(Received for publication, October 18, 1994)
From the
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
-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.
Stanniocalcin (STC) ()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,
-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.
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
-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.
Figure 1:
Concentration-related
effects of -amanitin on STC mRNA levels. CS cells in 1.2 mM Ca
were treated with increasing concentrations
of the polymerase II inhibitor
-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
-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.
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;
) or
high (1.9 mM;
) Ca
for 3 days. Then at
time 0, transcription was blocked with
-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
(
), the estimated half-life was
70
h. In the presence of 1.9 mM Ca
(
), STC
mRNA levels remained unchanged for the first 96 h of
-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.
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;
) or high (1.9
mM;
) Ca
for 24 h as described under
``Experimental Procedures.'' At time 0, transcription was
blocked with 2 µg/ml
-amanitin, and STC mRNA levels were
determined 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
150 h, whereas STC mRNA levels were unchanged in the presence of 1.9
mM Ca
(
), 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,
-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
-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
(
), 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
(
), 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.
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
-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 (
), 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 (
) or
with cycloheximide in the presence of normal (
) 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.
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
-amanitin had no major effects on cell viability.
However, as the levels of many mRNAs decline with prolonged exposures
to
-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
-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. (
)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.