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. Author manuscript; available in PMC: 2018 Jul 2.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Aug 20;35(10):2153–2160. doi: 10.1161/ATVBAHA.115.305750

Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract binding Protein 1 (PTB1)

Bing Yi 1,#, Maria Ozerova 1,#, Guan-Xin Zhang 1, Guijun Yan 1, Shengdong Huang 1, Jianxin Sun 1
PMCID: PMC6027644  NIHMSID: NIHMS714197  PMID: 26293469

Abstract

Objective

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function and its expression is regulated at posttranscriptional levels through a yet unknown mechanism. The purpose of this study is to elucidate the post-transcriptional factors regulating eNOS expression and function in endothelium.

Approaches and Results

To elucidate the molecular basis of TNF-alpha-mediated eNOS mRNA instability, biotinylated eNOS 3'-UTR was used to purify its associated proteins by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). We identified two cytosolic proteins, with molecular weight of 52 and 57 kDa, that specifically bind to eNOS 3'-UTR in response to TNF-α stimulation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis identified the 57-kDa protein as polypyrimidine tract binding protein 1 (PTB1). RNA gel mobility-shift and UV cross-linking assays demonstrated that PTB1 binds to a UCUU-rich sequence in eNOS 3'-UTR, and the C-terminal half of PTB1 is critical to this interaction. Importantly, PTB1 overexpression leads to decreased activity of luciferase gene fused with eNOS 3'-UTR as well as reduced eNOS expression and activity in human endothelial cells. In HUVECs, we show that TNF-α markedly increased PTB1 expression while adenovirus-mediated PTB1 overexpression decreased eNOS mRNA stability and reduced protein expression and endothelium dependent relaxation. Further, knockdown of PTB1 substantially attenuated TNF-alpha-induced destabilization of eNOS transcript and downregulation of eNOS expression.

Conclusions

These results indicate that PTB1 is essential for regulating eNOS expression at posttranscriptional levels and suggest a novel therapeutic target for treatment of vascular diseases associated with inflammatory endothelial dysfunction.

Keywords: eNOS, mRNA stability, TNF-α, PTB1

Introduction

Nitric oxide (NO) is a potent cell signaling and vasodilatory molecule that plays important and diverse roles in biological processes such as neurotransmission, inflammatory response, and vascular homeostasis 1,2. Because of its important biological effects, NO production by NO synthases is under complex and tight control. eNOS is a key enzyme involved in the regulation of vascular function and abnormality of eNOS activity and/or expression has been shown to cause several vascular diseases 3. Although eNOS was initially considered to be a constitutive enzyme, it was shown later that eNOS expression was regulated by a variety of exogenous stimuli. For instance, in cultured endothelial cells, cytokines, lipopolysaccaride, and oxidized LDLs have been shown to downregulate eNOS expression 4,5,6. In contrast, shear stress, estrogen, and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors have been demonstrated to upregulate eNOS expression 7,8,9. For many of these stimuli, modulation of eNOS mRNA stability plays an essential role in the regulation of eNOS expression 10. Although the mechanism for regulating the cellular stability of different genes has unique features, it seems that in each case, specific RNA sequences are required for the recognition of protein factors 11,12. Some of these sequences have been identified within the 3'-untranslated region (3'-UTR) of mRNA 13,14.

One of the most potent inhibitory stimuli for eNOS expression in vascular endothelial cells is tumor necrosis factor (TNF)-α. TNF-α –mediated inhibition of eNOS expression, via a mechanism of destabilization of eNOS mRNA, has been shown to be associated with vascular dysfunction in several disease states, such as atherosclerosis, diabetes, and heart failure 15,16. It is becoming increasingly clear that the 3'-UTR of eNOS mRNA plays an important role in the posttranscriptional regulation of mRNA stability 17,18,19. For example, in BAECs, TNF-α –induced eNOS mRNA destabilization is mediated, in part, by specific binding of a cytosolic 60-kDa protein to the 3'-UTR 17. Furthermore, a 51-kDa protein was identified to bind to the eNOS 3'-UTR and regulate eNOS mRNA half-life during cell growth 19. Similarly, in HUVECs, the binding of the 52- and 57-kDa proteins to the eNOS 3'-UTR has also been implicated in the modulation of mRNA stability by TNF-α 20,21. Previously, we have identified the 52 KDa protein as translation elongation factor 1-alpha 1 (eEF1A), which is critically involved in the destabilization of eNOS mRNA under both basal and inflammatory conditions 21. However, the identity of the 57-KDa protein and it role in regulating eNOS mRNA stability remain to be determined.

In the present study, by using a mass spectrometry (MS)-protein-sequencing approach, we identified the 56-kDa protein as polypyrimidine tract-binding protein 1 (PTB1). Furthermore, we demonstrated that PTB1 specifically interacts with eNOS mRNA 3'-UTR, and modulates eNOS mRNA stability under TNF-α stimulated conditions.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Identification of PTB1 as an eNOS 3'-UTR Binding Protein

Previous studies indicated that TNF-α decreases eNOS protein expression through destabilization of eNOS mRNA by cytosolic proteins, which bind to the 3'-UTR of eNOS mRNA 20,21. To identify these proteins, eNOS 3'-UTR binding proteins were purified from a cytosolic fraction of TNF-α-unstimulated and-stimulated HUVECs, using RNA affinity chromatography via biotinylated transcripts coupled to streptavidin-agarose. As shown in our previous report 21, we found that two proteins, with molecular weights at 57 and 52 kDa, respectively, significantly increased their bindings to the eNOS 3'-UTR in TNF-α–stimulated HUVECs. Furthermore, we successfully identified the 52-kDa as translation elongation factor 1-alpha 1 (eEF1A), which is critically involved in regulating eNOS mRNA stability in response to TNF-α stimulation 21.

The 57-kDa band was subjected to the MALDI-TOF mass spectrometric analysis. We identified two peptides (KLPGDVTEGEVISLGLPFGK, IAIPGLAGAGNSVLLVSNLNPER) that correspond to peptides deduced from polypyrimidine tract-binding protein-1 (PTB1). To determine whether PTB1 interacts specifically with eNOS 3'-UTR, UV cross-linking experiments were performed to investigate the binding of PTB1 to the biotinylated eNOS 3'-UTR. As shown in Figure 1A, recombinant PTB1, in a dose-dependent manner, exhibited a remarkable binding to the eNOS 3'-UTR, and this binding was specifically inhibited by an excessive unlabeled eNOS 3'-UTR transcript. In contrast, glutathione s-transferase (GST) had undetectable binding to the eNOS 3'-UTR. Furthermore, R-EMSA analysis using cytosolic extracts from HUVECs demonstrated a ribonucleoprotein (RNP) complex formation, which was retarded by anti-PTB1 antibody (Figure 1B), providing further evidence of the specificity of complex formation between eNOS 3'-UTR with PTB1 in HUVECs. The similar RNP complex formation was observed in in HUVECs transduced with adenovirus harboring PTB1 (Ad-PTB1) (Figure I in the online-only Data Supplement). In addition, immunoprecipitation using anti-PTB1 antibody, followed by RT-PCR assays, also demonstrated the interaction of PTB1 and eNOS mRNA 3'-UTR in both TNF-α-unstimulated and -stimulated HUVECs. However, TNF-α stimulation markedly increased the levels of eNOS mRNA associated with PTB1 (Figure 1C).

Figure 1.

Figure 1

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Interaction of PTB1 with eNOS 3'-UTR. A, UV cross-linking was performed by incubating the biotinylated eNOS 3'-UTR with increasing concentrations of recombinant His-PTB1 (0.2, 1, 3 μg) and a fixed concentration of glutathione s-transferase (GST) (3 μg). Competition with specific competitor (50 molar excess) corresponding to unlabeled eNOS 3'-UTR transcript was also performed in the presence of 3 μg of His-PTB1. B, EMSA was performed by incubating biotinylated eNOS 3'-UTR with 50 μg of cytosolic extracts from HUVECs. Arrows indicate the eNOS 3'-UTR ribonucleoprotein (RNP) complex formation and super-shift (SS) by PTB1 antibody, respectively. C, Immunoprecipitation by anti-PTB1 antibody, followed by RT-PCR assays, showing the interaction of PTB1 and eNOS mRNA 3'-UTR in HUVECs treated with or without TNF-α (20 ng/mL) for 1 hour.

Identification of Interacting Domains between PTB1 and eNOS 3'-UTR

As an RNA binding protein, PTB1 has four consensus RNA recognition motifs (RRM) distributed throughout the PTB1 molecule (Figure 2A). RRM1 and RRM2, which are in the N-terminal half of PTB1, are required for the PTB1 oligomerization and other protein-protein interactions, whereas RRM3 and 4, which are in the C-terminal half of PTB1, are necessary for the RNA binding activity 22. To characterize the specific eNOS mRNA binding domains, we will construct Flag-tagged proteins bearing various truncated PTB1 mutants that were expressed in HEK293T cells. As shown in Figure 2B, these Flag-tagged PTB1 mutants were abundantly expressed in HEK293T cells. Furthermore, UV cross-linking, using biotinylated eNOS 3'-UTR and cell lysates expressing PTB1 mutants, were performed to identify the domains responsible for the eNOS 3'-UTR binding activity. As shown in Figure 2C, eNOS 3'-UTR strongly interacted with the full length, the N-terminal half (RRM1+RRM2), and the C-terminal half (RRM3+RRM4) of PTB1, but not with any single RRM domains. In addition, the C-terminal half of PTB1 (RRM3+RRM4) exhibited a stronger association with eNOS 3'-UTR than the N-terminal half of PTB1. These results suggest that the C-terminal half of PTB1 is predominantly responsible for the eNOS 3'-UTR binding in PTB1.

Figure 2.

Figure 2

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Identification of the functional domains mediating the interaction of PTB1 with eNOS 3'-UTR. A, Schematic representation of human PTB1 functional domains. B Mammalian expression vectors bearing PTB1 RRM domains were transfected into HEK293T cells. 48 hours after transfection, cell lysates were collected to detect the expression of PTB1 mutants by western blot analysis. C, Identification of eNOS 3'-UTR binding domains in PTB. UV cross-linking was performed by incubating the biotinylated-eNOS 3'-UTR with cell lysates expressing PTB1 mutants. D, Construction of human eNOS 3'-UTR mutants. eNOS 3'-UTR mutants were generated by PCR and cloned at 3'-end of fire luciferase gene. E, Overexpression of PTB1 attenuates the activity of luciferase fused with eNOS 3'-UTR. BAECs in 6-well plates were cotransfected with Luc-eNOS-UTR reporter plasmids, together with either pFlag-CMV2 bearing PTB1 cDNA (pFlag-PTB1) expression or empty vector (EV). 48 hours after transfection, cell lysates were assayed for luciferase activities (n=5). *P<0.05 vs EV alone.

Based on the human eNOS mRNA sequence deposited in the GenBank database (accession no NM_000603), eNOS 3'-UTR is 418-nt long (3906 to 4323) 21. To localize the PTB1-binding site within the eNOS 3'-UTR, we constructed a series of reporter genes with the eNOS 3'-UTR mutants fused at the C-terminus of firefly luciferase gene (Figure 2D). As shown in Figure 2E, co-transfection pf PTB1 expression plasmid substantially inhibited the activity of the luciferase gene fused with the F5 mutant, however, a further deletion of its C-terminal 47 nt fragment completely abolished PTB1-mediated inhibition of luciferase activity, suggesting the functional domain responsible for the PTB1–mediated inhibition of the luciferase activity is located in the region between 4177 to 4223 (47 nt). Similarly, overexpression of PTB1 markedly inhibited the activity of firefly luciferase gene bearing the F7 mutant, but barely affected the activity of luciferase gene bearing F8 mutant, further indicating that the region between 4177 to 4223 (47 nt: 5’-UCUUAGUCGAAUGUUAGAUUCCUCUUGCCUCUCUCAGGAGUAUCUUA-3') is functionally involved in regulating eNOS expression by PTB1 in vascular ECs.

Overexpression of PTB1 Decreases eNOS mRNA Stability and Inhibits eNOS Expression

The interaction of PTB1 with the eNOS mRNA 3'--UTR prompted us to investigate whether PTB1 affects eNOS expression through regulating eNOS mRNA stability. To this end, Flag-tagged PTB1 cDNA, together with eNOS cDNA containing its 3'-UTR, were cotransfected into Cos-7 cells, and the expression of eNOS was determined by western blotting. As shown in Figure 3A and 3B, in a dose-dependent manner, cotransfection of PTB1 substantially inhibited both eNOS expression and the activity of the firefly luciferase gene bearing eNOS 3'-UTR. In contrast, overexpression of PTB1 had no effect on eNOS expression, when cotransfected with eNOS cDNA without its 3'-UTR sequence (data not shown). Moreover, the eNOS mRNA half-life is significantly reduced from 28.1 ± 3.2 hr to 15.2 ± 2.7 hr (P<0.01, n=4) when PTB1 is overexpressed (Figure 3C). In addition, overexpression of PTB1 dose-dependently inhibited the activity of fire luciferase gene fused with eNOS 3'-UTR (Figure 3D). Together, these results suggest that the binding of PTB1 to eNOS 3'-UTR may be functionally important for the regulation of eNOS expression.

Figure 3.

Figure 3

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Overexpression of PTB1 decreases eNOS expression and mRNA stability in COS-7 cells. A, Overexpression of PTB1 attenuates eNOS expression in Cos-7 cells. Cos-7 cells were transfected with eNOS cDNA containing 3'-UTR in combination of either empty vector or pFlag-PTB1. 48 hours after transfection, cell lysates were collected for western blot analysis. B, Quantitative analysis of eNOS expression in the presence of PTB1. *P<0.05 vs eNOS cDNA alone (n=5). C, Overexpression of PTB1 attenuates eNOS mRNA half-life in Cos-7 cells. Cos-7 cells were transfected with eNOS cDNA in combination with empty vector (EV) and pFlag-PTB1 vector. 48 hours after transfection, eNOS mRNA half-life was determined by q-RT-PCR. D, Cos-7 cells were transfected with firefly luciferase-eNOS-UTR reporter plasmid, together with either pFlag-PTB1 expression or empty vectors. 48 hours after transfection, cell lysates were assayed for luciferase activities (n=5). *P<0.05 vs pFlag-PTB1 at 0 μg.

PTB1 Is Critically Involved in Destabilization of eNOS mRNA by TNF-α in HUVECs

To determine the role of PTB1 in TNF-α-induced destabilization of eNOS mRNA in ECs, we examined the effect of TNF-α on PTB1 expression in HUVECs. As shown in Figure 4A, in a time-dependent manner, treatment of HUVECs with TNF-α (20 ng/mL) markedly increased PTB1 expression for up to 12 hours. In addition, treatment of HUVECs with IL-1β (10 ng/mL) also markedly increased PTB1 expression in HUVECs (Figure II in the online-only Data Supplement). To determine whether overexpression of PTB1 is able to mimic the inhibitory effect of TNF-α on eNOS expression, HUVECs were transduced with Ad-PTB1 for 48 hours. The eNOS expression was then determined by western blotting analysis. Indeed, overexpression of PTB1 markedly reduced eNOS protein expression by approximately 50% in HUVECs, which is comparable to that observed with TNF-α treatment (i.e.,60% reduction) (Figure 4B). Likewise, the eNOS activity, as determined by NO production (Figure 4C) and cGMP (Figure 4D), was significantly inhibited in both TNF-α-stimulated and PTB1 overexpressing HUVECs.

Figure 4.

Figure 4

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Overexpression of PTB1 mimics the downregulation of eNOS expression by TNF-α. A, TNF-α increases the expression of PTB1 in HUVECs. Cells were treated with TNF-α (20 ng/mL) for various time points, and expression of PTB1was determined by western blot. B, Overexpression of Flag-tagged PTB1 inhibits the expression of eNOS in HUVECs. HUVECs were transduced with Ad-LacZ and Ad-PTB1 at the multiplicity of infection of 50. 48 hours after transduction, cells were then treated with either vehicle (Veh) or TNF-α (20 ng/mL) for 24 hours. The expression of eNOS and PTB1 was detected by western blot and quantitated by densitometry. *P<0.05 vs Ad-LacZ. C, Overexpression of PTB1 decreases NO generation in the supernatants of ECs. *P<0.05 vs Ad-LacZ/Veh, n=4. D, Overexpression of PTB1 decreases cGMP levels as determined by ELISA. *P<0.05 vs Ad-LacZ, n=4. E. PTB1 overexpression decreases eNOS mRNA stability. Preconfluent HUVECs were transduced with Ad-LacZ and Ad-PTB1 at the multiplicity of infection of 50 for 48 hours. eNOS mRNA half-life was determined by qRT-PCR. Combined results were shown from 5 different experiments.

The posttranscriptional regulation of eNOS mRNA by PTB1 was determined in the presence of the transcriptional inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) (20 μg/mL). HUVECs were transduced with either Ad-PTB1 or Ad-LacZ for 48 hour. DRB was then added to stop transcription, and total RNAs were prepared 0 to 20 hours thereafter. As shown in Figure 4E, overexpression of PTB1 in HUVECs decreased the half-life of eNOS mRNA from 16 ± 3 to 7.5 ± 2 hours (P<0.05, n=5), further indicating the involvement of PTB1 in the posttranscriptional regulation of eNOS mRNA stability.

Overexpression of PTB1 Impairs Endothelium-Dependent Vasodilation

To examine whether PTB1 plays an important role in regulating endothelium-dependent vascular relaxation, we examined the expression of eNOS and Ach-induced vascular response in human internal mammary artery rings transduced with either Ad-PTB1 or Ad-LacZ as described previously 23. PTB1 is expressed in endothelium of mammary artery as determined by immunofluorescent staining (Figure IIIA in the online-only Data Supplement). As shown in Figure 5A and 5B, overexpression of PTB1 decreases eNOS expression and Ach-induced endothelium-dependent vasodilation to an extent similar to that seen in LacZ-transduced rings treated with TNF-α (Figure IIIB in the online-only Data Supplement). In the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), PTB1 overexpression caused a similar Ach-induced vasodilation in both Ad-LacZ and Ad-PTB1 transduced rings (Figure 6B). Furthermore, the endothelium-independent vasorelaxation, as induced by the NO donor sodium nitroprusside (SNP), was similar between Ad-PTB1 and Ad-LacZ transduced rings (Figure 5C). Together, these results suggested that PTB1 is essentially involved in the regulation of endothelium-dependent vascular relaxation via inhibiting eNOS expression.

Figure 5.

Figure 5

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PTB1 overexpression decreases endothelium-dependent relaxation in human internal mammary artery. A, Overexpression of PTB1 inhibited endothelial nitric oxide synthase (eNOS) expression in human internal mammary artery. The arteries were transfected with Ad-LacZ or Ad-PTB1 at 1011 viral particles per milliliter ex vivo. Twenty-four hours later, the tissue lysates were collected for Western blotting. n=4, *P<0.05 vs Ad-LacZ. B, adenovirus mediated PTB1 overexpression inhibited endothelium-dependent vasorelaxation. The arterial rings were infected with Ad-LacZ or Ad-PTB1 for 24 hours ex vivo. Endothelium-dependent vasodilation was determined by measuring acetylcholine (Ach)-induced relaxation in rings precontracted with phenylephrine. n=6, *P<0.01 vs Ad-LacZ. C, Endothelium-independent vasodilation to sodium nitroprusside was examined in rings transfected with Ad-LacZ or Ad-PTB1 for 24 hours.

Figure 6.

Figure 6

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PTB1 knockdown prevents TNF-α–induced destabilization of eNOS mRNA stability. A, PTB1 knockdown prevents TNF-α–induced inhibition of eNOS expression. HUVECs were transfected with PTB1-specific siRNA (siPTB) and control siRNA (siCTL). 72 hours after transfection, HUVECs were treated with either vehicle (Veh) or TNF-α (20 ng/mL), and the expression of eNOS and PTB1 was determined by western blot. B, Knockdown of PTB1 attenuates TNF-α–induced decrease in cGMP production, as determined by ELISA. C, Knockdown of PTB1 attenuates TNF-α–induced destabilization of eNOS mRNA. HUVECs were transfected with PTB1-specific siRNA (siPTB1) and control siRNA (siCTL). 72 hours after transfection, HUVECs were treated with vehicle or TNF-α (20 ng/mL), and eNOS mRNA half-life was determined by qRT-PCR. The data are representative of 5 independent experiments.

PTB1 Knockdown Prevents Destabilization of eNOS mRNA by TNF-α

To further substantiate the functional significance of PTB1 in the regulation of eNOS mRNA stability, we performed loss-of-function studies, using the RNA interference technique 21. Transfection of PTB1 siRNA substantially inhibited PTB1 expression by approximately 80% under both TNF-α-unstimulated and -stimulated conditions (Figure 6A). TNF-α treatment markedly inhibited the expression of eNOS and cGMP production in control siRNA (siCTL) transfected cells, and these inhibitory effects were significantly attenuated in PTB1 specific siRNA (siPTB) transfected cells (Figure 6A and 6B). To determine whether prevention of TNF-α-mediated eNOS downregulation by PTB1 siRNA is due to its effect on eNOS mRNA stability, we measured eNOS mRNA half-life by qRT-PCR in the presence and absence of TNF-α stimulation. As shown in Figure 6C, compared with control siRNA (siCTL), PTB1-specific siRNA (siPTB) modestly prolonged the half-life of eNOS mRNA under basal conditions. In HUVECs transfected with siCTL, TNF-α (20 ng/mL) shortened the half-life of eNOS mRNA from 15 ± 3 to 6 ± 2 hours (P<0.05, n=5). In contrast, knockdown of PTB1 by PTB1 siRNA substantially blocked the decrease in eNOS mRNA half-life in response to the TNF-α treatment (6 ± 2 to 12 ± 3 hours, P<0.05, n=5). Taken together, these results indicate that PTB1 is critically involved in the regulation of eNOS mRNA stability and expression by TNF-α in vascular ECs.

Discussion

In general, mRNA decay is mediated by interactions between a specific cis-acting element in the mRNA and its binding partners. The modulation of eNOS mRNA stability plays an important role in the regulation of eNOS expression in response to a variety of stimuli, including cytokines, lipopolysaccharide, and oxidized LDLs 10. Accumulating evidence suggests that the binding of cytosolic proteins to the cis-acting sequences within eNOS mRNA 3'-UTR was involved in the regulation of eNOS mRNA stability 17,20,21. Indeed, in our previous study, we have shown that at least two proteins, with molecular weight of 52 kDa and 57 kDa, directly associated with the eNOS 3'-UTR in TNF-α stimulated HUVECs 21. Subsequently, we have identified the 52 kDa protein as eEF1A, which is essentially involved in the regulation of eNOS expression at posttranscriptional levels 21. However, the identity and functional role of the 57KDa protein remained to be elucidated at that time. In the present study, we identified the 57KDa protein as PTB1 and the interaction of PTB1 with eNOS 3'-UTR was further confirmed by R-EMSA and UV cross-linking assays. Movevevr, we demonstrated that overexpression of PTB1 and siRNA-specific knockdown dramatically impact both eNOS mRNA stability and its activity in vascular ECs, further indicating the functional significance of PTB1 in the regulation of vascular function through its effects on eNOS expression.

PTB1, also known as hnRNP I, is a 57kDa protein that binds to pyrimidine-rich sequences, which are present mostly in the introns and untranslated regions of cellular and viral RNA 24. PTB1 has been shown to play an important role in regulating RNA metabolism, such as splicing, mRNA location, polyadenylation, and translation initiation 25. Importantly, increasing evidence suggests that PTB1 also plays essential roles in the regulation of RNA stability. For instance, the binding of PTB1 to the 3'-UTR of CD154, insulin, vascular endothelial growth factor, and inducible nitric oxide synthase has been shown to increase their mRNA stability 26,27,28,29. In contrast, PTB1 has been shown to decrease the mRNA stability through its binding to the 3'-UTRs of period 2 and human LDL receptor 30,31. In this study, we provided additional evidence implicating PTB1 as a critical regulator for eNOS mRNA destabilization in vascular ECs. At this point, the detailed molecular mechanism(s) underlying the differential effects of PTB1 on mRNA stability remains elusive, but it may be involved in the different structural properties of 3'-UTRs and/or the complex formation of PTB1 with other RNA binding proteins. Indeed, post-transcriptional regulation of gene expression involves various factors acting at the multiple steps of RNA processing. In vascular ECs, we and others have demonstrated that at least two proteins, with molecular sizes of 52 kDa and 57 kDa, are essentially associated with eNOS 3'-UTR 20,21, however, at present, their relative contributions to the regulation of eNOS expression are not fully understood. Since both proteins are up-regulated by TNF-α stimulation, it would be interesting to determine whether PTB1-mediated destabilization of eNOS mRNA transcript requires its functional interaction with eEF1A under TNF-α-stimulated conditions, or vice versa. It is plausible that both PTB1 and eEF1A bind to eNOS 3'-UTR in a cooperative manner to induce destabilization of eNOS mRNA. This hypothesis is under active investigation. At this time, the functional significance of PTB1 in endothelial biology remains largely unexplored. Comprehensive identification of PTB1 target genes in endothelial cells would likely provide significant novel insights into the molecular mechanisms underlying endothelial dysfunction under certain pathophysiological conditions.

The molecular mechanism(s) by which PTB1 regulates eNOS mRNA stability remains elusive, but certainly involves its ability to recognize specific RNA sequences in eNOS 3'-UTR. The consensus sequences for RNA-PTB1 binding are represented by sequences containing 15–25 pyrimidines, with a preference for pyrimidine tracts containing UCUU, CUCUCU 32,33. Indeed, the functional characterization of the PTB1 binding region in eNOS 3'-UTR, as demonstrated in this study, unveiled multiple consensus sequences for PTB1-RNA binding. PTB has the property to shuttle between nucleus and cytoplasm and the regulation of PTB1 on mRNA stability mainly occurs in the cytoplasm 24. In our study, we found that TNF-α treatment did not induce the translocation of PTB1 from the cytoplasm to the nucleus, but rather rapidly induces the expression of PTB1 in both nuclear and cytoplasmic fractions in HUVECs (data not shown). Thus, changes in PTB1 binding to eNOS 3'-UTR, under TNFα-stimulated condition, should be attributed to the relative abundance of PTB1 in endothelial cells. It remains possible that PTB1 posttranslational modification may be involved in regulating its binding to eNOS 3'-UTR. Indeed, statins have been shown to increase eNOS expression and prolong eNOS mRNA stability7, 10. However, in our study, we found that both simvastatin and fluvastatin did not significantly affect PTB1 expression under both basal and TNF-alpha stimulated conditions (data not shown), indicating that statins may affect PTB binding to eNOS 3'-UTR through post-translational mechanisms. Furthermore, it is plausible to speculate that the binding of PTB1 to the eNOS 3'-UTR may elicit multiple biological effects on eNOS mRNA metabolism in vascular ECs, including transport, polyadenylation, and mRNA translation. Indeed, eNOS localization and transport, possibly mediated by actin cytoskeleton, represent a major mechanism implicated in the posttranscriptional regulation of eNOS in response to various stimuli, including hypoxia, oxidized LDL, and cytokines such as TNF-α 34,5. For instance, inhibition of Rho and actin cytoskeletal inhibitors have been shown to prolong eNOS mRNA half-life and prevent the downregulation of eNOS by oxidized LDL and TNF-α under hypoxic conditions 34,6. Moreover, the interaction of actin with eNOS 3'--UTR has been shown to play an essential role in the posttranscriptional regulation of eNOS during cell growth 35. Interestingly, PTB1 has been shown to be associated with β-actin mRNA transcripts, which affects the abundance of the endogenous actin proteins and cytoskeletal assembly 36, 37. Thus, It is likely that knockdown of PTB1 in ECs may prolong the eNOS mRNA half-life through inhibiting the actin cytoskeleton, as it did in PC12 cells 37. This hypothesis certainly merits further investigation.

In summary, we have demonstrated a fundamental molecular mechanism underlying the regulation of eNOS expression at transcriptional levels. We identified PTB1 as an essential cytosolic protein that binds to eNOS 3'-UTR, leading to eNOS mRNA destabilization and impairment in endothelium dependent vasodilation. As the identification of new PTB1 targets is rapidly progressing by genome-wide investigations, future efforts might be focused on deciphering the pathophysiological significance of PTB1 in vascular disease and identifying small molecular modulators of PTB1 that could be potentially used for the treatment of cardiovascular diseases associated with endothelial dysfunction.

Supplementary Material

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2

Significance.

Endothelial dysfunction, as characterized by reduced nitric oxide (NO) production, is a hallmark of many cardiovascular diseases, such as atherosclerosis, diabetic vascular complication, and heart failure. eNOS is a key enzyme responsible for the NO generation in vascular endothelium. Understanding the molecular mechanism regulating eNOS expression and activity is essential for developing effective therapies to combat cardiovascular diseases. In the present study, we unveiled a fundamental mechanism governing eNOS expression at posttranscriptional levels. We identified a cytosolic protein, namely PTB1, as a critical modulator that binds to eNOS 3'-UTR and causes decreased eNOS mRNA stability and impairment in endothelium dependent vasodilation. Our results indicate that specific inhibition of PTB1 in vascular endothelium may represent a novel therapeutic approach for prevention and treatment of cardiovascular diseases.

Acknowledgments

Sources of Funding

This research was supported by the U.S. National Institutes of Health (HL103869) and the Chinese Natural Science Foundation (No.81470406) to S.H and (No. 81170114 and No. 81370418) to J.S.

Abbreviations

eNOS

endothelial nitric oxide synthase

PTB1

polypyrimidine tract binding protein 1

qRT-PCR

quantitative real-time PCR

HUVEC

human umbilical vein endothelial cell

EMSA

electrophoretic mobility shift assay

siRNA

small interference RNA

3'-UTR

3'-untranslational region

Luc

luciferase

RRM

RNA recognition motifs

TNF-α

tumor necrosis factor alpha

cGMP

cyclic guanosine monophosphate

Footnotes

Disclosures

None

References

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Supplementary Materials

1
NIHMS714197-supplement-1.pdf (329KB, pdf)
2
NIHMS714197-supplement-2.pdf (323.6KB, pdf)

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