(Received for publication, March 15, 1995; and in revised form, June 19, 1995)
From the
Hypertrophy of vascular smooth muscle cells (VSMC) is an important adaptive response of hypertension. Drug intervention studies have implicated a role for angiotensin II (A-II) in the mediation of VSMC hypertrophy in vivo, and A-II is a potent hypertrophic agent for VSMC in culture. Our laboratory has previously shown that A-II-induced hypertrophy of cultured VSMC is due in part to generalized increases in protein synthesis and increased content of rRNA. The aim of the present study was to determine if A-II stimulates rRNA gene synthesis and whether the rRNA transcription factor, upstream binding factor (UBF), is involved. Nuclear run-on analysis demonstrated that A-II induced a greater than 5-fold increase in rRNA gene synthesis within 6 h of stimulation. A-II also stimulated a rapid increase in UBF phosphorylation as well as nucleolar localization, but no changes in the content of UBF. Phosphoamino acid analysis showed that phosphorylation occurred only on serine residue(s). Results demonstrate that increased transcription of ribosomal DNA contributes to the A-II-induced increase in protein synthesis and VSMC hypertrophy, and suggest that an important regulatory event in this pathway may be the phosphorylation and/or nucleolar localization of UBF.
It is well established that arteries from hypertensive patients (1, 2) and animals (3, 4) are thicker
than those from their normotensive counterparts. The arterial medial
thickening is believed to represent an important adaptive response to
normalize the elevated wall stress that occurs secondary to the
increased blood pressure(3) . Previous studies in this and
other laboratories have demonstrated that medial thickening, at least
in large conduit vessels, is due in part to increased vascular smooth
muscle cell (VSMC) ()content or mass, which occurs primarily
by enlargement or hypertrophy of preexisting VSMC, with little to no
change in VSMC number(5, 6, 7) . As such,
there has been considerable interest in identifying cellular mechanisms
that mediate hypertrophic growth of vascular smooth muscle.
There is
clear evidence implicating a role for angiotensin II (A-II) in
mediation of VSMC hypertrophy during chronic hypertension(8) .
For example, angiotensin-converting enzyme inhibitors and A-II receptor
antagonists have been shown to be extremely effective in inhibiting
development of VSMC medial hypertrophy in a variety of hypertensive
animal models(9, 10, 11) . Importantly,
effects of angiotensin-converting enzyme inhibitors or A-II antagonists
do not appear to be due simply to blood pressure lowering, since other
antihypertensive drugs were not as efficacious in blocking hypertrophy
despite similar reductions in blood pressure. Consistent with in
vivo studies, several laboratories, including our own, have shown
that A-II stimulates increased protein synthesis and cellular
hypertrophy in cultured VSMC via stimulation of angiotensin AT receptors(12, 13) . The mechanism of this effect
is not clear. Moreover, our understanding in this area has been
confounded by observations implicating A-II in regulation of VSMC
mitogenesis following vessel injury in vivo(14) . In
general, however, A-II has been shown to have a very low efficacy as a
mitogen for cultured VSMC, and in cases where A-II is mitogenic, its
proliferative effects seem to be mediated by autocrine factors such as
platelet-derived growth factor-AA, transforming growth factor-
,
and/or b-fibroblast growth
factor(15, 16, 17) . In contrast, the
A-II-induced hypertrophy of VSMC appears to be
direct(12, 18) .
There has been considerable
interest in identifying the mechanism and cellular signaling pathways
whereby A-II stimulates VSMC hypertrophy. One approach has been to
attempt to identify which of the many signal transduction pathways
stimulated by A-II (e.g. Ca entry into
cells, increased phosphatidylinositol turnover,
Na
/K
exchange, increased activity of
protein kinase C, and increased mitogen-activated protein kinase
activity) (19, 20, 21, 22) are
required for the hypertrophic response, using various inhibitors of
these pathways. The problem with this approach is the marginal
specificity and unknown actions of many of the available signal
transduction pathway inhibitors and the fact that many of these factors (e.g. intracellular calcium chelators) are known to inhibit
key cellular processes required for growth, e.g. protein
synthesis, even in untreated cells. An alternative approach and one our
laboratory has pursued was to first identify the major structural
proteins that contribute to A-II-induced hypertrophy and then study
mechanisms whereby A-II stimulates their expression. To this end, we
have previously demonstrated that A-II-induced hypertrophy of cultured
VSMC was characterized by selective increases in the expression of a
number of cellular proteins such as smooth muscle (SM)
-actin, SM
-tropomyosin, and SM myosin heavy chain(24) . However, we
and others have also shown that A-II-induced hypertrophy was also
associated with generalized increases in protein synthesis and content,
as well as increased rRNA content(12, 25) . These
latter results indicate that A-II-induced hypertrophy of VSMC is
dependent on increases in the overall translational capacity of the
cell.
Whereas increased rRNA synthesis is absolutely essential for sustained growth of any cell, relatively little is known regarding signal transduction pathways whereby mitogens or hypertrophic agents stimulate such changes. Studies on the regulation of rRNA synthesis in eukaryotes have led to the model that RNA polymerase I activity is regulated by post-translational modification of RNA polymerase I and/or any of the four factors associated with the polymerase, including TIF-1A, TIF-1B, TIF-1C, and upstream binding factor (UBF). Whereas little information concerning the function of the TIF family is available, considerable progress has been made on the identification and characterization of UBF. UBF has been purified to homogeneity from a number of species, and the mammalian form consists of a protein doublet of 97 and 94 kDa referred to as UBF1 and UBF2. It is a member of the high mobility group family of proteins and appears to function both as an enhancer binding protein and transcription factor(26, 27) . It has also been shown to be involved in recruitment or stabilization of RNA polymerase I, as well as functioning as a transcription antirepressor by overcoming transcription inhibition caused by a repressor protein that competes with TIF-1B for DNA binding(28, 29) . Although the mechanism by which UBF regulates rRNA synthesis is unclear, it has been shown that removal of the hyperacidic C-terminal tail, or phosphatase treatment, resulted in a decreased ability of UBF to transactivate rDNA transcription in vitro(30) . O'Mahony et al.(31, 32) have demonstrated that serum deprivation of CHO cells resulted in decreased phosphorylation of UBF and translocation out of the nucleolus. Taken together, these studies strongly suggest that phosphorylation of UBF may be important in the regulation of rRNA gene transcription in response to serum-induced cellular proliferation. However, direct proof for this is currently lacking, and the kinase-signaling pathway that mediates serum-induced changes in UBF phosphorylation has yet to be identified. Moreover, no studies have been performed which investigate the effect of a single purified growth factor on UBF phosphorylation and rRNA gene transcription.
In this report, we demonstrate that stimulation of cultured VSMC with A-II resulted in increased rRNA synthesis and a notable increase in phosphorylation and nucleolar localization of UBF. Importantly, no alterations in cellular UBF content were found. These data provide evidence that the A-II-induced phosphorylation and/or nucleolar localization of UBF may be an important regulatory event in the induction of vascular smooth muscle hypertrophy.
Vascular
smooth muscle cells that are growth-arrested in this fashion show
[H]thymidine labeling induces <5%
incorporation, and no changes in cell number over extended time
periods. Cells remain viable and maintain high levels of expression of
multiple VSMC differentiation marker proteins including SM
-actin,
SM myosin heavy chain, and SM light
chain(33, 34, 35) . In addition, the growth
arrest state is reversible in that VSMC can be readily growth
stimulated with various purified mitogens such as platelet-derived
growth factor-BB or with serum(34, 35) .
Nuclear run-on
reactions utilized equal amounts of cellular DNA. The reaction mixture
contained 0.625 mM ATP, 0.312 mM GTP, 320 µCi of
[P]UTP (>3000 Ci/mMol), 40 mM Tris-HCl, pH 8.3, 150 mM NH
Cl, 7.5 mM MgCl
, and 200 units/ml RNasin. The reaction mix was
incubated for 35 min at 30 C, then DNase (30 units) and CaCl
(1.25 mM) were added and incubated for 30 min.
Extraction buffer (100 µl) was added and the reaction incubated for
2 h at 42 C. The reaction was phenol/chloroform-extracted and
ethanol-precipitated for 30 min. Following centrifugation, the pellet
was recovered in Tris-EDTA buffer, and unincorporated counts were
removed by Sephadex G-50 column chromatography. Equal volumes of eluate
were hybridized to a 5.8-kilobase pair insert of the human 18 S
ribosomal gene which was immobilized to a nylon membrane. Hybridization
was carried out for 24 h at 65 C in 5
SSPE, 10
Denhardt's solution, 1% SDS, 0.5 mg/ml herring sperm DNA, 0.05%
NaPP
. Blots were washed at high stringency according to the
Church and Gilbert (37) method, dried exposed to x-ray film,
and densitometric analysis was performed using a Visage 2000
(BioImage).
Figure 1:
Nuclear run-on analysis demonstrating
the effects of A-II on ribosomal RNA synthesis. Rat aortic VSMC were
treated with A-II or SFM vehicle and harvested for nuclear run-on
analysis. The relative rate of transcription of rRNA genes was measured
using a 5.8-kilobase pair insert of the human 18 S rRNA gene as
described under ``Experimental Procedures.'' Controls using
pBR322 plasmid DNA (or sense non-muscle -actin) showed no signal
above background. Activity was resistant to treatment with
-amanatin at a concentration of 80 µg/ml, at which selectively
inhibits RNA polymerases II and III. Activity was abolished by
treatment with actinomycin D (40 µg/ml), an inhibitor of RNA
polymerases I, II, and II (not shown). Blots were exposed to film for 3
h.
Figure 2:
PhosphorImage illustrating the kinetics of
UBF phosphorylation in response to A-II. Post-confluent growth arrested
VSMC were prelabeled with [P]orthophosphoric
acid and treated with A-II (10
M) or
vehicle (SFM). The cells were harvested and lysates immunoprecipitated
with an antibody to UBF. The immunoprecipitates were analyzed on a 4%
SDS-PAGE, and labeled bands were visualized and quantitated on a
PhosphorImager (6-h exposure). Increased phosphorylation above SFM was
detectable within 15 min and persisted up to 1 h after A-II
treatment.
To
determine if the concentration of exogenous A-II had an effect on the
magnitude of UBF phosphorylation, VSMC were stimulated with different
concentrations of A-II. Since the results from the preceding studies on
the kinetics of UBF phosphorylation showed maximal UBF phosphorylation
at 30 min, this time point was examined. Results demonstrated that the
largest increase in phosphorylation of UBF (241%) was observed at an
A-II concentration of 10M (Fig. 3).
Figure 3: The concentration dependence of A-II on phosphorylation of UBF. Prelabeled VSMC were stimulated for 30 min with different concentrations of A-II or the vehicle SFM. UBF was immunoprecipitated from cell lysates and run on 12% SDS-PAGE mini-gel. Labeled proteins from the immunoprecipitated material were visualized with a PhosphorImager (6-h exposure).
To test whether the A-II induced increase in 18 S rRNA synthesis and/or UBF phosphorylation were associated with increased content of UBF, Western blot analyses were performed using an anti-UBF antibody at 4, 8, and 24 h following A-II or vehicle treatment. Two immunoreactive proteins were detected corresponding to the 94- and 97-kDa UBF isoforms (Fig. 4). Densitometric analysis of the blots showed no significant difference in UBF protein content between A-II and SFM groups at any time point examined (n = 4).
Figure 4:
Western blot analysis of UBF1 and UBF2 in
A-II- and vehicle-treated VSMC. Post-confluent VSMC were
growth-arrested in SFM and stimulated for 4, 8, and 24 h with A-II
(10M) or SFM vehicle. Cells were
harvested, and equal amounts of cellular protein were analyzed by
SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with a
rabbit anti-UBF antibody. Two immunoreactive proteins corresponding to
UBF1 and UBF2 were detected. No reactivity was observed when the
membrane was immunoblotted with control rabbit
serum.
Figure 5: Phosphoamino acid analysis of UBF from A-II- and SFM-treated cells. UBF immunoprecipitates were transferred onto a PVDF membrane, and the labeled bands were cut out and acid hydrolyzed. The sample was mixed with phosphotyrosine (P-Tyr), phosphothreonine (P-Thr), and phosphoserine (P-Ser), spotted on a thin layer cellulose plate (origin), and electrophoresed toward the anode. The dashed circles indicate the migration of the phosphorylated standards.
Figure 6: Confocal images of VSMC immunostained for UBF after A-II and SFM treatment. Post-confluent growth arrested VSMC were treated with either SFM (a) or AII (b) for 15 min and fixed in 3% formaldehyde. The cells were immunostained for UBF and visualized under a confocal microscope.
Figure 7: CKII-induced phosphorylation of UBF in vitro. An in vitro kinase reaction (total volume of 200 µl) was set up as described under ``Experimental Procedures.'' At specific times, 20 µl from the reaction mixture were removed and added to a tube containing SDS-sample buffer to terminate the reaction. The samples were run on a 12% SDS-PAGE and exposed to film for 6 h. As can be seen, increasing the incubation time of the reaction resulted in increased phosphorylation of recombinant UBF (rUBF). No labeled proteins were observed when CKII was omitted from the reaction mixture (not shown).
To determine if A-II altered CKII activity in vivo, cultured VMSC were treated for various times with A-II and SFM, and assayed for in vitro CKII activity using casein as a substrate. No significant alteration in CKII activity was observed at any time point after A-II stimulation, nor between A-II and SFM (n = 4) (Fig. 8). Although this result does not rule out the possibility that CKII phosphorylates UBF in vivo, it suggests that CKII is not the kinase responsible for the A-II induced increase in UBF phosphorylation.
Figure 8: The effect of A-II treatment on endogenous CKII activity. Post-confluent growth-arrested VSMC were stimulated for various times with A-II or the vehicle SFM. The cells were harvested, and in vitro kinase reactions were performed using casein as a substrate. The amount of phosphorylated casein was determined by Cerenkov counting and normalized to total protein.
A-II-induced hypertrophy of vascular smooth muscle cells in
culture has been shown to be associated with a generalized increase in
protein synthesis as well as selective increases in synthesis of
cell-specific proteins such as SM -actin and SM myosin heavy chain (23, 24) . The former observation suggests that at
least part of the hypertrophic effect of A-II is due to an alteration
in the translational capacity and/or activity of the cell. A-II
stimulation has been shown to increase RNA
content(12, 25) , an obvious requisite step for
increasing the translational capacity of the cell. Results from nuclear
run-on assays in the present study demonstrate that A-II induces a
transient yet marked increase in 18 S rRNA synthesis, as well as
increased phosphorylation and nucleolar localization of the rRNA
transcription factor UBF. The preceding are three cellular processes
that might be expected to serve as a regulatory event for the enhanced
protein synthesis characteristic of VSMC hypertrophy.
Our results are consistent with those of O'Mahony et al.(31, 32) implicating an important role for UBF phosphorylation in regulation of serum-induced proliferation and increased rDNA transcription in CHO cells. Importantly, however, this study is the first to demonstrate such an effect in response to a known ligand (A-II), rather than serum. Moreover, an important distinction between our study and previous work is that A-II induces hypertrophy not hyperplasia under the conditions of our experiments(12) . As such, this is the first investigation to provide evidence for the involvement of UBF phosphorylation and/or nucleolar localization in the hypertrophic response of vascular smooth muscle to A-II treatment.
Although the cells used in these experiments were growth-arrested in serum-free medium, complete cessation of protein synthesis does not occur, thus the transcriptional and translation machinery of the cell must still be active. It is therefore reasonable to assume that there is a basal rate of rRNA transcription as evidenced by the run-on signal under control conditions (i.e. SFM). Also, the immunoprecipitates from prelabeled cells reveal there is some basal phosphorylation of UBF with SFM treatment. Phosphoamino acid analysis revealed that only serine residues are phosphorylated in both control and A-II stimulated conditions. However, we cannot ascertain from this experiment whether the increase in phosphorylation that occurs after A-II treatment is due to phosphorylation of a new site(s) or increased phosphate incorporation onto the basal site(s).
The kinase responsible for phosphorylation of UBF in vivo has not been determined. Casein kinase II is an attractive candidate for the UBF kinase for several reasons. First, CKII phosphorylates UBF obtained from a variety of sources. Second, this kinase has been shown to be associated with RNA polymerase I in the nucleolus and to phosphorylate several nuclear proteins(39, 40, 41) . Third, the activity of CKII has been reported to fluctuate with the growth rate of cells, and mirror the activity of rDNA transcription(42) . Results of the present studies show that although CKII is able to phosphorylate recombinant UBF in vitro, the activity of CKII in vivo is unchanged after A-II treatment. This does not necessarily eliminate a possible role for CKII-mediated phosphorylation of UBF in vivo, however, our results suggest that CKII is not the A-II-inducible kinase responsible for increased UBF phosphorylation. CKII may, however, be important as a kinase that is constitutively active, or its regulatory activity may be dependent upon localization of UBF. We are currently investigating whether CKII is actually the kinase that phosphorylates UBF in vivo by comparative two-dimensional phosphotryptic mapping of UBF phosphorylated in vivo (SFM and A-II) versus purified recombinant UBF phosphorylated in vitro with CKII.
The distribution of UBF at various stages of the cell cycle and in response to serum stimulation has been studied to investigate the relationship between UBF localization and rDNA transcription(32, 38) . In all cases, little or no UBF labeling was observed outside the nucleus. During stages of the cell cycle when transcription was elevated, UBF was found to accumulate within the nuclear organization regions, which are known to contain the rDNA and therefore are the sites of ribosomal synthesis. This group described the UBF staining as ``beads or granules'' within the nucleus, with each bead thought to represent a transcriptional unit. The immunolocalization studies presented in this report fit with these results in that, after A-II treatment, small clusters of UBF accumulated within the nucleus. Since we have also shown that A-II increases 18 S ribosomal gene transcription, these two pieces of evidence complement each other and add credence to the concept that the increased protein synthesis associated with A-II is due in part to increased ribosomal gene transcription mediated via the activation and/or localization of UBF.
The results from our experiments which determined the kinetics of UBF phosphorylation along with the immunolocalization data may provide some insight into the mechanism of UBF activation. Since UBF forms part of the transcription initiation complex at the nuclear organization region, it seems likely that translocation of UBF inside the nucleolus should precede its transcription regulatory activity. We have shown that nucleolar accumulation of UBF is observed as early as 15 min after A-II treatment. However, the phosphorylation data revealed that maximal phosphorylation of UBF did not occur until 30 min after A-II treatment. Taken together, these results suggest that phosphorylation of UBF is not necessary for nucleolar translocation into the nucleolus. The mechanisms whereby A-II stimulates nucleolar accumulation of UBF are not known. In addition, there is a lack of direct evidence that UBF phosphorylation regulates rDNA transcription in vivo, although there is strong evidence that phosphorylation of UBF regulates its transcriptional activity in vitro(31, 32) . The mechanisms whereby phosphorylation of UBF could regulate rDNA transcription are not clear, but may involve: (a) increasing the affinity of UBF for the upstream core element; (b) increasing its affinity for other transcription factors (e.g. TIF-1B); or (c) increasing its affinity for RNA polymerase I.
In summary, results from the present study demonstrate that A-II stimulates increased rRNA synthesis as well as increased phosphorylation and nucleolar localization of the rRNA transcriptional factor UBF. These findings are consistent with previous studies which have shown increased rDNA transcription and phosphorylation of UBF in response to serum-induced mitogenesis. However, the present studies are the first to demonstrate such effects utilizing a single well defined agonist which induces hypertrophic, rather than mitogenic, growth. Further studies are required to determine the signaling pathway that mediates phosphorylation of UBF and to determine the mechanisms whereby phosphorylation of UBF might regulate rRNA synthesis in vivo.