Regulation of ribosomal DNA transcription by insulin

Katherine M. Hannan1,2, Lawrence I. Rothblum1,2, and Leonard S. Jefferson1

1 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey 17033; and 2 Henry Hood Research Program, Sigfried and Janet Weis Center for Research, Danville, Pennsylvania 17822

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.

upstream binding factor; ribonucleic acid polymerase I; ribonucleic acid polymerase I-associated factor 53

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

INSULIN PLAYS A KEY ROLE IN the regulation of protein synthesis in a number of tissues and cell culture systems (1, 2, 6, 9, 16, 29). Its regulatory effects on protein synthesis appear to be mediated through multiple mechanisms. For example, insulin-mediated modulation of protein synthesis has been associated with 1) an increase in the efficiency of initiation and/or elongation of mRNA translation (16), 2) a change in transcription of specific genes (25), and 3) an increase in cellular RNA (14) resulting in a rise in the steady-state number of ribosomes (2, 6, 9). The degree to which each of these different mechanisms contributes to the regulation of protein synthesis is not well understood, since their relative roles appear to vary not only with the type of cell or tissue but also with the duration of insulin deprivation or resupplementation (16, 28).

Changes in the steady-state number of ribosomes can involve modulation of either ribosome biogenesis or degradation. Studies with cardiac and skeletal muscle preparations indicate that insulin can regulate both of these processes (2, 16). Cell culture systems have provided evidence that insulin enhances ribosome biogenesis, in some cases by stimulating transcription of the rRNA genes (rDNA). In primary cultures of resting chick embryo fibers, insulin was shown to stimulate total cell protein by 1.3- to 1.5-fold, whereas ribosome production was stimulated almost 4-fold (6). In undifferentiated mouse myoblasts, insulin stimulated translation of ribosomal proteins and rDNA transcription (9). More recent studies demonstrated that protein synthesis decreased by 40% in primary cultures of hepatocytes deprived of insulin, a change that was restored by insulin treatment (1). The changes in protein synthesis were accompanied by quantitatively and temporally similar changes in total RNA content, reflecting changes in the cellular content of ribosomes (1, 14). Insulin caused an elevation in the rRNA content of these cells by stimulating rDNA transcription rather than by altering rates of processing or stability of the rRNA (1). The mechanisms through which insulin causes these alterations in ribosome biogenesis are as yet not understood.

Ribosome biogenesis includes the transcription of the gene that encodes the 45S precursor of the 18S, 5.8S, and 28S rRNAs by RNA polymerase I and the transcription of the 5S RNA gene by RNA polymerase III. Theoretically, regulation of rDNA transcription can involve 1) changes in chromatin structure permissive for the formation of the initiation complex, 2) alterations in amounts, localization, or activity of RNA polymerase I, and/or 3) similar alterations in associated transcription factors (13, 28). In addition, recent studies suggest that the rDNA transcription apparatus can assemble (or colocalize) on the rDNA without actively transcribing, suggesting that there are mechanisms that inhibit transcription (15, 17) or prevent formation of productive initation complexes.

The present study was undertaken to investigate the molecular mechanism(s) through which insulin regulates rDNA transcription in serum-starved 3T6 cells and exponentially growing H4-II-E-C3 cells. The results show that rDNA transcription was markedly reduced in response to serum starvation, a response that was accompanied by a general decrease in content and/or activity of several components of the rDNA transcription complex. Addition of insulin was sufficient to restore rDNA transcription to the value observed in exponentially growing 3T6 cells. In addition, insulin treatment of exponentially growing H4-II-E-C3 cells was sufficient to stimulate rDNA transcription. In both cell types, stimulation of rDNA transcription was accompanied by an increase in the cellular contents of the transcription factor upstream binding factor (UBF) and the RNA polymerase I-associated factor (PAF) 53. Thus part of the mechanism through which insulin modulates rDNA transcription in 3T6 and H4-II-E-C3 cells is the regulation of factors that are involved in recruitment of RNA polymerase I to the rDNA promoter to form the active transcription initiation complex.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

3T6 and H4-II-E-C3 Cell Culture

Monolayer cultures of 3T6-Swiss albino cells (ATCC CCL-96) were maintained at 37°C in DMEM (GIBCO) containing 10% fetal bovine serum (FBS; GIBCO), in an atmosphere containing 5% CO2. The cells were plated on 100-mm tissue culture dishes at 0.25 × 106 cells/dish. After 24 h of culture, the medium was replaced with DMEM containing 0.5% FBS. After an additional 72 h, the medium was replaced with DMEM containing 10% FBS or else recombinant human insulin (Sigma) was added directly to the medium to a final concentration of 10 nM. Cells were harvested 3, 6, 12, and 24 h after treatment. Exponentially growing cells were plated at 6.25 × 104 cells/dish and were maintained in DMEM containing 10% FBS, ensuring that when cells were harvested the cell density was equivalent to that of the serum-starved cells.

Monolayer cultures of H4-II-E-C3 cells (ATCC CRL-1600) were maintained at 37°C in DMEM (GIBCO) containing 5% FBS (GIBCO), 5% newborn calf serum (GIBCO), 0.05% NaHCO3, and 6.3 mM HEPES (pH 7.3), in an atmosphere containing 5% CO2. The cells were plated on 100-mm tissue culture dishes at 1 × 106 cells/dish. After 24 h, human recombinant insulin was added directly to the medium to a final concentration of 10 nM. Cells were harvested at the times indicated.

The concentration of insulin used in these experiments was sufficient to elicit a maximal response in rDNA transcription (1).

Isolation of Nuclei

Cells were treated with trypsin-EDTA (GIBCO), washed once with ice-cold PBS, resuspended in 4 ml of nuclear isolation buffer (10 mM Tris · HCl, 10 mM NaCl, 10 mM MgCl2, and 0.5% NP-40, pH 7.4), and incubated on ice for 10 min. The swollen cells were vortexed, and nuclei were collected by centrifugation at 1,000 rpm for 5 min and resuspended in 200 µl of nuclear storage buffer (50 mM Tris, 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA, pH 8.3).

DNA Quantification

Twenty microliters of isolated nuclei were diluted in 1 ml of 1× saline sodium citrate-0.25% SDS. DNA was quantitated using the Hoechst dye assay of Cesarone et al. (5) with calf thymus DNA as the standard.

Nuclear Run-On Transcription Assay

Isolation of de novo synthesized RNA was carried out as described (7), using equivalent amounts of nuclei (DNA) for each time point. The isolated RNA was resuspended in 100 µl of TE buffer (100 mM Tris · HCl and 10 mM EDTA, pH 8.0), and unincorporated nucleotides were removed by centrifugation through STE Select-G(RF) columns (5 Prime-3 Prime). To control for recovery of the in vitro-transcribed RNA, a 3H-labeled actin RNA probe was added before extraction and purification, and the amount of 3H recovered was quantitated by liquid scintillation spectrometry.

Transcription from the rDNA and UBF gene promoters in isolated nuclei was measured by hybridization of in vitro- synthesized 32P-labeled run-on transcripts to slot blots of immobilized (Zetaprobe nylon membrane) plasmids containing either the mouse rDNA promoter (-168 to +292 with respect to the start site of transcription initiation) or a genomic clone of the mouse UBF promoter (-3124 to +404 with respect to the start site of transcription initiation). Two different concentrations of each DNA were used to ensure that the hybridization was quantitative. Slot blots of pBluescript DNA were included in the hybridization reaction as a control for nonspecific hybridization. Hybridization conditions and posthybridization washes were as described previously (7). Radioactive hybrids were detected and quantitated using a Molecular Dynamics PhosphorImager.

Western Blot Analysis

Samples were denatured in 2× Laemmli buffer, resolved by electrophoresis on 8% polyacrylamide gels, and then electroblotted onto Immobilon-P membrane (Millipore). The membranes were rinsed in PBS and blocked for 1 h in buffer A (PBS, 5% milk powder, and 0.1% Tween 20). The beta '- and beta -subunits of RNA polymerase I, UBF, and PAF53 were detected by incubating the membranes for 1 h with polyclonal rabbit antisera diluted 1:10,000 in buffer A (the original sample of PAF53 antiserum was kindly supplied by Dr. M. Muramatsu, Department of Biochemistry, Saitama Medical School, Japan). The membranes were then washed three times (for 10 min each time) with buffer A and exposed for 1 h to goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2,000; Sigma) diluted in buffer A. After three 10-min rinses in PBS containing 0.1% Tween 20, the immune complexes were visualized by enhanced chemiluminescence (Amersham). Western blots were quantitated by laser densitometry (Molecular Dynamics). Molecular sizes were verified by comparison with the migration of standard protein markers (Bio-Rad).

Transfections and Chloramphenicol Acetyltransferase Assays

After 2 days in culture, 3T6 cells were cotransfected with either pSMECAT, a wild-type rDNA promoter reporter, or pSMECAT-7, an inactive reporter containing a G-to-A substitution at -7 (12), and various concentrations of a vector for driving the expression of rat UBF1, pCDNA3UBF1 (12). All transfections were carried out using Lipofectamine and a constant amount of DNA (4 µg), achieved by including various amounts of pCDNA3. pCMV-beta Gal (1 µg) was included as an internal standard for the efficiency of transfection. Five hours after transfection, the culture medium was replaced with fresh DMEM plus 10% FBS. The cells were harvested 24 h later. Lysates were prepared as described (3) and frozen at -80°C until assayed for either chloramphenicol acetyltransferase (CAT) or beta -galactosidase activities (3). The synthesis of acetylated choramphenicol was measured by separating acetylated [14C]chloramphenicol from unmodified [14C]chloramphenicol by TLC (3). The results of the CAT assays were normalized with respect to beta -galactosidase activity to correct for variations in the efficiency of transfection.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Insulin Stimulates rDNA Transcription in Both Serum-Starved 3T6 and Exponentially Growing H4-II-E-C3 Cells

To investigate the modulation of transcription from the 45S precursor gene, the only gene transcribed by RNA polymerase I, nuclear run-on assays were performed in the presence of high concentrations of alpha -amanitin. Nuclei were harvested from exponentially growing and serum-starved 3T6 cells or from serum-starved cells that had been treated with insulin for the times indicated. Serum starvation of 3T6 cells has been shown to repress rDNA transcription to a "basal level" against which the effects of regulatory agents such as insulin can be compared (7). The results presented in Fig. 1A demonstrate that treatment of serum-starved 3T6 cells with insulin was sufficient to restore rDNA transcription to the values observed in exponentially growing cells. To control for the ability of the cells to respond to growth stimulatory agents, we measured their response to serum. In good agreement with previously published observations, serum stimulated rDNA transcription (data not shown; Refs. 7, 28). These results demonstrate that insulin alone is sufficient to stimulate rDNA transcription in serum-starved 3T6 cells.


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Fig. 1.   Insulin stimulates rDNA transcription in both serum-starved 3T6 and H4-II-E-C3 cells. A: nuclei were harvested from exponentially growing (E) 3T6 cells, serum-starved cells (SS), and serum-starved cells treated with insulin at times indicated. B: nuclei were harvested from exponentially growing (E) H4-II-E-C3 cells and cells treated with insulin at times indicated. A and B: specific RNA polymerase I transcription was measured by nuclear run-on assays as described in MATERIALS AND METHODS. Top: autoradiographs of hybridization product of in vitro-transcribed RNA to either immobilized rDNA (mouse 5' ETS) or Bluescript as a control for background. Bottom: results from 5 experiments were quantitated and plotted relative to control, i.e., value obtained from 3T6 cells serum starved for 72 h or exponentially growing H4-II-E-C3 cells (means ± SD). * P < 0.05 and ** P < 0.01 compared with control using a Dunnett multiple comparison test.

H4-II-E-C3 cells have proven useful in studies designed to examine the effects of insulin on regulation of metabolism and expression of genes involved in metabolism (16, 25). Insulin has also been reported to stimulate growth in H4-II-E-C3 cells (16, 25). Thus we examined the possibility that insulin might also regulate rDNA transcription in these cells. Treatment of H4-II-E-C3 with insulin for 6 h significantly stimulated rDNA transcription, and this effect was maintained for 24 h (Fig. 1B). These results confirm, in a different system and cell line, that insulin stimulates rDNA transcription.

Molecular Mechanisms Through Which Insulin Stimulates rDNA Transcription in Both Serum-Starved 3T6 and Exponentially Growing H4-II-E-C3 Cells

RNA polymerase I content. As an initial step toward identifying the mechanism(s) through which insulin stimulates rDNA transcription, we quantitated the relative contents of the beta '- and beta -subunits of RNA polymerase I in nuclei isolated from both 3T6 and H4-II-E-C3 cells. The results from a number of separate experiments were quantitated by laser densitometry and are presented in Fig. 2. It can be seen that after 72 h of serum starvation the nuclear contents of both the beta '- or beta -subunits were reduced by 70% compared with the values observed in exponentially growing 3T6 cells. Interestingly, the addition of insulin for up to 24 h did not significantly affect the content of the two subunits. Similarly, insulin had no effect on the cellular content of either the beta '- or beta -subunit in H4-II-E-C3 cells (Fig. 2B). If it is assumed that the cellular contents of these two subunits accurately reflect the remainder of the RNA polymerase I complex, then modulation of the amount of RNA polymerase I cannot account for the stimulation of rDNA transcription associated with insulin treatment of serum-starved 3T6 or exponentially growing H4-II-E-C3 cells.


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Fig. 2.   Cellular content of RNA polymerase I is not significantly regulated by insulin in either serum-starved 3T6 or H4-II-E-C3 cells. A: nuclei were harvested from exponentially growing (E) 3T6 cells, serum-starved cells (SS), and serum-starved cells treated with insulin at times indicated. B: nuclei were harvested from exponentially growing (E) H4-II-E-C3 cells and cells treated with insulin at times indicated. A and B: nuclear proteins were fractionated by SDS-PAGE, transferred to Immobilon-P membranes, and incubated with antiserum to beta '- and beta -subunits of RNA polymerase I. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) and quantitated by densitometry. Equal amounts of nuclear protein (1.2 µg) were loaded in each lane. Experiments were repeated 3 or more times, and results were quantitated by laser densitometry. Mean values are shown relative to control, i.e., value obtained from 3T6 cells serum starved for 72 h or exponentially growing H4-II-E-C3 cells (means ± SD). ** P < 0.01 compared with control using a Dunnett multiple comparison test.

PAF53 content. Recently, three PAFs were identified, PAF53, PAF51, and PAF49 (10). Immunolocalization studies indicate that PAF53 is present in the nucleoli of exponentially growing NIH/3T3 cells but not serum- starved cells (10). In addition, the amount of PAF53 associated with RNA polymerase I is reduced in quiescent cells (10). This observation led us to examine whether or not such changes might be contributing to the increased rDNA transcription observed during insulin treatment of serum-starved 3T6 and exponentially growing H4-II-E-C3 cells. In a series of experiments similar to those described above, nuclear and total cellular proteins were extracted from both 3T6 and H4-II-E-C3 cells. The amount of PAF53 in these samples was quantitated by SDS-PAGE and Western blot analysis using a monospecific polyclonal antibody that recognizes rodent PAF53. The results shown in Fig. 3A demonstrate that after 72 h of serum starvation the nuclear content of PAF53 in 3T6 cells had fallen to 15% of the value observed in exponentially growing cells. Treatment of serum-starved cells with insulin for 24 h resulted in restoration of the content of PAF53 to the value observed in exponentially growing cells. In addition, treatment of H4-II-E-C3 cells with insulin caused a significant increase in the content of PAF53 (Fig. 3B). Whole cell extracts were also prepared, and similar results were obtained (Fig. 3, A and B), suggesting that under these conditions the observed changes in PAF53 were due to alterations in content and not to subcellular localization as previously suggested (10). These results suggest that there may be a correlation between the content of PAF53 and rDNA transcription associated with insulin treatment of serum-starved 3T6 or exponentially growing H4-II-E-C3 cells.


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Fig. 3.   Cellular and nuclear contents of RNA polymerase I-associated factor 53 (PAF53) are significantly elevated by addition of insulin to both serum-starved 3T6 and H4-II-E-C3 cells. A: whole cell proteins or nuclear proteins were harvested from exponentially growing (E) 3T6 cells, serum-starved cells (SS), and serum-starved cells treated with insulin at times indicated. B: whole cell proteins or nuclear proteins were harvested from exponentially growing (E) H4-II-E-C3 cells and cells treated with insulin at times indicated. A and B: proteins were fractionated by SDS-PAGE and transferred to Immobilon-P. Cellular or nuclear content of PAF53 was determined by Western blot analysis with an anti-PAF53 antiserum. Immunoreactive protein was visualized by ECL and quantitated by densitometry. Equal amounts of whole cell (30 µg) or nuclear (1.2 µg) protein were loaded in each lane. Experiments were repeated 4 times, and results using nuclear samples were quantitated by laser densitometry. Mean values are shown relative to control, i.e., value obtained from 3T6 cells serum starved for 72 h or exponentially growing H4-II-E-C3 cells (means ± SD). * P < 0.05 and ** P < 0.01 compared with control using a Dunnett multiple comparison test.

UBF content. UBF is a nucleolar protein associated with increased efficiency of transcription from the rDNA promoter. For example, addition of UBF to cellfree transcription assays has been shown to augment rDNA transcription in a dose-dependent manner (18, 32). Moreover, overexpression of UBF in cardiomyocytes (12) and 3T6 cells (described below) has been shown to be sufficient to increase transcription from a coexpressed rDNA reporter construct. These findings led us to examine whether or not insulin-mediated changes in rDNA transcription in 3T6 cells might be associated with changes in the content of UBF.

Whole cell or nuclear protein was extracted from exponentially growing 3T6 cells and from cells that had been either serum starved or serum starved and treated with insulin. The amount of UBF1 was quantitated by SDS-PAGE and Western blot analysis using monospecific polyclonal antiserum that recognizes both isoforms of UBF. A number of experiments were quantitated by laser densitometry and are presented graphically in Fig. 4, A and B. After 72 h of serum starvation, the nuclear or cellular contents of UBF1 in 3T6 cells had decreased by 50% with respect to exponentially growing cells. By 24 h, insulin treatment increased the content of UBF1 to the value observed in exponentially growing cells (Fig. 4A). Similar results were obtained using whole cell protein samples (data not shown). Insulin treatment of H4-II-E-C3 cells also significantly increased the content of UBF1 (Fig. 4B), which correlated with an increase in rDNA transcription (Fig. 1B).


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Fig. 4.   Nuclear content of upstream binding factor (UBF) is significantly elevated by addition of insulin to both serum-starved 3T6 and H4-II-E-C3 cells. A: nuclei were harvested from exponentially growing (E) 3T6 cells, serum-starved cells (SS), and serum-starved cells treated with insulin at times indicated. B: nuclei were harvested from exponentially growing (E) H4-II-E-C3 cells and cells treated with insulin at times indicated. A and B: nuclear proteins were fractionated by SDS-PAGE, transferred to Immobilon-P membrane, and incubated with anti-UBF antiserum. Immunoreactive proteins were visualized by ECL and quantitated by densitometry. Equal amounts of nuclear protein (1.2 µg) were loaded in each lane. Experiments were repeated 3 or more times, and results were quantitated by laser densitometry. Mean values for UBF1 were calculated and are shown relative to control, i.e., value obtained from 3T6 cells serum starved for 72 h or exponentially growing H4-II-E-C3 cells (means ± SD). * P < 0.05 and ** P < 0.01 compared with control using a Dunnett multiple comparison test.

Alterations in the content of a protein can reflect changes in the turnover of that protein (posttranslational regulation) and/or alterations in the abundance of its mRNA (pretranslational regulation). As a first step in examining which of these mechanisms was responsible for the observed alterations in UBF content, we measured transcription of the UBF gene using nuclear run-on assays. The results from several experiments were quantitated and are presented graphically in Fig. 5, A and B. Transcription from the UBF gene was decreased significantly within 72 h of serum starvation of 3T6 cells, compared with exponentially growing cells, as was previously reported (7). Within 3 h of insulin treatment, UBF gene transcription was elevated to 60% of the value observed in exponentially growing cells (Fig. 5A). Similarly, UBF gene transcription was stimulated approximately twofold after 3 h of treatment of H4-II-E-C3 cells with insulin, and this level was maintained for the duration of the experiment, (24 h; Fig. 5B). These data demonstrate that insulin can regulate cellular UBF content at the level of transcription of the UBF gene in both cell types.


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Fig. 5.   Insulin stimulates UBF gene transcription in both serum-starved 3T6 and H4-II-E-C3 cells. A: nuclei were harvested from exponentially growing (E) 3T6 cells, serum-starved cells (SS), and serum-starved cells treated with insulin at times indicated. B: nuclei were harvested from exponentially growing (E) H4-II-E-C3 cells and cells treated with insulin at times indicated. A and B: specific transcription was measured by nuclear run-on assays and hybridization to a genomic clone of mouse UBF promoter as described in MATERIALS AND METHODS. Resultant 32P-labeled run-on transcripts were hybridized to a fragment of mouse UBF gene, and hybrids were visualized with a Molecular Dynamics PhosphorImager. Top: autoradiographs of hybridization product of in vitro-transcribed RNA to immobilized UBF promoter. Bottom: results from 5 experiments, for each cell type, were quantitated and plotted relative to control, i.e., value obtained from 3T6 cells serum starved for 72 h or exponentially growing H4-II-E-C3 cells (means ± SD). * P < 0.05 and ** P < 0.01 compared with control using a Dunnett multiple comparison test.

To determine whether an elevation of UBF cellular content might contribute to the insulin-mediated regulation of rDNA transcription, we utilized a modified rDNA reporter system first demonstrated by Palmer et al. (27). We had previously demonstrated that overexpression of UBF was sufficient to drive transcription from a cotransfected wild-type rDNA reporter (pSMECAT) but not from a mutant rDNA reporter (pSMECAT-7) in cardiomyocytes (12). As shown in Fig. 6, cotransfection of 3T6 cells with 1 µg of pSMECAT and increasing amounts of a UBF1 expression vector (pCDNA3UBF1) elevated transcription from pSMECAT (lanes 3-5). As expected, overexpression of UBF had no effect on transcription from the control (i.e., inactive) promoter, pSMECAT-7 (lanes 1 and 2). Thus, in 3T6 cells, as well as in primary cultures of cardiomyocytes (12), the overexpression of UBF1 drives rDNA expression. The results from a number of similar but independent experiments are summarized in Fig. 6B.


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Fig. 6.   Overexpression of UBF stimulates rDNA transcription. A: 3T6 cells were transfected with pSMECAT-7 (1 µg) or pSMECAT (1 µg) and either 0.5 µg (+) or 1 µg (++) of pCDNA3UBF1. All transfections were performed using Lipofectamine, at a constant DNA concentration (4 µg), and included pCMV-beta Gal (1 µg) as an internal standard for efficiency of transfection as described in MATERIALS AND METHODS. After 24 h, cell lysates were prepared and assayed for chloramphenicol acetyltransferase (CAT) activity and beta -galactosidase. Ac-Chlor, acetylated chloramphenicol; Chlor, chloramphenicol. B: results of CAT assay were adjusted for efficiency of transfection, i.e., beta -galactosidase activity, and are presented as average increase in multiples of control level (means ± SD; n = 3) in pSMECAT. ** P < 0.01 compared with control using a Dunnett multiple comparison test.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the studies presented here, we examined the molecular mechanism(s) through which insulin acts to regulate transcription of the rRNA genes. We used both 3T6 and H4-II-E-C3 cells for these studies because they exhibit a robust and reproducible response to insulin with respect to rDNA transcription. In addition, their ease of maintenance and rapid growth allowed for larger, more readily available amounts of material for delineation of the mechanisms involved in insulin action on rDNA transcription.

Insulin Stimulates rDNA Transcription in Both 3T6 and H4-II-E-C3 Cells

Previous studies demonstrated that serum starvation of 3T6 cells results in a significant reduction in rDNA transcription and that, in response to refeeding with 10% serum, rDNA transcription is restored to the value observed in exponentially growing cells (7, 28). We confirmed these observations and extended them to demonstrate that the addition of insulin is sufficient to stimulate rDNA transcription and therefore ribosome biogenesis in serum-starved 3T6 cells and exponentially growing H4-II-E-C3 cells. These results are supportive of previous studies suggesting that one of the mechanisms through which insulin regulates protein synthesis in vivo is by increasing ribosome biogenesis (i.e., increasing the cellular capacity of a cell to synthesize proteins) (1, 2, 6, 9).

Insulin Does Not Increase the Content of RNA Polymerase I in Either Serum-Starved 3T6 or Exponentially Growing H4-II-E-C3 Cells

The extent to which the various components of the rDNA transcription apparatus contribute to the stimulation of rDNA transcription observed in response to insulin is not known. The results of the present study demonstrate that serum starvation of 3T6 cells causes a decrease in the content of RNA polymerase I (Fig. 2A). Thus reduced rDNA transcription observed during serum starvation may stem from an overall decrease in the amount of RNA polymerase I available for transcription. However, the insulin-mediated stimulation of rDNA transcription was not associated with changes in the content of RNA polymerase I. Modulation of rDNA transcription in the absence of changes in content of RNA polymerase I appears to be a characteristic of several experimental models (31, 34). For example, alterations in rDNA transcription associated with norepinephrine- or contraction-induced hypertrophy of neonatal cardiomyocytes are not characterized by changes in the cellular content of RNA polymerase I (11, 13).

Insulin treatment also had no effect on the cellular content of RNA polymerase I in exponentially growing H4-II-E-C3 cells, even though rDNA transcription was stimulated in response to the hormone (Figs. 1B and 2B). Considered together, these results indicate that cellular content of RNA polymerase I is a poor correlate of rDNA transcription, which is in agreement with conclusions drawn from other studies (8, 29). Furthermore, the present results demonstrate that insulin stimulation of rDNA transcription must involve qualitative alterations in the polymerase, PAFs, or other components associated with transcription by RNA polymerase I. Factors that are thought to contribute to the ability of RNA polymerase I to form a stable initiation complex on the rDNA promoter include PAFs, transcription factor 1C/transcription imitation factor-1A, and rDNA transcription factors such as selectivity factor 1 (SL-1) and UBF (10, 28).

Stimulation of rDNA Transcription by Insulin Is Associated With Changes in the Content of Both PAF53 and UBF

PAF53 is a recently identified protein that has been shown to be closely associated with RNA polymerase I (10). Antibodies to PAF53 block specific transcription from the rDNA promoter but not nonspecific transcription (10). These findings suggest that PAF53 contributes to the formation of the initiation complex on the rDNA promoter. In addition, immunofluorescence assays demonstrated that the nucleolar content of PAF53 in exponentially growing NIH/3T3 cells is greater than that of quiescent cells (10).

In 3T6 cells, the whole cell and nuclear content of PAF53 fell dramatically following serum starvation. Moreover, after 24 h of treatment with insulin, the amount of PAF53 returned to the values observed for exponentially growing 3T6 cells (Fig. 3A). Insulin treatment of H4-II-E-C3 cells also increased the content of PAF53 concomitantly with the rate of rDNA transcription reaching maximal values by 6 h (Figs. 1B and 3B). These experiments demonstrate that the insulin-mediated increase in PAF53 content was not due to a change in localization but reflects the cellular content of PAF53 (Fig. 3, A and B). Thus we report, for the first time, regulation of PAF53 cellular content, in this case mediated by a single hormone, insulin. In addition, there is a greater correlation between changes in PAF53 and rDNA transcription than between RNA polymerase I content and rDNA transcription in 3T6 and H4-II-E-C3 cells. Future studies will be required to determine the mechanism through which PAF53 regulates RNA polymerase I activity and the importance of the regulation.

In addition to RNA polymerase I and PAF53, at least two transcription factors are required for efficient transcription by RNA polymerase I; these are SL-1 and UBF. However, to date, the activity of only one of these factors, UBF, has been shown to be regulated. SL-1, which consists of TATA-binding protein (TBP) and three TBP-associated factors specific for transcription by RNA polymerase I, is absolutely required for rDNA gene transcription in vitro (20-22). In contrast, UBF is not absolutely required for specific initiation of the rDNA promoter, although its addition to UBF-depleted extracts increases the efficiency of transcription in a dose-dependent manner (18, 28, 32). UBF is a DNA-binding phosphoprotein that contains four high mobility group (HMG) boxes, i.e., regions with homology to the DNA-binding domain of HMG1. Purified UBF consists of two polypeptides, referred to as UBF1 and UBF2. UBF2 is smaller than UBF1 by virtue of the deletion of HMG box 2 as a result of the alternative processing of the primary transcript of the UBF gene. Interestingly, UBF1 activates rDNA transcription in vitro and in vivo, but UBF2 is relatively inactive in such assays (18, 32).

A number of studies have linked the cellular content and/or activity of UBF to the rate of rDNA transcription. For example, the decreased rDNA transcription associated with differentiation of L6-myoblasts into myotubes is associated with a significant reduction in the cellular content of UBF protein and mRNA (19). More importantly, the directed overexpression of UBF1 in neonatal cardiomyocytes is sufficient to increase transcription from a cotransfected rDNA reporter construct (12). Similarly, as demonstrated in previous experiments, overexpression of UBF1 in 3T6 cells is sufficient to drive rDNA transcription (Fig. 6).

A previous study demonstrated that serum starvation of 3T6 cells is characterized by a decrease in UBF content, regulated at the level of transcription (7). Moreover, it has been shown that UBF is restored to the values observed in exponentially growing cells with the addition of serum (7). The present study confirms the results reported previously (data not shown; Ref. 7) and demonstrates that treatment of serum-starved 3T6 cells with insulin is sufficient to restore the content of UBF1 to the value observed in exponentially growing cells. Nuclear run-on experiments demonstrate that, at least in part, this response is due to changes in transcription from the UBF gene. A similar response was observed in insulin-treated H4-II-E-C3 cells.

Regulation of UBF at the level of transcription is poorly understood, and thus it will be of interest to determine which element(s) in the UBF promoter is responsive to insulin and/or serum. Cis-acting elements that are responsive to insulin or serum have been described (23-25). For example, the ability of beta -actin and c-fos genes to respond to insulin is, at least partly, due to the presence of insulin-responsive serum response elements in their respective promoters (26, 29).

It is possible that the increase in content of both PAF53 and UBF following insulin treatment may act to stimulate rDNA transcription in a cooperative manner. For instance, it was recently demonstrated that PAF53 can interact with UBF in vitro (10), and it is possible that this interaction may have a functional significance in vivo. Since UBF binds to the rDNA promoter, it is possible that PAF53 may interact with UBF to help recruit the core RNA polymerase I to the rDNA promoter. Thus increasing the nuclear content of both UBF and PAF53 would favor such an association.

In summary, the results reported here establish 3T6 and H4-II-E-C3 cells as suitable models in which to study the stimulation of rDNA transcription by insulin. We show that treatment of serum-starved 3T6 or exponentially growing H4-II-E-C3 cells with insulin does not act to increase the cellular content of RNA polymerase I, even though rDNA transcription is elevated. We conclude that insulin stimulates rDNA transcription in these systems at the level of formation of the initiation complex and at least partly by increasing the cellular contents of PAF53 and UBF. Further studies will be required to investigate the pathway(s) involved in insulin stimulation of the cellular content of PAF53 and UBF and to establish how these events mechanistically stimulate rDNA transcription.

We hypothesize that the effect of insulin on rDNA transcription in these systems is direct, i.e., it reflects a response to the signal transduction cascade initated by occupation of the insulin receptor. Supporting this hypothesis is the observation that maximal insulin-mediated stimulation of rDNA transcription was observed within 3-6 h. This stimulation preceded any effect on cell growth, i.e., an effect on DNA synthesis was not observed before 8-10 h, and cell division does not occur before 24 h in cells in which insulin has an effect on growth. Moreover, the same time course seen for insulin stimulation of rDNA transcription is observed in cells that do not divide, e.g., primary cultures of hepatocytes (1).

Insulin exerts pleiotrophic effects on intracellular signal transduction pathways and a diverse number of components that contribute to the regulation of rDNA transcription. We hypothesize that insulin may regulate several components of the rDNA transcription apparatus. This paper demonstrates that PAF53 and UBF are two of those targets.

    ACKNOWLEDGEMENTS

We thank Drs. Ross Hannan, Scot Kimball, and Howard Morgan for their helpful comments on the manuscript. We also acknowledge Dr. Muramatsu (Dept. of Biochemistry, Saitama Medical School, Japan) for graciously providing the original sample of PAF53 antiserum used in these studies.

    FOOTNOTES

This study was supported in part by National Institutes of Health Grants DK-15658 and DK-13499 (to L. S. Jefferson) and GM-48991 (to L. I. Rothblum), Juvenile Diabetes Foundation Grant JDFI195051 (to L. S. Jefferson), and an award from the Geisinger Foundation (to L. I. Rothblum).

Address for reprint requests: L. S. Jefferson, Dept. of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033.

Received 20 November 1997; accepted in final form 25 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(1):C130-C138
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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