRNA levels is due, in part, to mRNA stabilization.


EXPERIMENTAL PROCEDURES

Materials

Fine chemicals and electrophoretic reagents were purchased from BDH, restriction enzymes from Promega, chromatography supplies and labeling kits from Pharmacia Biotech Inc., blotting membranes and radioisotopes from Amersham Corp., tissue culture supplies from Life Technologies, Inc., alpha-amanitin from Boehringer Mannheim, and cycloheximide from Sigma.

Preparation of Cultured Cells

CS cells were prepared and cultured as described previously(14) . For each experiment, CS glands were collected from 80 adult, sexually maturing rainbow trout of mixed sex (0.25-0.40 kg). The glands were dissected free of surrounding renal and adipose tissue, teased apart with fine forceps, and trypsinized overnight in Leibovitz medium (L-15) containing 1.2 mM Ca, antibiotics (100 units/ml each penicillin and streptomycin and 2.5 µg/ml Fungizone), and 0.5% (w/v) trypsin. The dispersed cells were plated in L-15 medium containing 1.2 mM Ca, 10% (v/v) fetal bovine serum, and antibiotics (100 units/ml each penicillin and streptomycin) at a density of 0.5 times 10^6 cells/ml in 24-well culture plates and maintained at 15 °C in a normal atmosphere. All experiments commenced 4-5 days after plating. Prior to all experiments, cells were washed twice with serum-free L-15 medium containing 0.1% (w/v) bovine serum albumin to remove traces of fetal bovine serum. Cells were then maintained in serum-free L-15 medium containing 0.1% bovine serum albumin and supplemented with antibiotics for the duration of the experiment. All experiments were conducted in a normal atmosphere at 15 °C on quadruplicate wells of cells.

RNA Extraction and Northern Blot Analysis

Total RNA was isolated from plated cells according to Chomczynski and Sacchi(15) . RNA was resolved on 1% formaldehyde-agarose gels, transferred to nylon membrane (Hybond N) by capillary action, and cross-linked by UV irradiation. Prehybridization (2 h) and hybridization of blots were performed in 50% formamide, 6 times SSC, 1.25 times Denhardt's solution, 100 µg/ml salmon sperm DNA, and 0.1% SDS at 42 °C. Blots were incubated overnight with a random-primed P-labeled cDNA (2 times 10^6 dpm/ml) consisting of a 411-base pair fragment encoding residues Asn-Gln of coho salmon STC, which is also specific for STC mRNA in the rainbow trout (16) . Following hybridization, membranes were washed 5 times 10 min at room temperature in 2 times SSC, 0.1% SDS, followed by 2 times 30 min in 0.1 times SSC, 0.1% SDS at 65 °C and exposed to x-ray film. The blots were then stripped and reprobed under the same conditions as described above with a rabbit 18 S ribosomal RNA cDNA probe, which is also specific for the 18 S ribosomal subunit in trout. The relative intensities of the resulting autoradiographic images were determined by scanning densitometry, and the level of STC mRNA in each well of cells was expressed as a STC mRNA/18 S rRNA ratio.

Dose-related Effects of alpha-Amanitin on STC mRNA Levels

To determine the optimal concentration of alpha-amanitin required to fully inhibit transcription without compromising cell viability, cultured cells were maintained in L-15 medium containing 1.2 mM Ca and treated with 0, 0.5, 1, 2, 5, or 10 µg/ml alpha-amanitin for 48 h. The alpha-amanitin was dissolved in water and added to the cells from 100 times sterile stock solutions. STC/18 S RNA ratios were calculated and expressed as a percent of control (control being the STC/18 S RNA ratio in cells treated with no alpha-amanitin).

STC mRNA Half-life Estimates following 3-Day Exposures to High Ca

Exposing rainbow trout CS cells for 3 days to 1.9 mM Ca has consistently been shown to cause an induction of STC mRNA levels(14) . Therefore, to determine if the Ca-induced increase in STC mRNA abundance was due to increased stability of the transcript, the half-life of STC mRNA was measured in CS cells that had been incubated in control (1.2 mM) or high Ca (1.9 mM) medium for 3 days. CS cells were exposed to 1.2 or 1.9 mM Ca for 3 days, at which time the medium was replaced with the same medium containing 2 µg/ml alpha-amanitin. Total RNA was isolated at various time points up to 144 h after the addition of alpha-amanitin and subjected to Northern blot analysis as described above. STC/18 S RNA ratios were calculated and expressed as a percent of control (control being the STC/18 S RNA ratio in each treatment group at time 0 before the inhibition of transcription with alpha-amanitin).

Effect of Shorter Exposures to High Ca on STC mRNA Stability

High Ca can also induce an accumulation of STC mRNA in CS cells after 24 h(14) . Therefore, to determine the effects of a 24-h exposure to high Ca, primary cultured CS cells were treated with either 1.2 or 1.9 mM Ca medium for 1 day before the medium was replaced with the same medium containing 2 µg/ml alpha-amanitin. Total RNA was isolated at 0, 12, 24, 36, 48, and 60 h after the addition of alpha-amanitin and subjected to Northern blot analysis as described above. STC/18 S RNA ratios were calculated and expressed as a percent of control as described above.

To determine if Ca-mediated changes in transcription were required to stabilize the message, two groups of CS cells in 1.2 mM Ca medium were pretreated with 2 µg/ml alpha-amanitin for 2 h. Calcium chloride was then added to one group of cells to achieve a final concentration of 1.9 mM Ca. Total RNA was extracted at various time points after the addition of Ca and subjected to Northern blot analysis. STC/18 S RNA ratios were calculated and expressed as a percent of control as described above.

Effect of Inhibiting Protein Synthesis on Ca-induced STC mRNA Stabilization

Four plates of CS cells in 1.2 mM Ca medium were pretreated for 2 h either with alpha-amanitin alone (two plates) or in combination with 10 µg/ml cycloheximide (two plates) to inhibit translation. Calcium chloride was then added to one plate from each of the two treatment groups to achieve a final concentration of 1.9 mM calcium. Total RNA was isolated at 0, 12, 24, 36, 48, and 60 h after the addition of Ca and subjected to Northern blot analysis, and STC/18 S RNA ratios were calculated as described above. Under the conditions of this study, 10 µg/ml cycloheximide inhibited >95% protein synthesis in CS cells as assessed by [^3H]leucine uptake into trichloroacetic acid-precipitable protein, with no visible effects on cell viability (data not shown).

Statistical Testing and Data Analysis

Each STC/18 S RNA ratio was expressed as a mean ± S.E. of the four replicates taken at each time point. STC/18 S ratios were then transformed to common logs and subjected to linear regression analysis followed by analysis of variance. Regression equations were considered to be significantly different than zero or one another if p < 0.05.


RESULTS

Dose-related Effects of alpha-Amanitin on STC mRNA Levels

Treating CS cells with varying concentrations of alpha-amanitin for 48 h demonstrated that 1-2 µg/ml was sufficient to fully inhibit transcription under the conditions of the study. The results of the experiment are shown in Fig. 1. STC mRNA levels were moderately depressed in response to 0.5 µg/ml (89% of control) and maximally depressed by all other concentrations to 30-42% of control levels. Therefore, alpha-amanitin was used at a final concentration of 2 µg/ml for all subsequent studies.


Figure 1: Concentration-related effects of alpha-amanitin on STC mRNA levels. CS cells in 1.2 mM Ca were treated with increasing concentrations of the polymerase II inhibitor alpha-amanitin for 48 h. Total RNA was extracted, and STC mRNA levels were determined by Northern blotting as described under ``Experimental Procedures.'' STC mRNA levels decreased moderately in the presence of 0.5 µg/ml alpha-amanitin (89% of control), but were maximally decreased by all other concentrations. Each data point represents the mean of quadruplicate wells of cells normalized as a ratio of STC mRNA to 18 S rRNA and expressed as a percent of control untreated cells.



STC mRNA Half-life Estimates following 3-Day Exposures to High Ca

Following a 3-day exposure to either 1.2 or 1.9 mM Ca medium, further RNA synthesis was blocked with alpha-amanitin, and the decay rate of pre-existing STC mRNA was monitored. In the presence of 1.2 mM Ca, STC mRNA levels decreased progressively over time with an estimated half-life of 50 h (Fig. 2). In 1.9 mM Ca, however, STC mRNA levels remained unchanged for the duration of the experiment (60 h) such that the regression equation was not significantly different than zero, thus revealing the stabilizing effect of Ca on the message. However, it was apparent from this study that a more accurate estimate of half-life in the presence of 1.9 mM Ca could only be obtained with a longer sampling regime. Therefore, the experiment was repeated, and the sampling time was increased to 144 h. The results of this study are shown in Fig. 3. In the presence of 1.2 mM Ca, a revised half-life estimate of 70 h was obtained. Moreover, the message was completely stabilized for up to 96 h in the presence of 1.9 mM Ca, thereby confirming and extending the results of the previous experiment. Message levels in the high Ca-treated cells then dropped off rapidly thereafter. It was unlikely that this was due to cell death as the alpha-amanitin had no visible cytotoxic effects on the cells (they appeared normal under the microscope), and 18 S rRNA levels were consistent over the course of the experiment. However, the observed decrease in STC mRNA levels could have been due to the exhaustion of some essential component in the medium. To explore the latter possibility, the experiment was repeated again, except that the medium in both treatment groups was replaced at 72 h with fresh L-15 medium containing the same antibiotic supplements, Ca concentrations, and alpha-amanitin. Essentially the same results were obtained as those shown in Fig. 3(data not shown), thereby ruling out this alternative explanation.


Figure 2: STC mRNA stability measured over 60 h following a 3-day induction with 1.9 mM Ca. CS cells were first incubated in serum-free medium containing either normal (1.2 mM; bullet) or high (1.9 mM; circle) Ca for 3 days. Then at time 0, transcription was blocked with alpha-amanitin (2 µg/ml), and the decay rate of the STC mRNA pool was monitored over 60 h. Total RNA was extracted, and STC mRNA levels were determined by Northern blot analysis and scanning densitometry as described under ``Experimental Procedures.'' In the presence of 1.2 mM Ca, STC mRNA levels decreased progressively over 60 h with an estimated half-life of 50 h. In the presence of 1.9 mM Ca, STC mRNA levels remained unchanged such that the slope of the line was not significantly different from zero (p = 0.3532), indicating the stabilizing effect of high Ca. The slopes of the two decay curves were also significantly different than one another (p < 0.05). Each data point represents the mean of quadruplicate samples normalized as a ratio of STC mRNA to 18 S rRNA and expressed as a percent of time 0.




Figure 3: STC mRNA stability measured over 144 h following a 3-day induction with 1.9 mM Ca. CS cells were subjected to the same experimental protocol as described in the legend to Fig. 2, except that the decay rate of the STC mRNA pool was monitored over 144 h. a shows that in the presence of 1.2 mM Ca (bullet), the estimated half-life was 70 h. In the presence of 1.9 mM Ca (circle), STC mRNA levels remained unchanged for the first 96 h of alpha-amanitin treatment and then decreased rapidly between 96 and 144 h. The slopes of the two decay curves were significantly different than one another for the first 96 h (p < 0.001). Each data point represents the mean of quadruplicate samples normalized as a ratio of STC mRNA to 18 S rRNA and expressed as a percent of time 0. b is a representative autoradiogram from this experiment showing the relative intensities of the STC mRNA and 18 S RNA bands over the first 96 h.



Effect of Shorter Exposures to High Ca on STC mRNA Stability

Given the stimulatory effects of Ca on STC mRNA stability after a 3-day exposure to Ca, we decided to explore the effects of a 24-h exposure. The results revealed that 24 h was sufficient for 1.9 mM Ca to completely stabilize the message (Fig. 4) such that the regression equation was not significantly different than zero. However, the estimated half-life of STC mRNA in 1.2 mM Ca-treated cells was now 150 h, a much longer estimate than that obtained in the 3-day induction experiments.


Figure 4: STC mRNA stability following a 1-day induction with 1.9 mM Ca. CS cells were incubated in serum-free medium containing either normal (1.2 mM; bullet) or high (1.9 mM; circle) Ca for 24 h as described under ``Experimental Procedures.'' At time 0, transcription was blocked with 2 µg/ml alpha-amanitin, and STC mRNA levels were determined as described under ``Experimental Procedures.'' In the presence of 1.2 mM Ca (bullet), STC mRNA levels decreased progressively over 60 h with an estimated half-life of 150 h, whereas STC mRNA levels were unchanged in the presence of 1.9 mM Ca (circle), demonstrating that a 1-day exposure to Ca was sufficient to stabilize the message. The slopes of the two decay curves were significantly different (p < 0.05). Each data point represents the mean of quadruplicate samples normalized as a ratio of STC mRNA to 18 S rRNA and expressed as a percent of time 0.



To determine if ongoing transcription was required for Ca-induced stabilization of the message, in additional experiments, alpha-amanitin was added 2 h before raising the Ca concentration to 1.9 mM, and total RNA was extracted at various time points thereafter. In the presence of 1.2 mM Ca, the estimated half-life of STC mRNA was 43 h (Fig. 5). However, mRNA levels remained constant for 72 h in cells maintained in 1.9 mM Ca (regression equation was not significantly different than zero), indicating that the transcript had been stabilized by Ca even in the absence of transcription. Message levels then decreased rapidly between 72 and 144 h, as in the 3-day induction experiments.


Figure 5: Stabilizing effect of Ca on STC mRNA does not require ongoing transcription. Two groups of CS cells in 1.2 mM Ca were pretreated with alpha-amanitin (2 µg/ml) for 2 h, after which the media Ca concentration in one group was adjusted to 1.9 mM. Total RNA was extracted at various times after the addition of Ca, and STC mRNA levels were determined as described under ``Experimental Procedures.'' In the presence of 1.2 mM Ca (bullet), the estimated half-life of STC mRNA was 43 h, whereas STC mRNA levels remained unchanged over the first 72 h in 1.9 mM Ca (circle), suggesting that high Ca had a stabilizing effect on STC mRNA in the absence of transcription. Message levels in the high Ca-treated cells then decreased rapidly between 72 and 144 h. The slopes of the two decay curves were significantly different for the first 72 h (p < 0.001). Each data point represents the mean of quadruplicate samples normalized as a ratio of STC mRNA to 18 S rRNA and expressed as a percent of time 0.



Effect of Inhibiting Protein Synthesis on Ca-induced STC mRNA Stabilization

To determine if ongoing protein synthesis was required for Ca-induced stabilization of STC mRNA, four plates of cells in 1.2 mM Ca were pretreated for 2 h with alpha-amanitin alone (two plates) or in combination with cycloheximide (two plates). The media Ca concentration in one plate from each of these two groups was then raised to 1.9 mM, and total RNA was extracted at various time points thereafter. The decay curves for the four treatment groups are shown in Fig. 6and reveal that inhibiting protein synthesis had a stabilizing effect on the message in both normal and high Ca media. The estimated half-life of STC mRNA in 1.2 mM Ca was 42 h. However, in all other experimental groups (1.2 mM Ca + cycloheximide, 1.9 mM Ca, and 1.9 mM Ca + cycloheximide), the message was stabilized such that STC mRNA levels were relatively unchanged for the duration of the study. The regression equations calculated for these latter three groups were not significantly different than one another or zero and yet were all significantly different than the regression equation calculated for cells maintained in 1.2 mM Ca alone.


Figure 6: Stabilizing effect of Ca on STC mRNA does not require ongoing protein synthesis. Four groups of CS cells in 1.2 mM Ca were pretreated with alpha-amanitin alone (2 µg/ml) or in combination with cycloheximide (10 µg/ml) for 2 h, after which the media Ca concentration in two groups was adjusted to 1.9 mM. At the indicated times, total RNA was extracted, and STC mRNA levels were determined as described under ``Experimental Procedures.'' In the presence of 1.2 mM Ca alone (circle), STC mRNA levels decreased progressively with an estimated half-life of 42 h. However, message levels were completely stabilized in the groups treated with 1.9 mM Ca alone (bullet) or with cycloheximide in the presence of normal (down triangle) and high () calcium. This suggests that ongoing protein synthesis is not required to stabilize the transcript and that the polysomal complex may be protecting it from degradation. Each data point represents the mean of quadruplicate samples normalized as a ratio of STC mRNA to 18 S rRNA and expressed as a percent of time 0.




DISCUSSION

Recent studies have demonstrated that raising extracellular Ca levels has positive effects on STC synthesis at multiple levels. Calcium stimulation of CS cells increases de novo synthesis of STC from pre-existing mRNA and has pronounced effects on steady-state cellular levels of STC mRNA(13, 14) . The steady-state levels of any transcript represent a balance between its rate of nuclear synthesis, processing, and export to the cytoplasm and its rate of cytoplasmic degradation. Hence, the steady-state levels of functional mRNAs are determined, in part, by their rates of degradation. This study is the first to examine the mechanism by which STC mRNA levels are increased in response to Ca stimulation, specifically in relation to the possible effects of Ca on STC mRNA stability.

It is well established that the decay rates of mRNAs are altered in response to physiological signals, such as changing levels of hormones and blood metabolites. For instance, the half-life of liver vitellogenin mRNA is increased from 16 to 500 h in response to estrogen treatment(17) , and glucose is known to have a stabilizing effect on insulin mRNA(18) . Similarly, raising extracellular Ca levels from 1.2 to 1.9 mM had a pronounced effect on the stability of STC mRNA. In the presence of normal Ca levels, the half-life of the STC transcript ranged from 42 to 150 h and had a mean time of 71 h. By comparison, the half-lives of the insulin (19) , PTH, and chromogranin A (20) transcripts are all roughly 30 h under basal conditions. At this point in time, it is difficult to judge if the variations in our half-life estimates have any physiological significance. In spite of the fact that all cell preparations were treated equally and that viability was always >95%, it was unavoidable that the cells for each experiment were prepared at different times of the year. This may be important here as changing season is known to have profound effects on Ca homeostasis in fishes, reflecting the fact that fish growth rates are temperature-dependent and seasonally dependent(21, 22, 23) . Consequently, season also affects the physiology of the CS and STC, including the metabolic activity of CS cells(24) , the relative sensitivity of the STC gene to Ca(14) , and even the biological potency of circulating STC(8, 10, 11, 25) . Hence, it is reasonable to suggest that season should also be considered as a potential modifier of STC mRNA stability especially in view of the fact that the experiments reported here were conducted over a period of several months (October through March). More important, however, high Ca proved to have a pronounced stabilizing effect on the STC message. The results consistently showed that the STC message was completely stabilized in the presence of 1.9 mM Ca for as long as 96 h following the inhibition of transcription. Thereafter, message levels dropped off rapidly at a rate comparable to that of cells held in normal calcium. This rapid decline was not caused by cell death as 18 S rRNA levels remained constant throughout the study and the cells appeared healthy, indicating that alpha-amanitin had no major effects on cell viability. However, as the levels of many mRNAs decline with prolonged exposures to alpha-amanitin, some of which may encode stabilizing proteins, it seems reasonable to suggest that the factors responsible for stabilizing the STC transcript were completely depleted by 72-96 h, resulting in its rapid decline thereafter. Although transcriptional inhibitors are useful for estimating mRNA half-life under ``normal'' conditions, their limitations become apparent under circumstances where there is prolonged message stabilization, as in the case of this study. An unfortunate consequence of this was that we were unable to obtain a half-life estimate for the STC transcript in the presence of high calcium. To accomplish this in future studies, an alternative method that does not require transcriptional inhibitors will have to be employed (i.e. isotopic labeling).

At least two models can be used to explain the stabilization of cellular mRNAs. One proposes that covalent nuclear modifications to the transcript, involving alterations to the 5`- or 3`-untranslated regions, are responsible for changes in mRNA stability(26, 27, 28) . One such modification that has been extensively studied is poly(A) tail length. In the case of vasopressin mRNA, for example, the length of the poly(A) tail increases progressively from 250 to 400 nucleotides during the process of mRNA stabilization(29) . The second model proposes that mRNA stability can be regulated through protein-nucleic acid interactions in the cytoplasm(27, 28) , and in this study, both of these models were evaluated independently.

To determine if Ca-induced stabilization was conferred by covalent nuclear modifications to the STC message, transcription was blocked with alpha-amanitin 2 h prior to exposing the cells to high calcium. The results showed that STC mRNA stabilization was not dependent upon the production of new transcripts, thereby discounting the first model as a possible explanation. The alternative explanation, that message stability might be mediated by protein-nucleic acid interactions, was tested by comparing the message decay curves of cells maintained in the presence and absence of the translational inhibitor cycloheximide. A variety of transcripts are stabilized by cycloheximide (30, 31) , not only because it inhibits the synthesis of enzymes that normally degrade mRNA, but because cycloheximide also promotes polysomal aggregation, which in itself protects mRNA from degradation (32) . We found that the STC transcript was stabilized under high Ca conditions in the added presence of cycloheximide, proving that the stabilizing effects of high Ca were not mediated by de novo protein synthesis. Furthermore, the fact that cycloheximide had a stabilizing effect on STC mRNA under normal Ca conditions is consistent with its having promoted polysomal aggregation of the STC transcript as described above and thereby protecting it from degradation. Given that high Ca also promotes the translation of STC mRNA, in our view, the results of this study can best be explained by a model in which STC mRNA occurs in two forms: a polysome-bound form in which the mRNA is actively engaged in translation and protected from degradation, and a polysome-free form. Since the free form is far more susceptible to degradation, any treatment that promotes polysomal aggregation will ultimately protect the transcript. Hence, in cells treated with either cycloheximide or high Ca, any transcript that is already polysome-bound or subsequently driven into a polysomal complex by Ca is, in essence, shielded against degradation. Conversely, in CS cells maintained in normal Ca medium, there is little or no stimulus for STC secretion, and a proportionately greater amount of STC mRNA would exist in the free form, subject to degradation at a higher rate. The mechanism of Ca-induced stabilization appears to require neither transcription nor de novo protein synthesis and depends instead upon pre-existing factors in CS cells that remain to be identified. These factors are obviously labile, requiring constant renewal, as they are completely exhausted from the cells after 3-4 days of transcriptional inhibition.

Similar mechanisms to those described here have been identified in the pancreatic islets, where glucose stimulates both the synthesis and secretion of insulin(33, 34, 35, 36) . As in the case of Ca and STC, glucose stimulation increases the rate of de novo insulin synthesis from pre-existing mRNA as well as cellular levels of insulin mRNA(34, 35, 36) . Glucose induction of insulin mRNA has been attributed to increases in both the rate of gene transcription and the stability of the transcript(19, 37) . Furthermore, the mechanism of mRNA stabilization has been attributed to increased protein synthesis, whereby free insulin mRNA is driven into polysomal complexes in response to hyperglycemia(18) . Therefore, the mechanisms of Ca regulation in STC cells parallel, in many respects, those of glucose regulation in insulin cells at the various stages of hormone synthesis and secretion. There are also parallels in the biosynthetic pathways of STC and PTH, the principal Ca-regulating hormone in higher vertebrates. In the case of PTH, however, it is low levels of extracellular Ca that serve as a stimulus for secretion and gene transcription(38, 39) . Low Ca levels also encourage polysomal aggregation of PTH mRNA, yet interestingly enough, low Ca has no discernible effect on PTH mRNA stability(40) . In fact, to our knowledge, STC is the only transcript that is stabilized by the calcium ion. In addition to this, we have found that raising extracellular Ca levels has no noticeable effect on the Ca levels in CS cells. (^2)Hence, the cation does not appear to be directly involved in the stabilization process. Calcium has not yet been shown to increase the rate of STC gene transcription. However, given that STC mRNA levels are increased as much as 14-fold in response to Ca stimulation(14) , an effect that is unlikely to be due to message stabilization alone, we fully anticipate that STC gene transcription is activated by calcium. In support of this notion, in vivo studies have shown that sustained hypercalcemia in fishes causes nuclear swelling in STC cells, suggesting that gene transcription may indeed be affected by Ca(24) .

In summary, we have examined the post-transcriptional regulation of the STC gene by calcium. The results demonstrated that Ca had a pronounced effect on STC mRNA stability, indicating that the stimulatory effect of Ca on message levels is due, in part, to mRNA stabilization. The mechanism of STC mRNA stabilization does not involve ongoing transcription or translation and is possibly mediated by protein-nucleic acid interactions in the cytoplasm, whereby the polysomal complex protects the transcript from inactivation and degradation.


FOOTNOTES

*
This work was supported by a grant and scholarship from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Medical Research Council of Canada Scholar. To whom correspondence should be addressed. Tel.: 519-661-3966; Fax: 519-661-3827.

(^1)
The abbreviations used are: STC, stanniocalcin; CS, corpuscles of Stannius; PTH, parathyroid hormone.

(^2)
S. J. Dixon and G. F. Wagner, unpublished observations.


ACKNOWLEDGEMENTS

We thank Ewa Jaworski and Michel Haddad for assistance in obtaining the corpuscles of Stannius and the preparation of cell cultures.


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