Laminar Shear Stress and 3' Polyadenylation of eNOS mRNA
http://www.100md.com
循环研究杂志 2005年第6期
the Division of Cardiology, Department of Medicine, Emory University School of Medicine and the Atlanta Veterans Administration Medical Center, Atlanta, Ga.
Abstract
The 3' poly(A) tail is important in messenger RNA stability and translational efficiency. In somatic tissues, 3' polyadenylation of mRNAs has been thought to largely be a constitutively active process. We have reported that laminar shear stress causes a brief increase in endothelial nitric oxide synthase (eNOS) transcription, followed by a prolonged increase in eNOS mRNA stability. We sought to determine whether shear stress and other stimuli affected eNOS 3' polyadenylation in endothelial cells. Under basal (static) conditions, eNOS mRNA possessed short 3' poly(A) tails of <25 nt. In contrast, laminar shear stress increased expression of eNOS transcripts with long poly(A) tails. ENOS transcripts with longer poly(A) tails had prolonged half-lives (6 hours in static cells versus 18 hours in sheared cells). Polysome analysis revealed that eNOS mRNA from sheared cells was shifted into more translationally active polysome fractions compared with eNOS mRNA from static cells. Shear-induced lengthening of the eNOS 3' poly(A) tail was the result of increased nuclear polyadenylation. Furthermore, hydrogen peroxide and HMG Co-A reductase inhibitors, other stimuli known to modulate eNOS expression posttranscriptionally, also induced eNOS 3' poly(A) tail lengthening. These results support the concept that shear stress modulates eNOS mRNA stability and translation via increased 3' polyadenylation. We suggest that mRNA 3' polyadenylation is a posttranscriptional mechanism used by endothelial cells to regulate gene expression.
Key Words: endothelial nitric oxide synthase mRNA stability polyadenylation posttranscriptional regulation shear stress
Introduction
Laminar shear stress is a potent stimulus for the production of vascular nitric oxide (NO) because of its ability to increase both the activity and expression of the endothelial nitric oxide synthase (eNOS).1eC3 Endothelium-derived NO is crucial for maintenance of vascular homeostasis through its vasodilator activity;4 its ability to inhibit smooth muscle growth,5 platelet aggregation,6 and leukocyte adhesion,7 and its role in inhibiting lipid oxidation and regulating apoptosis in the vessel wall.8 Given these properties of NO, the shear stress-induced increase in eNOS expression may be important in preventing atherosclerosis. Indeed, areas of the vasculature exposed to high shear stress appear to be protected from the development of atherosclerosis, and areas exposed to low shear stress are prone to atherosclerotic lesion formation.9
Previously we have demonstrated that shear stress leads to increased eNOS mRNA expression via 2 separate mechanisms: a transient increase in eNOS transcription, and stabilization of eNOS mRNA.10 Posttranscriptional regulation of eNOS expression via modulation of eNOS mRNA stability is now recognized as an important response of endothelial cells to numerous biophysical and biochemical stimuli.11eC17 Although we have recently described a mechanism for the posttranscriptional regulation of eNOS expression during cell growth,18 the details for modulation of mRNA stability by other stimuli, including shear stress, remain poorly defined.
In mammalian cells, 3' poly(A) tails have been shown to regulate mRNA stability and translation.19,20 The cloned sequences of bovine and human eNOS mRNAs were reported to have 3' poly(A) tails 12 to 20 nucleotides in length.21,22 Although these studies did not specifically examine poly(A) tail length, the eNOS sequences were derived from RNA libraries of cultured cells not exposed to shear, suggesting that eNOS mRNA has a short 3' poly(A) tail under baseline conditions. We hypothesized that shear stress increases polyadenylation of eNOS mRNA. We report that shear stress led to an increase in eNOS transcripts with long poly(A) tails. We found that in cells subjected to shear stress there was a shift of eNOS mRNA from monosomes to polysomes, suggesting that a functional sequela of increased polyadenylation was increased translation. Furthermore, long poly(A) eNOS mRNA was more stable. These data provide evidence to support a novel mechanism for regulation of eNOS expression.
Materials and Methods
Materials
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) was obtained from Calbiochem. Cordycepin (3'-deoxyadenosine), cycloheximide, actinomycin D, hydrogen peroxide, mevastatin, and all primers were purchased from Sigma. Simvastatin was a generous gift from Merck (Rahway, NJ) and was activated before use, as described earlier.15
Tissue Culture
Bovine aortic endothelial cells (BAECs; Cell Systems, Kirkland, Wash) were cultured in Media 199 (M199; Cellgro, Mediatech) containing 10% fetal calf serum (FCS; Hyclone Labortories) as described earlier.23 Postconfluent BAECs between passages 4 to 8 were used for experiments. A cone-in-plate viscometer with a 1° angle was used to shear cells.24
RNA Isolation
Total cellular RNA was isolated using TRI-Reagent (Molecular Research Center Inc). For polysomal, cytosolic and nuclear fractionation, 2 e蘈 linear acrylamide (Ambion) was added as a carrier during RNA precipitation. The PARIS-Kit (Ambion) was used to separate cytosolic and nuclear RNA fractions.
Rapid Amplification of cDNA Ends-PolyA Test
To measure the 3' poly(A) tails of eNOS mRNA, Rapid Amplification of cDNA Ends-PolyA Test (RACE-PAT) was performed as described previously.25 The details of RACE-PAT are provided in the online data supplement at http://circres.ahajournals.org
Real Time RT-PCR
Real time RT-PCR was done with the Light Cycler (Roche) as described earlier.26 Reverse transcription was performed as described for RACE-PAT, using primers and conditions described in the online supplement.
Ribonuclease Protection Assays
A 600 nt band detected by eNOS RACE-PAT analysis was cloned into the pCRII-TOPO vector (Invitrogen) and its orientation was confirmed by DNA sequencing. A biotinylated antisense RNA probe was prepared by in vitro transcription, using T7 RNA polymerase (Mega Script kit, Ambion). Three to 10 e of total RNA from endothelial cells was hybridized with 374 pg of the biotinylated eNOS RNA-probe 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).
Polysomal Fractionation
The analysis of polysomes was performed as described previously.27,28 The details of polysome fractionation are described in the online supplement.
Results
eNOS mRNA Levels and Translational Activity in Response to Shear Stress
We used real time quantitative RT-PCR to compare eNOS mRNA levels in static control versus sheared BAECs. Shear increased eNOS mRNA levels by 9-fold compared with levels in nonsheared cells (Figure 1A). Because the increase in eNOS mRNA has been shown to be associated with increased eNOS protein levels,21 we hypothesized that translation of eNOS mRNA is also enhanced in response to shear. Polyribosomes from sheared and control endothelial cells were fractionated on the basis of size (Figure 1B), and the quantity of eNOS mRNA in each fraction was determined (Figure 1C). Polysome size reflects translation activity; transcripts that are associated with large polysomes are more translationally active. We found that large polysomal fractions (>3 ribosomes/mRNA) from sheared cells had 2.5- to 10.5-fold more eNOS mRNA than those from static cells. These data provide direct evidence that eNOS mRNA from sheared cells was translated with increased efficiency.
Identification of eNOS Transcripts With Long Poly(A) Tails With RACE-PAT
To determine the relationship between shear stress and eNOS mRNA polyadenylation, a PCR based method (RACE-PAT) was used to measure the poly(A) tail lengths of eNOS mRNA under shear and static conditions (Figure 2). The minimum predicted PCR product size was 237 nt: 225 nt of the eNOS 3' untranslated region (UTR) plus a poly(A) tail of 12 nt or less. The predominant RACE-PAT PCR product from both sheared and nonsheared BAECs was 250 nt, consistent with a poly(A) tail length of 25 nts (Figure 2B, lower arrow). As the amount of shear stress was increased, PCR products larger than 250 nt were observed, including a product that was 600 nt (Figure 2B, upper arrow). This finding was consistent with lengthening of the 3'poly(A) tail that was proportional to the amount of shear stress. To confirm that the larger PCR products were polyadenylated, RNA was subjected to oligo (dT) hybridization and RNase H digestion before RACE-PAT. RNase H digests RNA-DNA hybrids, and poly(A) tails hybridized with oligo (dT) are degraded by RNase H. When the RNase H digestion step was performed before RACE-PAT, the PCR products greater than 250 nts were not observed (Figure 2B), confirming that they were eNOS transcripts with different length poly(A) tails. The results of these RACE-PAT studies are compatible with shear increasing the eNOS mRNA 3' poly(A) tail length up to 300 nts.
The RACE-PAT PCR products of 250 nt and 600nt were cloned into pCRII (Invitrogen) and sequenced. Multiple clones from different experiments were sequenced, and both of the RACE-PAT products had the predicted eNOS 3'UTR sequence. As expected, the 250 nt PCR product had a relatively short poly(A) tail length of 12 to 30 bases. Although the entire sequence of the 600 nt product was not able to be obtained, a minimum poly(A) tail size of 112 bases was identified, confirming that it had a lengthened poly(A) tail. Furthermore, the inability to obtain 3' sequence beyond 112 adenines is consistent with its composition being poly(A).
Identification of eNOS Transcripts With Long Poly(A) Tails With RNase Protection Assay
To further examine shear-induced polyadenylation of eNOS mRNA, RNase protection assays (RPAs) were performed using a riboprobe that was antisense to the cloned 600 nt RACE-PAT product. Total RNA from cells exposed to increasing dynes/cm2 of shear stress was isolated and subjected to RPA analysis (Figure 3A). Consistent with the RACE-PAT analysis, the predominant protected fragment in nonsheared cells was 250 nt, which had a poly(A) tail calculated to be 25 nt. Importantly, RNA from cells exposed to shear stress had a significant, dose-dependent increase in longer protected fragments, specifically fragments of 300 and 385 nt. These longer fragments had poly(A) tails calculated to be 75 and 160 nt in length, respectively, reflecting increased polyadenylation with shear. To confirm that these protected fragments were polyadenylated, cells were exposed to 100 eole/L cordycepin (3'-deoxyadenosine) for one hour before 6 hours of shear. Cordycepin inhibits the formation of mRNA with long poly(A) tails but allows for the correct splicing and transport of transcribed mRNA.29 The longer protected fragments (300 and 385 nt) were absent after cordycepin pre-treatment (Figure 3C), verifying that they were long poly(A) eNOS transcripts.
A time course experiment was performed to correlate the duration of laminar shear stress with the quantity of long poly(A) eNOS fragments (Figure 3B). The longer fragments were observed after 2 hours of shear, and their levels progressively increased up to 6 hours. Interestingly, the appearance of long eNOS poly(A) transcripts at 2 hours of shear stress temporally coincided with the reported onset of increased eNOS mRNA stability,10 suggesting a role for shear-induced polyadenylation in modulating eNOS mRNA stability.
Stability of Polyadenylated eNOS mRNA
DRB chase studies were performed to examine the stability of polyadenylated eNOS mRNA. Before the addition of DRB to the media, cells were exposed to shear stress for 6 hours. An RPA analysis of polyadenylated eNOS mRNA was performed on cells that had been exposed to DRB for various periods of time (Figure 4A). At the 0 hour time point, the short poly(A) fragment was the predominant species in control cells. In sheared cells, both short and long poly(A) fragments were present at 0 hours. Over the next 18 hours, there was a gradual decay in eNOS mRNA from control cells. In contrast, the long poly(A) transcripts from sheared cells did not decay until 6 hours, indicating that these transcripts were more stable for at least 6 hours. After 6 hours, polyadenylated eNOS transcripts from sheared cells had a similar decay rate as those from control cells, but the calculated overall half-life for the transcripts was longer (18 hours in sheared cells versus 6 hours in static cells). These findings support the hypothesis that shear stress-induced polyadenylation increases eNOS mRNA stability.
The Role of Nuclear Polyadenylation in eNOS 3' mRNA Processing
Transcription and polyadenylation are intimately coupled processes.30,31 Genes undergoing enhanced transcription would be expected to have increased nuclear polyadenylation. However, lengthening of the 3' poly(A) tail can also occur in the cytoplasm through a process known as cytoplasmic polyadenylation.32,33 Because shear stress increases the rate of eNOS mRNA transcription,10 we hypothesized that shear-induced polyadenylation was a nuclear process involving the nuclear transcription and polyadenylation machinery.
RNA was isolated from both nuclear and cytosolic fractions of sheared and static cells and subjected to RPA analysis (Figure 5A). In sheared cells, nuclear extracts contained predominantly long poly(A) eNOS transcripts, and cytosolic extracts had both long and short poly(A) transcripts. In nonsheared cells, eNOS transcripts were faintly detected in nuclear extracts. Cytosolic extracts from control cells had predominantly short poly(A) transcripts. These findings suggest that shear-induced polyadenylation of eNOS mRNA is a nuclear process.
Relationship Between Shear-Induced Transcription and Polyadenylation
To further examine the relationship between eNOS mRNA transcription and polyadenylation, we determined the effect of blocking transcription on shear-induced eNOS polyadenylation. BAECs underwent a 1 hour preincubation with DRB (60 eol/L) and were then exposed to shear stress. RPA analysis was performed on nuclear and cytosolic extracts obtained various times after shear stress (Figure 5B). DRB pretreatment led to a dramatic decrease in the amount of long poly(A) eNOS transcripts in nuclear extracts. Under these conditions, the predominant protected fragment in cytosolic extracts was that of the short poly(A) transcript. Thus, blocking transcription with DRB severely attenuated shear-induced polyadenylation, signifying that eNOS transcription and polyadenylation are linked.
DRB indirectly inhibits RNA polymerase II (RNAPII); it modulates the activity of the regulatory kinases CDK 7 and CDK 9. We also determined the effect of actinomycin D, a direct inhibitor of RNAPII. BAECs underwent a 1 hour preincubation with actinomycin D (5 e/mL) and were subsequently exposed to shear stress. After 2 hours of shear stress there was a slight increase in the long polyadenylated eNOS transcripts in nuclear extracts (Figure 5C). Greater amounts of these protected fragments were seen after 4 to 6 hours of shear. Virtually no long poly(A) transcripts were detected in cytosolic extracts from cells treated with actinomycin D and shear stress. These results confirm that transcription is important for long poly(A) eNOS transcripts to be exported to the cytoplasm. However, transcription is not completely essential for polyadenylation to occur in sheared cells, as evidenced by the progressive increase in long poly(A) fragments after actinomycin D treatment. The virtual absence of long poly(A) fragments in cytosolic extracts of both actinomycin D- and DRB-treated cells suggests that cytoplasmic polyadenylation does not play a role in shear-induced lengthening of eNOS poly(A) tail.
The Influence of Hydrogen Peroxide and Statins on eNOS mRNA 3' Processing
Hydrogen peroxide (H2O2) and 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors (statins) are known regulators of eNOS expression.13,15 H2O2 increases eNOS expression through both transcriptional and posttranscriptional mechanisms, whereas statins increase eNOS expression through an entirely posttranscriptional mechanism. We examined whether changes in eNOS 3' polyadenylation also occur after treatment with H2O2 or statin.
BAECS were exposed to either H2O2 (100 eol/L for 24 hours) or statin (simvastatin, 10 eol/L for 24 hours) and RPA analysis was used to quantify changes in polyadenylated eNOS mRNA (Figure 6). H2O2 treatment resulted in an increase in long poly(A) eNOS transcripts. Statin treatment also led to increased eNOS polyadenylation, but the response was not as robust as that seen with shear stress or H2O2. These data show that other stimuli can induce changes in eNOS 3' polyadenylation, suggesting that this process may be a common mechanism by which endothelial cells regulate eNOS expression.
Discussion
The current studies provide insight into how laminar shear stress increases eNOS mRNA stability and translational activity by identifying a role for 3' polyadenylation in the posttranscriptional regulation of eNOS. In endothelial cells exposed to shear stress, there was dramatic increase in expression of eNOS transcripts with long 3' poly(A) tails that was dependent on the magnitude and duration of the shear stress stimulus. These transcripts were more stable than those from nonsheared cells, whose poly(A) tails were predominantly short (<25 nt). Furthermore, eNOS mRNA from sheared cells was found to be more actively translated. Finally, we found evidence that modulation of eNOS mRNA polyadenylation occurs in response to other stimuli (H2O2 and statins) known to increase eNOS mRNA stability. Interestingly, modulation of 3'polyadenylation does not appear to be unique to the endothelial isoform of NOS. A recent report describes a role for increased 3' polyadenylation in the regulation of the inducible isoform of NO synthase, iNOS.34
The initial step in mRNA degradation is removal of the poly(A) tail, followed by removal of the 5' 7-methylguanosine cap and rapid exonuclease digestion.35eC38 A long 3' poly(A) tail is important to mRNA stabilization because it facilitates binding of multiple poly(A) binding protein (PABP1) molecules, which protects against ribonucleolytic attack.39 The poly(A) tail is also important in allowing mRNA-ribosome interactions.40,41 In this regard, PABP1 is involved in the formation of a "closed-loop" structure where the 5' and 3' ends of the mRNA interact. We found that eNOS translational activity was increased in sheared endothelial cells, suggesting that this is a functional sequela of increased polyadenylation. This concept is supported by our observation of long poly(A) RACE-PAT products in large polysome fractions (data not shown).
In eukaryotic cells, transcription stimulates the assembly of a protein complex that is involved in both transcription and polyadenylation.30,31 Nuclear polyadenylation occurs immediately following 3' cleavage of a newly-transcribed mRNA precursor. In mammalian cells, the core polyadenylation signal requires the presence of a hexanucleotide sequence, 10 to 30 nucleotides upstream from the 3' cleavage site. The published sequences for human22 and bovine21 eNOS both appear to have hexanucleotide sequences that are single nucleotide variants of the canonical sequence (AAUAAA). These hexamer variants have been shown to be inefficient signals for 3' cleavage and polyadenylation. It is conceivable that shear makes it possible for cells to overcome this relatively inefficient polyadenylation signal.
Recently, it has become evident that some eukaryotic mRNAs undergo cytoplasmic polyadenylation.19 As an example, the neuronal NMDA receptor mRNA has been shown to undergo cytoplasmic polyadenylation in response to glutamate.42 We therefore considered the possibility that the eNOS mRNA underwent cytoplasmic polyadenylation in response to shear. The distribution of transcripts with long poly(A) tails in nuclear and cytosolic extracts suggested, however, these transcripts were synthesized primarily in the nucleus. In nuclear extracts of sheared cells, the eNOS transcripts predominantly had long poly(A) tails. In cytoplasmic extracts, both long and short poly(A) tails were observed. This is compatible with the concept that long poly(A) transcripts were synthesized in the nucleus, transported to the cytoplasm and subsequently underwent some degree of degradation.
The ability of DRB to attenuate shear-induced eNOS 3' poly(A) tail lengthening further supports the concept that eNOS polyadenylation is a nuclear process. DRB prevents phosphorylation of RNAP II, a critical step in assembly of the nuclear polyadenylation machinery.30,31,43 DRB and actinomycin D affect the phosphorylation state of RNAP II differently,44 and this may account for their somewhat dissimilar effects. Actinomycin D is known to inhibit transcription, but it can alter RNAP II phosphorylation and promote polyadenylation activity.44 This may explain the increase in long poly(A) eNOS transcripts observed in the nuclear extracts after actinomycin D treatment. Alternatively, actinomycin D could be inhibiting the nuclear export of polyadenylated eNOS mRNA.
The eNOS mRNA half-life determined in the present study is consistent with our previous report.10 However, compared with our previous analysis, there appeared to be a relatively rapid decline in eNOS mRNA levels after 6 hours of DRB treatment. The Northern analysis used in our previous assessment of stability showed a more gradual decline in eNOS transcript levels. The RPA analysis used in the current studies targeted the eNOS 3'UTR and therefore may more accurately reflect acute changes in eNOS mRNA processing. We believe that the pattern of protected fragments observed in our eNOS mRNA stability studies shows that long poly(A) transcripts become degraded into shorter transcripts over time.
The precipitous rate of decline in polyadenylated eNOS mRNA stability 6 hours after the addition of DRB may reflect the loss or degradation of factors that either enhance polyadenylation or stabilize the processed RNA. Indeed, the absence of polyadenylated transcripts in cytoplasmic extracts of cells pretreated with DRB or actinomycin D may be due to reduced expression of a poly(A) mRNA stabilizing factor. Although we have established that polyadenylation of eNOS mRNA is increased by shear stress, we have not identified cis- and trans-acting factors involved in this process. It is possible, that shear stress increases transcription of factors responsible for polyadenylation. Shear may also reduce expression of cytoplasmic factors involved in mRNA deadenylation and degradation. Future studies will need to address this issue.
Although the present study was performed using in vitro shear conditions, it is possible that in vivo, in the presence of constant blood flow, eNOS mRNA always has a long poly(A) tail. Two findings from our studies indicate that modulation of eNOS mRNA polyadenylation likely occurs in vivo. First, there was a dose-dependent effect of shear stress on eNOS polyadenylation. This would indicate that in vivo, sites of the circulation with low shear stress would have eNOS transcripts with short poly(A) tails, whereas sites with high shear stress would have transcripts with longer poly(A) tails. Interestingly, a dose-dependent effect of shear on eNOS mRNA levels has been observed in vivo.45 Second, polyadenylation was dependent on the duration of shear, suggesting that brief periods of increased flow in vivo would have less of an impact on eNOS polyadenylation than sustained increases in flow. Furthermore, in preliminary studies, we have used RACE-PAT on segments from mouse aorta and pig atrium and have identified eNOS transcripts of varying poly(A) tail lengths in these tissues (data not shown).
Shear stress is known to increase eNOS expression in human endothelial cells,46,47 and we believe that our study is relevant to the regulation of human eNOS. Like the bovine polyadenylation signal, the human hexanucleotide sequence appears to be a single nucleotide variant of the canonical sequence. This would suggest that at baseline, polyadenylation of human eNOS is relatively inefficient, which is consistent with the short poly(A) tail that is published for human eNOS mRNA.22 In preliminary studies, we have found increased polyadenylation of eNOS mRNA from sheared human aortic endothelial cells (data not shown), suggesting that a similar mechanism of regulation exists for human eNOS.
Shear stress, H2O2, and statins are diverse stimuli, yet they appear to share the ability to increase eNOS polyadenylation. Shear stress and H2O2 are both known to increase the rate of eNOS transcription and we found that they enhanced polyadenylation of eNOS in an analogous fashion. Statins, whose mechanism for increased eNOS expression is entirely posttranscriptional, appeared to be a relatively weaker stimulus for 3' polyadenylation. These data support our observation that transcriptional activation is important for polyadenylation, but, as evidenced by the response to statin, it is not completely necessary.
Recently, we described a mechanism for the posttranscriptional regulation of eNOS mRNA during cell growth that involves binding of monomeric actin to the eNOS 3'UTR.18 Binding of monomeric actin was associated with decreased eNOS mRNA stability, and H2O2 treatment led to a significant attenuation of binding activity. Interestingly, we found only modest decrease in actin binding with statin treatment and virtually no change in binding in response to shear. Taken together, these data suggest that some features of regulatory mechanisms for eNOS expression are common to diverse stimuli, but there are also aspects of these mechanisms that are distinct for a given stimulus. Further work will need to define the interaction between common regulatory elements and those that are specific to a particular stimulus.
Acknowledgments
This study was supported by National Institutes of Health (NIH) grants HL04062-01, HL39006, PO1-58000, PO1-075209-01, CA63640, and Merit Grant from the Veterans Administration. We thank Tove Goldson and Choi Youkyung Hwang for their help with the polysome analysis and RACE-PAT, respectively.
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Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, Nerem RM, Harrison DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol. 1995; 269: C1371eCC1378.
Topper JN, Cai J, Falb D, Gimbrone MA, Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively upregulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996; 93: 10417eC10422.(Martina Weber, Curt H. Ha)
Abstract
The 3' poly(A) tail is important in messenger RNA stability and translational efficiency. In somatic tissues, 3' polyadenylation of mRNAs has been thought to largely be a constitutively active process. We have reported that laminar shear stress causes a brief increase in endothelial nitric oxide synthase (eNOS) transcription, followed by a prolonged increase in eNOS mRNA stability. We sought to determine whether shear stress and other stimuli affected eNOS 3' polyadenylation in endothelial cells. Under basal (static) conditions, eNOS mRNA possessed short 3' poly(A) tails of <25 nt. In contrast, laminar shear stress increased expression of eNOS transcripts with long poly(A) tails. ENOS transcripts with longer poly(A) tails had prolonged half-lives (6 hours in static cells versus 18 hours in sheared cells). Polysome analysis revealed that eNOS mRNA from sheared cells was shifted into more translationally active polysome fractions compared with eNOS mRNA from static cells. Shear-induced lengthening of the eNOS 3' poly(A) tail was the result of increased nuclear polyadenylation. Furthermore, hydrogen peroxide and HMG Co-A reductase inhibitors, other stimuli known to modulate eNOS expression posttranscriptionally, also induced eNOS 3' poly(A) tail lengthening. These results support the concept that shear stress modulates eNOS mRNA stability and translation via increased 3' polyadenylation. We suggest that mRNA 3' polyadenylation is a posttranscriptional mechanism used by endothelial cells to regulate gene expression.
Key Words: endothelial nitric oxide synthase mRNA stability polyadenylation posttranscriptional regulation shear stress
Introduction
Laminar shear stress is a potent stimulus for the production of vascular nitric oxide (NO) because of its ability to increase both the activity and expression of the endothelial nitric oxide synthase (eNOS).1eC3 Endothelium-derived NO is crucial for maintenance of vascular homeostasis through its vasodilator activity;4 its ability to inhibit smooth muscle growth,5 platelet aggregation,6 and leukocyte adhesion,7 and its role in inhibiting lipid oxidation and regulating apoptosis in the vessel wall.8 Given these properties of NO, the shear stress-induced increase in eNOS expression may be important in preventing atherosclerosis. Indeed, areas of the vasculature exposed to high shear stress appear to be protected from the development of atherosclerosis, and areas exposed to low shear stress are prone to atherosclerotic lesion formation.9
Previously we have demonstrated that shear stress leads to increased eNOS mRNA expression via 2 separate mechanisms: a transient increase in eNOS transcription, and stabilization of eNOS mRNA.10 Posttranscriptional regulation of eNOS expression via modulation of eNOS mRNA stability is now recognized as an important response of endothelial cells to numerous biophysical and biochemical stimuli.11eC17 Although we have recently described a mechanism for the posttranscriptional regulation of eNOS expression during cell growth,18 the details for modulation of mRNA stability by other stimuli, including shear stress, remain poorly defined.
In mammalian cells, 3' poly(A) tails have been shown to regulate mRNA stability and translation.19,20 The cloned sequences of bovine and human eNOS mRNAs were reported to have 3' poly(A) tails 12 to 20 nucleotides in length.21,22 Although these studies did not specifically examine poly(A) tail length, the eNOS sequences were derived from RNA libraries of cultured cells not exposed to shear, suggesting that eNOS mRNA has a short 3' poly(A) tail under baseline conditions. We hypothesized that shear stress increases polyadenylation of eNOS mRNA. We report that shear stress led to an increase in eNOS transcripts with long poly(A) tails. We found that in cells subjected to shear stress there was a shift of eNOS mRNA from monosomes to polysomes, suggesting that a functional sequela of increased polyadenylation was increased translation. Furthermore, long poly(A) eNOS mRNA was more stable. These data provide evidence to support a novel mechanism for regulation of eNOS expression.
Materials and Methods
Materials
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) was obtained from Calbiochem. Cordycepin (3'-deoxyadenosine), cycloheximide, actinomycin D, hydrogen peroxide, mevastatin, and all primers were purchased from Sigma. Simvastatin was a generous gift from Merck (Rahway, NJ) and was activated before use, as described earlier.15
Tissue Culture
Bovine aortic endothelial cells (BAECs; Cell Systems, Kirkland, Wash) were cultured in Media 199 (M199; Cellgro, Mediatech) containing 10% fetal calf serum (FCS; Hyclone Labortories) as described earlier.23 Postconfluent BAECs between passages 4 to 8 were used for experiments. A cone-in-plate viscometer with a 1° angle was used to shear cells.24
RNA Isolation
Total cellular RNA was isolated using TRI-Reagent (Molecular Research Center Inc). For polysomal, cytosolic and nuclear fractionation, 2 e蘈 linear acrylamide (Ambion) was added as a carrier during RNA precipitation. The PARIS-Kit (Ambion) was used to separate cytosolic and nuclear RNA fractions.
Rapid Amplification of cDNA Ends-PolyA Test
To measure the 3' poly(A) tails of eNOS mRNA, Rapid Amplification of cDNA Ends-PolyA Test (RACE-PAT) was performed as described previously.25 The details of RACE-PAT are provided in the online data supplement at http://circres.ahajournals.org
Real Time RT-PCR
Real time RT-PCR was done with the Light Cycler (Roche) as described earlier.26 Reverse transcription was performed as described for RACE-PAT, using primers and conditions described in the online supplement.
Ribonuclease Protection Assays
A 600 nt band detected by eNOS RACE-PAT analysis was cloned into the pCRII-TOPO vector (Invitrogen) and its orientation was confirmed by DNA sequencing. A biotinylated antisense RNA probe was prepared by in vitro transcription, using T7 RNA polymerase (Mega Script kit, Ambion). Three to 10 e of total RNA from endothelial cells was hybridized with 374 pg of the biotinylated eNOS RNA-probe 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).
Polysomal Fractionation
The analysis of polysomes was performed as described previously.27,28 The details of polysome fractionation are described in the online supplement.
Results
eNOS mRNA Levels and Translational Activity in Response to Shear Stress
We used real time quantitative RT-PCR to compare eNOS mRNA levels in static control versus sheared BAECs. Shear increased eNOS mRNA levels by 9-fold compared with levels in nonsheared cells (Figure 1A). Because the increase in eNOS mRNA has been shown to be associated with increased eNOS protein levels,21 we hypothesized that translation of eNOS mRNA is also enhanced in response to shear. Polyribosomes from sheared and control endothelial cells were fractionated on the basis of size (Figure 1B), and the quantity of eNOS mRNA in each fraction was determined (Figure 1C). Polysome size reflects translation activity; transcripts that are associated with large polysomes are more translationally active. We found that large polysomal fractions (>3 ribosomes/mRNA) from sheared cells had 2.5- to 10.5-fold more eNOS mRNA than those from static cells. These data provide direct evidence that eNOS mRNA from sheared cells was translated with increased efficiency.
Identification of eNOS Transcripts With Long Poly(A) Tails With RACE-PAT
To determine the relationship between shear stress and eNOS mRNA polyadenylation, a PCR based method (RACE-PAT) was used to measure the poly(A) tail lengths of eNOS mRNA under shear and static conditions (Figure 2). The minimum predicted PCR product size was 237 nt: 225 nt of the eNOS 3' untranslated region (UTR) plus a poly(A) tail of 12 nt or less. The predominant RACE-PAT PCR product from both sheared and nonsheared BAECs was 250 nt, consistent with a poly(A) tail length of 25 nts (Figure 2B, lower arrow). As the amount of shear stress was increased, PCR products larger than 250 nt were observed, including a product that was 600 nt (Figure 2B, upper arrow). This finding was consistent with lengthening of the 3'poly(A) tail that was proportional to the amount of shear stress. To confirm that the larger PCR products were polyadenylated, RNA was subjected to oligo (dT) hybridization and RNase H digestion before RACE-PAT. RNase H digests RNA-DNA hybrids, and poly(A) tails hybridized with oligo (dT) are degraded by RNase H. When the RNase H digestion step was performed before RACE-PAT, the PCR products greater than 250 nts were not observed (Figure 2B), confirming that they were eNOS transcripts with different length poly(A) tails. The results of these RACE-PAT studies are compatible with shear increasing the eNOS mRNA 3' poly(A) tail length up to 300 nts.
The RACE-PAT PCR products of 250 nt and 600nt were cloned into pCRII (Invitrogen) and sequenced. Multiple clones from different experiments were sequenced, and both of the RACE-PAT products had the predicted eNOS 3'UTR sequence. As expected, the 250 nt PCR product had a relatively short poly(A) tail length of 12 to 30 bases. Although the entire sequence of the 600 nt product was not able to be obtained, a minimum poly(A) tail size of 112 bases was identified, confirming that it had a lengthened poly(A) tail. Furthermore, the inability to obtain 3' sequence beyond 112 adenines is consistent with its composition being poly(A).
Identification of eNOS Transcripts With Long Poly(A) Tails With RNase Protection Assay
To further examine shear-induced polyadenylation of eNOS mRNA, RNase protection assays (RPAs) were performed using a riboprobe that was antisense to the cloned 600 nt RACE-PAT product. Total RNA from cells exposed to increasing dynes/cm2 of shear stress was isolated and subjected to RPA analysis (Figure 3A). Consistent with the RACE-PAT analysis, the predominant protected fragment in nonsheared cells was 250 nt, which had a poly(A) tail calculated to be 25 nt. Importantly, RNA from cells exposed to shear stress had a significant, dose-dependent increase in longer protected fragments, specifically fragments of 300 and 385 nt. These longer fragments had poly(A) tails calculated to be 75 and 160 nt in length, respectively, reflecting increased polyadenylation with shear. To confirm that these protected fragments were polyadenylated, cells were exposed to 100 eole/L cordycepin (3'-deoxyadenosine) for one hour before 6 hours of shear. Cordycepin inhibits the formation of mRNA with long poly(A) tails but allows for the correct splicing and transport of transcribed mRNA.29 The longer protected fragments (300 and 385 nt) were absent after cordycepin pre-treatment (Figure 3C), verifying that they were long poly(A) eNOS transcripts.
A time course experiment was performed to correlate the duration of laminar shear stress with the quantity of long poly(A) eNOS fragments (Figure 3B). The longer fragments were observed after 2 hours of shear, and their levels progressively increased up to 6 hours. Interestingly, the appearance of long eNOS poly(A) transcripts at 2 hours of shear stress temporally coincided with the reported onset of increased eNOS mRNA stability,10 suggesting a role for shear-induced polyadenylation in modulating eNOS mRNA stability.
Stability of Polyadenylated eNOS mRNA
DRB chase studies were performed to examine the stability of polyadenylated eNOS mRNA. Before the addition of DRB to the media, cells were exposed to shear stress for 6 hours. An RPA analysis of polyadenylated eNOS mRNA was performed on cells that had been exposed to DRB for various periods of time (Figure 4A). At the 0 hour time point, the short poly(A) fragment was the predominant species in control cells. In sheared cells, both short and long poly(A) fragments were present at 0 hours. Over the next 18 hours, there was a gradual decay in eNOS mRNA from control cells. In contrast, the long poly(A) transcripts from sheared cells did not decay until 6 hours, indicating that these transcripts were more stable for at least 6 hours. After 6 hours, polyadenylated eNOS transcripts from sheared cells had a similar decay rate as those from control cells, but the calculated overall half-life for the transcripts was longer (18 hours in sheared cells versus 6 hours in static cells). These findings support the hypothesis that shear stress-induced polyadenylation increases eNOS mRNA stability.
The Role of Nuclear Polyadenylation in eNOS 3' mRNA Processing
Transcription and polyadenylation are intimately coupled processes.30,31 Genes undergoing enhanced transcription would be expected to have increased nuclear polyadenylation. However, lengthening of the 3' poly(A) tail can also occur in the cytoplasm through a process known as cytoplasmic polyadenylation.32,33 Because shear stress increases the rate of eNOS mRNA transcription,10 we hypothesized that shear-induced polyadenylation was a nuclear process involving the nuclear transcription and polyadenylation machinery.
RNA was isolated from both nuclear and cytosolic fractions of sheared and static cells and subjected to RPA analysis (Figure 5A). In sheared cells, nuclear extracts contained predominantly long poly(A) eNOS transcripts, and cytosolic extracts had both long and short poly(A) transcripts. In nonsheared cells, eNOS transcripts were faintly detected in nuclear extracts. Cytosolic extracts from control cells had predominantly short poly(A) transcripts. These findings suggest that shear-induced polyadenylation of eNOS mRNA is a nuclear process.
Relationship Between Shear-Induced Transcription and Polyadenylation
To further examine the relationship between eNOS mRNA transcription and polyadenylation, we determined the effect of blocking transcription on shear-induced eNOS polyadenylation. BAECs underwent a 1 hour preincubation with DRB (60 eol/L) and were then exposed to shear stress. RPA analysis was performed on nuclear and cytosolic extracts obtained various times after shear stress (Figure 5B). DRB pretreatment led to a dramatic decrease in the amount of long poly(A) eNOS transcripts in nuclear extracts. Under these conditions, the predominant protected fragment in cytosolic extracts was that of the short poly(A) transcript. Thus, blocking transcription with DRB severely attenuated shear-induced polyadenylation, signifying that eNOS transcription and polyadenylation are linked.
DRB indirectly inhibits RNA polymerase II (RNAPII); it modulates the activity of the regulatory kinases CDK 7 and CDK 9. We also determined the effect of actinomycin D, a direct inhibitor of RNAPII. BAECs underwent a 1 hour preincubation with actinomycin D (5 e/mL) and were subsequently exposed to shear stress. After 2 hours of shear stress there was a slight increase in the long polyadenylated eNOS transcripts in nuclear extracts (Figure 5C). Greater amounts of these protected fragments were seen after 4 to 6 hours of shear. Virtually no long poly(A) transcripts were detected in cytosolic extracts from cells treated with actinomycin D and shear stress. These results confirm that transcription is important for long poly(A) eNOS transcripts to be exported to the cytoplasm. However, transcription is not completely essential for polyadenylation to occur in sheared cells, as evidenced by the progressive increase in long poly(A) fragments after actinomycin D treatment. The virtual absence of long poly(A) fragments in cytosolic extracts of both actinomycin D- and DRB-treated cells suggests that cytoplasmic polyadenylation does not play a role in shear-induced lengthening of eNOS poly(A) tail.
The Influence of Hydrogen Peroxide and Statins on eNOS mRNA 3' Processing
Hydrogen peroxide (H2O2) and 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors (statins) are known regulators of eNOS expression.13,15 H2O2 increases eNOS expression through both transcriptional and posttranscriptional mechanisms, whereas statins increase eNOS expression through an entirely posttranscriptional mechanism. We examined whether changes in eNOS 3' polyadenylation also occur after treatment with H2O2 or statin.
BAECS were exposed to either H2O2 (100 eol/L for 24 hours) or statin (simvastatin, 10 eol/L for 24 hours) and RPA analysis was used to quantify changes in polyadenylated eNOS mRNA (Figure 6). H2O2 treatment resulted in an increase in long poly(A) eNOS transcripts. Statin treatment also led to increased eNOS polyadenylation, but the response was not as robust as that seen with shear stress or H2O2. These data show that other stimuli can induce changes in eNOS 3' polyadenylation, suggesting that this process may be a common mechanism by which endothelial cells regulate eNOS expression.
Discussion
The current studies provide insight into how laminar shear stress increases eNOS mRNA stability and translational activity by identifying a role for 3' polyadenylation in the posttranscriptional regulation of eNOS. In endothelial cells exposed to shear stress, there was dramatic increase in expression of eNOS transcripts with long 3' poly(A) tails that was dependent on the magnitude and duration of the shear stress stimulus. These transcripts were more stable than those from nonsheared cells, whose poly(A) tails were predominantly short (<25 nt). Furthermore, eNOS mRNA from sheared cells was found to be more actively translated. Finally, we found evidence that modulation of eNOS mRNA polyadenylation occurs in response to other stimuli (H2O2 and statins) known to increase eNOS mRNA stability. Interestingly, modulation of 3'polyadenylation does not appear to be unique to the endothelial isoform of NOS. A recent report describes a role for increased 3' polyadenylation in the regulation of the inducible isoform of NO synthase, iNOS.34
The initial step in mRNA degradation is removal of the poly(A) tail, followed by removal of the 5' 7-methylguanosine cap and rapid exonuclease digestion.35eC38 A long 3' poly(A) tail is important to mRNA stabilization because it facilitates binding of multiple poly(A) binding protein (PABP1) molecules, which protects against ribonucleolytic attack.39 The poly(A) tail is also important in allowing mRNA-ribosome interactions.40,41 In this regard, PABP1 is involved in the formation of a "closed-loop" structure where the 5' and 3' ends of the mRNA interact. We found that eNOS translational activity was increased in sheared endothelial cells, suggesting that this is a functional sequela of increased polyadenylation. This concept is supported by our observation of long poly(A) RACE-PAT products in large polysome fractions (data not shown).
In eukaryotic cells, transcription stimulates the assembly of a protein complex that is involved in both transcription and polyadenylation.30,31 Nuclear polyadenylation occurs immediately following 3' cleavage of a newly-transcribed mRNA precursor. In mammalian cells, the core polyadenylation signal requires the presence of a hexanucleotide sequence, 10 to 30 nucleotides upstream from the 3' cleavage site. The published sequences for human22 and bovine21 eNOS both appear to have hexanucleotide sequences that are single nucleotide variants of the canonical sequence (AAUAAA). These hexamer variants have been shown to be inefficient signals for 3' cleavage and polyadenylation. It is conceivable that shear makes it possible for cells to overcome this relatively inefficient polyadenylation signal.
Recently, it has become evident that some eukaryotic mRNAs undergo cytoplasmic polyadenylation.19 As an example, the neuronal NMDA receptor mRNA has been shown to undergo cytoplasmic polyadenylation in response to glutamate.42 We therefore considered the possibility that the eNOS mRNA underwent cytoplasmic polyadenylation in response to shear. The distribution of transcripts with long poly(A) tails in nuclear and cytosolic extracts suggested, however, these transcripts were synthesized primarily in the nucleus. In nuclear extracts of sheared cells, the eNOS transcripts predominantly had long poly(A) tails. In cytoplasmic extracts, both long and short poly(A) tails were observed. This is compatible with the concept that long poly(A) transcripts were synthesized in the nucleus, transported to the cytoplasm and subsequently underwent some degree of degradation.
The ability of DRB to attenuate shear-induced eNOS 3' poly(A) tail lengthening further supports the concept that eNOS polyadenylation is a nuclear process. DRB prevents phosphorylation of RNAP II, a critical step in assembly of the nuclear polyadenylation machinery.30,31,43 DRB and actinomycin D affect the phosphorylation state of RNAP II differently,44 and this may account for their somewhat dissimilar effects. Actinomycin D is known to inhibit transcription, but it can alter RNAP II phosphorylation and promote polyadenylation activity.44 This may explain the increase in long poly(A) eNOS transcripts observed in the nuclear extracts after actinomycin D treatment. Alternatively, actinomycin D could be inhibiting the nuclear export of polyadenylated eNOS mRNA.
The eNOS mRNA half-life determined in the present study is consistent with our previous report.10 However, compared with our previous analysis, there appeared to be a relatively rapid decline in eNOS mRNA levels after 6 hours of DRB treatment. The Northern analysis used in our previous assessment of stability showed a more gradual decline in eNOS transcript levels. The RPA analysis used in the current studies targeted the eNOS 3'UTR and therefore may more accurately reflect acute changes in eNOS mRNA processing. We believe that the pattern of protected fragments observed in our eNOS mRNA stability studies shows that long poly(A) transcripts become degraded into shorter transcripts over time.
The precipitous rate of decline in polyadenylated eNOS mRNA stability 6 hours after the addition of DRB may reflect the loss or degradation of factors that either enhance polyadenylation or stabilize the processed RNA. Indeed, the absence of polyadenylated transcripts in cytoplasmic extracts of cells pretreated with DRB or actinomycin D may be due to reduced expression of a poly(A) mRNA stabilizing factor. Although we have established that polyadenylation of eNOS mRNA is increased by shear stress, we have not identified cis- and trans-acting factors involved in this process. It is possible, that shear stress increases transcription of factors responsible for polyadenylation. Shear may also reduce expression of cytoplasmic factors involved in mRNA deadenylation and degradation. Future studies will need to address this issue.
Although the present study was performed using in vitro shear conditions, it is possible that in vivo, in the presence of constant blood flow, eNOS mRNA always has a long poly(A) tail. Two findings from our studies indicate that modulation of eNOS mRNA polyadenylation likely occurs in vivo. First, there was a dose-dependent effect of shear stress on eNOS polyadenylation. This would indicate that in vivo, sites of the circulation with low shear stress would have eNOS transcripts with short poly(A) tails, whereas sites with high shear stress would have transcripts with longer poly(A) tails. Interestingly, a dose-dependent effect of shear on eNOS mRNA levels has been observed in vivo.45 Second, polyadenylation was dependent on the duration of shear, suggesting that brief periods of increased flow in vivo would have less of an impact on eNOS polyadenylation than sustained increases in flow. Furthermore, in preliminary studies, we have used RACE-PAT on segments from mouse aorta and pig atrium and have identified eNOS transcripts of varying poly(A) tail lengths in these tissues (data not shown).
Shear stress is known to increase eNOS expression in human endothelial cells,46,47 and we believe that our study is relevant to the regulation of human eNOS. Like the bovine polyadenylation signal, the human hexanucleotide sequence appears to be a single nucleotide variant of the canonical sequence. This would suggest that at baseline, polyadenylation of human eNOS is relatively inefficient, which is consistent with the short poly(A) tail that is published for human eNOS mRNA.22 In preliminary studies, we have found increased polyadenylation of eNOS mRNA from sheared human aortic endothelial cells (data not shown), suggesting that a similar mechanism of regulation exists for human eNOS.
Shear stress, H2O2, and statins are diverse stimuli, yet they appear to share the ability to increase eNOS polyadenylation. Shear stress and H2O2 are both known to increase the rate of eNOS transcription and we found that they enhanced polyadenylation of eNOS in an analogous fashion. Statins, whose mechanism for increased eNOS expression is entirely posttranscriptional, appeared to be a relatively weaker stimulus for 3' polyadenylation. These data support our observation that transcriptional activation is important for polyadenylation, but, as evidenced by the response to statin, it is not completely necessary.
Recently, we described a mechanism for the posttranscriptional regulation of eNOS mRNA during cell growth that involves binding of monomeric actin to the eNOS 3'UTR.18 Binding of monomeric actin was associated with decreased eNOS mRNA stability, and H2O2 treatment led to a significant attenuation of binding activity. Interestingly, we found only modest decrease in actin binding with statin treatment and virtually no change in binding in response to shear. Taken together, these data suggest that some features of regulatory mechanisms for eNOS expression are common to diverse stimuli, but there are also aspects of these mechanisms that are distinct for a given stimulus. Further work will need to define the interaction between common regulatory elements and those that are specific to a particular stimulus.
Acknowledgments
This study was supported by National Institutes of Health (NIH) grants HL04062-01, HL39006, PO1-58000, PO1-075209-01, CA63640, and Merit Grant from the Veterans Administration. We thank Tove Goldson and Choi Youkyung Hwang for their help with the polysome analysis and RACE-PAT, respectively.
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