Regulation of rDNA Transcription During Endothelin-1Induced Hypertrophy of Neonatal Cardiomyocytes
Hyperphosphorylation of Upstream Binding Factor, an rDNA Transcription Factor
Abstract Treatment of cultured neonatal cardiomyocytes with endothelin-1 and phorbol 12-myristate 13-acetate (PMA) results in cardiomyocyte hypertrophy. However, the signal transduction pathways involved in this process are poorly understood. Because increased ribosome biogenesis is a requisite for hypertrophy, we sought to (1) confirm the hypothesis that these two hypertrophic agents did indeed induce rRNA synthesis and (2) examine the mechanism through which this induction was accomplished. In this study, hypertrophy of contraction-arrested neonatal cardiomyocytes induced by treatment with either endothelin-1 or PMA was associated with increased rDNA transcription. Western blots demonstrated that the enhanced rates of rDNA transcription were not mediated by increased amounts of either RNA polymerase I or upstream binding factor (UBF), an rDNA transcription factor. However, immunoprecipitation of [32P]orthophosphate-labeled UBF from hypertrophying neonatal cardiomyocytes suggested that the increased rate of rDNA transcription may be due to the hyperphosphorylation of UBF, which would increase the activity of UBF. The increase in UBF phosphorylation occurred within 3 to 6 hours after exposure to either agent, was maximal at 12 hours, and was sustained for at least the first 24 hours of exposure. Phosphoamino acid analysis of UBF immunoprecipitated from control and treated cardiomyocytes demonstrated that UBF was phosphorylated exclusively on serine residues. Our previous studies have shown that the cellular UBF content increased in adrenergic- and contraction-induced models of cardiac hypertrophy. This study with endothelin-1 and PMA demonstrates that the modulation of UBF phosphorylation is an additional pathway by which ribosome biogenesis may be regulated in neonatal cardiomyocytes. These results support the hypothesis that UBF is an important regulatory factor during the initiation and maintenance of the accelerated rate of rDNA transcription observed during neonatal cardiomyocyte hypertrophy mediated by both phorbol esters and endothelin-1.
Endothelin-1 is a potent endogenous vasoconstrictor expressed in a wide variety of tissues.1 The physiological effects of endothelin-1 are mediated by G protein–coupled serpentine receptors,2 and endothelin-1–specific receptors, EtA, can be found in the right atrium and left ventricle of neonatal and adult hearts.3 4 5 Studies, both in vivo and in tissue culture, have suggested that endothelin-1 may play a role in mediating cardiac growth and function.6 7 For example, endothelin-1 has been shown to induce a number of immediate-early response genes (c-fos and Egr-1) and structural genes (myosin light chain-2, α-actin, and troponin 1) as well as the hypertrophic growth of cultured neonatal cardiomyocytes.6 7 Furthermore, it has also been suggested that endothelin-1 may mediate the angiotensin II–induced hypertrophy of cultured neonatal myocytes.8
The signal transduction pathways through which endothelin-1 exerts its effects on growth and gene expression have not been clearly delineated. However, several lines of evidence suggest that PKC is a component of this pathway. For example, events in cardiomyocytes that are coupled to occupation of the endothelin-1 receptor include the stimulation of phospholipase Cβ and phosphoinositide hydrolysis, resulting in the generation of diacylglycerol and inositol 1,4,5-tris-phosphate and the subsequent stimulation of PKC.6 7 Phorbol esters (such as PMA), which activate PKC, can induce neonatal cardiomyocyte hypertrophy.9 10 Conversely, H-7, a PKC inhibitor, has been shown to block endothelin-1–induced hypertrophy of neonatal cardiomyocytes.11 By analogy, activation of PKC, subsequent to the binding of endothelin-1 to its receptor, may be sufficient to mediate the hypertrophic effects of endothelin-1. More recently, endothelin-1 has been demonstrated to activate mitogen-activated kinases and S6 kinase in neonatal myocytes.12 13 14 15 Considered together, these findings suggest a potential role for endothelin-1 in the development of cardiac hypertrophy.
Cardiomyocyte hypertrophy requires an increase in protein accumulation, which results from an accelerated rate of protein synthesis. Approximately 90% of the cardiomyocyte pool of existing ribosomes are engaged in protein synthesis.16 Thus, in the absence of evidence of a significant increase in the efficiency of utilization of these existing ribosomes, de novo ribosome biogenesis is required to increase the cell’s protein synthetic capacity.
Transcription of the 45S pre-rRNA genes (rDNA) has been identified as the rate-limiting step for ribosome biogenesis in a variety of mammalian cells, including cardiac myocytes. For example, studies of norepinephrine- and contraction-induced hypertrophy of neonatal myocytes have demonstrated that increased ribosome biogenesis was accompanied by an elevated rate of rDNA transcription, whereas the fractional rate of pre-rRNA processing was unchanged.17 18 19 These results implicate rDNA transcription as a common target of the various stimuli of cardiac hypertrophy. The elucidation of the molecular events that regulate this process in response to the individual hypertrophic stimuli should therefore contribute to our understanding of both normal and pathological cardiac growth of the myocardium.
At least two trans-acting factors, UBF and SL-1, in addition to active RNA polymerase I, are required for the efficient transcription of rDNA.20 21 SL-1, which consists of TATA binding protein and three TATA binding protein–associated factors, is absolutely required for the recruitment of RNA polymerase I to the promoter and for maintaining basal levels of transcription. UBF is a phosphoprotein that, at least in vitro, is not absolutely required for transcription initiation. However, the addition of UBF to nuclear extracts increases the rate of rDNA transcription by increasing the efficiency of template utilization.22 This ability is reduced when UBF is treated with phosphatase or the phosphorylated carboxy terminal of the molecule is removed,23 24 25 indicating that phosphorylated UBF is likely the more active form of the transcription factor.
Numerous studies indicate the involvement of UBF in the regulation of growth.24 26 We have recently shown that in neonatal cardiac myocytes undergoing hypertrophy in response to norepinephrine and contractile activity, the increase in rDNA transcription was accompanied by increased amounts of UBF protein and mRNA.19 27 Endothelin-1 can also induce neonatal cardiomyocyte hypertrophy.6 7 Accordingly, the goals of the present study were to determine if treatment with endothelin-1 resulted in an increased rate of rDNA transcription and to determine if this was associated with modulation of the amount and/or activity of UBF. In addition, since part of the signal transduction pathway that mediates the effects of endothelin-1 involves the activation of PKC, we also determined if the activation of PKC by a phorbol ester (PMA) would mimic the effects of endothelin-1 on cardiac hypertrophy, rDNA transcription, and UBF.
Both endothelin-1 and PMA caused the treated myocytes to grow and accelerated the rate of rDNA transcription. Interestingly, this increase of the rDNA transcription rate was not accompanied by an elevated content or either RNA polymerase I or UBF. However, there was a marked increase in the phosphorylation of UBF. Inhibitors of PKC activity were able to block the endothelin-1–induced hyperphosphorylation of UBF. Phosphoamino acid analysis of UBF immunoprecipitated from control cells and cells treated with either PMA or endothelin-1 demonstrated phosphorylation on serine residue(s).
Materials and Methods
Neonatal cardiomyocytes were isolated from the ventricles of 1-day-old Sprague-Dawley rat pups as described previously.17 18 28 After the cardiomyocytes were attached overnight in MEM containing 10% newborn calf serum and 0.1 mmol/L 5-bromo-deoxyuridine, the cardiomyocytes were transferred to defined serum-free media29 containing 0.1 mmol/L bromo-deoxyuridine. KCl (50 mmol/L) was added to the medium to prevent the spontaneous contraction characteristic of neonatal cardiomyocytes plated at high density (4×106 cells per 60-mm dish).17 18 28 All experiments were initiated 3 days after the cardiomyocyte dispersion.
Induction of Cardiomyocyte Hypertrophy
Control contraction-arrested cells were maintained in media containing 50 mmol/L KCl. After 2 days in defined media (day 3 of culture), hypertrophy was induced by adding endothelin-1 (10−7 mol/L) or PMA (10−7 mol/L). Controls for PMA treatment were exposed to the inactive phorbol ester PDD (10−7 mol/L). After the appropriate length of treatment, cells were harvested for analysis.
Protein and DNA Determination
Cardiomyocytes were washed in ice-cold PBS and scraped directly into lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 137 mmol/L NaCl, 2 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 0.3% SDS, 0.4% sodium deoxycholate, 1% NP-40, 1 μg/mL aprotinin, and 10% glycerol) and frozen at −20°C until analysis. DNA determinations were performed by the fluorometric method of Cesarone et al.30 Protein was assayed using the BioRad DC assay kit. Growth was expressed as the percentage increase in the protein-to-DNA ratio of contraction-arrested cells treated with either endothelin-1 or PMA over time compared with time-matched contraction-arrested (control) cells treated with either dimethyl sulfoxide or PDD. Experiments were repeated a minimum of five times from separate cardiomyocyte preparations.
Cardiomyocytes were rinsed in ice-cold PBS and then scraped directly into lysis buffer and stored at −20°C until analysis. Western blots with anti-UBF and anti–RNA polymerase I antibodies were carried out as described previously.19 The anti-UBF antibody used in these experiments recognizes both UBF1 and UBF2. The molecular sizes of the immunoreactive proteins were verified by comparison with the migration of standard protein markers (BioRad) that were electrophoresed in parallel lanes.
Nuclear Run-On Transcription
Transcription from the rDNA promoter in isolated cardiomyocyte nuclei was measured by the hybridization of in vitro–synthesized 32P-labeled run-on transcripts to a 45S rDNA clone (pU5.1E/X), as described previously.31 Equivalent amounts of nuclei from control and treated cardiomyocytes were assayed per time point. The hybridization conditions and the posthybridization washes were the same as described previously.32 Control pUC19 DNA was also immobilized on the filters to control for nonspecific hybridization. Hybridization was detected by autoradiography and quantified using either a Molecular Dynamics laser densitometer or an Ambis 4000 radioanalytic imager.
Measurement of the phosphorylation status of UBF was carried out by immunoprecipitation of [32P]orthophosphate-labeled UBF protein from cardiomyocyte extracts using polyclonal anti-UBF antisera.24 Two conditions were used to metabolically label UBF. In some experiments, contraction-arrested neonatal cardiomyocytes were prelabeled with [32P]orthophosphate (1 mCi per 60-mm dish) for 16 hours, after which the media was replaced with fresh media containing 1 mCi per dish of [32P]orthophosphate, 50 mmol/L KCl, and the agent to be tested. After the time indicated, the cells were washed three times in PBS and scraped directly into 500 μL of modified RIPA buffer.33 In other experiments, the cells were not prelabeled but were exposed to PMA or endothelin-1 at the same time that [32P]orthophosphate was added to the media. UBF was immunoprecipitated from the extracts by incubation overnight at 4°C with 50 μL of anti-UBF antisera coupled to 20 μL of protein A–agarose beads. After four 1-mL washes in modified RIPA buffer,33 the beads were boiled for 10 minutes in the presence of 2× SDS sample buffer. When indicated, the various protein kinase inhibitors were added to the cultured cardiomyocytes 2 hours before the addition of endothelin-1 and [32P]orthophosphate. The inhibitors were added from freshly made stock solutions in either water or dimethyl sulfoxide. Bisindolylmaleimide I, HCl (bis), RO-31, herbimycin A, and H-7 were added to final concentrations of 10−6 , 5×10−7 , 10−6, and 5×10−6 mol/L, respectively. Phosphorylated UBF1 and UBF2 were resolved by SDS-PAGE and visualized by autoradiography. Autoradiograms were quantified by laser densitometry (Molecular Dynamics), or gels were analyzed using a MolecularDynamics Phosphorimager.
Phosphoamino Acid Analysis
Phosphoamino acid analysis was carried out as described previously.24 34 The positions of authentic phosphoamino acids (Sigma) were coelectrophoresed with the samples to serve as standards and were detected with ninhydrin.
Preparation of Figures
Autoradiograms obtained from Western, Northern, and nuclear run-on analyses were scanned and converted to 8-bit TIFF Bitmap files using a laser densitometer and ImageQuant software (Molecular Dynamics). The TIFF Bitmap files were subsequently imported directly and unmodified into CorelDraw, where labels and headings were attached. Completed figures were output to print film on a Montage FRL Lasergraphics.
Induction of Cardiac Hypertrophy and rDNA Transcription
Neonatal cardiomyocytes were maintained at a density of 4×106 cells per 60-mm dish in media containing 50 mmol/L KCl to arrest spontaneous contraction for 3 days before the initiation of experiments to allow the cells to adapt to the culture conditions. During the subsequent 4 days in culture, there were no significant changes in protein-to-DNA ratios in contraction-arrested neonatal cardiomyocytes. These results are similar to those obtained by others using this system.17 18
After 3 days in culture, the cardiomyocytes were exposed to endothelin-1 (10−7 mol/L). Within 24 hours, the cardiomyocytes accumulated 18% more protein than did the time-matched control cells (Fig 1⇓). By the second day of treatment, the cardiomyocytes exhibited a 31% increase in total protein compared with time-matched contraction-arrested cells (Fig 1⇓). The increases in protein content of the cells occurred in the absence of changes in DNA content, indicating that the growth was due to hypertrophy rather than hyperplasia.
Previous studies demonstrated that endothelin-1–induced hypertrophy in cardiomyocytes is associated with increases in PKC activity,6 12 13 14 15 and Allo and colleagues9 10 have demonstrated that treatment with PMA, a membrane-permeable activator of the PKC family, results in cardiomyocyte hypertrophy. Thus, we studied the effects of PMA treatment as a potential model for the effects of endothelin-1 on our preparations of neonatal cardiomyocytes. As shown in Fig 1⇑, treatment of cultured neonatal cardiomyocytes with PMA (10−7 mol/L) resulted in increases in protein accumulation of 27% and 28% after 24 and 48 hours, respectively, compared with time-matched control cells. Cells treated with the inactive phorbol ester PDD did not accumulate protein. The results obtained with PMA are in good agreement with those reported previously.10
Although the hypertrophy associated with treatment of neonatal cardiomyocytes with PMA has been shown to be associated with accelerated ribosome biogenesis as the result of increased rDNA transcription,17 18 28 this has not been established for endothelin-1. To characterize this process in our cultures, we used nuclear run-on analysis to measure the rate of rDNA transcription in contraction-arrested neonatal cardiomyocytes treated with either endothelin-1 or PMA. Nuclear run-on assays demonstrated that the rates of rDNA transcription in nuclei derived from either endothelin-1–treated or PMA-treated cardiomyocytes are greater than the rates observed in nuclei obtained from time-matched contraction-arrested cardiomyocytes (Fig 2⇓). Significant increases in the rates of rRNA synthesis were observed within 12 hours after exposure to endothelin-1 or PMA (45±5%) and reached maximal levels within 24 to 48 hours (80±21%, Fig 2⇓). These results confirm that hypertrophy of neonatal cardiomyocytes induced by treatment with either PMA or endothelin-1 is associated with significant increases in the rate of synthesis of rRNA as the result of accelerated rates of transcription of the 45S pre-rDNA.
Analysis of RNA Polymerase I and UBF Levels in Hypertrophic Cardiomyocytes
In theory, the regulation of rDNA transcription in neonatal cardiomyocytes can be effected by a number of different mechanisms, including alterations in the chromatin structure, and/or by the amount/activity of RNA polymerase I and/or the amount/activity of the rDNA transcription factors.20 21 We have previously reported that hypertrophy induced by contraction or treatment with norepinephrine is associated with increases in the cardiomyocyte content of UBF but not RNA polymerase I.19 27 Accordingly, we determined whether the accelerated rates of rDNA transcription observed in neonatal cardiomyocytes treated with PMA or endothelin-1 might also be characterized by changes in the amounts of either of these two factors. As shown in Fig 3⇓, the amounts of UBF protein and the β′ subunit of RNA polymerase I protein were identical in hypertrophic cells and in control time-matched contraction-arrested cells at any of the time points examined (12 to 72 hours). The relative mobility of the detected proteins was confirmed by coelectrophoresis with a nuclear extract of rat Novikoff hepatoma ascites cells and molecular mass markers.
Immunoprecipitation of Phosphorylated UBF From Neonatal Cardiomyocytes
UBF1 and UBF2 are phosphoproteins, and the degree of UBF phosphorylation affects its ability to activate transcription in cell-free extracts.23 24 These findings led us to examine whether, in the absence of alterations in the UBF content, hyperphosphorylation of UBF might account for the differences in rDNA transcription between control contraction-arrested and PMA-treated or endothelin-1–treated neonatal cardiomyocytes. [32P]Orthophosphate-labeled UBF was immunoprecipitated from extracts of contraction-arrested cardiomyocytes and from cardiomyocytes that had been treated with endothelin-1 or PMA for 12 and 24 hours and analyzed by SDS-PAGE (Fig 3⇑). After 12 hours of treatment, we observed threefold and fourfold increases in the phosphorylation of UBF in the cardiomyocytes that had been exposed to either endothelin-1 or PMA, respectively, relative to the radioactivity recovered from an equal number of arrested cardiomyocytes. Even after 24 hours of treatment, there were twofold to threefold increases in the incorporation of [32P]orthophosphate into UBF. Additional studies demonstrated that the increased phosphorylation of UBF could be observed as early as 3 hours after exposure of the cardiomyocytes to endothelin-1 (data not shown). Western analysis of the immunoprecipitates and on parallel extracts did not demonstrate an increase in the mass of UBF (data not shown, see Fig 3⇑). In these experiments, the cultured neonatal cardiomyocytes were exposed to the [32P]orthophosphate for 16 hours before the exposure to either PMA or endothelin-1, to preclude possible effects of alterations of the internal phosphorous pool. However, essentially identical results were obtained when the cells were simultaneously exposed to the hypertrophic agents and [32P]orthophosphate (Fig 4⇓, top right and bottom left [autoradiogram and graph, respectively]). Thus, it would appear that there is a net change in the phosphorylation state of UBF when cells are exposed to either PMA or endothelin-1 but no change in the cellular content of the factor.
We have previously shown that cardiomyocytes stimulated with α1-adrenergic agents such as phenylephrine exhibit increased levels of UBF protein.19 Since some of the cellular effects of α1-adrenergic stimulation have been attributed to PKC activation,35 we examined whether phenylephrine might also increase the phosphorylation status of UBF (Fig 4⇑, bottom right). Interestingly, although cardiomyocytes that had been treated with phenylephrine (10−4 mol/L) for 12 hours exhibited a 3.5-fold increase in [32P]orthophosphate incorporation into UBF (Fig 4⇑, bottom right; immunoprecipitated [Immunoppt]), this was not significantly different from the increased amounts (3.4-fold) of UBF protein recovered from those cells (Fig 4⇑, bottom right; Western). These results demonstrate that in contrast to endothelin-1, there was no net change in the phosphorylation status of UBF between phenylephrine-stimulated cardiomyocytes and control cells.
UBF Is Phosphorylated on Serine Residues
As a first step in elucidating the kinases responsible for the phosphorylation of UBF in response to treatment with endothelin-1 and PMA and hence the signal transduction pathways that activate those kinases, we analyzed the phosphoamino acids of UBF from control and neonatal cardiomyocytes treated with endothelin-1 and PMA. As shown in Fig 5⇓, lane 1, UBF immunoprecipitated from control cardiomyocytes contained only phosphoserine residues. Similarly, phosphoamino acid analysis of UBF purified by immunoprecipitation from endothelin-1–treated (lane 2) and PMA-treated (lane 3) cardiomyocytes demonstrated only phosphoserine residues.
Selective Inhibitors of PKC Block the Endothelin-1–Induced Phosphorylation of UBF
Previous studies have placed PKC in the signal transduction pathway(s) activated by endothelin-1.6 7 11 12 13 14 15 Further, the observation that PMA, an activator of PKC, also induced UBF hyperphosphorylation is also consistent this model. To more fully substantiate this correlation, we examined the possibility that inhibitors of PKC would inhibit the endothelin-1–induced hyperphosphorylation of UBF. H-7, a compound that can inhibit both PKA and PKC and cardiomyocyte hypertrophy11 at the concentration used in the present study,36 37 significantly inhibited UBF phosphorylation induced by endothelin-1 (Fig 6⇓, top). Further, both bisindolylmaleimide and RO-31 also inhibited the endothelin-1–induced phosphorylation of UBF (Fig 6⇓, bottom). Both bisindolyl maleimide and RO-31 are also inhibitors of PKC and PKA. However, at the concentrations used in these experiments, 10−6 and 5×10−7 mol/L, respectively, these compounds are more selective for PKC than PKA.38 39 40 Thus, the effect of these PKC inhibitors on the endothelin-1–induced phosphorylation of UBF correlates with the observation that PMA also induces UBF phosphorylation and suggests that the PKC family plays a role in this process.
Numerous studies have implicated the potent vasoconstricting peptide endothelin-1 in the regulation of cardiac-specific gene expression hypertrophic growth6 7 11 and in the growth and function of the myocardium.6 7 However, the molecular signals and pathways by which endothelin-1 regulates these processes are poorly understood. Moreover, it remains to be established whether cardiac hypertrophy occurs as a primary effect of endothelin-1 or is secondary to the effects of endothelin-1 on cardiac contractility.
The consensus of recent studies using isolated perfused heart preparations and neonatal cardiomyocytes in culture is that increased ribosome biogenesis is essential for the accelerated protein synthesis associated with cardiac hypertrophy.16 At least in response to contraction and adrenergic agents, this process appears to be regulated at the level of transcription of the rRNA genes (rDNA).18 19 27 28 Consistent with this view, we have shown here that endothelin-1–induced cardiomyocyte hypertrophy is characterized by increased rates of rDNA transcription. These findings provide strong support for the importance of rDNA transcription in the regulation of cardiomyocyte growth induced by distinct stimuli.
Importantly, by using contraction-arrested neonatal cardiomyocyte cultures, we have clearly established that endothelin-1 can effect changes in rRNA synthesis and protein accumulation (hypertrophy) independent of its actions on cardiomyocyte contractility. By contrast, previous studies examining the effect of endothelin on cardiomyocyte growth were carried out in spontaneously contracting cardiomyocytes.6 7 Since contractile activity alone is a determinant of cardiac growth and since endothelin-1 strongly influences cardiac contractility, it was not possible in those earlier studies to dissociate the direct effects of endothelin on cardiac growth from those secondary to increased contractility.
It is interesting to compare and contrast the cellular and molecular changes associated with cardiac hypertrophy observed in response to endothelin-1 and another naturally occurring vasoactive agent, norepinephrine. Both agents induce cardiac hypertrophy independent of their effects on contractile activity, and both hormones augment the rate of rDNA transcription to a similar extent. Moreover, neither of these stimuli modulates the cardiomyocyte levels of RNA polymerase I (as reflected by measurement of the β′ subunit of RNA polymerase I). Indeed, at least in response to these two stimuli, alterations in the amount of RNA polymerase I cannot account for the changes in rDNA transcription. However, in distinct contrast to the effects of norepinephrine, which elicited hypertrophic growth responses in cardiomyocytes that were accompanied by an increased cellular content of the rDNA transcription factor UBF19 in the absence of changes in the degree of phosphorylation, endothelin-1 had no effect on UBF mRNA or protein levels. Instead, endothelin-1–mediated cardiomyocyte hypertrophy was characterized by a sustained increase in the phosphorylation of UBF on serine residues. Thus, in neonatal cardiomyocytes, both the absolute content and the degree of phosphorylation of UBF can be modulated, depending on the stimuli. It is possible that the differential regulation of UBF in neonatal cardiomyocytes may be one mechanism to ensure diversity and specificity of response to distinctly different hypertrophic stimuli and to allow for fine tuning of the final growth response.
Given the importance of rDNA transcription to cellular growth, the strong correlation between the degree of phosphorylation of UBF and the rate of cellular growth is suggestive of a cause-and-effect relationship. In vitro studies directly support this idea, since experiments with cell-free nuclear extracts have shown that hyperphosphorylated UBF is significantly more effective than hypophosphorylated UBF at initiating transcription from the 45S rDNA promoter.23 24 25 By analogy, it is likely that endothelin-1–mediated hyperphosphorylation of UBF in neonatal cardiomyocytes results in a significant enhancement by UBF of rDNA transcription.
Many intracellular effects of endothelin-1 have been linked to endothelin-1 receptor–mediated phosphoinositol hydrolysis, activation of the serine-threonine kinase family, PKC,6 7 12 13 14 15 and tyrosine kinase activation, and the subsequent activation of mitogen-activated protein kinases and the 90-kD S6 kinase.12 13 14 15 In the present study, activation of PKC isoforms by PMA induced cardiomyocyte hypertrophy and increased the rate of rDNA transcription to levels similar to those observed in response to endothelin-1. These findings are in agreement with previous studies demonstrating that phorbol esters were potent activators of rDNA transcription and cardiomyocyte growth.9 10 At the molecular level, the parallel effects of PMA and endothelin-1 on the rDNA transcription apparatus were extended to RNA polymerase I and UBF. Treatment of cardiomyocytes with PMA had no affect on the cellular content of RNA polymerase I but did result in hyperphosphorylation of UBF in the absence of changes in UBF protein levels. Moreover, phosphoamino acid analysis of the UBF residues that phosphorylated in response to PMA and endothelin-1 demonstrated only phosphoserine residues. Thus, in broad terms, PMA was able to mimic the endothelin-1–induced posttranslational modification of UBF both in a quantitative and qualitative fashion.
Since many of the physiological effects of endothelin-1 have been linked to endothelin-1 receptor–mediated phosphoinositol hydrolysis and the activation of PKC, these results strongly implicate PMA-sensitive PKC-dependent pathways in the regulation of UBF phosphorylation by endothelin-1. The correlation between a potential PKC-dependent pathway and UBF phosphorylation is substantiated by the effects of bisindolyl maleimide and RO-31 on the endothelin-1–induced phosphorylation of UBF. These compounds can inhibit both PKA and PKC. However, the concentrations used in these studies were less than the reported IC50 values for PKA,38 39 arguing that the effect is specific for a PKC-dependent pathway. Obvious effector sites downstream from PKC activation that may be involved in the phosphorylation of UBF include the mitogen-activated protein kinase–related pathways.12 13 14 15 However, there is significant evidence for signal cross talk between the endothelin-1 G protein–coupled receptor and protein tyrosine kinase activation.7 11 15 In apparent agreement with these reports, preliminary experiments demonstrated that herbimycin A, a specific tyrosine kinase inhibitor,41 inhibited UBF hyperphosphorylation (results not shown). This would appear to support the report of Sadoshima et al,15 who linked hypertrophic stimuli induced by Gαq-coupled receptors with protein-tyrosine kinases and mitogen-activated kinase and S6 kinase. However, those authors found evidence that intracellular Ca2+, but not PKC, was involved in mediating the activation of the downstream threonine-serine kinases. In contrast, both the present study and others11 14 provide evidence for the involvement of PKC in the hypertrophic response induced by endothelin-1.
If indeed endothelin-induced UBF phosphorylation is mediated via PKC-dependent pathway(s), then these studies also raise the interesting question of why phenylephrine, a known activator of PKC (References 9 and 10 and reviewed in Reference 35), failed to hyperphosphorylate UBF. One possible answer may lie in the ability of phenylephrine and endothelin-1 to differentially activate certain members of the diverse PKC family.12 13 14 15 35 For example, at least in the short term, endothelin-1 receptor activation is characterized by a rapid translocation of PKC-δ in neonatal cardiomyocytes, whereas translocation of this isoform is barely detected in response to phenylephrine.14 It is possible that UBF phosphorylation in response to endothelin stimulation may be mediated by pathways that are distal to, and specific for, PKC-δ activation.
Perhaps the most striking outcome of these studies is that in the face of challenges from diverse hypertrophic stimuli, one of the mechanisms by which neonatal cardiomyocytes regulate rDNA transcription may be by the differential activation of a single transcription factor, UBF. As such, these experiments serve to further emphasize that the regulation of rDNA transcription is a pivotal point in the regulation of the cardiomyocyte hypertrophy observed in response to distinctly different stimuli.
Selected Abbreviations and Acronyms
|PKA||=||protein kinase A|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
|UBF||=||upstream binding factor|
This study was supported in part by National Institute of Health grants HL-47638 (Dr Rothblum) and GM-48991 (Dr Rothblum) and an award from the Geisinger Foundation (Dr Rothblum). Dr Hannan was supported by a Fellowship of the American Heart Association. Dr Luyken was a fellow of the Deutsche Forschungsgemeinschaft. The authors would like to thank Drs David Carey, Hal Singer, and Howard Morgan for their helpful comments on the manuscript.
- Received September 8, 1995.
- Accepted November 30, 1996.
- © 1996 American Heart Association, Inc.
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