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Vol. 18, Issue 4, 1457-1463, April 2007
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Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084
Submitted October 11, 2006;
Revised January 30, 2007;
Accepted February 5, 2007
Monitoring Editor: Jean Schwarzbauer
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSThe effect of ERK...DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSThe effect of ERK...DISCUSSIONREFERENCES |
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; Sherr and Roberts, 1999). Active cdk4/6 and cdk2 phosphorylate the pocket proteins, comprising the retinoblastoma protein Rb, p107, and p130 (Weinberg, 1995; Takahashi et al., 2000). Phosphorylated pocket proteins release associated E2Fs that, in turn, allows for transcription of the E2F-dependent genes required for further cell cycle progression. Cyclin A is one of those E2F-dependent genes; the induction of cyclin A and formation of active cyclin A-cdk2 complexes is required for S phase entry. This established sequence of events emphasizes the central role of cyclin D1 as a primary target of mitogenic signaling.
ERK activity is also required for cell cycle progression through G1 phase. Early studies indicated that the persistence of ERK activity in mid-G1 phase correlated with efficient S phase entry (Meloche et al., 1992). Subsequent studies showed that sustained ERK signal of 5–6 h is associated with the induction of cyclin D1 mRNA and protein in mid-G1 phase (
8–9 h after mitogenic stimulation of quiescent cells; Albanese et al., 1995; Lavoie et al., 1996; Weber et al., 1997; Balmanno and Cook, 1999; Welsh et al., 2001). Additionally, early G1 phase ERK activity stimulates a transient induction of p21cip1 (Bottazzi et al., 1999), an effect that may contribute to assembly of cyclin D-cdk4/6 complexes (Sherr and Roberts, 1999). Interestingly, intense ERK signals (e.g., resulting from overexpression of activated Raf) can lead to a persistent up-regulation of p21cip1 and G1 phase arrest (Sewing et al., 1997; Woods et al., 1997).
Finally, several studies have indicated that ERK activity also contributes to the down-regulation of p27 (Kerkhoff and Rapp, 1997; Rivard et al., 1999; Delmas et al., 2001; Kortylewski et al., 2001; Foster et al., 2003; Bhatt et al., 2005). In this report, we used mitogen stimulation of quiescent fibroblasts to study the role of endogenous ERK activity in G1 phase progression, map its temporal effects, identify its essential targets, and resolve its primary versus secondary cell cycle effects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSThe effect of ERK...DISCUSSIONREFERENCES |
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). For experimentation, serum-starved cells were trypsinized, resuspended in DMEM-BSA, and collected by centrifugation. MEFs (106 cells in 10 ml) and human fibroblasts (1.5 x 106 cells in 20 ml) were then resuspended in DMEM-BSA and preincubated in suspension with 50 µM U0126 (Promega, Madison, WI) or DMSO (vehicle) for 30 min at 37°C before being reseeded in 100-mm (MEFs) or 150-mm (human fibroblasts) dishes and stimulated with 10% FBS in the continued presence of DMSO or 50 µM U0126. When indicated, U0126 was washed-out by rinsing monolayers twice with cold DMEM and then refeeding the cultures fresh DMEM-10% FBS. MEFs expressing a tetracycline-repressible cyclin D1 (Tet-D1-MEFs) were prepared as described (Zhu et al., 2000) and cultured similarly except that they were maintained in the presence of 2 µg/ml tetracycline and then serum-starved and stimulated in the absence or presence of 2 µg/ml tetracycline. Primary MEFs from wild-type and p27T187A knockin mice (Malek et al., 2001) were maintained in 10% FCS and used at passages 2–5.
Adenoviral Infection and Small Interfering RNA
For adenoviral infection, confluent MEFs were incubated in serum-free DMEM-1 mg/ml BSA for 12 h, infected overnight with adenoviruses encoding LacZ or cyclin D1 (kind gift of Jeffrey Albrecht) and then incubated in fresh DMEM-BSA for an additional 24 h. For cyclin D1 knockdown, MEFs (106) were seeded in 100-mm dishes with antibiotic-free DMEM-10% FBS and incubated overnight. The cultures were washed three times with serum-free/antibiotic free OptiMEM (Invitrogen, Carlsbad, CA) before transfection with 100 nM of an irrelevant small interfering RNA (siRNA; human E cadherin; GAGUGAAUUUUGAAGAUUGtt with 4/19 mismatches relative to the homologous mouse sequence) or siRNA specific to mouse cyclin D1 (GCGGUAGGGAUGAAAUAGUtt). The transfection used Lipofectamine 2000 (Invitrogen) and a final volume of 4 ml OptiMEM and generally followed the manufacturer's protocol. After a 4–6 h, the medium was replaced with DMEM-BSA, and the cells were starved for 2 d.
Analysis of mRNA and Protein Expression
Cell pellets were extracted in Trizol (Invitrogen; 1 ml/1–1.5 x 106 cells) for isolation of total RNA. Northern blotting of total RNA used random-primed cDNA probes and standard procedures. Quantitative real-time RT-PCR (QPCR) was performed as described (Stewart et al., 2004). Mouse p27 and cyclin E mRNAs were quantified using Assay on Demand primer-probe sets Mm00438167_g1 and Mm00432367_ml, respectively (Applied Biosystems, Foster City, CA). The primers and probe used to detect Skp2 mRNA have been described (Stewart et al., 2004), and those used for 18S rRNA were as follows: 5'-CCT GGT TGA TCC TGC CAG TAG (forward primer, 150 nM), 5'-CCG TGC GTA CTT AGA CAT GCA (reverse primer, 150 nM), and VIC-TGC TTG TCT CAA AGA TTA-MGB-NFQ (probe, 100 nM). QPCR results show the mean ± SD of duplicate PCR reactions.
When cells were analyzed by Western blotting, they were washed with cold PBS, 1 µM Na3VO4, scraped, collected by centrifugation, and quick-frozen in dry ice before lysis. Western blotting used antibodies specific for ERK1/2 (BD Transduction Laboratories, Lexington, KY; M12320 [GenBank] ), dually phosphorylated (active) ERK1/2 (pERK1/2; Cell Signaling, Danvers, MA; 9101S), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA; SC-8396), p27 (BD Transduction Laboratories; K25020), Skp2 (Zymed Laboratories, South San Francisco, CA; 32-3400), cdk4 (Santa Cruz, SC-260), Rb (Zymed Laboratories; 28-0007), AKT1 (gift of Morris Birnbaum), phospho-AKTSer473 (Cell Signaling; 9271S), and actin (Santa Cruz; SC-8432). The rabbit polyclonal cyclin A antibody was prepared in this laboratory using recombinant cyclin A as immunogen. Western blot signals were detected by enhanced chemiluminescence. The images were digitized by scanning, and figures were assembled using Adobe Photoshop (San Jose, CA).
Analysis of Cyclin D1-cdk4 Complexes
Cell pellets were lysed in 100 µl of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20, 1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 mM NaF, and 10 mM Na3VO4. Equal amounts of lysate (200 µg) were incubated with 5 µg of anti-cdk4 (BioSource, Camarillo, CA; AH20202) for 2 h at 4°C followed by incubation with 50 µl washed anti-mouse conjugated agarose (Invitrogen) with rocking for 2 h at 4°C. Collected immunoprecipitates were washed five times with the lysis buffer and resuspended in 50 µl of reducing SDS sample buffer. The immunoprecipitates were fractionated on SDS polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-cyclin D1 (Santa Cruz; SC-8396) and anti-cdk4 (Santa Cruz; SC-260).
Immunostaining
S phase entry was determined by performing the serum stimulation in dishes containing coverslips and 3 µg/ml bromodeoxyuridine (BrdU; GE Healthcare, Piscataway, NJ). Coverslips were removed at selected times, washed with PBS, fixed (15 min) in 3.7% paraformaldehyde, incubated (10 min) in 50 mM ammonium chloride, and permeabilized (5 min) with 0.2% Triton X-100 in PBS. Permeabilized cells were incubated at room temperature sequentially with DNase (500 U/ml) and 2% BSA in PBS for 1 h, anti-BrdU (BIODESIGN, Saco, ME; M20105S; 250-fold dilution) and DNAse in PBS for 1 h, and TRITC-conjugated donkey anti-sheep antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 200-fold dilution) in PBS for 1 h. Cell nuclei were stained with DAPI (Sigma, St. Louis, MO; 2 µg/ml in PBS) for 10 min. BrdU-positive cells were identified using epifluorescence microscopy, usually counting 150–200 DAPI-positive cells per sample.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSThe effect of ERK...DISCUSSIONREFERENCES |
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; Mody et al., 2001). Although all three ERK isoforms have been linked to the induction of cyclin D1, the roles of endogenous ERK1 and ERK2 are well established, but the role of ERK5 is less clear (Squires et al., 2002; Mulloy et al., 2003).
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9 h after serum stimulation of quiescent fibroblasts) (Welsh et al., 2001; Figure 1B; DMSO). U0126 blocked the induction of cyclin D1 mRNA and protein when added between 0 and 6 h after mitogenic stimulation, but addition of U0126 at 6 h and beyond was without effect (Figure 1, B and C; U0126). Although this result supports previous studies showing that a sustained ERK signal of 5–6 h is associated with the mid-G1 phase induction of cyclin D1 (Weber et al., 1997; Balmanno and Cook, 1999; Welsh et al., 2001), U0126 washout experiments showed that cyclin D1 mRNA and protein were expressed normally even when ERK activity was inhibited through the first 3 h after mitogenic stimulation (Figure 1, E and F). Controls showed that ERK activity was blocked within 5 min after addition of U0126 and restored within 5 min of removing U0126; these effects were specific (Supplementary Figure 1, A and B, respectively). Thus, although a sustained ERK signal of 5–6 h does correlate with mid-G1 phase induction of cyclin D1 mRNA, the first 3 h of ERK signaling are, in fact, dispensable. The critical ERK signaling period is 3–6 h after mitogenic stimulation of quiescent cells. Remarkably, inhibition of MEK/ERK signaling with U0126 for the first 3 h of G1 phase did not affect maximal S phase entry in quiescent MEFs stimulated with serum (Figure 2A; U0 washout [wo] 3 h) and only had a small effect on the kinetics of S phase entry (Figure 2B). In contrast, inhibition of MEK/ERK signaling for the first 5 h of G1 phase effectively prevented S phase entry (Figure 2A; U0 wo 5 h). These results reveal a tight correlation between ERK-dependent cyclin D1 induction and ERK-dependent S phase entry. This finding was not restricted to MEFs; U0126 washout experiments showed that the first 3 h of ERK signaling are also dispensable for S phase entry and cyclin D1 induction in early passage human fibroblasts (Supplementary Figure 2). Thus, cyclin D1 induction and S phase entry share the requirement for mid-G1 phase ERK activity, raising the possibility that cyclin D1 represents the major ERK cell cycle target in G1 phase.
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; Treinies et al., 1999) have reported that ERK is involved in assembly of cyclin D1-cdk4 complexes, but we found that ectopically expressed cyclin D1 bound to cdk4 in serum-stimulated MEFs, both in the absence and presence of U0126 (Figure 3D). In fact, the absence of an ERK requirement for cyclin D1-cdk4 complex formation was expected given the normal kinetics of Rb phosphorylation in the U0126-treated cyclin D1 expressors. Thus, ERK activity is dispensable for G1 phase progression of mitogen-treated MEFs expressing cyclin D1.
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The effect of ERK on p27 Levels Is a Downstream Consequence of Cyclin D1 Induction |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSThe effect of ERK...DISCUSSIONREFERENCES |
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; Rivard et al., 1999; Delmas et al., 2001; Kortylewski et al., 2001; Foster et al., 2003; Bhatt et al., 2005), and p27 down-regulation, in turn, contributes to S phase entry by facilitating the activation of cyclin-cdk2 complexes. Some of these studies concluded that the ERK effect is posttranscriptional, but the exact mechanism by which ERK regulates p27 has not been resolved.
We first examined the effect of endogenous ERK inhibition on the levels of p27 as serum-stimulated MEFs progressed from G0 to S phase. U0126 inhibited the down-regulation of p27 protein in MEFs (Figure 5A), but this effect was incomplete and was not detected until 18 h, when serum-stimulated MEFs are progressing through S phase (refer to Figure 2B). Thus, the ERK effect on p27 is less pronounced than the ERK effect on cyclin D1. U0126 did not block the mid- to late G1 phase down-regulation of p21 (data not shown), consistent with our previous studies, which concluded that p21 down-regulation is ERK-independent (Bottazzi et al., 1999).
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) and SCFSkp2 (Carrano et al., 1999; Tsvetkov et al., 1999). KPC is thought to act in G0/G1 phase, whereas Skp2 acts in late G1/S phase (Kamura et al., 2004). Because ERK inhibition selectively affected p27 levels in late G1/S phase (refer to Figure 5A), we asked if the ERK effect on p27 required the action of SCFSkp2. Indeed we found that late G1/S phase p27 levels were unaffected by U0126 in knockin MEFs that express p27T187A (Malek et al., 2001), a p27 mutant that cannot be recognized by SCFSkp2 (Figure 5B; compare 21 h ± U0126 in WT and T187A cells). Thus, when SCFSkp2-dependent degradation of p27 is precluded, ERK loses its ability to regulate p27. We conclude that the ERK effect on p27 is mediated by SCFSkp2. Note that down-regulation of p27 persists in T187-MEFs (Figure 5B; G0 vs. 21 h), even after ERK inhibition. This result implies that SCFSkp2-independent degradation of p27 is also independent of ERK activity (see Discussion).
SCFSkp2 is comprised of core components (Skp1, Cul-1, and Rbx/Roc1 or Ro52) and the F-box protein, Skp2, which is the substrate-recognizing component of the complex (Carrano et al., 1999; Tsvetkov et al., 1999; Sabile et al., 2006). Because the core components are typically constitutively expressed, whereas Skp2 is an E2F-regulated gene (Markey et al., 2002; Vernell et al., 2003; Zhang and Wang, 2006 and our unpublished results), we reasoned that the effect of U0126 on p27 degradation might be a downstream consequence of a primary ERK effect on cyclin D1, with consequent inactivation of Rb (or other pocket proteins), release of E2Fs, and induction of Skp2. Indeed, we found that the inhibitory effect of U0126 on G1/S phase down-regulation of p27 correlated well with the inhibitory effect of U0126 on Rb hyperphosphorylation and Skp2 expression (Figure 5C). Moreover, U0126 blocked the induction of Skp2 mRNA but did not affect the levels of p27 mRNA (Figure 5D), indicating that the ERK effect on p27 is posttranscriptional.
To directly test whether the ERK effects on cyclin D1, Skp2, and p27 were causally related, we used siRNA and ectopic expression to manipulate cyclin D1 levels downstream of ERK activation. Cyclin D1 knockdown inhibited, although not completely, the mitogen-dependent induction of Skp2 mRNA (Figure 6A) and protein (Figure 6B), and it similarly inhibited the down-regulation of p27 in late G1/S phase (50% relative to actin; Figure 6C). Conversely, ectopic cyclin D1 rescued the expression of Skp2 mRNA (Figure 7A) and protein (Figure 7B) in a dose-dependent manner in U0126-treated MEFs, and the rescue (40–85% of endogenous expression; see Figure 7 legend) could be detected with near normal levels of ectopic cyclin D1. This effect was transcriptional and consistent with cyclin D1–dependent Rb inactivation and E2F release because cyclin E (a prototype E2F1-induced gene; DeGregori et al., 1995) and Skp2 mRNA were similarly rescued by ectopic cyclin D1 expression (Figure 7A). Moreover, the forced expression of cyclin D1 restored p27 down-regulation in MEK/ERK-inhibited cells (Figure 7C; LZ+U0126 vs. D1+U0126). On the basis of the results in Figures 6 and 7, we conclude that the ERK effects on Skp2 and p27 are secondary consequences of the ERK effect on cyclin D1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSThe effect of ERK...DISCUSSIONREFERENCES |
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) and another concluded that ERK activity was required throughout most of G1 phase (Yamamoto et al., 2006). Our results, indicating that only mid-G1 phase ERK activity is required for mitogenesis in fibroblasts, agree well with Fassett et al. (2003), who studied the relationships between ERK activity, cyclin D1 expression, and G1 phase progression in rat hepatocytes. Similarly, others have shown that the induction of cyclin D1 and DNA synthesis correlates with sustained, mid-G1 phase ERK activity and the mid-G1 phase expression of Fra-1 and Fra-2 rather than transient/early G1 phase ERK activity and expression of c-fos (Balmanno and Cook, 1999; Cook et al., 1999). Finally, the correlation we observe between the inhibitory effect of U0126 on cyclin D1 expression and S phase entry is consistent with studies (Peeper et al., 1997; D'Abaco et al., 2002), showing that Ras and ERK activities are dispensable for S phase entry in Rb-null cells. Thus, although a sustained ERK signal correlates with cyclin D1 induction and S phase entry in fibroblasts, only the mid-G1 phase ERK signal is required. This finding can explain why cyclin D1 gene expression in fibroblasts requires coordinated signaling by growth factor receptor tyrosine kinases and integrins, because growth factors do not sustain the ERK signal into mid-G1 phase in the absence of a concomitant integrin signal (Roovers et al., 1999).
Two studies (Cheng et al., 1998; Treinies et al., 1999) using conditional expression of activated MEK have concluded that MEK/ERK activity is sufficient to stimulate complex formation between cyclin D1 and cdk4. Although our results do not contradict these findings, our data with conditional cyclin D1 expression shows that MEK/ERK activity is not required for assembly of a cyclin D1-cdk4 complex in mitogen-treated MEFs. Other kinases may facilitate assembly in these conditions, or assembly might be mediated by p21cip1 or p27, both of which are present in MEK/ERK-inhibited MEFs. These cdk inhibitors are established mediators of cyclin D1-cdk4 complex formation (reviewed in Sherr and Roberts, 1999).
In addition to cyclin D1, several studies have indicated that p27 accumulates when endogenous ERK activity is inhibited (Rivard et al., 1999; Delmas et al., 2001; Kortylewski et al., 2001; Foster et al., 2003; Bhatt et al., 2005). In most of these studies, the effect of ERK on p27 was posttranscriptional. Our data extend these observations by showing that the ERK effect on p27 is restricted to late G1/S phase, is mediated by SCFSkp2, and is due (at least in large part) to an ERK-dependent induction of Skp2 gene expression. However, we also show that the ERK effect on p27 is incomplete (because p27 continues to down-regulate in MEK/ERK-inhibited cells) and secondary to its effect on cyclin D1. In particular, we find that ERK regulates p27 levels after it stimulates cyclin D1 expression. Cyclin D1 depletion is sufficient to inhibit late G1/S phase Skp2 induction and p27 down-regulation, whereas ectopic cyclin D1 expression rescues these events in MEK/ERK-inhibited cells. We note, however, two studies that reported ERK-mediated effects on p27 that were independent of Skp2 (Delmas et al., 2001; Foster et al., 2003).
Our findings can explain why ERK selectively regulates p27 degradation in late G1/S phase: its effect is secondary to cyclin D1 expression, pocket protein inactivation, and Skp2 induction. G1 phase p27 levels decreased in U0126-treated MEFs and even in T187A-MEFs, showing that SCFSkp2-independent p27 down-regulation persists in ERK-inhibited cells. KPC is thought to regulate p27 degradation in the G1 portion of the cell cycle (Kamura et al., 2004). The overall ERK effect on p27 levels may therefore be partial because KPC-dependent p27 degradation is ERK independent.
The degree of Skp2 and S phase inhibition by cyclin D1 siRNA was somewhat less than that seen after inhibition of ERK with U0126. This result was expected because cyclin D1 siRNA also had a somewhat reduced inhibitory effect on Rb phosphorylation, relative to U0126. Incomplete transfection, residual cyclin D1, or the cyclin D2, which is ERK-dependent and expressed in MEFs (Dey et al., 2000; Piatelli et al., 2002; Huang et al., 2006), most likely account for the residual hyperphosphorylation of Rb and induction of Skp2 in MEFs transfected with cyclin D1 siRNA. Nevertheless, our results cannot exclude the possibility that other component of SCFSkp2 complexes, such as Ro52 (Sabile et al., 2006) or cks1 (Ganoth et al., 2001) may also be involved in ERK-dependent p27 degradation.
Overall, we conclude that early G1 phase ERK activity is dispensable for both cyclin D1 gene expression and S phase entry when quiescent fibroblasts are mitogen-stimulated to reenter the cell cycle. In stark contrast, sustained ERK activity in mid-G1 phase is required for these events. We also conclude that cyclin D1 is a primary and essential cell cycle target of mid-G1 phase ERK signaling during cell cycle reentry of mitogen-stimulated fibroblasts. In contrast, the effects of ERK on Skp2 and p27 levels are, at least in large part, downstream consequences of ERK-dependent cyclin D1 induction. Our results clarify the importance of sustained ERK signaling in G1 phase and suggest that the primary ERK-regulated cell cycle targets required for mitogenesis in mesenchymal cells may be more limited than generally thought.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
* These authors contributed equally to this work.
Address correspondence to: Richard K. Assoian (rka{at}pharm.med.upenn.edu)
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