Statin Treatment and 3‘ Polyadenylation of eNOS mRNA

【摘要】  Objective— Statins have been shown to increase endothelial nitric oxide synthase expression via enhanced mRNA stability. Because the poly(A) tail is an important determinant of transcript stability, we sought to characterize the effect of statins on eNOS mRNA 3' polyadenylation.

Methods and Results— Endothelial cells treated with statins had a time- and dose-dependent increase in eNOS transcripts with long poly(A) tails (75 to 160 adenosines). This effect was dependent on 3-hydroxy-3-methylglutaryl (HMG)-coenxyme A (CoA) reductase inhibition and was observed with both lipophilic (simvastatin) and hydrophilic (rosuvastatin) statins. In mRNA stability assays, polyadenylated eNOS transcripts from statin-treated cells were 2- to 3-fold more stable than transcripts from untreated cells. The effect of statins on eNOS polyadenylation was related to cytoskeleton organization; there was increased eNOS mRNA polyadenylation after Rho inhibition and cytochalasin D treatment. Further, we found increased phosphorylation of RNA polymerase II in statin-treated cells, suggesting that statin-induced polyadenylation involved modulation of RNA polymerase II activity.

Conclusions— Our data provide insight into a mechanism by which statins enhance eNOS mRNA stability and increase eNOS protein: statins increase eNOS mRNA polyadenylation through Rho-mediated changes in the actin cytoskeleton.

We examined the effect of statins on eNOS mRNA polyadenylation, a process known to increase mRNA stability and translation. Statins increased polyadenylation in a time- and dose-dependent manner through a mechanism that appears to involve Rho-induced changes in the actin cytoskeleton.

【关键词】  endothelial nitric oxide synthase mRNA stability polyadenylation posttranscriptional regulation statin

Introduction

Clinically, statins have been used for their ability to potently lower plasma lipid levels, but these agents also possess other significant antiatherogenic properties. These include improvement of endothelial function, 1–3 enhancement of atherosclerotic plaque stability, 4–6 attenuation of oxidative stress and inflammation, 7,8 and inhibition of thrombogenesis. 9,10 One property central to their pleiotropism is their ability to increase the bioavailability of nitric oxide (NO · ). 11 NO · plays a significant role in vascular homeostasis, and its bioavailability is dependent on several factors: the expression of endothelial nitric oxide synthase (eNOS), 12 the presence of substrate and cofactors for eNOS, 13 the phosphorylation of eNOS, 14,15 and reaction with reactive oxygen species. 16

Time- and dose-dependent upregulation of eNOS protein by statins has been observed in both cell culture and animal studies. 17–22 The mechanism responsible for this upregulation is largely posttranscriptional; statin treatment prolonged eNOS mRNA half-life. 20,23 Importantly, posttranscriptional regulation of eNOS expression is known to be an endothelial cell response to numerous biophysical and biochemical stimuli. 24

Recently, we described a mechanism for shear stress–induced eNOS expression that involved changes in mRNA 3' polyadenylation. 25 Cells exposed to laminar shear stress had an increase in eNOS transcripts with long 3' poly(A) tails, which was associated with increased eNOS mRNA stability and enhanced translation. In the current study, we sought to characterize the effect of statins on eNOS mRNA 3' polyadenylation.

Materials and Methods

Materials

5,6-dichloro-1-β- D -ribofuranosylbenzimidazole (DRB), toxin B, and cytochalasin D were obtained from Calbiochem. FFP, GGPP, and mevalonate were purchased from Sigma. Rosuvastatin was provided by AstraZeneca. Simvastatin was provided by Merck and was activated before use. 20

Tissue Culture

Bovine aortic endothelial cells (BAECs; Cell Systems) were cultured in Media 199 (M199; Cellgro, Mediatech) containing 10% fetal calf serum (FCS; Hyclone Laboratories) as previously described. 26 Postconfluent BAECs between passages 4 to 7 were used for experiments.

RNA Isolation

Total cellular RNA was isolated using TRI-Reagent (Molecular Research Center, Inc). The PARIS-Kit (Ambion) was used to isolate cytosolic and nuclear RNA fractions.

Assessment of eNOS mRNA Polyadenylation

Ribonuclease protection assays (RPA) were performed using a biotinylated, antisense RNA probe prepared by in vitro transcription (T7 RNA polymerase and Mega Script kit, Ambion). 25 6 to 10 µg of total RNA from endothelial cells was hybridized with 300 pg of the biotinylated riboprobe overnight at 42°C. Unprotected RNA was digested with RNaseA/T1 (Ambion) in digestion buffer for 30 minutes at 37°C; the reaction was stopped by RNA precipitation. The samples were run on a 9% polyacrylamide gel containing urea. RNA was electroblotted onto a positively charged nylon membrane, which was developed using the Bright Star Biodetect Kit (Ambion).

Assessment of RNA Polymerase II Expression

Western analysis for RNA polymerase II (RNAP II) was performed with primary antibodies against phosphorylated RNAP II (H5 and H14) and unphosphorylated RNAP II (8WG16). RNAP II antibodies were obtained from Covance and the antibody against actin was from Santa Cruz. Western analyses included goat anti-mouse secondary antibody, conjugated to horseradish peroxidase (Bio-Rad). Antigen detection was performed with a chemiluminescent detection system (ECL; Amersham).

Results

Statin Treatment Increases 3' Polyadenylation of eNOS mRNA

RNase protection assays (RPAs) were used to assess the effect of statins on eNOS mRNA 3' poly(A) tail length. The RPA riboprobe was targeted to protect the last 225 nt of the bovine eNOS 3'UTR plus a poly(A) tail up to 375 A. The riboprobe was specific for bovine eNOS mRNA; it did not protect yeast RNA ( Figure 1 ) or human endothelial cell RNA (not shown). In untreated cells, the predominant protected fragment was approximately 250 nt in length, with a poly(A) tail calculated to be 25 A ( Figure 1A and 1 B). In endothelial cells exposed to rosuvastatin or simvastatin for 24 hours, there was a dose-dependent increase in longer protected eNOS fragments (300 and 385 A), with poly(A) tails calculated to be 75 and 165 nt in length, respectively. The longer fragments were absent from RPAs of cells pretreated with cordycepin (not shown), an agent that inhibits poly(A) tail lengthening, confirming that these fragments represented eNOS mRNA with long poly(A) tails.

Figure 1. Statin treatment and eNOS mRNA polyadenylation. A and B, RPAs of BAECs treated with rosuvastatin or simvastatin (24 hours). Densitometric data (mean±SEM, n=4) expressed as ratio of long poly(A) eNOS (300, 350 nt bands) to total (250, 300, 350 nt bands). * P <0.05, ** P <0.01, *** P <0.001, statin vs untreated (1-way ANOVA, Bonferroni test). C and D, RPAs of BAECs treated with statin for indicated durations. Rosuvastatin vs 0 hour: * P <0.01; simvastatin vs 0 hour: * P <0.05.

Endothelial cells were exposed to statins for 12 to 24 hours to examine the effects of statin treatment duration on eNOS mRNA polyadenylation ( Figure 1C and 1 D). Statin treatment had no effect on cell viability (please see http://atvb. ahajournals.org). A modest increase in eNOS polyadenylation was observed after 12 hours of treatment with either rosuvastatin (10 micromol/L) or simvastatin (20 micromol/L). Detection of long poly(A) fragments appeared to be greatest after 24 hours of statin treatment.

Polyadenylated eNOS mRNA From Statin-Treated Cells Is More Stable

DRB chase studies (described at http://atvb.ahajournals.org) were performed to determine whether statin-induced polyadenylation conferred increased transcript stability. Endothelial cells were treated with rosuvastatin (10 micromol/L) for 24 hours before DRB (60 micromol/L) treatment. RPAs were performed on RNA extracted from cells at different times after DRB ( Figure 2 ). Using this method, we found that polyadenylated eNOS transcripts from statin-treated cells were markedly more stable than those from untreated cells. The half-life of transcripts from untreated cells was 8 hours, whereas the half-life of those from statin-treated cells was 18 hours. It should be noted that the RPA analysis used in these studies targeted the 3' end of eNOS mRNA, whose stability may differ from that of the entire message. However, the stability of these protected fragments was consistent with our previous findings 25 and suggests that statin-induced polyadenylation prolongs eNOS mRNA half-life.

Figure 2. Stability of polyadenylated eNOS mRNA after statin treatment (DRB chase assay). A, RPAs of control cells and statin-treated cells (rosuvastatin, 10 micromol/L, 24 hours) at various time points after DRB. B, Densitometric analysis (mean±SEM, n=3) of protected eNOS fragments, expressed as percent of 0 hour value.

Nuclear Versus Cytoplasmic Polyadenylation in eNOS 3' mRNA Processing

In eukaryotic cells, 3' polyadenylation of newly transcribed RNA occurs in the nucleus, after 3' cleavage of the primary transcript. However, lengthening of the 3' poly(A) tail can also occur in the cytoplasm through a process known as cytoplasmic polyadenylation. 27 To examine whether statin-induced eNOS mRNA poly(A) tail lengthening involves cytoplasmic polyadenylation, RNA was isolated from both nuclear and cytoplasmic fractions of rosuvastatin-treated cells and subjected to RPA analysis ( Figure 3 A). In nuclear extracts from statin-treated cells, the predominant protected fragments were long poly(A) eNOS transcripts (300 and 385 nt). In cytosolic extracts of statin-treated cells, both long and short polyadenylated eNOS transcripts were detected. Compared with untreated cells, the effect of statins on eNOS polyadenylation appeared to be more prominent in nuclear fractions, suggesting that statin-induced eNOS polyadenylation is mainly a nuclear process. We propose that both long and short poly(A) transcripts are synthesized in the nucleus and are subsequently exported to the cytosol where they undergo degradation. However, these experiments do not completely exclude the possibility that some poly(A) tail lengthening also occurs in the cytosol.

Figure 3. Polyadenylated eNOS in different cell fractions. A, Representative RPA (n=3) of control or rosuvastatin-treated cells (10 micromol/L, 24 hours). B, DRB pretreatment (60 micromol/L, 1 hour) attenuated statin-induced polyadenylation (rosuvastatin, 10 micromol/L, 24 hours). Left, RPA; right, pooled analysis (n=3) vs control, * P <0.05, ** P <0.001.

Because nuclear 3' polyadenylation of mRNA is linked to transcription initiation and elongation, 28 we assessed the effect of blocking transcription on eNOS polyadenylation. Endothelial cells were exposed to DRB for 1 hour before 24 hours of statin treatment. Pretreatment with DRB attenuated, but did not abolish the long poly(A) transcripts ( Figure 3 B), suggesting that statin-induced expression of these transcripts was only partially dependent on active transcription.

Statins Modulate Phosphorylation of RNA Polymerase II Carboxy-Terminal Domain

In eukaryotic cells, RNA polymerase (RNAP) II plays a critical role in coordinating pre-mRNA processing events, including capping, splicing, and 3' end formation. This processing function is mediated primarily by phosphorylation of the carboxy-terminal domain (CTD) of the enzyme. 29,30 To examine the effect of statins on RNAP II phosphorylation, Western analysis was performed on endothelial cells exposed to rosuvastatin (10 micromole/L) for up to 24 hours, using antibodies specific for different phosphoisoforms of RNAP II. After 24 hours of treatment, a 2-fold increase in phosphorylated RNAP II was observed, with no change in expression of unphosphorylated RNAP II ( Figure 4 ). Further, the increase in RNAP II phosphorylation was primarily at serine 2 of the heptapeptide repeat (YSPTSPS) of CTD. In contrast, there was no effect on CTD phospho-serine 5 (not shown). This is important because RNAP II CTD phospho-serine 2 is associated with transcription elongation and mRNA 3'-end processing. 31

Figure 4. Rosuvastatin treatment and RNAP II phosphorylation. A, Western analysis of phosphorylated (CTD serine 2 and serine 5) and unphosphorylated RNAP II in BAECs treated with rosuvastatin (10 micromol/L) for the indicated time. B, Grouped densitometric data (mean±SEM, n=5) for phosphorylated RNAP II. * P <0.05, vs 0 hour time point.

Dependence of eNOS mRNA Polyadenylation on HMG-CoA Reductase Inhibition, Rho GTPase Activity, and Actin Cytoskeleton Organization

BAECs were cotreated with rosuvastatin and mevalonate, the product formed by the reduction of HMG-CoA. L-mevalonate (400 micromole/L) attenuated rosuvastatin-induced eNOS mRNA polyadenylation by 64% ( Figure 5 ). Alone, mevalonate had no effect on expression of long poly(A) eNOS (not shown). Geranylgeranylpyrophosphate (GGPP, 10 micromole/L), an isoprenoid intermediate in the cholesterol biosynthesis pathway, also markedly reduced rosuvastatin-induced eNOS polyadenylation. In contrast, farnesyl pyrophosphate did not significantly affect eNOS polyadenylation. Together, these data suggest that the effect of statins on eNOS mRNA polyadenylation is mediated through inhibition of HMG-CoA reductase.

Figure 5. Effect of isoprenoid intermediates on eNOS 3' polyadenylation. A, RPA: BAECs exposed to rosuvastatin alone (10 micromol/L, 24 hours), rosuvastatin plus mevalonate (400 micromol/L) or rosuvastatin plus GGPP (10 micromol/L). B, Densitometric analysis (n=3 to 6), * P <0.05 (unpaired t test) compared with rosuvastatin alone. C, RPA: Rosuvastatin alone (10 micromol/L, 24 hour) or rosuvastatin plus FPP (10 micromol/L). D, Densitometric analysis (n=3).

GGPP is a lipid attachment that is important for the posttranslational modification of signaling peptides in the Rho GTPase family. 32 Statins upregulate eNOS expression by blocking Rho geranylgeranylation. 23 We examined whether inhibition of Rho signaling increased eNOS polyadenylation. As a first step, we examined RhoA activity in cells treated with rosuvastatin. In a GTP-RhoA pull down assay, rosuvastatin dose-dependently decreased the expression of active RhoA ( Figure 6 A). Subsequently, cells treated with an inhibitor of Rho GTPase, C. difficile toxin B (10 ng/mL), had a dramatic increase in eNOS polyadenylation ( Figure 6 B).

Figure 6. eNOS 3' polyadenylation in response to Rho GTPase inhibition, cytochalasin D, TNF-, and DFO. A, Representative Western (n=3) of BAEC Rho-GTP levels after different rosuvastatin treatments (24 hours). B and C, Representative RPAs of BAECs treated with toxin B (10 ng/mL, 6 hours; n=3), or cytochalasin D (5 µmol/L, 6 hours; n=6). D, RPA of cells exposed to TNF- (10 ng/mL, 24 hours) or DFO (100 micromol/L, 24 hours) then rosuvastatin (10 micromol/L, 24 hours). Densitometric data (n=5 to 8) analyzed by unpaired t test.

The actin cytoskeleton is a downstream sensor of Rho GTPase, and direct alteration of cytoskeleton organization with cytochalasin D can increase eNOS expression. 19 Similarly, we found an increase in long poly(A) eNOS transcripts in cells treated with cytochalasin D (5 micromole/L, Figure 6 C). Together, these data indicate that statin-induced expression of long poly(A) transcripts is mediated through the inhibition of RhoA signaling and changes in actin cytoskeleton organization.

Rosuvastatin Increases eNOS Polyadenylation in Cells Exposed to Tumor Necrosis Factor- or Hypoxia

Both tumor necrosis factor (TNF)- and hypoxia downregulate eNOS expression, 33–35 and statins can block these effects. 21,36 Endothelial cells were exposed to either TNF- or the hypoxia mimetic desferrioxamine (DFO 37 ) and then treated with rosuvastatin to determine whether these stimuli would attenuate statin-induced eNOS polyadenylation ( Figure 6D and 6 E). In cells treated with TNF-, the level of polyadenylated eNOS was similar to that for unstimulated cells. Subsequent treatment with rosuvastatin enhanced eNOS 3' polyadenylation 2-fold. Similarly, rosuvastatin increased eNOS 3' polyadenylation in cells that had been subjected to DFO (4-fold). Thus, in keeping with the ability of statins to restore eNOS expression in the presence of TNF- (please see http://atvb.ahajournals.org) or hypoxia, statins increase 3' polyadenylation in cells exposed to these stimuli.

Discussion

Although there is disagreement as to whether the pleiotropic effects of statins are clinically relevant, the importance of statin-induced upregulation of eNOS expression has been demonstrated in animal models of stroke and myocardial infarction. 19,38 In vitro studies have indicated that statins increase eNOS expression by prolonging eNOS mRNA half-life. 20,21 In this study, we have provided insight into the posttranscriptional mechanism by which statins might regulate eNOS expression. We believe that this mechanism involves changes in nuclear 3' polyadenylation of eNOS mRNA.

The 3' poly(A) tail is an important determinant of mRNA stability and translational efficiency. 39 Transcripts with short 3' poly(A) tails are less stable and less translationally active than those with long tails. We have found that eNOS mRNA has predominantly a short 3' poly(A) tail ( 25 nt) at baseline in bovine 25 and human endothelial cells (please see http://atvb.ahajournals.org). Based on this finding and the nature of the 3' sequence for both human and bovine eNOS mRNA, 40,41 we suspect that eNOS has an inherently inefficient polyadenylation signal.

Statin treatment increased eNOS 3' polyadenylation in a dose- and time-dependent manner. This is similar to what we have observed with laminar shear stress, 25 although long poly(A) transcripts were observed earlier in the course of stimulation compared with statins (2 hours versus 12 hours). This may be related to the differences in the rate of eNOS transcription: transcription is transiently increased with shear but not with statin treatment. Recently, laminar shear stress was shown to inhibit HMG-CoA reductase activity and expression. 42 It is possible that both shear- and statin-induced eNOS polyadenylation involve inhibition of HMG-CoA reductase. Although our data indicate that statin-induced eNOS polyadenylation was dependent on inhibition of HMG-CoA reductase, we did not determine the time course for enzyme inhibition in our cell cultures. Thus, the delay in the effect of statins compared with laminar shear may be related to the ability of the drug to penetrate the cell and inhibit HMG-CoA reductase.

The structural features of rosuvastatin that enhance its interaction with HMG-CoA reductase also make the drug more hydrophilic and less likely to enter endothelial cells by passive diffusion. The hydrophilic statins are taken up by hepatocytes through an organic anion transporter (OATP-C), 43 but it is uncertain as to whether a similar mechanism exists for their entry into extrahepatic cells. It has been suggested that statins upregulate eNOS expression by blocking hepatic production of mevalonate, rather than direct inhibition of HMG-CoA reductase in endothelial cells. 44 Our data show that inhibition of endothelial HMG-CoA reductase by either hydrophilic or lipophilic statins can result in eNOS poly(A) tail lengthening.

The polyadenylated eNOS transcripts from statin-treated cells were more stable than those from untreated cells. We have observed a similar association between eNOS mRNA polyadenylation and message stability previously. 25 Our results vary quantitatively from earlier reports of eNOS mRNA stability, 45 but we suspect that this may be attributable to different methods of analysis.

In eukaryotic cells, polyadenylation mostly occurs in the nucleus as part of pre-mRNA processing. Transcription initiation, elongation, and pre-mRNA processing are regulated by a multi-protein complex whose main component is RNA polymerase (RNAP) II. Statin treatment was associated with increased phosphorylation of RNAP II, suggesting modulation of the processing activity of RNAP II. In addition, we found that statin treatment primarily increased serine 2 phosphorylation of the heptapeptide repeat of CTD. Changes in RNAP II phosphorylation in response to external stimuli have been described in yeast and different mammalian cell lines, 46 but mRNA 3'-end processing has not been characterized in endothelial cells. We conclude that statin treatment altered RNAP II phosphorylation status in a manner that would enhance mRNA 3' polyadenylation.

The Rho GTPase signaling pathway is known to be a negative regulator of eNOS expression. 19 Inhibition of this pathway ultimately leads to changes in actin cytoskeleton organization, and eNOS expression was increased by an agent that directly alters the actin cytoskeleton, cytochalasin D. 19 These findings suggest that the actin cytoskeleton can posttranscriptionally regulate eNOS expression and are consistent with our observation that cytochalasin D increased expression of long poly(A) eNOS transcripts. Recently, an association between actin and eNOS transcription has been described. 47 Our data suggest that actin may also be important in 3' processing of eNOS mRNA.

Actin has been shown to have a role in mRNA transcription, processing, and nucleocytoplasmic transport. 48 Actin may influence RNAP II processing activity through its interaction with small nuclear ribonucleoproteins, or through chromatin remodeling. 49 Previously, we have found that monomeric actin can interact with cytoplasmic eNOS mRNA, 50 but the nature of this interaction in the context of 3' processing is unknown at this time.

In this study, we have provided insight into how an agent that is widely used in the prevention and treatment of vascular disease increases the expression of a gene that is essential for vascular health. We have extended findings from our previous studies by demonstrating that eNOS mRNA poly(A) tail lengthening is associated with changes in RNAP II phosphorylation and actin cytoskeleton organization. Because statins can increase NO bioavailability by posttranscriptional and posttranslational mechanisms, an issue that arises concerns the relative importance of the mechanism described in this study to overall statin-induced NO bioavailability. This issue has not been examined directly and is the focus of ongoing studies.

Acknowledgments

Sources of Funding

This study was supported by National Institutes of Health Grant R01HL077274 and a grant from the Investigator-Sponsored Study Program of Astra-Zeneca.

Disclosures

Dr Searles is the principal investigator for a grant from the Investigator-Sponsored Study Program of Astra-Zeneca and NIH grant Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase (RO1HL077274-01).

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作者单位：Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.