(Received for publication, October 27, 1994; and in revised form, January 4, 1995)
From the
Ribosomal DNA transcription is important to the regulation of
cardiomyocyte ribosome content and, as a consequence, the rate of
protein synthesis and accumulation during cardiac hypertrophy. We
studied the regulation of ribosomal RNA synthesis and the levels of RNA
polymerase I and the ribosomal DNA transcription factor, UBF, during
norepinephrine-induced hypertrophy of contraction-arrested neonatal
cardiomyocytes in culture. Nuclear run-on assays and Western blots
demonstrated that, concomitant with hypertrophy, norepinephrine (1
µM) increased the rate of ribosomal DNA transcription,
without causing an increase in the amount of RNA polymerase I. However,
the elevated rate of rRNA synthesis was accompanied by an increased
cellular content of UBF protein as determined by Western analysis.
Northern blots demonstrated norepinephrine-induced increases in UBF
mRNA in neonatal cardiomyocytes indicating that the response was
regulated, at least in part, at the pretranslational stage. Both -
and
-adrenergic agents increased the level of UBF mRNA. The
-adrenergic response was mimicked by forskolin (1 µM)
and the cyclic AMP analog dibutyryl cAMP (10 µM). However,
activation of protein kinase C by phorbol 12-myristate 13-acetate (0.1
µM) did not increase expression of UBF. These results
implicate UBF as a possible regulatory factor of the accelerated rDNA
transcription observed during norepinephrine-mediated cardiomyocyte
hypertrophy.
The cardiomyocytes of the adult heart respond to growth stimuli
by increasing in size in the absence of cellular division
(hypertrophy). A variety of model systems have been employed to examine
myocyte hypertrophy. Using these systems, the stimuli which either
directly or indirectly modulate cardiomyocyte growth have been
identified. These include mechanical stimuli such as active or passive
stretch, hormones such as norepinephrine (NE), ()endothelin,
angiotensin, or thyroid hormone, and various peptide growth
factors(1, 2) . One finding common to these reports is
that cardiomyocyte growth results mainly from an increase in general
protein synthesis and total RNA accumulation in the absence of changes
in the fractional rate constants of protein and RNA
degradation(3) . Experiments in several systems indicate that
the rate of general protein synthesis is limited by the protein
synthetic capacity of the cardiomyocytes, i.e. the cellular
ribosome content(4, 5, 6) . This suggests
that ribosome biogenesis is the major rate-limiting step in the
accumulation of protein during hypertrophy. In fact, McDermott et
al.(6, 7) demonstrated that the increased rates
of ribosomal RNA (rRNA) synthesis during beatinginduced hypertrophy of
cultured, neonatal cardiomyocytes resulted from elevated transcription
of the rRNA genes (rDNA) that encode the 45 S precursor of the 18 S,
5.8 S, and 28 S rRNAs. Taken together, these studies implicate
transcription of the ribosomal RNA genes (rDNA transcription) as a
control point in the regulation of cardiomyocyte protein synthesis and
consequently cardiac growth. As ribosomal DNA transcription constitutes
approximately 40-60% of the total nuclear RNA synthesis, even a
relatively small increase in this activity would represent a
significant increase in total cellular protein synthetic capacity.
Therefore, the elucidation of the molecular mechanisms by which rDNA
transcription is regulated will be of significant importance to the
general understanding of cardiac hypertrophy during both normal and
disease states.
Despite species-specific variations in their utilization, mammalian cells appear to utilize a set of highly homologous, if not identical, factors for transcription of the ribosomal DNA genes(8, 9) . At least two DNA-binding proteins, referred to as UBF and SL-1, as well as RNA polymerase I, are required for efficient transcription of ribosomal DNA promoters in vitro. The ability of RNA polymerase I to recognize the protein-DNA complexes which form on the promoter requires the presence or activity of at least one polymerase-associated factor referred to as either Factor C*, TF-1C, or TIF-IA. SL-1 is absolutely required for transcription, while UBF appears to be an auxiliary transcription factor. That is, UBF is not required for basal levels of transcription in vitro(10) . However, the addition of UBF to an in vitro transcription system increases the efficiency of template utilization(11) , and over-expression of UBF in NIH3T3 cells leads to increased expression of a co-transfected reporter gene under the control of the rDNA promoter(12) . UBF is a dimeric, DNA-binding protein. SDS-PAGE analysis of UBF purified from mammalian cells demonstrates two proteins of molecular sizes 97 and 94 kDa, referred to as UBF1 and UBF2, respectively(8, 9) . These two proteins are coded for by two different mRNAs, which result from alternative processing of a single transcript(13) . Both UBF1 and UBF2 can bind to DNA and form homo- and heterodimers. However, only UBF1 has been shown to activate rDNA transcription(10, 12) .
UBF is subject to regulation by at least two different mechanisms. O'Mahony et al.(14, 15) and Voit et al.(16) demonstrated that UBF is a phosphoprotein and that the activity of UBF was reduced when the protein was dephosphorylated in vitro. Further, our laboratory demonstrated that when CHO cells were serum-starved, and rDNA transcription significantly reduced, the phosphorylation state of UBF was also reduced(15) . We have also found that cells might regulate UBF and, by analogy, the rate of rDNA transcription, by altering the cellular content of UBF. When L6 myoblasts are induced to differentiate, there is a decrease in the rate of rDNA transcription of approximately 80%(17) . The amount of UBF present in these cells decreased in parallel with the decrease in the rate of rDNA transcription. Interestingly, the level of the UBF mRNA decreased with more rapid kinetics, suggesting that at least one rate-limiting step in regulating rDNA transcription during this process was transcription of the UBF gene(17) .
In the studies
presented here, we have examined rDNA transcription and the levels of
both the RNA polymerase I ` subunit and an rDNA transcription
factor, UBF, during norepinephrine-induced cardiomyocyte hypertrophy.
Specifically, we sought to 1) characterize the changes in ribosomal
transcription during norepinephrine-induced cardiac hypertrophy, 2)
assess whether the levels of RNA polymerase I and/or UBF protein were
increased in response to norepinephrine treatment, and, if so, 3)
determine whether the quantitative and temporal patterns of expression
of these proteins were consistent with the hypothesis that they might
be involved in the modulation of the observed increases in rDNA
transcription and consequent protein accumulation.
We show here that
when neonatal cardiomyocytes undergo hypertrophy as the result of
norepinephrine treatment, they exhibit increased rates of rDNA
transcription as well as increased amounts of UBF protein. In contrast,
the amount of RNA polymerase I (reflected by the levels of the
polymerase I ` subunit) was not altered by norepinephrine. The
increased UBF protein levels were regulated at least partially at the
pretranslational level since cardiomyocytes also demonstrated transient
increases in UBF mRNA. Both
- and
-adrenergic agents
increased the level of UBF mRNA. The
-adrenergic response could be
mimicked by forskolin and cyclic AMP (cAMP) analogs. On the other hand,
activation of protein kinase C by phorbol 12-myristate 12-acetate
(PMA), which has been implicated in the
-adrenergic
response(1, 2) , did not result in increased levels of
UBF. These represent the first experiments linking stimulation of
cardiomyocyte growth to the induction of a factor known to be
intimately involved in rDNA transcription. These results are consistent
with the hypothesis that UBF is an important regulatory factor in
ribosome biogenesis during norepinephrine-induced neonatal
cardiomyocyte growth.
Specific rDNA transcription was determined by the hybridization of run-on transcripts to 45 S rDNA (clone pU5.1E/X) immobilized on Zeta-Probe nylon membranes. The hybridization conditions and the post-hybridization washes were the same as described(24) . Control pUC19 template was also immobilized on the filters to control for nonspecific hybridization. Hybridization was detected by autoradiography and quantified using a laser densitometer (Molecular Dynamics) and an Ambis 4000 radioanalytic imager (Ambis Systems).
Neonatal
cardiomyocytes were prepared and cultured at a relatively high density
(4 10
cells/60-mm dish) in the presence of 50
mM KCl as described under ``Materials and Methods.''
Experiments were initiated after 3 days in culture, to allow the
cardiomyocytes to adapt to the culture
conditions(6, 7) . During the subsequent 4 days in
culture, the control cardiomyocytes demonstrated no significant growth.
The protein to DNA ratios of control cultures varied by 4.3% ±
2.9 over this time period. These findings are in accordance with those
obtained by others using similar culture
conditions(6, 7) . However, cells plated at high
density and exposed to 1 µM norepinephrine for 1-4
days demonstrated a significant accumulation of total cellular protein,
as reflected by increased protein to DNA ratios with respect to control
cells. After 2, 3, and 4 days of treatment with norepinephrine, total
cellular protein levels had increased by 27.9% ± 4.9, 25.71%
± 4.33, and 25.89% ± 5.72 respectively, as compared to
time-matched control cells. Treatment with norepinephrine did not
significantly increase the DNA content of the cardiomyocytes (data not
shown) indicating that growth was the result of hypertrophy rather than
hyperplasia. The growth of these cardiomyocytes was not due to
norepinephrine-induced contractile activity since the cardiomyocytes
were contraction-arrested with 50 mM KCl.
Similar increases
in total protein and protein to DNA ratios (31.2% ± 5.6 increase
in protein to DNA ratio after 4 days) in response to norepinephrine
were observed in nonbeating cardiomyocytes plated at a relatively low
density (0.5 10
cells/60-mm plate). These results
are in agreement with the reports of others(18, 26) .
In order to confirm that the norepinephrine-induced increases in cellular protein levels were accompanied by increased 45 S rRNA synthesis, we measured the rate of rDNA transcription in nuclei isolated from control and norepinephrine-stimulated neonatal cardiomyocytes. The results presented in Fig. 1A demonstrate that the rate of RNA polymerase I transcription in nuclei derived from norepinephrine-treated neonatal cardiomyocytes maintained at either high density or low density was significantly greater than that derived from control cells. Further, the increased RNA polymerase I transcriptional activity in nuclei isolated from norepinephrine-treated neonatal cardiomyocytes maintained at low density was independent of contractile activity since inclusion of KCl (50 mM) in the media did not significantly reduce the norepinephrine-induced increase in the hybridization signal (Fig. 1A, slots 4 and 5). The specificity of hybridization was verified by the lack of hybridization to pUC19 DNA. The maximal increase in the rate of rDNA transcription occurred after 24-48 h of continuous norepinephrine treatment (57% ± 10.5 and 58% ± 8.8, respectively) and remained significantly above basal levels (49% ± 10.1) after 3 days of exposure to the agent (Fig. 1B).
Figure 1:
rDNA
transcription in cultured neonatal cardiomyocytes following
norepinephrine (NE) stimulation. A, neonatal
cardiomyocytes, cultured as indicated, were stimulated for 6-72 h
with NE (1 µM), and nuclei were isolated from 16
10
cells per time point. When indicated, KCl was added to a
final concentration of 50 mM. RNA polymerase I transcription
was measured by incubating the isolated nuclei at 37 °C in the
presence of [
P]UTP and
-amanitin as
described under ``Materials and Methods.'' Radiolabeled rRNA
transcripts were purified from the reaction mixture and hybridized to
45 S rDNA (clone pU4.5E/X) or control pUC19 DNA, and, after stringent
washes, the hybrids were visualized by autoradiography. B, the
radioactivity of 45 S run-on transcripts obtained from a number of
separate experiments were quantified by laser densitometry and are
presented as the percent increase in rDNA transcription in response to
NE over the transcription observed in time-matched control cells. For
these experiments, the cells were cultured at 4
10
cells/60-mm plate, and contraction was arrested by the addition
of 50 mM KCl. The vertical lines indicate the
standard deviation from the mean of 5 or more separate
experiments.
These results demonstrate that norepinephrine-induced hypertrophy of neonatal cardiomyocytes is accompanied by a sustained elevation in the rate rDNA transcription and that this response is independent of the density at which the cells were plated. These findings are in accordance with the model that increased rDNA transcription is a pivotal point in the regulation of ribosome accumulation and the increased rate of protein synthesis prerequisite for the growth of cardiomyocytes(3) .
Figure 2:
RNA polymerase I ` subunit (rPOLI
`) levels in neonatal cardiomyocytes following
norepinephrine (NE) stimulation. A, total cellular
protein extracted from exponentially growing rat Novikoff hepatoma
cells (lanes 1 and 2) was fractionated by SDS-PAGE
(25 µg/lane), transferred to a nylon membrane, and incubated with
either preimmune antisera (lane 1) or antisera obtained from
the same rabbit after immunization with recombinant rPOLI
` (lane 2). rPOLI
` was subsequently visualized by ECL as
described under ``Materials and Methods.'' B, total
cellular protein was extracted from contraction-arrested, neonatal
cardiomyocytes cultured at 4
10
cells/60-mm plate
at the times indicated after treatment with vehicle (0.1% ascorbic
acid, lanes 1 and 3) or NE (1 µM, lanes 2 and 4). After SDS-PAGE and Western transfer,
rPOLI
` protein was detected with a rabbit anti-rPOLI
`
antibody and visualized by ECL as described under ``Materials and
Methods.'' Equal amounts of protein (25 µg) were loaded per
lane.
Total protein was extracted
from arrested cardiomyocytes treated with vehicle or norepinephrine for
24 or 48 h, and the levels of rPOLI` protein were determined by
SDS-PAGE and Western analysis. As shown in Fig. 2B,
after 24 and 48 h of treatment with norepinephrine, the level of
rPOLI
` protein in neonatal cardiomyocytes was not significantly
altered relative to time-matched control cells. In fact, the level of
rPOLI
` did not change in response to norepinephrine at any time
point examined (12-96 h, data not shown). These results indicate
that it is unlikely that an increase in the amount of the RNA
polymerase I enzyme contributes to the elevated rate of rDNA
transcription observed during norepinephrine-induced hypertrophy of
neonatal cardiomyocytes.
The addition of UBF to cell-free transcription assays has been shown to increase rDNA transcription in a dose-dependent manner(11) . Moreover, overexpression of UBF in cells has been shown to augment transcription from a co-transfected ribosomal DNA promoter(12) . Accordingly, we ascertained whether the increased rDNA transcription observed during norepinephrine-induced hypertrophy of neonatal cardiomyocytes, was associated with increased amounts of UBF protein.
Total protein was extracted from arrested cardiomyocytes treated with vehicle or norepinephrine for 6-72 h, and the levels of UBF1 and UBF2 protein were determined by SDS-PAGE and Western analysis. As shown in Fig. 3, A and D, cardiomyocytes treated with norepinephrine exhibited elevated levels of UBF protein as compared to time-matched control cells. The UBF levels were maximal after 12-24 h. However, even after 48 h of treatment with norepinephrine, the absolute amount of UBF in the treated cells was greater than that in the control cells. Since equal amounts of protein were loaded per lane, the changes in the level of UBF were not merely the consequence of the general increase in protein content which is observed in norepinephrine-treated cells.
Figure 3:
Treatment of neonatal cardiomyocytes with
norepinephrine (NE) leads to increased levels of UBF protein
and mRNA. A, total cellular protein was extracted from
contraction-arrested, neonatal cardiomyocytes cultured at 4
10
cells/60-mm plate at the times indicated after treatment
with vehicle (0.1% ascorbic acid, lanes 1 and 3) or
NE (1 µM, lanes 2 and 4). After SDS-PAGE
and Western transfer, UBF1 and UBF2 protein was detected with a rabbit
anti-UBF antibody and visualized by ECL as described under
``Materials and Methods.'' Equal amounts of protein (25
µg) was loaded per lane. B, total RNA was extracted from
contraction-arrested, neonatal cardiomyocytes cultured at 4
10
cells/60-mm plate at the times indicated after treatment
with vehicle (0.1% ascorbic acid, lanes 1, 3, and 5) or NE (1 µM, lanes 2, 4, and 6). After electrophoresis and Northern blotting, the RNA (30
µg) was hybridized to
P-labeled UBF cDNA, and the UBF
mRNA transcripts were visualized by autoradiography. C, total
RNA was extracted from neonatal cardiomyocytes cultured at a density of
0.5
10
cells/plate at the various times indicated
following exposure to vehicle (0.1% ascorbic acid, lanes 1, 2, and 5) or NE (1 µM, lanes 3, 4, and 6). After electrophoresis and Northern
blotting, the RNA was hybridized to
P-labeled UBF cDNA and
UBF mRNA transcripts visualized by autoradiography. Where indicated,
KCl was included in the media to a final concentration of 50
mM. D, the experiments depicted in A and B were repeated a number of times, and the results were
quantified by laser densitometry. After normalization for load, the
results were expressed as the -fold increase in signal obtained from
NE-treated cells over the signal obtained from time-matched control
cells. Vertical lines represent the standard deviation from
the mean of 5 or more separate experiments.
The alteration in the cellular content of a protein can reflect changes in the turnover of that protein (post-translational) and/or alterations in the levels of its mRNA (pretranslational). In order to determine whether the norepinephrine-induced increase in UBF protein reflected either of these stages of regulation, UBF mRNA levels were measured in the total RNA extracted from control and norepinephrine-treated high density cardiomyocyte cultures. The results presented in Fig. 3B demonstrate that cardiomyocyte UBF mRNA levels were rapidly and significantly (3-4-fold) increased after 12 h of treatment with norepinephrine compared to the level found in time-matched control cells (Fig. 3B, lanes 1 and 2). Northern blots do not resolve the two mRNAs for UBF1 and UBF2(13) . Interestingly, and in contrast to the pattern demonstrated by the UBF protein levels, the increase in UBF mRNA levels was transient. After 24 h of treatment, the norepinephrine-treated cells exhibited UBF mRNA levels similar to those found in untreated control cells. The integrity of the RNA samples and the amounts loaded were determined by quantifying the fluorescence pattern of the 18 S ribosomal RNA observed after transfer to the Zeta Probe nylon membrane (Fig. 3B).
Recent studies have revealed that the
alterations of specific transcription events observed in response to
norepinephrine may vary with the density at which the cardiomyocytes
are cultured(27) . In order to determine if the observed
induction of UBF by norepinephrine was density-dependent, we also
examined UBF mRNA levels in cardiomyocytes maintained at a relatively
low density. As shown in Fig. 3C, cardiomyocytes
cultured at 0.5 10
cells per 60-mm culture plate
and treated with norepinephrine exhibited increases in UBF mRNA levels
that were quantitatively similar to those observed in high density
cultures (Fig. 3C, lanes 1-4). Once
again, the observed induction cannot be explained by
norepinephrine-induced increases in contractile activity of the
cardiomyocytes as the inclusion of 50 mM KCl in the media did
not significantly alter the increased UBF mRNA levels (Fig. 3C, lanes 5 and 6). The
norepinephrine-induced increases in UBF protein and mRNA levels
exhibited by 4-5 separate, high density cardiomyocyte
preparations were quantitated by laser densitometry and are presented
graphically in Fig. 3D. These results suggested that in
high and low density cultures of cardiomyocytes, at least part of the
response that leads to increased levels of UBF protein is
pretranslational.
We also found that cardiomyocyte cultures purified by centrifugation through Percoll gradients (28) exhibited the same response, i.e. the induction of UBF mRNA in response to NE, as observed in less rigorously prepared cultures (results not shown). Thus, we conclude that the observed increase in UBF protein and mRNA levels reflect a direct affect of norepinephrine upon the cardiomyocytes themselves and is not mediated by other cell types.
Figure 4:
Accumulation of UBF mRNA in neonatal
cardiomyocytes in response to - and
-adrenergic stimulation. A, total RNA was extracted from arrested neonatal
cardiomyocytes at the times indicated following exposure to vehicle (CTL, 0.1% ascorbic acid, lanes 1 and 2),
norepinephrine, 1 µM (NE, lane 3), a
combination of the
-adrenergic agonist phenylephrine,
10 µM, and the
-adrenergic antagonist propranolol, 5
µM (Phe + Prop, lane 4),
phenylephrine alone (lane 5), or the
-adrenergic agonist
isoproterenol, 1 µM (Iso, lane 6). All
other culture conditions were as described in the legend to Fig. 1. After electrophoresis and Northern blotting, the RNA (30
µg) was hybridized to
P-labeled UBF cDNA, and the UBF
mRNA transcripts were visualized by autoradiography. B, the
experiments in A were repeated a number of times, and the UBF
hybridization signals were quantitated by laser densitometry. After
normalization for loading, the results were expressed as the -fold
increase in signal obtained from NE-treated cells over the signal
obtained from time-matched control cells. Vertical lines represent the standard deviation from the mean of 3 or more
separate experiments.
Activation of the
-adrenergic receptors on neonatal cardiomyocytes leads to
activation of adenylyl cyclase and the subsequent activation of protein
kinase A in response to the increased cellular content of
cAMP(1) . Since pathways distal to cAMP have been implicated in
the regulation of nuclear trans-acting factors(29) ,
we examined the effect of forskolin, a drug known to increase cellular
cAMP levels, on the expression of UBF. Treatment of cardiomyocytes with
forskolin (1 µM) led to an increase in the level of
cardiomyocyte UBF mRNA after 12 h of treatment (Fig. 5, lane
3), similar to that observed in response to norepinephrine (Fig. 5, lane 2). Additional studies demonstrated that
the effect of
-adrenergic activation on UBF expression could be
mimicked by addition of the membrane-soluble cAMP analog, dibutyryl
cAMP (10-100 µM) to the cells (results not shown).
These studies demonstrate that cellular signals downstream of cAMP may
regulate induction of UBF in response to
-adrenergic agonists.
Figure 5:
UBF mRNA levels in neonatal cardiomyocytes
following treatment with norepinephrine, forskolin, or PMA. Total RNA
was extracted from arrested neonatal cardiomyocytes after 12 h of
treatment with vehicle (0.1% ascorbic acid, 0.001% dimethylsulfoxide, lane 1), norepinephrine (NE, 1 µM, lane 2), forskolin (1 µM, lane 3), or
phorbol 12-myristate 13-acetate (PMA, 0.1 µM, lane 4). After electrophoresis and blotting, the RNA (30
µg) was hybridized to P-labeled UBF cDNA, and the UBF
mRNA transcripts were detected by autoradiography. The experiment was
repeated three times, and a representative autoradiogram is
shown.
Activation of the -adrenergic receptors leads to the release of
inositol 1,4,5-trisphosphate and sn-(1,2)-diacylglycerol via
phospholipid hydrolysis(30, 31) . Diacylglycerol is
the endogenous activator of the phospholipid-dependent protein kinase C
family; a group of kinases that have been variously implicated in the
activation of nuclear trans-acting
factors(26, 32, 33) . Accordingly, we also
examined whether PMA, a synthetic activator of protein kinase C, might
also increase UBF mRNA levels in neonatal cardiomyocytes.
Interestingly, treatment of cells with PMA (0.1 µM) for 12
h did not increase UBF mRNA above basal levels (Fig. 5, lane
4) nor was an elevation of UBF mRNA level observed at any other
time point investigated (3-72 h). In addition, treatment with PMA
did not affect cardiomyocyte UBF protein levels at any time studied
although, in agreement with the findings of others(26) , this
treatment did lead to significant activation of protein kinase C in
these cells (results not shown).
Cardiomyocyte hypertrophy requires increased ribosome
synthesis in order to produce more protein since the pool of existing
ribosomal subunits are already engaged in synthesizing
proteins(3) . Hence, studies examining the mechanism of
ribosome biogenesis in cardiomyocytes are of importance to the general
understanding of cardiac growth. We have examined the hypothesis that
the adrenergic mediated hypertrophy of cultured neonatal cardiomyocytes
is associated with increased rates of ribosomal RNA synthesis.
Secondly, we examined possible mechanisms through which the
cardiomyocytes might increase the rate of rDNA transcription.
Specifically, we have demonstrated that norepinephrine-induced
cardiomyocyte hypertrophy is accompanied by increases in the rate of
rDNA transcription. Significantly, the amount of RNA polymerase I (as
reflected by the amount of the ` subunit of the enzyme) does not
change, but the amount of the rDNA transcription factor, UBF, does.
Furthermore, we have demonstrated that the levels of UBF mRNA in
neonatal cardiomyocytes increase in response to both
- and
-adrenergic stimulation and have presented preliminary data toward
defining the signal transduction pathways involved in this process.
We next examined the levels of the rDNA transcription factor UBF during norepinephrine-induced cardiac hypertrophy. We have previously demonstrated that the addition of UBF to cell-free extracts depleted of UBF increases transcription from the ribosomal DNA promoter in a dose-dependent manner(11) . Further, overexpression of UBF has been shown to increase transcription from a co-transfected 45 S gene construct(12) . These two lines of evidence suggest that alterations in the amount of UBF is one mechanism by which rDNA transcription by polymerase I can be regulated. We report here that increased ribosomal RNA synthesis observed in neonatal cardiomyocytes following norepinephrine stimulation is associated with elevated levels of both UBF1 and UBF2 mRNA and protein. The maximal increase in UBF protein occurred within 12-24 h of norepinephrine treatment and was coincident with the time when the rate of rDNA transcription rate reached its maximum. Thus, the temporal profile of UBF protein induction is consistent with the hypothesis that this nucleolar transcription factor is important to both the initiation and maintenance of accelerated rDNA transcription observed during norepinephrine-induced hypertrophic growth of neonatal cardiomyocytes. During the first 12 h of norepinephrine treatment, there was a significant accumulation of UBF mRNA. We are currently using nuclear run-on experiments to determine whether alterations in the rate of transcription can account for the observed increased in UBF mRNA levels(36) .
Interestingly, and in contrast to the observed increase in UBF protein, the induction of UBF mRNA in response to norepinephrine was transient. While the UBF protein levels remained elevated for 48-72 h, the UBF mRNA levels returned to the level found in control cells within 24 h. This pattern contrasted with our previous observations of the regulation of UBF in differentiating myoblasts(17) . When L6 cells differentiated, there was a direct correlation between the reduction in UBF mRNA and UBF protein since they decreased in parallel. Comparing that study with the results presented here suggests that UBF expression during hypertrophy may also be subject to post-translational regulation, i.e. protein stabilization.
Stimulation of -receptors in
cardiomyocyte membranes leads to activation of protein kinase A in
response to an increased cellular content of cAMP(29) . In
turn, activated protein kinase A phosphorylates a number of
transcription factors including cAMP response element binding protein,
cAMP response element modulator, and activating transcription factor 1 (29) which bind to their respective response elements in the
nucleus and result in altered patterns of gene transcription. In the
present study, the
-adrenergic induction of UBF mRNA could be
mimicked by treatment of the cells with the membrane-permeable cAMP
analog, dibutyryl cAMP, or with forskolin, a drug which elevates
cellular cAMP levels. Thus, it is likely that pathways distal to cAMP
accumulation, such as protein kinase A activation, mediate
-adrenergic increases in UBF mRNA levels in neonatal
cardiomyocytes.
The protein kinase C family has been implicated in
the hypertrophic response of neonatal cardiomyocytes to
-adrenergic agents, although their exact role is still
controversial(38) . However, in these experiments, the
-adrenergic induction of UBF mRNA and protein could not be
reproduced by exposure of neonatal cardiomyocytes to PMA, a
membrane-permeable synthetic activator of the protein kinase C family.
These results suggest that activation of protein kinase C does not, or
is not sufficient by itself, to increase UBF levels in neonatal
cardiomyocytes. However, one cannot extrapolate the effects of
synthetic activators of protein kinase C, such as PMA, to those of the
endogenous activators of protein kinase C, such as norepinephrine,
without caution(38) . For example, chronic treatment of
cardiomyocytes with PMA down-regulates certain protein kinase C
isoforms, while chronic treatment with norepinephrine up-regulates
others(38, 39) . Furthermore, some members of the
protein kinase C family are ``atypical'' in that they are not
Ca
-dependent or are phorbol
ester-insensitive(39, 40) . Thus, differences in the
exact subset of protein kinase C isoforms which are activated and/or
their intracellular translocation may also contribute to discrepancies
in the results obtained from norepinephrine- and PMA-treated cells.
It is possible that the regulation of the amount or activity of UBF
is a conserved phenomenon important to the increased rate of ribosome
biogenesis induced by diverse hypertrophic stimuli. In support of this
hypothesis, we have preliminary data indicating that UBF mRNA and
protein levels are up-regulated during neonatal cardiac hypertrophy
stimulated by contraction and serum. However, to
definitively determine the effect of elevated levels of UBF on the rate
of rDNA ribosomal transcription and protein synthesis in cardiomyocytes in vivo will require further experiments. We are currently
assessing the effect of overexpressing UBF in neonatal cardiomyocytes
on transcription of a co-transfected reporter gene under the control of
the rDNA promoter. Further studies concerning the regulation of the UBF
gene may provide clues as to the pathways by which
- and
-adrenergic receptors contribute to cardiac hypertrophy.
The studies described in this report emphasize rDNA transcription as an important step in the regulation of ribosome content and, subsequently, protein synthesis during norepinephrine-induced hypertrophy of neonatal cardiomyocytes. Furthermore, this is the first report linking adrenergic stimulation of neonatal cardiomyocyte growth to the regulation of a nuclear factor, UBF, known to be intimately associated with rDNA transcription. These findings should now allow us to define the regulatory pathways which connect hypertrophic stimuli to an increase in ribosome biogenesis during more physiologically relevant settings such as the hypertrophy of adult cardiomyocytes in vivo.