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HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage

Gerd Pääjärvi^*, Emilie Roudier^*, Milita Crisby, Johan Högberg^* and Ulla Stenius^*,1

^* Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden;
Neurotec Department, Division of Geriatric Medicine, Karolinska Institutet and Karolinska University Hospital, Huddinge, Stockholm, Sweden

¹Correspondence: Institute of Environmental Medicine, Karolinska Institutet, Box 210, Stockholm 17177, Sweden. E-mail: ulla.stenius{at}imm.ki.se

SPECIFIC AIMS

HMG-CoA reductase inhibitors (statins) are widely used cholesterol lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53-specific ubiquitin ligase. The question we posed was whether statins induce Akt activation in hepatocytes and whether this activation may induce changes in Mdm2/p53 regulation. We studied the effect of statins on Mdm2/p53 regulation in unstressed and in DNA-damaged HepG2 cells. Employing immunohistochemistry, we analyzed the effect of pravastatin on constitutive Mdm2 expression in rat liver in situ and on the p53 response to DNA damage.

PRINCIPAL FINDINGS

1. Pravastin induces Mdm2 phosphorylation at Ser166 and mTOR phosphorylation at Ser2448
3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are widely used cholesterol-lowering drugs. The liver is the primary target organ and they affect the rate-limiting step in cholesterol synthesis. Recent studies indicate that statins may have anticancer effects and in several in vitro models, statins sensitize human tumor cells to cytostatic drugs. Earlier studies have shown that statins induce Akt activation in endothelial cells and that Akt may phosphorylate Mdm2 at Ser166 and Ser186. Therefore, the effect of pravastatin on Akt and Mdm2 phosphorylation was studied. We found that incubation of serum starved cells with pravastatin induced Mdm2 phosphorylation at Ser166 (Fig. 1 A). Ser166 phosphorylation was induced rapidly and a minor increase was registered within 0.5 h. As shown in Fig. 1B , this increase was dose-dependent. The effect of PI3K inhibitors LY294002 and wortmannin and the mTOR inhibitor, rapamycin were tested. As shown in Fig. 1C , rapamycin inhibited the pravastatin-induced Mdm2 phosphorylation at Ser166 whereas the PI3-kinase inhibitors LY294002 and wortmannin had no effect. These data indicate that Ser166 phosphorylation was induced by a PI3K-independent pathway and suggest an involvement of mTOR. Next, the effect of pravastatin on mTOR phosphorylation was studied. Figure 1D shows that pravastatin induced mTOR phosphorylation at Ser2448, the active form of mTOR.

View larger version (31K): [in this window] [in a new window]   Figure 1. Pravastatin induces rapid Ser166 and 2A10 specific phosphorylations of Mdm2 as well as Ser2448 phosphorylation of mTOR. Serum starved HepG2 cells were incubated with (A) pravastatin for times indicated or (B) with pravastatin in concentrations indicated for 2 h. C) Cells were incubated with kinase inhibitors LY294002 (25 µM), wortmannin (100 nM), or rapamycin (20 nM) for 45 min before addition of pravastatin (2 µM) or insulin (1 µg/mL) for 1 h or (D) with pravastatin (2 µM) or insulin (1 µg/mL) for 1 h. Samples were analyzed by Western blot with phospho Ser166 Mdm2, 2A10 Mdm2, phospho Ser473 Akt, phospho Ser2448 mTOR. Pase: Western blots with alkaline phosphatase pretreatment. Cdk2 was used as a loading control.

The effect of statins on levels of pAkt was examined in serum-starved HepG2 cells. Incubation with pravastatin (2 µM) for 0.5–3 h did not induce any significant increase in Ser473 phosphorylated Akt (Fig. 1A-C ). Insulin was used as a positive control and induced a rapid Akt phosphorylation at Ser473 (Fig. 1C, D ) and this effect was suppressed by the PI3K inhibitors LY294002 and wortmannin (Fig. 1C ). It has been reported that the 2A10 Mdm2 antibody inefficiently recognizes phosphorylated Mdm2 but that alkaline phosphatase (Pase) treatment increases the detectability of Mdm2 protein. The data in Fig. 1A indicate that pravastatin induced 2A10 specific phosphorylation(s) of Mdm2 within 0.5 h incubation. Increased phosphorylation of Mdm2 was still found 24 h after pravastatin addition. This indicates the long-lasting effects of statins.

Mdm2 mRNA levels (measured by RT-PCR and related to 18S as internal standard) were analyzed. However, statin treatment did not induce any changes in HepG2 cells, indicating that alterations induced by statins were restricted to posttranscriptional modifications of Mdm2 protein.

2. Pravastatin alters Mdm2 expression and the p53 response to DNA damage in rat liver in vivo
We previously reported that midzonal hepatocytes constitutively express high levels of cytoplasmic Mdm2. In sections from rats treated with pravastatin, midzonal cells exhibited stained nuclei and decreased cytoplasmic staining. There was an increased staining for Mdm2 in centrilobular areas. We challenged pravastatin pretreated rats with a dose of diethylnitrosamine (DEN) and analyzed the effects 24 h after DEN treatment. Figure 2 shows that a larger proportion of hepatocytes exhibited a nuclear staining for Mdm2 in pretreated rats than in rats not pretreated with pravastatin. We found that in all pravastatin pretreated rats there was a weaker p53 staining than in rats challenged by DEN only. Pravastatin gave a less intense staining, fewer hepatocytes were positive, and the induction of apoptosis was attenuated. As high Mdm2 levels are known to facilitate p53 degradation, it is reasonable to assume that the low p53 response was due to a rapid degradation of p53 leading to an attenuated p53 response. In primary cultures of hepatocytes, the DEN-induced p53 response was inhibited dose-dependently by pretreatment with pravastatin.

View larger version (116K): [in this window] [in a new window]   Figure 2. Pravastatin pretreatment increases the Mdm2 response and decreases the p53 response following DEN treatment. Liver sections stained for Mdm2 (SMP-14) and p53 are shown. Rats were given a single dose of DEN (0.9 mmol/kg i.p.) 24 h before sacrifice. Pravastatin was given (4 mg/kg p.o.) 48 and 25 h before sacrifice. Many stained nuclei in pericentral areas (central vein: cv) can be seen.

3. Pravastatin decreases the half-life of Mdm2 and p53 in DNA-damaged HepG2 cells and decreases the p21 response to genotoxicity
Phosphorylation of Mdm2 on Ser166 has been shown to enhance its nuclear localization and to increase p53 degradation in stressed cells. Because pravastatin induced Ser166 phosphorylation, we examined the effect of statins on the half-lives of Mdm2 and p53 in cells stressed by DNA damaging agents. It was found that in cells pretreated with pravastatin and stressed by 5-Fu or leptomycinB, Mdm2 and p53 were degraded more rapidly than in cells not pretreated with pravastatin. In coimmunoprecipitation experiments an increased binding between p53 and Mdm2 was found in pravastatin and simvastatin pretreated cells. These data suggest that statins induced posttranscriptional changes in Mdm2 that facilitated the formation and the degradation of the Mdm2/p53 complex. This increased degradation was associated with attenuated p53 response and attenuated p21^WAF1 stabilization.

CONCLUSIONS AND SIGNIFICANCE

Our data show that statins in micromolar concentrations induced a rapid and long lasting phosphorylation of Mdm2 at Ser166 as well as phosphorylation at 2A10 specific epitopes in HepG2 cells. Immunohistological observations indicated a nuclear translocation of Mdm2. This is in line with in vitro studies showing that Ser166 phosphorylation is involved in activation and nuclear translocation of Mdm2. The data presented here suggest that statins induce Mdm2 phosphorylations in hepatocytes in vitro and in situ.

We found that statins attenuated the p53 response following DNA damage. These effects can be explained by the alterations in Mdm2 phosphorylations and the increased activity of Mdm2. Mdm2 undergoes extensive phosphorylations in response to various types of stress, including DNA damage, and Mdm2 function in the DNA damage response is regulated by these modifications. Nucleo-cytoplasmic shuttling as well as the Mdm2-dependent degradation of p53 is regulated by phosphorylations. Previous studies show that Ser166 and Ser186 phosphorylations activated Mdm2 and enhanced the ubiquitination-promoting function. We thus conclude that statins can induce Ser166 phosphorylation and other phosphorylations of Mdm2 and that the attenuated p53 response can be seen as a downstream consequence to these phosphorylations. The finding that statins affected both leptomycin B- and DNA-damage-induced p53 accumulations argues against the possibility that statins primarily affected DNA damage signaling.

Akt has been implicated in Ser166 phosphorylation of Mdm2 and a physical interaction between Akt and Mdm2 was shown. However, we did not find evidence that Akt was activated by statins in hepatocytes. Ser166 phosphorylation was not inhibited by the PI3K inhibitors LY294002 and wortmannin. These results indicate that the statin induced Ser166 phosphorylation in hepatocytes was not mediated by PI3K.

Statin-induced phosphorylation of mTOR and the effect of rapamycin, a selective inhibitor, implicate mTOR. It was recently noted that mTOR, in collaboration with p38 MAPK, can increase Mdm2 levels and induce a rapid degradation of p53 in DNA-damaged cells. In another study, MAPK was activated by insulin-like growth factor and shown to enhance Mdm2-dependent degradation of p53 in DNA damaged cells. Thus, mTOR and MAPK have been implicated in the same type of Mdm2/p53 alterations as described here. Our data indicate that statins activate this signaling pathway and suggest that mTOR phosphorylates Ser166. Further studies will characterize this pathway in more detail.

We have shown that statins, well-characterized and established pharmaceuticals, affect Mdm2 expression in unstressed cells as well as the Mdm2/p53 response to DNA damage. The significance of these findings for possible anticancer effects of statins should be characterized in more detail. Our data may have implications for both cancer therapy and chemopreventive strategies.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the effect of statins on Mdm2 phosphorylation and its consequences on the p53 response to DNA damage.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2745fje;

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