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Originally published In Press as doi:10.1074/jbc.M307735200 on December 19, 2003

J. Biol. Chem., Vol. 279, Issue 10, 8911-8918, March 5, 2004
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Phosphatidylinositol 3-Kinase and mTOR Signaling Pathways Regulate RNA Polymerase I Transcription in Response to IGF-1 and Nutrients*

Martyn J. James{ddagger} and Joost C. B. M. Zomerdijk§

From the Division of Gene Regulation and Expression, Wellcome Trust Biocentre, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom

Received for publication, July 17, 2003 , and in revised form, December 17, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of ribosomal RNA gene transcription by RNA polymerase I (Pol I) is fundamental to ribosome biogenesis and therefore protein translation capacity and cell growth, yet little is known of the key signaling cascades involved. We show here that insulin-like growth factor-1 (IGF-1)-induced Pol I transcription in HEK293 cells is entirely dependent on phosphatidylinositol 3-kinase (PI3K) activity and, additionally, is modulated by the mammalian target of rapamycin (mTOR), which coordinates Pol I transcription with the availability of amino acids. The mitogen-activated protein kinase (MAPK) pathway is weakly stimulated by IGF-1 in these cells and partly contributes to Pol I transcription regulation. Activation of Pol I transcription by IGF-1 results from enhancement of the activity of the Pol I transcription machinery and increased occupancy by SL1 of the endogenous tandemly repeated ribosomal promoters in vivo. The inputs from PI3K, mTOR, and MAPK pathways converge to direct appropriate rRNA gene expression by Pol I in the nucleolus of mammalian cells in response to environmental cues, such as growth factors and nutrients.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell growth and proliferation, which are separable yet coordinately regulated processes, necessitate appropriate production of proteins. The level of protein synthesis is proportional to ribosome abundance in cells, and the biogenesis of ribosomes is in part dependent upon the production of ribosomal RNA. Hence, the level of transcription of the rRNA genes, which is relatively high and accounts for about half of all nuclear transcription (1), is coordinated with changing demands for protein synthesis and nutrient supplies (2, 3). Ribosomal RNA genes are transcribed by RNA polymerase I (Pol I),1 which is recruited to the rDNA promoters in human cells by the essential core promoter-binding factor SL1, a TBP-TAFI complex (4), and transcription is activated by the transcription factor UBF, an architectural protein with multiple HMG boxes (5).

Yeast cell growth and proliferation is modulated primarily by the availability of nutrients in their environment, and rRNA synthesis appears to be regulated accordingly (6). Pol I transcription in multicellular eukaryotes has also been shown to respond to nutritional conditions (7). Additionally, ribosomal RNA synthesis in these cells is influenced by other extracellular signals, such as hormones, mitogens, and growth factors (1). Differentiation of cells generally decreases rRNA gene expression, and Pol I transcription is completely repressed during mitosis (8). Finally, regulation of Pol I transcription is linked to cellular hypertrophy (9, 10). Despite these findings, little is known of the signaling pathways involved in the control of Pol I transcription. We have investigated the signal transduction pathways that regulate the rRNA gene expression in proliferating mammalian cells (HEK293) in their rapid response to insulin-like growth factor IGF-1 and nutrient availability.

Here, we provide evidence for the essential role of PI3K, a modulating role for the mTOR pathways and a minor role for the MAPK cascade, in the regulation of rRNA gene expression by Pol I in response to nutrients and growth factor stimulation. Furthermore, our results demonstrate that the control of rRNA gene expression can be achieved by modulation of the activity of the Pol I transcription machinery, including an increased promoter occupancy by the essential Pol I transcription factor SL1 following IGF-1 induction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Inhibitory Compounds—HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum until reaching 70–80% confluency (% field area) and then serum-starved (DMEM without serum) for 16–20 h, prior to stimulation with 2 nM IGF-1 (Invitrogen). Inhibitory compounds LY294002 (50 µM), PD98059 (50 µM), and rapamycin (50 nM) (Calbiochem) were added 30 min prior to the addition of IGF-1 to cells. Amino acid starvation was for 1 or 2 h in Earle's Balanced Salt Solution, to which glucose was added to 4.5 g/liter to match that in DMEM. 3-Methyladenine (Sigma) was used at 5 mM.

Cell Extracts and Immunoblotting—HEK293 cell extracts were prepared and then analyzed by immunoblotting as described (11), with primary antibodies: mouse monoclonal specific for TBP, rabbit polyclonal specific for human TAFI63 (12, 13), affinity-purified rabbit anti-peptide polyclonal specific for largest subunit of human Pol I A190 (14), rabbit polyclonal specific for UBF, mouse polyclonal specific for PAF53, rabbit polyclonal specific for p70/p85 S6 kinase, sheep polyclonal specific for MAPKAP-K1{alpha} (phosphothreonine-360), and a mouse monoclonal specific for ERK (phosphotyrosine-204; Santa Cruz Biotechnology).

Total RNA Isolation and Analysis of pre-rRNA by S1 Nuclease Protection—Total RNA was isolated using the RNeasy mini kits (Qiagen) according to the manufacturer's instructions. The concentration of RNA was determined spectrophotometrically, and integrity of the RNA was checked on agarose gels. Pre-rRNA in total RNA (0.5 µg) was analyzed using an S1 nuclease protection assay using an excess of an S1 oligonucleotide identical to –20 to +40 of the human rDNA template strand, and thus complementary to the first 40 nucleotides of the 45 S rRNA primary transcript (14, 15). The 5'-end-labeled S1 oligonucleotide (0.1 pmol) was annealed for 4–16 h at 50 °C to 0.5 µg of HEK293 cell total RNA in 40 mM PIPES (pH 6.4), 0.4 M NaCl, 1 mM EDTA (pH 8), and 3 µg/µl Escherichia coli tRNA (Roche Applied Science), and then digested with 350 U of S1 Nuclease (Amersham Biosciences) as described (14). Products were separated on 7.5 M urea/8% polyacrylamide gels (National Diagnostics). Signals were quantified with the aid of a phosphorimager (Fuji).

Nuclear Extracts and in Vitro Transcription—HEK293 cell nuclear extracts (NE) were prepared as follows. HEK293 cells, after aspiration of media, were removed from the tissue culture dish by pipetting with cold PBS (10 ml per dish) and collected by centrifugation (1000 x g for 5 min (Beckman GS-6R)). The cells were then washed once with ice-cold PBS and resuspended in one packed cell volume (PCV) of hypotonic buffer (10 mM HEPES (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, phosphatase inhibitors (50 mM {beta}-glycerophosphate, 1 mM benzamide, 0.5 mM sodium vanadate) and protease inhibitors (1x EDTA-free protease inhibitor mixture (Roche Applied Science), 0.1 mM AEBSF (Calbiochem), and 1.5 mM EGTA)), left on ice for 15 min, and mechanically sheared by five passes through a 23-gauge syringe needle. Nuclei were collected by microcentrifugation at full speed for 20 s. The supernatant was then removed and the nuclear pellet was resuspended in two-thirds PCV of nuclei extraction buffer (20 mM HEPES, pH 8.0, 1.5 mM MgCl2, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA (pH 8), 1 mM dithiothreitol, and protease and phosphatase inhibitors). The nuclei were then left for 30 min at 4 °C with constant mixing (Dynal rotator), and then centrifuged in a microcentrifuge for 5 min at 14,000 rpm at 4 °C to remove the nuclear debris. Protein concentration in the supernatant (nuclear extract, NE) was determined and normalized using the Bradford reagent (BioRad) as suggested by the manufacturer's instructions using bovine serum albumin as a standard.

In vitro transcription reactions were performed as described previously (14–16) in a total volume of 25 µl with 5–10 µg NE in 50–75 mM NaCl, 5% glycerol and supercoiled prHu3 plasmid DNA (100 ng), which contains the human rRNA gene promoter from –515 to +1548 in pBR322 (17), for 30 min at 30 °C. The resulting transcripts were isolated by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation, and analyzed in an S1 nuclease protection assay after annealing the RNA to an excess of a 5'-end-labeled S1-oligonucleotide, as described above for the detection of pre-rRNA in total cellular RNA.

Chromatin Immunoprecipitation (ChIP) Assay—Chromatin immunoprecipitation was performed essentially as described (18, 19). Immunoprecipitation of cross-linked chromatin was performed with TBP-specific antibodies (Mouse monoclonal 3G3, a kind gift from L. Tora) coupled to protein G-Sepharose. Immunoprecipitated chromatin-derived DNA was analyzed in a quantitative PCR with primer pairs specific for the ribosomal promoter (forward primer rDNA promoter region from –182 5'-TGTCCTTGGGTTGACCAG-3', reverse primer +23 5'-TCGCCAGAGGACAGCGTG-3'), the internal transcribed spacer-1 of the rDNA (forward primer rDNA +7060 5'-CCGAGTTCCCGTGGCCGCCGCCTGCG-3', reverse primer rDNA +7299 5'-CGGAACCGCGGCGACCGGGACGCGCT-3') or the intergenic spacer region of the rDNA (forward primer rDNA +28098 5'-TAGACTCTTCTACTTGGGCTTTGGGA-3', reverse primer rDNA +28321 5'-ATGTCAGCCTGGCAAGAATGAGATCG-3'). Reactions were carried out in a volume of 25 µl and contained in addition to DNA, 0.4 pM primers, 1.5 mM MgCl2,40 µM dNTPs (Ultrapure, Amersham Biosciences), 2.5 units of TaqDNA Polymerase (Promega), and 1 µCi of 3000 Ci/mmol [{alpha}-32P]dCTP (ICN). Cycling was for 5 min at 95 °C, followed by 18 cycles of 45 s at 95 °C, 45 s at 63 °C, and 2 min at 72 °C, and then a final 10 min at 72 °C. PCR products were resolved on a 6% non-denaturing polyacrylamide gel in 1x Tris borate/EDTA and quantified with the aid of a Fuji Phosphorimager. We also prepared DNA from the cross-linked chromatin solution prior to immunoprecipitation and used this DNA, at the equivalent of 1% of the chromatin solution used for an immunoprecipitation, as an input control in the PCR reactions. As a control for the linearity of the PCR, we used 0–5 ng of plasmid prHu3 (rDNA promoter).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RNA Polymerase I Transcription Is Stimulated by IGF-1—To study the signaling of IGF-1 to rRNA synthesis by Pol I, 45 S nascent rRNA levels were assessed in HEK293 cells. The cells were grown to about 80% confluency (% field area) and cultured for a further 16 h in medium with 10% or no serum. The cells were then stimulated with IGF-1 and, as a sensitive and quantitative measure for Pol I transcription in vivo, pre-rRNA levels in total cellular RNA were determined by S1 nuclease protection of the extreme 5'-end of the nascent rRNA. The 5'-end of the 45 S rRNA is rapidly processed within cells (20), and therefore the steady-state level of this pre-rRNA primarily reflects the rate of synthesis by Pol I. Decreased processing, to explain any increase in pre-rRNA in response to growth factor is unlikely, since this would actually lower the amount of 18, 5.8, and 28 S rRNA, which would be counterproductive to ribosome biogenesis during cell growth. Note that if the rate of rDNA transcription increases but the rate of rRNA processing does not increase with the same kinetics then the S1 nuclease assay described may over- or underestimate the actual increase in transcription.

Pol I transcription was stimulated by IGF-1 in HEK293 cells cultured with and without serum. The relative stimulation was higher in the serum-starved cells since serum-starvation had reduced the level of Pol I transcription 2-fold (Fig. 1A). IGF-1, at concentrations of 2–50 nM, produced a 3–3.5-fold increase in the abundance of pre-rRNA (Fig. 1B), with a half-maximal increase at 0.35 nM, which suggests a specific response since this concentration is within the range of other IGF-1-induced cellular responses (21). IGF-1 has been shown to activate the PI3K/PKB/p70 S6K pathway and the phosphorylation of the 70 and 85 kDa isoforms of S6K served as a control for activation of this pathway (Fig. 1B). The response of Pol I transcription to IGF-1 was rapid (Fig. 1C) and reached a maximum of 3.5–4-fold over basal rRNA synthesis after about 3 h. Thus, in HEK293 cells physiological concentrations of IGF-1 induce an early response in Pol I transcription. Nuclear extracts isolated from these cells displayed Pol I transcription activity that was ~1.5–2-fold greater than that of nuclear extracts from serum-starved cells (Fig. 1D). Unfortunately, the relatively modest increases did not permit a reliable identification of the target(s) of the signaling pathways following fractionation of the extracts. None of the tested components of the Pol I transcription machinery in the whole cell extracts was found to increase in abundance consistently after IGF-1 treatment (Fig. 1E).



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  		FIG. 1.
Ribosomal RNA synthesis by Pol I is rapidly stimulated in response to the growth factor IGF-1. A, stimulation by IGF-1 of Pol I transcription in HEK293 cells cultured with or without serum. HEK293 cells were cultured in DMEM for 16 h with or without 10% fetal calf serum, and then stimulated with 2 nM IGF-1 for up to 2 h. Pre-rRNA (5'-ETS) levels were determined by S1 nuclease protection, and representative autoradiographs are shown. The levels of pre-rRNA of two experiments were quantified and are represented in the bar graph above the gel, with the levels of pre-rRNA in unstimulated serum-starved cells set at 1.0. Brackets represent the highest and lowest values for the duplicate experiments. B, concentration-dependent stimulation of Pol I transcription by IGF-1. IGF-1 was added to serum-starved HEK293 cells for 90 min at concentrations between 0 and 50 nM (lanes 1–6). Total RNA and cell extracts were isolated and subjected to S1 nuclease protection to detect the 5'-end of the pre-rRNA (in duplicate). Immunoblot analysis of p70 and p85 isoforms of S6K are shown with open and black triangles pointing to the hypo- and hyperphosphorylated forms, respectively. C, time-dependent stimulation of Pol I transcription by IGF-1. Serum-starved HEK293 cells were stimulated with 2 nM IGF-1 for 0 to 4 h (lanes 1–5). Total RNA and cell extracts were isolated and subjected to S1 nuclease protection to detect the 5'-end of the pre-rRNA (in duplicate) and immunoblotting for p70/85 S6K, respectively. Bar graphs are as for A. D, nuclear extract isolated from IGF-1-stimulated HEK293 cells displayed increased Pol I transcription initiation in vitro. Transcript levels were determined by S1 nuclease protection and quantified from in vitro transcription reactions with 5 µg of nuclear extracts derived from cells exposed for 0–3 h to 2 nM IGF-1 and the rDNA promoter (top panel). The levels of in vivo synthesized pre-RNA in these nuclear extracts were assessed in a control in vitro transcription reaction in which the rDNA promoter template was absent (bottom panel). E, immunoblot analysis with antisera against A190 (Pol I), UBF, TAFI63 (SL1), PAF53 and TBP of whole cell extract isolated from serum-starved HEK293 cells, which had been stimulated with 2 nM IGF-1 for 0–4 h.

 
IGF-1 acts as a mitogen, promoting activation of the Ras-MAPK pathway and stimulating proliferation of growth arrested cells. However, FACS analysis for DNA content revealed that HEK293 cells, serum-starved for 20 h, did not accumulate in the resting G0 phase of the cell cycle. The fraction of cells in G1 and S decreased with a corresponding 20% increase of cells in G2/M. When these cells were then stimulated with IGF-1, the proportion of cells in G2/M decreased by 10%, whereas those in G1 and S increased by about 5% after 4 h (data not shown). The observed 2.5 to 3.5-fold increase in Pol I transcription following serum-starvation and IGF-1 exposure are therefore unlikely to be accounted for by changes in the cell cycle distribution.

PI3K-dependent Stimulation of Pol I Transcription in Response to IGF-1 Is Modulated by mTOR—Cell-permeable inhibitors were used to elucidate the signaling pathways involved in regulating Pol I transcription in response to IGF-1. PI3K is known to act almost immediately downstream of IGF-1 via interactions with insulin receptor substrate (IRS) proteins (22) (Fig. 5). Preincubation of cells for 30 min with a PI3K inhibitor, LY294002 (23, 24), completely blocked IGF-1 stimulation of rRNA synthesis in vivo and the phosphorylation of p70/85 S6K (Fig. 2A). This suggests that PI3K activity is essential for mediating the transcriptional response in the nucleolus to extracellular IGF-1. Since other mammalian members of the PIK family are sensitive to LY294002 (25), a role for these kinases in Pol I transcription cannot be fully excluded in this experiment, however.



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  		FIG. 5.
Signaling pathways which control cellular growth and cell proliferation in response to IGF-1 and amino acids target rRNA gene transcription by RNA polymerase I in the nucleolus. The scheme summarizes the important connections within and between signaling cascades, which target Pol I transcription (thick lines), identified in this study in the context of known or probable signaling events affecting protein translation (thin lines; see references in text). Pol I transcription is stimulated by IGF-1 and external amino acids and this is mediated primarily by PI3K and its downstream targets which may not exclusively signal through the mTOR kinase alone, and secondarily by the ERK/MAPK cascade. The amino acids effect is primarily mTOR-dependent, with an apparent influence of amino acid withdrawal on ERK/MAPK signaling. Stimulation of Pol I transcription by IGF-1 is entirely dependent on the availability of amino acids. The sites of action of inhibitors used here are shown. The coherent signaling through these cascades leads to stimulation of rRNA gene transcription by activating components of the Pol I transcription machinery during initiation of transcription and increased occupancy of rDNA promoters by the essential transcription initiation factor SL1, which coordinates preinitiation complex formation.

 



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  		FIG. 2.
The Pol I transcription response to IGF-1 is dependent on PI3K activity and involves the mTOR and MAPK signaling pathways. A, serum-starved HEK293 cells were incubated for 30 min with 50 µM LY294002 (lanes 6–10) or drug carrier alone (DMSO; lanes 1–5) and then stimulated with 2 nM IGF-1 for 0–120 min. Total RNA and cell extracts were isolated from these treated cells and subjected to S1 nuclease protection (in duplicate) and immunoblotting for p70/85 S6K, respectively. Triangles at the S6K immunoblot are as for Fig. 1B. Bar graphs are as for Fig. 1A. B, serum-starved HEK293 cells were preincubated with either 50 nM rapamycin (lanes 4–6), 50 µM PD98059 (lanes 7–9), a mixture of 50 nM rapamycin and 50 µM PD98059 (10–12) or drug carrier alone (DMSO; lanes 1–3), and exposed to 2 nM IGF-1 for 0–2 h. Total RNA and cell extract were isolated and subjected to S1 nuclease protection, to detect the 5'-end of the pre-rRNA (in duplicate), and immunoblot analysis for p70/85 S6K and phospho-Thr360-specific antibodies for MAPKAP-K1{alpha}, respectively. Bar graphs are as for Fig. 1A.

 
Mammalian TOR regulates cell growth and protein translation in response to mitogens or growth factors and in accordance with nutrient availability (26–28). mTOR downstream signaling in vivo can be blocked by rapamycin (Fig. 5). At 50 nM rapamycin specifically inhibited the IGF-1-induced stimulation of Pol I transcription by 46 and 38% after 1 or 2 h IGF-1 exposure, respectively (Fig. 2B, compare lanes 2 and 3 with 5 and 6, respectively), while not significantly altering basal Pol I transcription (Fig. 2B, compare lane 4 with 1). The IGF-1-induced phosphorylations of both isoforms of S6K were completely inhibited by the immunosuppressant.

IRS couples to the IGF-1 receptors and not only activates PI3K but also guanine nucleotide exchange factors (GEFs), like SOS, which convert small GTPases, like Ras, into an active GTP-bound state and consequently activate the Ras-MAPK pathway (29, 30) (Fig. 5). When the Ras-MAPK pathway was specifically inhibited with the compound PD98059, which blocks MAPK/ERK kinase (MEK) activation by Raf (31, 32), the IGF-1 induced stimulation of rRNA synthesis was reduced by 25 and 40% (Fig. 2B, lanes 9 and 8). This partial inhibition is consistent with the relatively weak signaling from IGF-1 through the Ras-MAPK cascade in HEK293 cells (33), as reflected by the low levels of MAPKAP-K1{alpha} or p90rsk phosphorylation (Fig. 2B, lanes 2 and 3).

Preincubation with a mixture of rapamycin and PD98059, which inhibited both the mTOR and Ras-MAPK pathways, resulted in an almost complete block (92% reduction) of Pol I activation within the first hour of IGF-1 exposure (see Fig. 2B, lane 11). Intriguingly, after 2 h of exposure to IGF-1, and with the mTOR and MAPK pathways blocked, the cells were still showing a partial (37%) Pol I transcription response (Fig. 2B, lane 12). These data suggest that in addition to the mTOR and Ras-MAPK pathways, which mediate the early Pol I response to growth factor stimulation of PI3K, alternative downstream effectors of the PI3K pathway may contribute to the delayed Pol I response to IGF-1.

IGF-1 Stimulation of rRNA Synthesis Is Dependent upon Amino Acids—In mammalian cells, mTOR signals to S6K and 4E-BP1, but not in the absence of amino acids, and hence allows activation of translation only under conditions that are favorable to cell growth. Thus mTOR is suggested to act as the molecular modulator of cell growth and to link this to nutrient sensing (27, 28). Withdrawal of amino acids did not affect rRNA synthesis in HEK293 cells (Fig. 3A, lanes 1–3), yet addition of essential amino acids to amino acid-starved cells produced a concentration-dependent stimulation of rRNA synthesis (~2-fold; Fig. 3A, lanes 4–6), concomitant with a detectable S6K phosphorylation (Fig. 3B, lane 4). This stimulation of transcription is entirely dependent on PI3K and in part requires mTOR activities (Fig. 3B, lanes 6 and 5, respectively). The addition of both IGF-1 and amino acids resulted in an even stronger response (Fig. 3A, compare lanes 8–10 with 11).



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  		FIG. 3.
The response of Pol I transcription to IGF-1 is dependent on the availability of amino acids. A, IGF-1 and essential amino acids up-regulate Pol I transcription in an additive manner. Serum-starved HEK293 cells were amino acid-starved for 1 or 2 h (lanes 2–10), then DMEM (lanes 4 and 8), a 0.5x stock or 1x stock of essential amino acids (lanes 5 and 9, and 6 and 10, respectively) or no amino acid stock (lanes 2, 3, and 7) was added and the cells were incubated with (lanes 7–10) or without (lanes 2–6) 2 nM IGF-1 for 1 h. Analysis and representation of transcript levels from two independent experiments are as for Fig. 1A. B, amino acid stimulation of Pol I transcription is dependent on the activities of PI3K and mTOR. Serum-starved HEK293 cells were amino acid-starved for 1 or 2 h (lanes 2–6), then stimulated with a 1x stock of essential amino acids (lanes 4–6) in the presence of the inhibitors rapamycin (50 nM, lane 5) or LY294002 (50 µM, lane 6), or in the absence of inhibitors (lane 4). Analysis and representation of transcript levels from two independent experiments are as for Fig. 1A. Triangles for S6K immunoblot are as for Fig. 1B. C, IGF-1 stimulation of Pol I transcription is completely blocked in the absence of both autophagy and extracellular amino acid supply. Serum-starved HEK293 cells were either amino acid-starved for 1 or 2 h (lanes 3–8) or not amino acid-starved (lanes 1 and 2), then exposed to 5 mM 3-methyladenine for 45 min to inhibit autophagy (lanes 4, 6, and 8) or left unexposed (lanes 1–3, 5, and 7), and then incubated with (lanes 2, 7, and 8) or without (lanes 3–7) IGF-1 for 1 h. Pre-rRNA levels from two independent experiments were determined and are presented as for Fig. 1A. Triangles for S6K immunoblot are as for Fig. 1B. An immunoblot with phosphospecific ERK antibodies is presented.

 
Exposure of amino acid-starved cells to IGF-1 yielded a Pol I transcriptional response, albeit incomplete (41%; Fig. 3A, compare lanes 7 and 11), but in amino acid-deprived HEK293 cells now supplemented with 3-methyladenine, which inhibits autophagy (34) and hence amino acid salvage pathways (35), the IGF-1-induced response of rRNA synthesis was completely lost (Fig. 3C, compare lanes 7 and 8). The low level signaling through the Ras-MAPK pathway appears, at least in part, to be dependent upon these nutrients as well (Fig. 3C, lane 8). We conclude that the growth factor stimulation of Pol I transcription is dependent on amino acids, and this is likely to involve the established role of mTOR in sensing amino acid availability (27, 28).

Ribosomal Promoter Occupancy by SL1 Increases in IGF-1-stimulated Cells—Next, we asked whether the stimulated transcription may in part result from an increased occupancy of the multicopy rDNA promoters in vivo by the essential TBP-TAF complexes, SL1, which recruit Pol I (11). We therefore assessed the steady-state numbers of SL1 complexes associated with rDNA promoters in IGF-1-stimulated cells, using the chromatin immunoprecipitation assay. Immunoprecipitation was performed with an antibody specific for TBP, which itself does not bind the rDNA promoter (36), and is more efficient in the immunoprecipitation than the antibodies specific for TAFIs currently available to us. Moreover, the activator of Pol I transcription, UBF, appears unsuitable in such studies since its binding to the rDNA repeat is not restricted to active promoter sequences (37).

Linear PCR reactions (Fig. 4B, lanes 1–4) for rDNA promoter sequences (–182 to +23, Fig. 4A), and normalized amounts of input chromatin (Fig. 4B, lanes 5–7) were established. Strikingly, stimulation of cells with IGF-1 reproducibly resulted in an ~50% rise in rDNA promoter-TBP complexes and, by inference, of promoter-bound SL1 (Fig. 4B, lanes 9 and 10, and Fig. 4C, lanes 1 and 2). Incidentally, incubation of serum-starved cells for 4 h in PBS resulted in an ~40% drop in the recovery of SL1-rDNA promoter complexes compared with that from untreated serum-starved cells (Fig. 4B, lanes 8 and 9, respectively) and a corresponding decrease in rRNA synthesis (data not shown). This illustrates that there is a correlation between the level of SL1 promoter occupancy and Pol I transcription. Importantly, the IGF-1-induced increase in promoter occupancy of SL1 is the result of signaling through PI3K, since LY294002 blocked this response (Fig. 4C, lane 3). No PCR products were synthesized from immunoprecipitated DNA using primers specific for the internal transcribed spacer 1 or for the intergenic spacer (Fig. 4, A and D, lanes 4–10). Taken together, the data indicate that IGF-1 signals to increase steady-state levels of SL1 at rDNA promoters, which, given the important role of SL1 in directing preinitiation complex formation, likely contributes to increased rRNA synthesis in HEK293 cells.



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  		FIG. 4.
IGF-1 stimulation of HEK293 cells leads to an increased ribosomal promoter occupancy of the TBP-TAFI complex SL1. A, schematic representation of one of the tandem human rDNA repeats, with the transcription start site (+1) and the black boxes representing the 18, 5.8, and 28 S rRNA genes. Location of primer sets used in the PCR of the ChIP: internal transcribed spacer 1 (rDNA +7060 to +7299) and interGenic spacer (+28098 to +28321) and rDNA promoter (–182 to +23). B, IGF-1 stimulates rDNA promoter occupancy of SL1. Serum-starved HEK293 cells (lanes 5–14) were incubated for 4 h in PBS (lanes 5, 8, and 12), stimulated for 3 h with 2 nM IGF-1 (lanes 7, 10, and 14) or untreated (lanes 6, 9, and 13). The cells were then treated with formaldehyde prior to ChIP. In control PCRs, 5–1.25 ng of prHu3 plasmid DNA (containing the ribosomal promoter, lanes 1–3) and 1.0% chromatin input DNA (lanes 5–7) was used as template to amplify promoter rDNA by PCR. TBP-specific monoclonal antibodies (lanes 8–11) and control mouse IgG antibodies (12–14) were used in the ChIP. The PCR products for the rDNA promoter are shown, quantified with a phosphorimager, and the yields (corrected for the slight variations in the input DNA, see lanes 5–7) have been calculated and expressed relative to the level in un-stimulated cells (lane 9, 100%). *, in lane 11, twice the amount of chromatin solution was immunoprecipitated with the TBP-specific antibody, and since twice the amount of PCR product was observed this indicated that not only the PCR but also the immunoprecipitation was reproducible and within the linear range. C, increased SL1 promoter occupancy is dependent on IGF-1-induced activation of PI3K. Chromatin immunoprecipitations were performed as in B, from cells, which were IGF-1-induced with (lane 3) or without (lane 2) 50 µM LY294002. D, TBP subunit of SL1 did not bind detectably to regions in the internal transcribed sequences and intergenic spacer. Analysis as in Fig. 4B, but PCR analysis of immunoprecipitated DNA was performed with primers for the ITS1 (+7060 to +7299) and IGS (+28098 to +28321) (see Fig. 4A). Asterisk (lane 7) denotes the same procedure as that in lane 11 of Fig. 4B.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PI3K, mTOR, and MAPK Coordinate Growth Factor Signaling to Pol I Transcription with Nutrient Availability—In this study we set out to determine which signaling cascades regulate Pol I transcription in response to the growth factor IGF-1 and amino acid availability in HEK293 cells.

IGF-1 regulates cell growth, survival, proliferation, aging, and longevity (38, 39), processes which, when deregulated, may be conducive to tumor growth. We have demonstrated that PI3K activity is essential in the response of Pol I transcription to the growth factor IGF-1. Moreover, rapamycin inhibition studies suggested that downstream signaling through mTOR contributes to stimulated Pol I transcription. In HEK293 cells, signaling through Ras-MAPK contributes weakly to stimulated Pol I transcription (pathways have been summarized in Fig. 5). Moreover, since inhibitors of both the mTOR and MAPK pathways did not entirely block growth factor-stimulated Pol I transcription after 2 h, while almost completely inhibiting the early response, PI3K signals to other pathways that impinge on Pol I transcription by mechanisms with distinct kinetics. One such pathway downstream of PI3K may include PDK1 and PKB (reviewed in Refs. 40 and 41). The recently reported translocation to the nucleolus of IRSs following growth factor stimulation may also contribute to the regulation of Pol I transcription (42).

mTOR coordinates nutritional and mitogenic signals to control mammalian cell growth even when cell cycle progression is blocked (43). This is consistent with a genetically demonstrable role for Drosophila TOR in cell growth control (44, 45). We found that the mTOR inhibitor rapamycin attenuates the rapid response of ribosomal transcription to IGF-1 also independently of cell cycle effects in HEK293 cells. This is consistent with a report that prolonged treatment of fibroblasts with rapamycin reduced the steady-state levels of 18 and 28 S rRNAs during okadaic acid-induced cellular hypertrophy or cell growth (46). By contrast, the long-term (24 h) effects of rapamycin on rRNA synthesis in mammalian cells have been associated with cell cycle arrest (47). Although the mTOR inhibitor rapamycin attenuated the rapid response of ribosomal transcription to IGF-1, it did not reduce the pre-rRNA levels in IGF-1-stimulated cells to those of unstimulated cells. In addition to invoking rapamycin-insensitive pathways in this response, it is also feasible that rapamycin inhibits not only rRNA synthesis but also pre-rRNA processing in HEK293 cells, as has been demonstrated in yeast (48), such that the steady-state levels of pre-rRNA would drop only partially.

The involvement of the mTOR pathway in the response of ribosomal transcription to IGF-1 is particularly relevant since its activity is modulated by nutrients, for example, amino acid availability and, here, we have demonstrated that amino acids generate signals which stimulate Pol I transcription and, further, that depletion of the cell external and internal pools of essential amino acids, by a combination of amino acid starvation and inhibition of autophagy, specifically abolished the response of Pol I transcription to IGF-1. Thus, the IGF-1 signal is entirely dependent on the availability of amino acids. Therefore, the mTOR pathway is likely to be part of a control mechanism to prevent wasteful synthesis of rRNA during inappropriate growth conditions. The control of Pol I transcription by mTOR may explain the reported negative effects of amino acid starvation on rRNA synthesis (7, 49).

The mTOR pathway to Pol I transcription appears functionally conserved from mammals to yeast, because in yeast rapamycin treatment also leads to repression of Pol I (and Pol III) transcription (48, 50). The control by mTOR of the transcription by Pol I of the rRNA genes, shown here and by Hannan et al. (51), the transcription of the 85 ribosomal protein genes by Pol II (52, 53), the synthesis of many small RNAs, such as 5 S rRNA and tRNAs by Pol III (50), the translation of specific mRNAs, for example of translation factors and ribosomal proteins and of global translation (reviewed in Ref. 54; see Fig. 5), all highlight the pivotal role of mTOR in rapidly coordinating ribosome biogenesis and translational capacity important for mammalian cell growth in response to both appropriate nutrient supply and growth factors.

The low level of activation by IGF-1 of the Ras-MAPK signaling to Pol I, which occurs even in the absence of a significant cell proliferation response, also appeared impaired when the availability of amino acids was restricted, suggesting a coordinate response and cross-talk between growth factor-induced signaling pathways under nutritionally restrictive conditions. Perhaps the absence of amino acids generates additional signals distinct from those involving mTOR, as also revealed in the overlapping but distinct gene expression profiles of rapamycin-treated versus amino acid-starved lymphocytes (55).

Therefore, our data show that the PI3K, mTOR and MAPK pathways converge to coordinately regulate appropriate rRNA gene expression in response to environmental cues, such as growth factors and nutrients, and so tune the protein translation capacity and mass accumulation of a mammalian (Fig. 5). We believe that these fundamental signaling pathways, associated with both cell growth and proliferation, are likely to operate in the control of Pol I transcription in many cell types and that the relative contribution of each pathway will vary according to cell type and the specific environmental conditions.

Stimulated Transcription by Pol I—While the focus of this study is on the signaling pathways involved in regulating Pol I transcription in response to IGF-1 in rapidly growing cells, our data, together with that of others, point to multiple mechanisms that operate in parallel with different kinetics. The abundance of UBF or any of the other tested components, such as SL1 and the Pol I subunits A190 and PAF53, had not changed in the IGF-1-induced HEK293 cells and yet an increased rRNA synthesis by the Pol I machinery was observed in cell-free transcription assays. This may indicate post-translational modifications that rapidly affect the components of the Pol I machinery at transcription initiation. Given the complexity of the multiple pathways stimulated in response to IGF-1 in HEK293 cells, several targets may be affected, each contributing to some degree to stimulated transcription. Unfortunately, the relatively modest increases observed in our cell-free Pol I transcription assays did not permit, by fractionation of cell extracts, the reliable identification of the target(s) in the Pol I machinery affected by the signaling pathways implicated here. Perhaps in future studies to identify these targets other cell types that have a single predominant signaling pathway activated by a growth factor may be more suitable. For example, EGF-stimulated Pol I transcription in human neuroepithelioma cells appears almost exclusively to involve UBF activation by the ERK/MAPK pathway (56), and in mouse NIH3T3 cells the MAPK pathway has recently been shown to control RRN3/TIF-IA phosphorylation under varying growth conditions (57). We may speculate that the PI3K, mTOR and MAPK signaling cascades operate to control Pol I-RRN3 complex formation in HEK293 cells, given that the association of hRRN3/TIF-IA with Pol I, to generate Pol I{beta} (11), has been demonstrated to involve phosphorylations (58, 59). In yeast, it has recently been reported that the Tor signaling pathway regulates the Rrn3p-dependent recruitment of Pol I (60). Other studies have suggested various complementary scenarios affecting Pol I factors, further increasing the complexity and network of regulation, and these include regulated phosphorylation of UBF (51, 56, 61–63), altered abundance of UBF (9, 64, 65), and the expression of an inhibitor of transcription (66).

In addition to the observed increase in rRNA synthesis by the Pol I machinery in cell-free transcription assays, we have demonstrated a significantly increased promoter occupancy of the endogenous and tandemly repeated rRNA genes by the essential TBP-TAF complex SL1, which nucleates productive Pol I preinitiation complex formation, in cells stimulated with IGF-1. The increased promoter occupancy by SL1 was dependent on signaling through PI3K and correlated with levels of Pol I transcription in both IGF-1 stimulated and PBS-treated cells. The steady-state promoter occupancy is determined by the equilibrium between on and off rates for SL1 at an individual rDNA promoter and the number of promoters bound by SL1 in the tandem repeats. While the ChIP assay cannot discriminate between these two possibilities, it is possible that the increased promoter occupancy reflects a significant increase in the number of rDNA promoters bound by SL1, since SL1 was found to remain stably bound at rDNA promoters through multiple rounds of transcription in a cell free system (14)2 and throughout mitosis in vivo (67–69). Indeed, in mitogen-stimulated lymphocytes, an increase in rRNA synthesis is reportedly concomitant with a rise in the number of active rRNA genes (70, 71) and also, in yeast, a change in the proportion of non-nucleosomal (active and perhaps potentially active) and nucleosomal (inactive) rRNA gene copies in response to variations in environmental conditions and hence cell growth has been observed (72). Engagement of new rRNA gene promoters in transcription might correlate with the observed delayed (2 h) PI3K-dependent response of HEK293 cells to IGF-1, as this might require chromatin remodeling activities to increase rDNA promoter accessibility to SL1 prior to initiation. The PI3K, mTOR, and MAPK signaling pathways activated in response to growth factors have all been shown to stimulate these activities (73–75). A recent study in yeast suggests that the Tor signaling pathway modulates Pol I transcription without altering the number of active rRNA gene repeats (60).

It is possible that not all of the mechanisms leading to IGF-1-stimulation of rRNA synthesis in vivo are reconstituted in vitro given that the kinetics and extent of stimulation differ. Stimulation of the activity of the Pol I machinery in response to IGF-1 may be rapid and therefore reflected in the rapid increase in transcription in the first hour which occurs both in vivo and in vitro. Chromatin remodeling activities, on the other hand, which might be required for the regulation of nucleolar structure by mTOR (76) as well as for increased rDNA promoter occupancy by SL1 in response to IGF-1, cannot contribute to the increased transcription which occurs in vitro since the template DNA is naked but could be involved in the delayed increase in transcription which occurs only in vivo.

The importance of Pol I transcription, which is at the heart of ribosome biogenesis, as a downstream target in the physiological response of cells to growth factors and nutrients is underscored by our findings that multiple signaling pathways with well established roles in both cell growth and proliferation coordinate the regulation of Pol I transcription in response to the growth factor IGF-1 and amino acid availability, probably through multiple mechanisms operating in parallel with distinct kinetics.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a Medical Research Council Ph.D. studentship. Current address: Institute of Biotechnology, Viikki Biocenter, P. O. Box 56, FIN-00014 University of Helsinki, Finland. Back

§ A Wellcome Trust Senior Research Fellow in the Basic Biomedical Sciences. To whom correspondence should be addressed. Tel.: 44-1382-344242; Fax: 44-1382-348072; E-mail: j.zomerdijk{at}dundee.ac.uk.

1 The abbreviations used are: Pol I, RNA polymerase I; DMEM, Dulbecco's modified Eagle's medium; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; IGF, insulin-like growth factor; HEK, human embryonic kidney; IRS, insulin receptor substrate; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; ChIP, chromatin immunoprecipitation assay. Back

2 J. K. Friedrich, K. I. Panov, and J. C. B. M. Zomerdijk, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank A. Sparks for technical assistance with the FACS analysis. We thank L. Tora for monoclonal antibodies specific for human TBP, B. McStay for UBF antibodies, M. Muramatsu for PAF53 antibodies, P. Cohen for phosphospecific MAPKAP-K1{alpha} antibodies, and C. Proud for p70/p85 S6 kinase antibodies. We thank Dario Alessi, Chris Proud, Calum Sutherland, Jackie Russell, Taciana Kasciukovic, Kostya Panov, and other members of the Zomerdijk laboratory for support and critical reading of the manuscript, and in particular Jackie Russell for help with writing the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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Inhibition of PI-3 kinase/Akt/mTOR, but not calcineurin signaling, reverses insulin-like growth factor I-induced protection against glucose toxicity in cardiomyocyte contractile function
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J. K. Friedrich, K. I. Panov, P. Cabart, J. Russell, and J. C. B. M. Zomerdijk
TBP-TAF Complex SL1 Directs RNA Polymerase I Pre-initiation Complex Formation and Stabilizes Upstream Binding Factor at the rDNA Promoter
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 J. Biol. Chem.Home page
J. Chen, A. Wu, H. Sun, R. Drakas, C. Garofalo, S. Cascio, E. Surmacz, and R. Baserga
Functional Significance of Type 1 Insulin-like Growth Factor-mediated Nuclear Translocation of the Insulin Receptor Substrate-1 and {beta}-Catenin
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 Mol. Cell. Biol.Home page
C. Zhang, L. Comai, and D. L. Johnson
PTEN Represses RNA Polymerase I Transcription by Disrupting the SL1 Complex
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 BloodHome page
Y. Taguchi, T. Kondo, M. Watanabe, M. Miyaji, H. Umehara, Y. Kozutsumi, and T. Okazaki
Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase
Blood, November 15, 2004; 104(10): 3285 - 3293.
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 DiabetesHome page
J. E. Kim and J. Chen
Regulation of Peroxisome Proliferator-Activated Receptor-{gamma} Activity by Mammalian Target of Rapamycin and Amino Acids in Adipogenesis
Diabetes, November 1, 2004; 53(11): 2748 - 2756.
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