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. Author manuscript; available in PMC: 2014 Jul 31.
Published in final edited form as: Oncogene. 2010 Jan 18;29(13):1976–1986. doi: 10.1038/onc.2009.485

ARF-INDUCED DOWNREGULATION OF Mip130/LIN-9 PROTEIN LEVELS MEDIATES A POSITIVE FEEDBACK THAT LEADS TO INCREASED EXPRESSION OF p16Ink4a AND p19Arf

Julie Song 1, Raudel Sandoval 1, Mark A Pilkinton 1, Xinyong Tian 2, Pradip Raychaudhuri 3, Oscar R Colamonici 1,*
PMCID: PMC4116813  NIHMSID: NIHMS597960  PMID: 20101237

Abstract

The ARF-MDM2-p53 pathway constitutes one of the most important mechanisms of surveillance against oncogenic transformation, and its inactivation occurs in a large proportion of cancers. Here, we demonstrate that ARF regulates Mip130/LIN-9 by inducing its translocation to the nucleolus and decreasing the expression of the Mip130/LIN-9 protein through a post-transcriptional mechanism. The knockdown of Mip130/LIN-9 in p53-/- and Arf-/- MEFs mimics some effects of ARF, such as the downregulation of B-Myb, impaired induction of G2/M genes, and a decrease in cell proliferation. Importantly, although the knockdown of Mip130/LIN-9 reduced the proliferation of p53 or Arf null MEFs, only p53-/- MEFs showed a senescence-like state and an increase in the expression of ARF and p16. Interestingly, the increase in p16 and ARF is indirect since the Mip130/LIN-9 knockdown decreased the transcription of negative regulators of the Ink4a/Arf locus, such as BUBR1 and CDC6. Chromatin immunoprecipitation assays also reveal that Mip130/LIN-9 occupies the promoters of the BubR1 and cdc6 genes, suggesting that Mip130/LIN-9 is necessary for the expression of these genes. Altogether, these results indicate that there is a feedback mechanism between ARF and Mip130/LIN-9 in which either the increase of ARF or the decrease in Mip130/LIN-9 causes a further increase in the expression of ARF and p16.

Keywords: LIN-9, ARF, p53, senescence and cell cycle

INTRODUCTION

Immortalization or the escape from the natural process of senescence is an obligatory step during cancer development (Campisi & d’Adda di Fagagna, 2007; Collado et al., 2007; Dimri, 2005). Experiments performed with mouse embryonic fibroblasts (MEFs) isolated from mice with mutations of Arf (also known as p19ARF), p53, or the pocket proteins (pRB, p107 and p130) have demonstrated that these genes play a pivotal role in the regulation of senescence (reviewed in (Serrano, 2000; Sharpless, 2004; Sherr & Weber, 2000; Zhang, 2007). MEFs derived from these mice lack the ability to undergo senescence or apoptosis in response to oncogenic stimuli (Blagosklonny, 2006; Campisi & d’Adda di Fagagna, 2007; Dimri, 2005). For example, oncogenic RAS, which in wild-type MEFs induces senescence, transforms MEFs deficient in Arf, p53, or the entire pocket protein family (Serrano et al., 1997; Sharpless & DePinho, 2004). Notably, ARF-p53 act upstream of the pocket proteins and their partners, the E2F family of transcription factors, since MEFs lacking all pocket proteins are resistant to ARF- and p53-induced senescence (Cobrinik, 2005; Dannenberg et al., 2000; Du & Pogoriler, 2006; Frolov & Dyson, 2004; Sage et al., 2000; Stevaux & Dyson, 2002). The regulation of the Ink4a/Arf locus is complex, as different transcription factors have been linked to changes in Arf and/or p16 expression (reviewed in Gil & Peters, 2006). It is of particular interest that members of the polycomb group (Gil et al., 2004; Isono et al., 2005; Jacobs et al., 1999a; Jacobs et al., 1999b; Voncken et al., 2003) and CDC6 directly repress the expression of p16 and Arf (Gonzalez et al., 2006), while other proteins, such as BUBR1, inhibit their expression by unknown mechanisms (Baker et al., 2006; Baker et al., 2008a; Baker et al., 2008b; Hartman et al., 2007). As expected, deletion or insufficiency of these transcription factors triggers early senescence in MEFs.

It is worth mentioning that p53-independent functions of ARF (reviewed in Sherr 2006) contribute to the response to oncogenic RAS, as illustrated by the finding that the growth rate of papillomas due to RAS activation is lower in Arf+/+/p53-/- than Arf-/-p53-/- mice (Kelly-Spratt et al., 2004). Moreover, the p53-independent pathway plays an active role in senescence as suggested by data indicating that expression of p53 did not induce senescence in p53-/- MEFs unless ARF was induced through co-expression of oncogenic RAS or activated MEK1 (Ferbeyre et al., 2002).

The convergence of the senescence pathway on E2F-regulated genes suggests that events that regulate the activation of pocket proteins, such as phosphorylation by CDK4 and CDK2, can also affect the normal senescence process. Interestingly, deregulation of CDK4 by mutation of its Ink4a (p16) binding site resulted in tumor formation and immortalization of MEFs (Sotillo et al., 2001a; Sotillo et al., 2001b). It is worth mentioning that CDK4, unlike CDK2, phosphorylates/inactivates not only pRB, but also p107 and p130 (Beijersbergen et al., 1995; Cobrinik, 2005; Hansen et al., 2001; Leng et al., 2002; Xiao et al., 1996). Phosphorylation of p107 and p130 by CDK4 inactivates the repressor complex they form with E2F4/5,DP1/2 by inducing relocalization of E2F4/5 to the cytoplasm (Lindeman et al., 1997; Muller et al., 1997; Verona et al., 1997). Importantly, p107/p130,E2F4/5 not only repress genes required for the entrance into S-phase, but also those responsible for the G2/M transition (Zhu et al., 2004).

It has been recently reported that p107/p130, but not pRB, form a complex in G0 and early G1 with a novel group of proteins representing the mammalian homologs of the so-called dREAM complex in Drosophila (Litovchick et al., 2007). One of the proteins in this complex, Mip130/LIN-9, directly interacts with p107/p130 in G0/G1 (Sandoval et al., 2009). CDK4 phosphorylation of these pocket proteins releases Mip130/LIN-9 from p107/p130 and repressor E2Fs, which allows its interaction with B-Myb in late-G1 and S-phase (Pilkinton et al., 2007a ; Sandoval et al., 2009). Along the cell cycle, Mip130/LIN-9 is in a complex, termed MCC/LINC, which also contains other proteins, such as Mip40/LIN-37, Mip120/LIN-54, and LIN52 (Litovchick et al., 2007; Schmit et al., 2007; Sandoval et al., 2009). The MCC/LINC, after its separation from p107/p130, recruits B-Myb during S-phase and is responsible for the induction of genes required for the G2/M progression (Osterloh et al., 2007; Pilkinton et al., 2007b). The role of CDK4 in the release of Mip130/LIN-9, as well as other components of the MCC/LINC, is demonstrated by the finding that the deletion of the p107/p130-binding domain of Mip130/LIN-9 results in a partial correction of the CDK4 null phenotype (Sandoval et al., 2009; Sandoval et al., 2006). Thus, a decrease in CDK4 activity, perhaps due to an increase in p16 for example, can sequester Mip130/LIN-9 preventing its interaction with B-Myb, which is subsequently degraded (Sandoval et al., 2009).

The finding that CDK4 activity is required for the release of Mip130/LIN-9 from p107/p130 and the induction of G2/M genes (Sandoval et al., 2009), and for immortalization produced by the deletion of Arf (Zou et al., 2002) suggests these pathways may intersect. For example, ARF can produce a G2 arrest and block expression of E2F-regulated genes responsible for G2/M transition, such as cyclin A, Cdc25, and Cdk1, in the absence of p53 (Datta et al., 2005; Eymin et al., 2003; Gazzeri et al., 1998; Hemmati et al., 2005; Modestou et al., 2001; Normand et al., 2005; Quelle et al., 1995; Saadatmandi et al., 2002) (reviewed in ref. (Sherr, 2006)).

We report here that while the expression of ARF increases in MEFs approaching senescence, levels of Mip130/LIN-9 protein decrease, and the trend is reversed as cells become immortalized. ARF regulates the expression of Mip130/LIN-9 by inducing its translocation to the nucleolus and degradation via a proteasome-independent mechanism, suggesting that downregulation of Mip130/LIN-9 could be part of the tumor suppressor effects of ARF. Although the expression of p53 does not mimic the effect of ARF, the presence of p53 is required for degradation of Mip130/LIN-9 in response to ARF, suggesting pathways involving ARF-p53 and ARF alone mediate this effect. Importantly, downregulation of Mip130/LIN-9 by siRNA or antisense decreases the proliferation of Arf-/- and p53-/- MEFs. However, the knockdown of Mip130/LIN-9 in p53-/-, but not Arf-/- MEFs, triggered a senescence-like state, and significant increases in the expression of ARF and p16. This effect is likely mediated by a decrease in two known repressors of the Ink4a/Arf locus, CDC6 and BUBR1, which are directly regulated by Mip130/LIN-9. Thus, these data support a senescence pathway that involves a positive feedback in which decreased Mip130/LIN-9 further increases the expression of the Ink4a/Arf locus. This model could explain the progressive accumulation of ARF and p16 as wild type MEFs approach senescence.

MATERIALS AND METHODS

Tissue culture, constructs, retroviral infections and transformation assays

NIH3T3 and MEF cells were grown in DMEM supplemented with 10% FBS, glutamine and non-essential amino acids (Pilkinton et al., 2007b). MEF cells null for Arf or p53 were obtained from Drs. Charles Sherr and Martine Roussel of St. Jude Children’s Research Hospital. Senescence of MEFs was assessed using a classical 3T3 or 3T9 protocol (Todaro & Green, 1963). Retroviral plasmids for the indicated shRNAs were obtained from Open Biosystems (Huntsville, AL) and retroviruses for pBabe-puro, pBabe-puro-Mip130/LIN-9 antisense were produced in an ecotropic packaging cell line previously described (Pear et al., 1993).

Protein half-life

For the determination of the half-life of Mip130/LIN-9 protein, NIH3T3 cells were transfected with pCMV-ARF-GFP or infected with the indicated retroviral stocks for 24 hours, and incubated with cycloheximide for the last 8 hours of the experiment. X-Ray films were processed and analyzed using the computer program ImageJ. Protein levels were assessed by Western blotting and normalized by expression of tubulin or CDK4, and the mean and standard deviation of 4 separate experiments was used for graphical representation of the data.

Immunofluorescence

Cells were seeded at a density of 1 × 104 on 12-mm glass coverslips coated with 0.1% gelatin 24 hours prior to transfection with 0.4 μg of plasmid DNA. The following constructs were transfected using TurboFect Tranfection Reagent (Fermentas Inc.): GFP-tagged Mip130/LIN-9 (pEGFP-C3- Mip130/LIN-9-L), myctagged Mip130/LIN-9 (pCMV4-myc- Mip130/LIN-9), pCMV-GFP-ARF, pCMV-p53 or vector control (pEGFP-C3). Twenty-four hours post-transfection, cells were incubated in complete phenol-red free growth medium, fixed with 2% paraformaldehyde, and stained with mouse monoclonal antibodies (mAb) against myc tag (4A6, Upstate Biochemicals) and p53 (DO-1, Santa Cruz Biotechnology) as well as goat polyclonal antibody for B23 (C-19, Santa Cruz Biotechnology). Cells were washed, incubated with Alexa 488, 568 goat anti-mouse and Alexa 633 donkey anti-goat IgGs (Molecular Probes from Invitrogen), washed, and permanently mounted on glass slides using the ProLong Gold antifade reagent (Molecular Probes from Invitrogen). Images presented in this study were acquired with Zeiss LSM 510 confocal microscope.

Immunoblotting

MEFs were lysed in Triton X-100 lysis buffer, and proteins (100 μg) were resolved by SDS-PAGE, transferred to PVDF membranes and immunoblotted as previously described (Sandoval et al., 2006). The following primary antibodies were used: anti- Mip130/LIN-9 mAb was previously described (51); anti-pRB (554136) and - CDK1 (610038) antibodies were obtained from Pharmingen; and anti-myc (4A6) and anti-tubulin (DM1A + DM1B) monoclonal antibodies (mAb) were obtained from Millipore and Abcam Inc., respectively. Antibodies against CDK2 (M2), CDK4 (C-22) and B23 (C-19) were obtained from Santa Cruz Biotechnology.

RT-PCR and Chromatin Immunoprecipitation

They are described in detail in Supplemental Materials.

RESULTS

The expression of Mip130/LIN-9 is down regulated in senescent cells

To determine if Mip130/LIN-9 was affected during senescence, its expression was monitored in wild-type (WT) MEFs maintained in culture following a 3T3 or 3T9 protocol (Todaro & Green, 1963). Fig. 1A shows that WT MEFs stop proliferating after 5-6 passages and is accompanied by an increase in the expression of ARF, p53, and p21 (Fig 1B). Interestingly, Mip130/LIN-9 expression decreased as expression of these senescence markers accumulated with particular prominence at passage 6 (Fig. 1B). Moreover, when the expression of Mip130/LIN-9 was followed in MEFs that underwent immortalization during a classical 3T3 protocol, it reappeared as cells escaped senescence (Fig. 1C and D). The escape from senescence was also accompanied by very low expression of ARF. As expected, expression of B-Myb, which depends on concomitant expression of Mip130/LIN-9 (Pilkinton et al., 2007b), paralleled this pattern since it decreased as cells approached senescence and reappeared when cells underwent immortalization. Total expression of retinoblastoma protein did not have major differences at the passages studied. Detection of Mip130/LIN-9 in early passages (Fig. 1B, P2) was variable in different MEF preparations and likely depended on the degree of confluence at which cells were harvested before passage 2 plating, as Mip130/LIN-9 expression is reduced in confluent cells (data not shown).

Figure 1.

Figure 1

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Inverse correlation of the expression of Mip130/LIN-9 and ARF in MEFs maintained in a 3T9 or 3T3 protocol. The population doubling (A and C) and expression of senescence markers (B and D) of wild type MEFs maintained following 3T9 (A and B) or 3T3 protocols (C and D, respectively) were assessed as described in Materials and Methods.

ARF induces nucleolar translocation and decreases expression of Mip130/LIN-9

The data described above raised two possible scenarios: i) the decrease of Mip130/LIN-9 could be linked to activation of the ARF-p53 pathway, or ii) the fluctuations in Mip130/LIN-9 are a mere consequence of changes in the proliferation rate since transcription and protein expression of Mip130/LIN-9 progressively increase along the cell cycle (Osterloh et al., 2007; Pilkinton et al., 2007b). Since Mip130/LIN-9 localizes diffusely in the nucleus or in perinucleolar areas, yet always excluding the nucleolus (Sandoval et al., 2006), and ARF induces the nucleolar translocation of proteins, such as MDM2 (reviewed in refs. (Sherr, 2000; Sherr & McCormick, 2002), we next tested whether ARF was able to induce localization of Mip130/LIN-9 to nucleoli. Such a scenario would argue in favor of a direct effect of ARF on Mip130/LIN-9 rather than a simple decrease in expression due to cell cycle arrest. Fig. 2 shows that co-transfection of RFP-ARF with GFP- or myc-tagged Mip130/LIN-9 (panels A and B, respectively) induces localization of a significant proportion of Mip130/LIN-9 to nucleoli in NIH3T3 cells. Moreover, expression of ARF in NIH3T3 cells induces a decrease of endogenous Mip130/LIN-9 protein and a concomitant decrease in CDK1 (Fig. 2C), which has been previously demonstrated to be dependent on Mip130/LIN-9 (Osterloh et al., 2007; Pilkinton et al., 2007b). Since transcription of Mip130/LIN-9 increases as the cells progress along the cell cycle (Osterloh et al., 2007; Pilkinton et al., 2007b), we explored whether its decreased expression was due to ARF-induced cell cycle arrest produced in NIH3T3 cells. Figure 2D shows the expression of ARF did not affect mRNA levels of Mip130/LIN-9, strongly suggesting that the decrease of Mip130/LIN-9 protein is not a mere consequence of the inhibition of cell proliferation produced by ARF. It also indicates ARF regulates the expression of Mip130/LIN-9 by a post-transcriptional mechanism.

Figure 2.

Figure 2

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ARF induces nucleolar localization and down regulation of Mip130/LIN-9. The subcellular localization of Mip130/LIN-9 was assessed by confocal microscopy using NIH3T3 cells co-transfected with GFP- or myc-tagged versions of Mip130/LIN-9 (A and B, respectively) and RFP-ARF or RFP alone. The B23/Nucleophosmin protein was used a nucleolar marker. Nuclei were stained with Hoechst. C) The protein expression of Mip130/LIN-9 and components of the ARF-p53 pathway (ARF, p53, MDM2 and p21) was determined by Western blotting with specific antibodies. The expression of CDK1 was used as marker for the expression of G2/M genes known to be downregulated by ARF and the decrease in Mip130/LIN-9. D) ARF does not affect the transcription of Mip130/LIN-9. NIH3T3 cells were transfected with GFP or GFP-ARF and the expression of the Mip130/LIN-9 mRNA was assessed by RT-PCR as described in Materials and Methods. The levels of MDM-2 mRNA, whose transcription is enhanced by ARF via the activation of p53, was used as a positive control.

p53 does not replicate the effect of ARF, but is necessary for ARF-driven downregulation of Mip130/LIN-9

The previous data would predict that Mip130/LIN-9 should be expressed at higher levels in cells that do not express ARF. Indeed, expression of Mip130/LIN-9 protein is higher in passage 4 MEFs that lack Arf alone, or in combination with p53 and Mdm2, (Fig 3A) when compared to wild-type counterparts. However, it was also elevated in cells that lacked p53, or p53 with Mdm2, which, incidentally, express extremely high levels of ARF (Fig 3A). These results and the finding that transfection of ARF in p53-/- MEFs did not decrease the expression of Mip130/LIN-9 (Fig. 3B) suggest that p53, which is the main effector of ARF, is involved in the down-regulation of Mip130/LIN-9. To confirm the role of p53 in this process, we transfected ARF or p53 in Arf-/- and p53-/- MEFs. The transfection of p53 alone failed to reproduce the ARF effect on Mip130/LIN-9 in either genetic background (i.e., the induction of nucleolar localization and decreased protein expression; data not shown and Fig.3C), which was especially surprising in p53-/- MEFs since they express very high levels of ARF. However, as shown in NIH3T3 cells (Arf-null), exogenous ARF reduced the expression of Mip130/LIN-9 in Arf-/-, but not p53-/- MEFs. This result is even more dramatic if taken into account that the efficiency of ARF transfection in Arf-/- MEFs was lower than endogenous levels of ARF detected in p53-null MEFS. The minor decrease in Mip130/LIN-9 observed in p53 or ARF transfected cells (lanes 5 and 6) is due to slightly less protein loaded in these lanes, as suggested by comparing levels of CDK4. Therefore, ARF-induced down-regulation of Mip130/LIN-9 requires p53. However, the finding that the expression of p53 alone does not mimic the effects of ARF suggests that down-regulation of Mip130/LIN-9 by ARF is mediated by activation of p53-dependent and p53-independent pathways.

Figure 3.

Figure 3

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The requirement of p53 in the ARF-induced decrease in Mip130/LIN-9 expression. A) Expression of Mip130/LIN-9 in MEFs deficient for the different genes involved in the ARF-MDM2-p53 pathway. MEFs derived from mice lacking p53, p53 and MDM2, Arf, Arf and p53, all 3 genes (TKO), and wild type were assessed for the expression of Mip130/LIN-9 by Western blot as described in Materials and Methods. The expression of ARF and CDK4 (control loading) was also determined using specific antibodies (see Materials and Methods). B) The expression of ARF does not affect Mip130/LIN-9 in p53-/- MEFs. MEFs null for p53 were transfected with GFP-ARF or control GFP and immunoblotted with Mip130/LIN-9, ARF, and CDK4 antibodies. C) Arf-/- or p53-/- MEFs were transfected with plasmids encoding GFP, GFP-ARF, or GFP-p53. Immunoblotting was performed with antibodies against ARF, human p53 (DO-1), CDK4 or Mip130/LIN-9. The migration of endogenous ARF (endo. ARF) and GFP-ARF is indicated.

Importantly, co-transfection of p53 and ARF into Arf-/-p53-/- MEFs, failed to reduce the expression of Mip130/LIN-9 (data not shown). These results are consistent with results obtained with p53-/- MEFs (Fig. 3C), and raise the possibility that once deletion of p53 takes place, it may produce irreversible changes in the cell, which is likely through genome instability associated with its absence, and cannot be overcome by reconstitution of this tumor suppressor. This finding is also in agreement with a previous report indicating that reintroduction of p53 into p53 null MEFs did not induce senescence but rather a reversible cell cycle arrest despite high levels of ARF present in these cells (Ferbeyre et al., 2002) (see Discussion).

ARF induces degradation of Mip130/LIN-9 through a proteasome-independent mechanism

Since nucleolar translocation of MDM2 induced by ARF results in its degradation, we next tested whether the decrease in Mip130/LIN-9 protein was due to a similar mechanism. To determine whether the half-life of Mip130/LIN-9 protein was affected by ARF, we transfected NIH3T3 cells with ARF or control GFP, and assessed the expression of Mip130/LIN-9 after treatment with cycloheximide. These experiments demonstrated that ARF decreased the half-life of the Mip130/LIN-9 protein (Fig. 4A). Interestingly, the mechanism that leads to the decrease in Mip130/LIN-9 was not blocked by proteasomal inhibitor MG132, although this compound inhibited degradation of p53, MDM2, and p21 (Fig. 4B). Altogether, these data suggest that ARF accelerates the degradation of Mip130/LIN-9 by a mechanism not involving the proteasome.

Figure 4.

Figure 4

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ARF induces the degradation of the Mip130/LIN-9 protein through a proteasome-independent pathway. A) The effect of ARF on the half-life of the Mip130/LIN-9 protein was assessed by Western blot with the anti- Mip130/LIN-9 mAb#6 at different time points after cycloheximide addition to the cultures. The level of the Mip130/LIN-9 protein detected was normalized by the level of CDK4, and plotted to show the accelerated decay of this protein in cells expressing ARF. Intensity of the bands was determined using the image processing software program ImageJ. Results are expressed as the mean ± standard deviation obtained from 4 different experiments. B) The effect of ARF and p53 on Mip130/LIN-9 expression in the presence of the proteasome inhibitor MG132 (MG) was assessed in NIH3T3 cells 24 hours after transfection by Western blotting. The expression of MDM2, p21, and p53 were used as control for the effect of MG-132, which is known to increase their expression through the inhibition of the proteasome.

The knockdown of Mip130/LIN-9 reduces proliferation in Arf and p53 null MEFs, but only induces a senescence-like state in p53-/- MEFs

Since previous experiments suggest that the decline in Mip130/LIN-9 in senescent MEFs is the consequence of the increase in ARF, we next tested whether a knockdown of Mip130/LIN-9 would mimic, at least in part, the senescence process. MEFs null for Arf or p53 were infected with shRNAs against murine Mip130/LIN-9 or a control represented by shRNA against the human sequence with 2 mismatches. Clones derived from Arf-/- and p53-/- MEFs infected with shRNA against mu Mip130/LIN-9 grew significantly slower than controls (Fig. 5A and B) and were resistant to transformation by oncogenic RAS (Supplemental Fig. 1). As expected, these clones showed a clear decrease in levels of Mip130/LIN-9 protein expressed (Fig. 6, top panels). Interestingly, a significant proportion of p53 null MEFs infected with muMip130/LIN-9 shRNA exhibited a senescent morphology and were positive for senescence associated β-galactosidase staining (Fig. 5C). Surprisingly, although Arf-/- MEFs were infected with the same shRNA and grew significantly slower than controls, they showed no signs of senescence. It is worth mentioning that the effect of the knockdown of Mip130/LIN-9 in wild type MEFs was even more severe, since cells did not survived infection with either shRNA nor antisense against Mip130/LIN-9 (data not shown).

Figure 5.

Figure 5

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The knockdown of Mip130/LIN-9 inhibits cell proliferation and induces a senescence-like state in p53-/- MEFs. A and B) Arf-/- or p53-/- MEFs were infected with empty retrovirus or encoding the Mip130/LIN-9 shRNA. Cells were seeded at 10,000/well and counted every day in duplicate for 6 days as previously described for cells expressing a Mip130/LIN-9 antisense (Pilkinton et al., 2007b). C) The expression of SA-ß-galactosidase (ß-Gal) was performed as described in Materials and Methods and expressed as the mean ± standard deviation of two separate experiments using duplicate samples.

Figure 6.

Figure 6

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Mip130/LIN-9 knockdown induces the expression of p16 and ARF. p53-/- and Arf-/- MEF stable clones expressing an shRNA (A and C, respectively) or an antisense (B) against Mip130/LIN-9 were studied for the expression of the indicated senescence markers by Western blotting. D) The expression of the pockets proteins and phospho-RB795 were determined in the p53-/- MEFs with a knockdown of Mip130/LIN-9. E) The levels of mRNA encoding p16 and ARF were determined by RT-PCR in p53-/- MEFs expressing antisense against Mip130/LIN-9 or pBabe-puro vector.

Mip130/LIN-9 regulates the expression of p16 and Arf

To determine the molecular basis for the induction of senescence in p53-/- but not in Arf-/- null cells by Mip130/LIN-9, we next examined the expression of different cell cycle and senescence regulators in both cell types. Fig. 6 shows that the knockdown of Mip130/LIN-9 by shRNA or antisense led to very high levels of p16 and ARF protein expression in p53-/- MEFs (Fig. 6A and B). Although p16 is expressed in Arf-/- MEFs, its expression is not affected by the knockdown of Mip130/LIN-9 (Fig. 6C). These high levels of p16 and ARF protein expression in p53-/- MEFS are paralleled by an increment in mRNA encoding these genes, suggesting that Mip130/LIN-9 negatively regulates the Ink4a/Arf locus (Fig. 6E). Thus, an important difference that could explain the induction of senescence in p53-/-, but not in Arf-/- MEFs, in response to Mip130/LIN-9 shRNA, is the increased expression of ARF and p16. Moreover, in p53-/- MEFs the knockdown of Mip130/LIN-9 leads to an increase in the expression of p130, as expected in cells in which part of the population exited the cell cycle, and a modest decrease in phosphorylation of serine 795 of pRB (Fig. 6D). These results suggest that downregulation of Mip130/LIN-9 can contribute to inhibition of G1 progression and repression of E2F-regulated genes in p53-/- MEFs undergoing senescence.

As expected, the knockdown of Mip130/LIN-9 in Arf-/- and p53-/- MEFs resulted in a reduction in B-Myb and E2F-regulated G2/M genes, such as CDK1 (Fig. 6A-C) (Osterloh et al., 2007; Pilkinton et al., 2007b). It is worth mentioning that these effects are specific since cell cycle proteins not regulated by Mip130/LIN-9, such as CDK4, were not significantly affected (Osterloh et al., 2007; Pilkinton et al., 2007b).

To further determine the mechanism that leads to transcriptional activation of the Ink4a/Arf locus, we first studied whether Mip130/LIN-9 was able to bind to this locus. The scan of the locus using ChIP assays with several sets of primers previously described (Bracken et al., 2007) failed to show specific binding of Mip130/LIN-9 to the Ink4a/Arf promoters (data not shown). We next explored whether Mip130/LIN-9 indirectly affected transcription of Arf and p16 through regulation of known transcription modulators of the Ink4a/Arf locus, such as BMI-1, a polycomb protein, CDC6, and BUBR1. Although no alterations in the expression of bmi-1 were observed, knockdown of Mip130/LIN-9 decreased transcription of BubR1 and cdc6 by 40-50% and 65-70%, respectively (Fig. 7A), suggesting that Mip130/LIN-9 is involved in the regulation of these genes. Moreover, chromatin immunoprecipitation assays revealed that Mip130/LIN-9 bound to the promoters of BubR1 and cdc6, but not Actin, which served as a negative control (Fig.7B). Together, these data strongly suggest that Mip130/LIN-9 is necessary for the expression of regulators of the Ink4a/Arf locus, such as BUBR1 and CDC6, and therefore indirectly affects the expression of ARF and p16.

Figure 7.

Figure 7

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The knockdown of Mip130/LIN-9 inhibited the expression of CDC6 and BubR1 while enhancing the transcription of the Ink4a/Arf locus products, p16 and ARF. A) The mRNA expression for the indicated genes was determined using quantitative real-time PCR as described in Materials and Methods in two Mip130/LIN-9 antisense clones and compared with control clones expressing empty vector. Message levels were normalized by the expression of GAPDH and the effect of the Mip130/LIN-9 antisense was expressed as fold change over control empty vector. B) The direct binding of Mip130/LIN-9 to the cdc6 and BubR1 gene promoters was tested by ChIP assays with specific anti- Mip130/LIN-9 antibodies and primers for the specific amplification of the BubR1 and cdc6 promoters.

DISCUSSION

The ARF-p53 pathway regulates the cell’s entrance into senescence. This mechanism is not only important as part of the normal life cycle of many cells, but also as a key element to neutralize oncogenic events (Campisi & d’Adda di Fagagna, 2007; Collado et al., 2007; Dimri, 2005). The activation of the senescence pathway leads to cell cycle arrest via the Arf-p53-p21 and p16 pathways, respectively. The finding that the activation of the senescence pathway via ARF can repress expression of G2/M genes and produce G2/M arrest even in the absence of p53 (Eymin et al., 2003; Hemmati et al., 2005; Modestou et al., 2001; Normand et al., 2005; Quelle et al., 1995; Saadatmandi et al., 2002; Stott et al., 1998) led us to test whether the mechanism may involve Mip130/LIN-9 and B-Myb, which are critical for the induction of G2/M genes (Osterloh et al., 2007; Pilkinton et al., 2007b). This was further supported by the finding that Mip130/LIN-9 expression decreases while ARF increments as cells approach senescence, and that the converse occurs when MEFs become immortal (Fig. 1). Moreover, two lines of evidence indicate that the decrease in Mip130/LIN-9 is not a mere reflection of the proliferation status, but rather the direct effect of ARF. First, ARF triggers nucleolar translocation of Mip130/LIN-9, which is normally nucleoplasmic. And, second, ARF did not affect transcription of Mip130/LIN-9 as it would be expected if the effect was due to a cell cycle arrest, since Mip130/LIN-9 transcription and protein expression augment along the cell cycle (Osterloh et al., 2007; Pilkinton et al., 2007b). Finally, ARF was shown to increase the rate of decay of Mip130/LIN-9. It is worth noting that the effect of ARF is not mediated by a direct interaction between Mip130/LIN-9 and any of the components of the pathway: ARF, p53 or MDM2 (data not shown).

The effect of ARF on Mip130/LIN-9 is complex and cannot be explained by activation of either the ARF-MDM2-p53 axis or p53-independent function of ARF alone. First, expression of p53 does not reproduce the effect of ARF when expressed in NIH3T3 cells, Arf-/- nor in p53-/- MEFs, which express very high levels of ARF, clearly indicating that stabilization of p53 by itself is not sufficient for degradation of Mip130/LIN-9. Second, this effect is not explained either by the so-called p53-independent function of ARF, since expression of ARF in p53-/- MEFs did not affect the expression of Mip130/LIN-9. Thus, it is likely that both pathways participate in nucleolar translocation and degradation of Mip130/LIN-9 downstream of ARF. Surprisingly, attempts to reconstitute p53 in cells that did not express this tumor suppressor failed to restore the ability of ARF to down-regulate Mip130/LIN-9, suggesting that once the integrity of this gene is compromised, secondary events lead to permanent inactivation of this pathway. This is in agreement with a previous report that indicated the restitution of p53 in MEFs null for this tumor suppressor triggered a reversible cell cycle arrest, but not senescence despite very high levels of ARF (Ferbeyre et al., 2002).

A negative effect of the ARF-p53 pathway on positive regulators of the G2/M progression, such as Mip130/LIN-9 and B-Myb, would not have been unexpected as part of the cell cycle arrest required for the senescence process. However, our attempt to mimic the downregulation of Mip130/LIN-9 by Arf using shRNAs against Mip130/LIN-9 clearly indicated that its participation in the senescence process was beyond a role in cell cycle regulation. The knockdown of Mip130/LIN-9 triggered a senescence-like phenotype in p53-/-, but not in Arf-/- MEFs. Moreover, we provide strong evidence that the knockdown of Mip130/LIN-9 triggers a feedback loop that increases transcription of both members of the Ink4a/Arf locus, p16 and Arf itself, which could result in a permanent exit from the cell cycle (Fig. 8). The potential importance of this mechanism is suggested by the fact that although the decrease in Mip130/LIN-9 reduces proliferation of both Arf-/- and p53-/- MEFs, it does not induce senescence in the former since the absence of ARF would interrupt the feedback loop. Thus, there is a dissociation between the antiproliferative effect triggered by the decrease in Mip130/LIN-9, observed in both genetic backgrounds, which is likely mediated by impairment in the induction of G2/M genes (Osterloh et al., 2007; Pilkinton et al., 2007b), and the induction of senescence. In the latter, down-regulation of Mip130/LIN-9 may trigger a “perfect storm” scenario: i) it would stop cells from traversing the G2/M boundary due to its effect on the induction of G2/M genes in association with B-Myb (Osterloh et al., 2007; Pilkinton et al., 2007b), and ii) up-regulation of the Ink4a/Arf locus leads to a complete G1/S arrest through the blockade of phosphorylation of pocket proteins, which is due to activation of ARF-p53-p21 and p16. This model is also in agreement with a report by Carnero et al., which suggested that ARF exerts its effect via pocket proteins in addition to p53 (Carnero et al., 2000). In this context, the increase in p16 produced by the down regulation of Mip130/LIN-9 blocks inactivation of pocket proteins by CDK4/6, while a further increase in ARF will activate p53 leading to inhibition of CDK2 by p21.

Figure 8.

Figure 8

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The induction of ARF leads to the degradation of Mip130/LIN-9 by a process that is p53-dependent. The degradation of Mip130/LIN-9 protein has two effects. First, B-Myb becomes degraded, which results in a lack of G2-phase gene induction and this contributes to cell cycle arrest. Second, the decrease in Mip130/LIN-9 leads to a decrease in the transcription of negative regulators of the Ink4a/Arf locus, BUBR1 and CDC6, and a subsequent increase in ARF and p16 expression.

Our results also suggest that Mip130/LIN-9 does not directly regulate the Ink4a/Arf locus, but rather through two known Ink4a/Arf repressors, BUBR1 and CDC6. Mip130/LIN-9 binds to the BubR1 and cdc6 promoters, and triggers their transcriptional activation, as suggested by the dramatic decrease in BubR1 and cdc6 transcripts in cells treated with shRNA targeting Mip130/LIN-9. As shown in Fig. 8, Mip130/LIN-9 and B-Myb are not only critical for the transcriptional activation of G2/M genes as previously reported (Osterloh et al., 2007; Pilkinton et al., 2007b), but Mip130/LIN-9 also participates in a feedback loop within the ARF-MDM2-p53 pathway. The activation of the senescence pathway downregulates the level of Mip130/LIN-9 protein that results in a G2/M arrest, and a downregulation of CDC6 and BUBR1, repressors of the Ink4a/Arf locus, that further increase the levels of p16 and ARF. Once this feedback loop is triggered, it may lock the fate of the cell by making the senescence process irreversible.

Supplementary Material

Supp1
Supp2

Acknowledgments

We would like to thank Drs. Charles Sherr and Martine Roussel, St. Jude Children’s Research Hospital, for the ARF and p53 plasmid constructs and Arf-/- and p53-/- MEFs.

This work was supported by Public Health Service grants RO1 GM81562 (ORC) and NCI-KO1 CA127862 (RS) from the National Institutes of Health. JS and MP were supported by NIH Institutional T32 training grants, Ruth L. Kirschstein National Research Service Award NOT-OD-06-093 and Training Program in Signal Transduction and Cellular Endocrinology DK07739, respectively.

Abbreviations

MEFs

Mouse Embryonic Fibroblasts

CDK

cyclin-dependent kinase

Mip

Myb-interacting protein

WT

wild type

GFP

green fluorescence protein

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