Statins are lipid-lowering drugs widely used in the treatment of hypercholesterolemia. Several intervention trials have demonstrated that statins decrease the incidence of cardiovascular events and improve survival rates both in secondary and primary prevention. In addition to lowering LDL cholesterol, statins reduce triglyceride levels and increase HDL cholesterol. HDL and its major protein constituent apoA-I play a critical role in cholesterol metabolism due to their capacities to eliminate excessive amounts of cholesterol from peripheral arteries and return it back to the liver, the so-called reverse-cholesterol transport pathway. HDL cholesterol levels are inversely correlated with CHD (47
), and statins increase HDL cholesterol and apoA-I levels (6
). In the present study we studied the molecular mechanism of statin action on apoA-I production.
Statin treatment of human hepatoma cells resulted in a time- and dose-dependent increase of apoA-I mRNA levels. A similar induction of hepatic apoA-I mRNA after statin treatment has been demonstrated previously in vivo in rats (48
). In vivo experiments in transgenic apoA-I mice treated with statins revealed no effect on apoA-I mRNA (data not shown), which is likely due to the rapid and almost complete catabolism of cerivastatin in this species (49
). The effects of statins on apoA-I expression occur at the transcriptional level since actinomycin D pretreatment blocked the induction. Furthermore, the statin effect depends on downstream products of the mevalonate pathway since mevalonate reversed the increase of apoA-I mRNA levels. To determine the molecular mechanisms involved, transient transfection experiments were performed, and a direct effect on human apoA-I promoter activity was demonstrated. Furthermore, a statin response element was mapped between –256 bp and –128 bp. Interestingly, a previous study in HepG2 cells demonstrated that cholesterol loading, either by LDL-uptake or addition of free cholesterol, led to an increase in apoA-I mRNA levels (50
). Since high levels of exogenous cholesterol lead to the downregulation of HMG-CoA reductase, common mechanisms may be the basis of the apoA-I induction by statins and cholesterol loading. This induction of apoA-I gene expression by statins extends the results from previous in vivo studies demonstrating an increase in apoA-I production rate (6
) after statin administration in humans. Increased expression and production of apoA-I likely contribute, therefore, to the increase of apoA-I and HDL plasma levels observed after statin therapy.
A statin response element was mapped to the A site, which also contains a functional PPRE (31
). The induction of human apoA-I gene expression by fibrates, another class of hypolipidemic drugs, occurs by activation of PPARα, which binds to this element. Interestingly, fibrates and statins share a number of pharmacological properties. Both classes of drugs reduce triglyceride and increase HDL levels and exert antithrombotic and anti-inflammatory actions in the vascular wall (25
). These observations led us to test whether statins could activate PPARα. Results from cotransfection experiments with PPARα in HepG2 cells demonstrate that statins act directly on the apoA-I A site via PPARα. In addition, in RK13 cells that are devoid of PPARα, statins activate a synthetic PPRE-driven promoter only in the presence of cotransfected PPARα. These results suggest a role for this transcription factor in the statin regulation of apoA-I gene expression and clearly establish a cross-talk between the PPARα and statin-signaling pathways. Since mevalonate addition reverses the PPARα activation by statins, it can be concluded that statins are not direct ligands, but rather that downstream products of the mevalonate pathway inhibit PPARα activity. Transfection experiments using the LBD of PPARα fused to the yeast transcription factor Gal4 DBD indicate that statins induce the transactivation capacity of PPARα in a general, promoter-independent manner and suggest that statin treatment may result in the generation of PPARα ligands or may increase the activity of the DBD of PPARα.
Mevalonate is a key intermediate in the de novo synthesis of both sterol and nonsterol isoprenoids. The majority of mevalonate is converted to cholesterol, which is a precursor of steroid hormones, bile acids, vitamin D, and a wide variety of oxysterols. Furthermore, a variety of nonsteroidal isoprenoid products are formed from mevalonate. Cholesterol depletion induced by statins triggers the cleavage of the cholesterol-sensitive transcription factors, called SREBPs. Studies in adipocytes revealed that ADD1/SREBP-1, a transcription factor participating in adipose tissue differentiation, leads to the production of endogenous ligands for PPARγ (40
). These intermediates are lipid molecules that bind directly to PPARγ, since they displaced the binding of synthetic ligands such as thiazolidinediones. Interestingly, SREBP activation leads to the induction of lipogenic enzymes such as FAS and thus may lead to the production of fatty acids, which are ligands of PPARα (23
). In addition, PPAR ligands may be produced by oxidation of endogenous fatty acids by lipoxygenases (24
), or cyclooxygenases (43
), or by degradation of phospholipids by phospholipase A2, which liberates fatty acids (46
). This raised the question of whether PPARα ligands might be generated through activation of these pathways. However, experiments to reverse statin action on PPARα activity using potent inhibitors of these enzymes were without effect on statin-induced PPARα activity. Furthermore, compounds derived from the cholesterol biosynthetic pathway, including sterols, squalene, or cholesterol metabolites such as bile acids or oxysterols, did not modulate statin-induced PPARα activity. Thus these data provide evidence that statins activate PPARα via a pathway other than the sterol or SREBP pathways.
Mevalonate is not only a precursor for cholesterol synthesis but also is a precursor of nonsteroidal isoprenoid compounds. Fpp and GGpp are substrates for the posttranslational prenylation of proteins (53
). Farnesol is a Fpp-derived metabolite that has been shown recently to induce PPARα activity and thereby influence keratinocyte differentiation (56
). However, our results demonstrate that statin action on PPARα was prevented by GGpp, but not Fpp nor farnesol, suggesting that geranylgeranyl-modified intermediates may antagonize PPARα. Fpp and GGpp are implicated in membrane translocation, leading to the activation of a variety of proteins, including Ras and Rho GTP-binding proteins, respectively (57
). Rho A, Rho B, Rac, and Cdc42 are the major substrates for posttranslational modification by geranylgeranylation, which leads to their activation and membrane translocation. This process has been shown to be inhibited by statins in SMCs indicating a direct effect of statins on the vascular wall via inhibition of Rho geranylgeranylation (59
). After posttranslational modification by geranylgeranylation (18
), the Rho family of small GTP-binding proteins can be inactivated by treatment with C3 exoenzyme, which selectively ADP-ribosylates low-molecular-weight G proteins of the Rho A and B subfamily, rendering them biologically inactive (60
). C3 exoenzyme treatment enhanced statin-induction of PPARα transactivation (data not shown), further pointing to the implication of Rho proteins in the statin activation of PPARα. Since Rho A, but not Cdc42 or Rac dominant negative proteins, enhances PPARα activity and, more specifically, the PPARα LBD, it is likely that the effects of statins on PPARα are mediated by Rho A. Downstream targets of Rho family proteins have just begun to be identified, and the molecular mechanisms by which Rho proteins may regulate gene expression are not clearly understood. Posttranslationally modified Rho proteins control cytoskeletal reorganization, motility, and cell growth (61
). Rho, Rac, and Cdc42 have been reported to regulate the c-Jun
terminal kinase (JNK), and the p38 MAP kinase (MAPK) cascades (62
). PPARα activity is modulated by phosphorylation, resulting in either enhanced or lowered transcriptional activity (65
). In this study, we show that statins decrease the phosphorylation of PPARα. Interestingly, a recent study demonstrated downregulation of PPARα activity after activation of the MAPK pathway (66
). However, MAPK sites in PPARα were mapped in the NH2
-terminal part of the protein (67
). Since statins induce PPARα LBD by its activity, they likely act through a novel mechanism. Further studies are required to delineate the molecular mechanism of PPARα regulation by Rho A. The GGpp pathway has already been implicated in mediating the antithrombotic and anti-inflammatory effects of statins in SMCs and macrophages (16
). Our data provide evidence that the GGpp pathway is also implicated in the effects of statins acting on PPARα. Since PPARα exerts potent anti-inflammatory activities in vascular cells (27
), we speculate that the reported anti-inflammatory activities of statins are, at least in part, mediated by PPARα activation (Figure ).
Figure 9 Cross-talk between the statin and PPARα pathways. C3 T, C3 transferase; DN, dominant negative. The pathway in black is that implicated in PPARα regulation by statins. The other pathways of mevalonate metabolism are depicted in gray. PP, (more ...)
Previous studies have demonstrated induction of PPARγ activity by statins through the generation of ligands after SREBP activation (40
). Furthermore, PPARγ transcription has been shown to be induced by statins by a SREBP response element in the PPARγ promoter (69
). However, this is the first time that a cross-talk of the PPARα and statin-signaling pathways is shown. Furthermore, we show that PPARα activation by statins occurs through a completely different molecular mechanism, implicating the GGpp pathway and prenylation of Rho family proteins. Moreover, we demonstrate that simultaneous treatment with statins and fibrate PPARα ligands results in a synergistic effect on PPARα transactivation. Thus PPARα is an important molecular target for the two major classes of hypolipidemic drugs. Together, these data provide a molecular rationale for combination therapy with statins and fibrates in the treatment of CHD.