Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2010 Mar;30(3):561–567. doi: 10.1161/ATVBAHA.109.199554

Laminar Shear Stress Modulates Phosphorylation and Localization of RNA Polymerase II on the Endothelial Nitric Oxide Synthase Gene

Jeffrey P Moore 1, Martina Weber 1, Charles D Searles 1,*
PMCID: PMC2948754  NIHMSID: NIHMS172806  PMID: 20167666

Abstract

Objective

In endothelial cells exposed to unidirectional, laminar shear stress (LSS), eNOS transcription, mRNA stability, and protein levels are enhanced. We have previously demonstrated that these changes are associated with increased 3′ polyadenylation of eNOS mRNA. Here, we investigated the effect of LSS on the phosphorylation and localization RNA Polymerase (Pol) II, the enzyme primarily responsible for coordinating transcription and posttranscriptional processing.

Methods and Results

Using Western and chromatin immunoprecipitation (ChIP) analyses, Pol II phosphorylation and localization on the eNOS gene were assessed in bovine aortic endothelial cells (BAECs) exposed to LSS. Total Pol II (phosphorylated and unphosphorylated) levels were increased 65% in response to LSS. This was associated with an increase in Pol II phospho-serine 2, but no change in levels of the unphosphorylated or phospho-serine 5 isoforms. Quantitative ChIP analysis showed that LSS enhanced binding of Pol II phospho-serine 2 to the 3′ end of the eNOS gene, particularly exon 26, which encodes the 3′UTR. Treatment of cells with DRB attenuated LSS-induced Pol II phosphorylation, eNOS 3′ polyadenylation, and eNOS expression.

Conclusion

These data suggest that LSS enhances eNOS mRNA 3′ polyadenylation by modulating phosphorylation and localization of Pol II.

Keywords: laminar shear stress, endothelial nitric oxide synthase, RNA Polymerase II, mRNA processing, gene expression

INTRODUCTION

Mechanical forces created by pulsatile pressure and blood flow are important modulators of cellular function in the cardiovascular system. In the blood vessel wall, endothelial cells are particularly adept at changing their properties in response to mechanical forces 1. One of these forces is unidirectional, laminar shear stress (LSS), the dragging frictional force created by blood flow that acts parallel to the luminal surface of the vessel. Endothelial cells respond to LSS by modulating gene expression, cell metabolism, cell morphology, and the release of vasoactive substances2, including nitric oxide (NO·). NO· is produced by endothelial NO synthase (eNOS), and LSS has been shown in both in vitro and in vivo studies to increase the activity and expression of eNOS3-5.

LSS-induced up regulation of eNOS occurs by two mechanisms: a transient increase in eNOS transcription followed by a prolonged stabilization of eNOS mRNA6. Although both mechanisms are dependent on the activity of c-Src, downstream signaling events for transcriptional and posttranscriptional regulation of eNOS differ 6. Previously, we described a posttranscriptional mechanism by which LSS induces enhanced eNOS mRNA stability and translation that involves enhanced 3′ polyadenylation. Under static conditions, eNOS transcripts have short 3′ poly(A) tails, but these tails dramatically lengthen in response to LSS7.

In eukaryotic cells, mRNA synthesis is performed by a transcription elongation complex whose main component is RNA Polymerase II (Pol II). This enzyme plays a critical role in coordinating chromatin remodeling and posttranscriptional mRNA processing, including 5′ capping, splicing, and 3′ polyadenylation. The processing functions of Pol II are mediated primarily by its carboxy-terminal domain (CTD)8, 9, which serves as a platform for factors required for mRNA processing. Pol II CTD contains repeats of a highly conserved seven-amino-acid consensus sequence, YSPTSPS10, and serines at positions 2 and 5 are targets for phosphorylation11. Transcription initiation is preferentially associated with binding of unphosphorylated Pol II to promoter sequences, while Pol II phospho-serine 5 is associated with release of the transcription complex from the promoter, 5′ capping and splicing12. Pol II phospho-serine 2 is responsible for transcript elongation, termination and 3′polyadenylation13.

Transcript elongation and posttranscriptional mRNA processing are highly regulated14, and distinct signal transduction pathways have been shown to modulate Pol II activity through CTD phosphorylation15, 16. We hypothesized that mechanotransduction of LSS in endothelial cells leads to changes in Pol II phosphorylation, which in turn enhances eNOS mRNA 3′ polyadenylation. In this study, we focused on changes in Pol II phosphorylation and localization on the eNOS gene in endothelial cells exposed to LSS. We found that LSS enhanced expression of Pol II phospho-serine 2 and its localization to sequences encoding the eNOS 3′UTR, adjacent to the transcription termination site. These findings provide further insight into mechanisms by which transduction of mechanical forces can modulate endothelial gene expression.

MATERIALS AND METHODS

Cell Culture

Bovine aortic endothelial cells (BAECs; Cell Systems) were cultured in Media 199 (Mediatech) containing 10% fetal calf serum (Hyclone), penicillin-streptomycin, glutamine and non-essential amino acids. Postconfluent BAECs between passages 4 to 8 were used for all experiments. A cone-in-plate viscometer with a 1 ° angle was used to create unidirectional laminar shear stress of 15 dynes/cm2 for 6 hours17. The magnitude and 6-hour period of shear stress was selected based on previous experiments, in which eNOS polyadenylation was found to be maximally increased after 6 hours of LSS7.

Western analysis

Western analysis was performed as previously described 18. Antibodies against the different Pol II phosphoisoforms were obtained from Covance. These include H5 (Pol II phospho-serine 2, 1:500), H14 (phospho-serine 5, 1:500), and 8WG16 (unphosphorylated Pol II CTD, 1:000). Antibodies against total Pol II (targeting N-terminal, 1:10000) and eNOS (1:2500) were from BD Pharmingen. The antibody against cyclin-dependent kinase 9 was purchased from Santa Cruz (1:500). Anti-α-Actin antibody (Sigma, 1:1000) was used as a loading control. All antibodies were prepared in 3% blocking milk.

RNA isolation and real time PCR

Total cellular RNA was isolated from cells using TRIzol-Reagent (Invitrogen). Light Cycler (Roche) was used for real time PCR, as described earlier19. Prior to PCR, DNA was purified with Micro Bio-Spin 30 Chromatography Columns (Bio Rad). PCR was performed on 2 μl of cDNA using Platinum Taq Polymerase (Invitrogen) and buffer provided with the enzyme. For continuous fluorescence monitoring of DNA specific binding, 0.5% SYBR Green Dye (Roche) was used.

Chromatin Immunoprecipitation (ChIP)

BAECs were sheared for 6 hrs (15 dynes/cm2), with or without DRB (5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole, 60 μmol/L, Sigma) pretreatment (1 hour), or treated with cytochalasin D (5 μmol/L, Sigma) for 6 hrs. Control cells were kept under static conditions or treated with DMSO. Protein was crosslinked to DNA with 1% formaldehyde for 10 min at 37°C. The ChIP assay was performed following the Upstate ChIP Assay protocol with slight modifications. Briefly, cells were washed with 1 x PBS and lysed for 10 min on ice in SDS Lysis-Buffer (Upstate). Cells were sonicated using a Sonic Dismembrator 60 (Fisher Scientific). The DNA was sheared 3 times (4 watts, 10 pulses at 1 second each), into approximately 200 bp long fragments (online supplemental Figure 1A). Protein was immunoprecipitated overnight using, Pol II phospho-serine 2 antibody (H5) or IgG as control, then pulled down with Salmon Sperm DNA/ Protein G/A- Agarose (Upstate). Crosslinking was reversed overnight and protein digested with proteinase K (Invitrogen) for one hour. DNA was extracted using phenol/chloroform and ethanol precipitation. Primers for PCR are listed in the Online Data Supplement. Primers were subjected to Basic Local Alignment Search Tool (BLAST) analysis (National Center for Biotechnology Information) and none had 100% homology with any other bovine genomic sequence besides eNOS. In fact, exon 26 primers did not have homology with any other sequence in the bovine genome. Primer efficiency was determined using 0-0.6 ng/ul genomic DNA standard curves, analyzed by linear regression. Sample values were calculated by subtracting the y-intercept and then dividing by the slope. Each sample was normalized to the individual amount of input DNA.

RNase Protection Assay

RNA was isolated using TRIzol (Invitrogen). To detect the length of Polyadenylated eNOS mRNA, we used a special riboprobe generated from a 3′ RACE-PAT of eNOS as described previously 7.

RESULTS

LSS preferentially increases expression of Pol II phospho-serine 2

BAECs subjected to LSS (6 hours, 15 dynes/cm2) had a 65% increase in total Pol II protein expression compared to static control cells (Figure 1A). The antibody used to assess total Pol II expression targets the N-terminal domain of the enzyme and therefore detected both phosphorylated and unphosphorylated Pol II. Because the mRNA processing activities of Pol II are linked to phosphorylation of its CTD, further Western analysis was performed to assess expression of the three specific Pol II phosphoisoforms in response to LSS. Endothelial cells exposed to LSS had a 40 to 50% increase in expression of Pol II phospho-serine 2 (Figure 1B) compared to control cells. In contrast, there was no significant effect of LSS on expression of unphosphorylated Pol II (Figure 1C) or Pol II phospho-serine 5 (Figure 1D). Thus, the LSS-induced upregulation of total Pol II appeared to be largely associated with enhanced expression of the Pol II phospho-serine 2 isoform.

Figure 1.

Figure 1

Open in a new tab

Pol II levels in BAECs exposed to LSS or nonsheared conditions. Western blot and densitometric analysis for A, total Pol II; B, Pol II phospho-serine 2; C, Pol II phospho-serine 5; and D, unphosphorylated Pol II, n = 4-6, * p = 0.01, unpaired t-test.

The 6-hour time point was used for these studies because, after 6 hours of LSS, maximum 3′ polyadenylation of eNOS mRNA was observed7. A shear time course (1-6 hours) showed that expression of Pol II phospho-serine 2 is increased over baseline levels as early as 1 hour after the cells are subjected to LSS and remains increased for the duration of LSS (Online Supplemental Figure 2A). In comparison, eNOS protein is increased after 4-6 hours of LSS.

Localization and quantitative assessment of Pol II phospho-serine 2

ChIP analysis was performed to determine the effect of LSS on Pol II phospho-serine 2 binding to different regions of the eNOS gene. Chromatin from sheared and static control cells was immunoprecipitated with the antibody against Pol II phospho-serine 2 and quantitative PCR was performed with primers targeting sequences in the promoter area, exons 19-26, and sequences downstream of the transcription termination site (Figure 2A). The different efficiencies of the primer pairs were determined and subsequently used to quantify binding of Pol II phospho-serine 2 to the eNOS gene (supplemental Figure 1B). The DNA sequences that were studied were chosen because of the importance of their interaction with Pol II in demonstrating the different activities of Pol II during the transcription cycle. These activities include transcription initiation, demonstrated by Pol II binding to promoter sequences, and transcript elongation, termination and 3′ processing, demonstrated by Pol II binding to exonic sequences at the 3′ end of the gene as well as sequences downstream of the polyadenylation site.

Figure 2.

Figure 2

Open in a new tab

Pol II localization on the eNOS gene. A, Location of ChIP PCR primer pairs. B, quantitative ChIP analysis of Pol II phospho-serine 2 binding to promoter area and later exons (n=3-8), S=static, L=LSS, *p=0.038.

The Pol II CTD is phosphorylated at serine 2 during elongation of the primary RNA transcript, and Pol II phospho-serine 2 is responsible for coordinating 3′ polyadenylation by recruiting factors necessary for 3′ processing. High amounts of DNA (~6 ng) were found to bind Pol II phospho-serine 2 on exon 26 (eNOS 3′UTR) in cells exposed to LSS. Compared to control cells, LSS induced a significant 11.4 fold increase in Pol II binding to exon 26. We did not observe a lot of binding of Pol II phospho-serine 2 in the promoter area; this region of the gene should be associated with unphosphorylated Pol II or Pol II phospho-serine 5, respectively. We detected binding of Pol II phospho-serine 2 to exons 19, 21 and 22, but the values for binding to these sequences were still much lower compared to binding of Pol II phospho serine 2 to exon 26 (less than 1 ng DNA). Together, these data indicate that LSS modulates Pol II by increasing phosphorylation of CTD serine 2 and enhancing localization of the serine 2 phosphoisoform to eNOS sequences encoding the 3′ UTR. Further, quantitative ChIP analysis of the actin 3′UTR did not show increased Pol II phospho-serine 2 binding in response to LSS, (supplemental Figure 2B), suggesting that enhanced binding of Pol II phospho-serine 2 to eNOS 3′UTR in response to LSS is specific.

DRB attenuates LSS-induced Pol II phosphorylation and eNOS 3′ Polyadenylation

Positive transcription elongation factor b (P-TEFb) is responsible for the transition of Pol II from the initial phases of transcription into the productive elongation phase 20. P-TEFb is a heterodimer composed of CDK9 and a regulatory cyclin (T1, T2, or K), and Pol II serine 2 is a specific substrate for phosphorylation by CDK920. We have observed that DRB, an agent that blocks transcription by inhibiting CDK9 21, 22, attenuated LSS-induced changes in eNOS 3′polyadenylation7. Figure 3A shows a ribonuclease protection assay (RPA) of RNA from control and sheared cells, using a riboprobe that targets the 3′ end of eNOS mRNA. The RPA of control cells shows one protected fragment of 250 nt, which correlates with a short eNOS 3′ poly(A) tail of 25 nucleotides in length. Additional protected fragments were detected for RNA from sheared cells; these fragments correlate with poly(A) tails of 75 to 160 nucleotides in length. Pretreatment of cells with DRB (60 μmol/L) for 1 hour prior to LSS dramatically reduced eNOS polyadenylation (Figure 3A, right panel). Subsequently, we examined the effect of DRB pretreatment on LSS-induced changes in Pol II phosphorylation. DRB pretreatment attenuated the observed LSS-induced increase in total Pol II (Figure 3B), Pol II phospho-serine 2 expression (Figure 3C), and eNOS expression (Figure 4B). Together, these data suggest that the LSS-induced increase in Pol II serine 2 phosphorylation and eNOS 3′polyadenylation is mediated by CDK9.

Figure 3.

Figure 3

Open in a new tab

Effect of DRB on LSS-induced changes in expression of Pol II. A, Representative RPA of eNOS transcripts showing inhibition of 3′ polyadenylation after DRB pretreatment (n = 3). Western analysis for B, total Pol II and C, Pol II phospho-serine 2, n = 3, * p < 0.05.

Figure 4.

Figure 4

Open in a new tab

A, Effect of DRB on Pol II phospho-serine 2 binding to exon 26, quantitative ChIP analysis of cells pretreated with DRB (60uM, 1 hour) and exposed to LSS, compared to static control cells and LSS cells (n=6-8, *p=0.038). B, Western analysis of eNOS protein in static and LSS cells with or without DRB pretreatment. C, Western analysis of total Pol II and its phosphoisoforms after cytochalasin D (Cyt D) treatment, D, Quantitative ChIP of Pol II phospho-serine 2 binding to exon 26 after 6 hours of Cyt D treatment, compared to DMSO static control cells. n = 6-7, ns.

DRB modulates LSS-induced binding of Pol II phospho-serine 2 to eNOS gene

To examine the effect of DRB on localization of Pol II phospho-serine 2 on the eNOS gene, we performed additional ChIP analysis on cells that were pretreated with DRB for 1 hour and subsequently subjected to LSS for 6 hours. Quantitative assessment of Pol II phospho-serine 2 binding to exon 26 showed that DRB prevented LSS-induced binding of Pol II phospho-serine 2 (Figure 4A). We hypothesized that a consequence of this effect would be less 3′ processing and therefore less eNOS mRNA stability and translation. Western analysis of eNOS protein in static and LSS treated cells with and without DRB pretreatment demonstrated the well known increase of eNOS protein in response to LSS (Figure 4 B) and it revealed that this response is prevented in the presence of DRB, most likely because of the lack of Pol II phospho-serine 2 binding to eNOS 3′ UTR.

Cytochalasin D treatment does not alter expression or localization of Pol II on eNOS

Cytochalasin D is an agent that directly alters actin cytoskeleton organization, and, like LSS, increases eNOS 3′ polyadenylation23 and expression24. The effect of cytochalasin D on Pol II phosphorylation and localization in BAECs was studied using Western analysis and quantitative ChIP analysis (Figure 4C). We did not observe any change in expression of total Pol II levels or any of the Pol II phosphoisoforms. Quantitative ChIP analysis of Pol II phospho-serine 2 binding to eNOS exon 26 did not reveal any significant differences between cytochalasin D-treated and control endothelial cells (Figure 4D). Since this finding differs from the response to LSS, we conclude that not all stimuli that increase eNOS polyadenylation will necessarily have enhanced Pol II phospho-serine 2 binding to the 3′ UTR. Interestingly, stimulus-dependent changes in Pol II serine 2 phosphorylation and localization are not unique to our model system; this phenomenon has been noted in other mammalian model systems16.

DISCUSSION

Shear stress forces generated by blood flow are important modulators of signaling and gene expression in the vessel wall25. In the current study, we sought to extend our previous work on LSS-induced changes in eNOS expression by examining how LSS increases 3′ polyadenylation of eNOS mRNA. Previous studies of eNOS expression have focused on the early phases of the transcription cycle, such as transcription initiation. However, the cell’s transcription machinery is not solely associated with promoter activity, and other aspects of mRNA synthesis, such as capping, splicing, and 3′ polyadenylation, are dynamic and highly regulated14. These posttranscriptional processing reactions are influenced by changes in phosphorylation of Pol II, whose CTD coordinates mRNA processing reactions by providing key molecular contacts throughout transcriptional elongation and termination (Figure 5)26. The central finding of this study is that LSS alters expression, phosphorylation and localization of Pol II on the eNOS gene. Thus LSS, besides stimulating recruitment of Pol II to the eNOS promoter for transcription initiation6, also enhances recruitment of Pol II phospho-serine 2 to the 3′ region of the eNOS gene important for transcription termination and polyadenylation.

Figure 5.

Figure 5

Open in a new tab

Schema of LSS and Pol II activity. LSS increases transcription initiation of eNOS. LSS also increases phosphorylation of Pol II CTD serine 2 and enhanced localization to the sequences encoding the 3′UTR. This mechanism appears to be mediated by CDK9 and leads to enhanced eNOS 3′ polyadenylation and expression. As depicted, the bovine eNOS polyadenylation hexamer is AATAAT.

In quiescent endothelial cells, the expression of eNOS is constitutive and restricted to the endothelial cell layer of medium- to large-caliber vessels. Basal eNOS transcription is controlled by two positive regulatory domains (PRD I and II) in the proximal promoter27, and the endothelial cell-specific nature of eNOS transcription has recently been linked to the chromatin structure in this region28, 29. It has been shown that active elongation and 3′ processing of eNOS transcripts by phosphorylated Pol II in quiescent endothelial cells is relatively hindered, analogous to the elongation block observed in immediate early gene (IEG) transcription30. Interestingly, both eNOS mRNA from human and bovine quiescent endothelial cells7 have short 3′ poly(A) tails, suggesting that 3′ mRNA processing is impeded in nonstimulated cells.

Consistent with this concept, we found very little Pol II phospho-serine 2 binding to eNOS DNA in nonsheared control cells, but, in response to LSS, there was increased binding of Pol II phospho-serine 2 to DNA on exon 26. This observation supports our hypothesis that mechanotransduction of LSS in endothelial cells leads to upregulation of eNOS expression in part by altering phosphorylation of Pol II, thus relieving the baseline impediment to active elongation and 3′ polyadenylation. Indeed, we found that LSS specifically enhanced expression of the Pol II phospho-serine 2, the phosphoisoform associated with transcript elongation and 3′ polyadenylation (Figure 5). In contrast, we also found that cytochalasin D, which, like LSS, is known to increase eNOS 3′ polyadenylation and expression23, 24, had no effect on Pol II expression, phosphorylation, or localization to the eNOS 3′UTR. This result indicates that LSS and cytochalasin D increase eNOS 3′ polyadenylation by different mechanisms, which may not be unexpected given that LSS also increases transcription initiation whereas cytochalasin D does not.

The recruitment of P-TEFb (consisting of CDK9 and a cyclin subunit) to the transcription unit is important for stimulus-driven synthesis of mRNA 15, 30, 31. Fujita et al. demonstrated that DRB, an adenosine analogue DRB that specifically blocks Pol II elongation by inhibiting P-TEFb 22, 32, inhibited phosphorylation of Pol II and subsequent progression of Pol II to downstream regions of the MKP-1 gene in neuroendocrine cells15. Similarly, we observed that, in endothelial cells, DRB inhibited LSS-induced changes in Pol II phosphorylation and localization, as well as eNOS 3′ polyadenylation and protein expression. The relationship between shear stress and P-TEFb has not been studied previously, although CDK9 has been shown to be responsive to stress signals associated with cardiac myocyte hypertrophy, such as aortic banding31. These signals did not change expression CDK9 or cyclin T, but did alter their association with an inhibitory small nuclear RNA, 7SK; stress-induced disinhibition of CDK9 was mediated by calcineurin and Gq. Whether this regulatory pathway is responsible for LSS-induced changes in endothelial Pol II phosphorylation is unknown. Figure 5 depicts a schema of how LSS-induced changes in Pol II phosphorylation correlate with its location and synthesis of eNOS mRNA; a question mark has been inserted between LSS and P-TEFb because we are not certain whether LSS directly or indirectly modulates CDK9.

Steady state levels of eNOS mRNA are subject to modest, but likely important degrees of regulation by different stimuli that have been implicated in vascular pathophysiology. Recent work has demonstrated that expression of this gene is complex and involves multiple different cis and trans-acting factors, as well as changes in promoter activity, chromatin remodeling, mRNA processing, mRNA stability, and translational activity. In addition, in humans and mice, an antisense mRNA that is complementary to the 3′end of eNOS has been shown to posttranscriptionally downregulate eNOS in nonendothelial cells33. This antisense mRNA, known as sONE, is derived from a transcription unit on the DNA strand opposite eNOS, oriented in a tail-to-tail configuration with eNOS. However, we do not think our ChIP analysis of the bovine 3′UTR is confounded by Pol II binding to the 3′UTR of bovine sONE. First, we have not been able to detect sONE mRNA in bovine endothelial cells. Second, the predicted bovine sONE does not overlap to the extent that the human sONE does, so the sequences encoding bovine eNOS and sONE 3′UTRs appear to be different.

In this study, we have used a well-established in vitro model of unidirectional shear stress forces, the cone-in-plate viscometer1, to show that transduction of prolonged shear stress (6 hours) in endothelial cells leads to changes in Pol II phosphorylation and localization, which then has an impact on eNOS 3′ polyadenylation and expression. The detailed mechanism of how LSS-induced modulation of the general transcription machinery leads to changes in 3′ processing of specific mRNAs is not completely clear, but we believe that this issue is related to cis-acting elements recognized by Pol II, including the polyadenylation signal. Both bovine and human eNOS 3′UTRs contain relatively inefficient polyadenylation signals and hexamers (AAUAAU – bovine, ACUAAA-humans), which are associated with short poly(A) tails under non- or low shear conditions7. The bovine polyadenylation hexamer/signal is depicted in Figure 5. We hypothesize that phosphorylation of Pol II serine 2 facilitates 3′ polyadenylation of genes, like eNOS, that have inefficient polyadenylation signals. Interestingly, LSS did not induce enhanced localization of Pol II phospho-serine 2 to the beta-actin gene, which contains an efficient (AAUAAA, canonical) polyadenylation signal. Importantly, we have observed increased Pol II phospho-serine 2 levels relative to the other Pol II phosphoisoforms in human endothelial cells (HUVECs) subjected to LSS for 6 hours, suggesting that the effect of LSS is not species specific (online Supplemental Figure 2C). Current efforts are focused on identifying other endothelial genes that demonstrate similar LSS-induced changes in 3′polyadenylation and localization of Pol II phospho-serine 2.

Condensed Abstract.

We examined the effect of shear stress on RNA Polymerase II in endothelial cells. Compared to unsheared cells, sheared cells had altered Pol II phosphorylation and localization on the eNOS gene. Our data indicate that mechanotransduction induces changes in Pol II that are important for eNOS mRNA expression.

Supplementary Material

Supp1

Acknowledgments

SOURCES OF FUNDING

This work was supported by National Institutes of Health Grant R01HL077274.

Footnotes

DISCLOSURES None.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  • 1.Chien S. Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. Am J Physiol Heart Circ Physiol. 2007;292:H1209–1224. doi: 10.1152/ajpheart.01047.2006. [DOI] [PubMed] [Google Scholar]
  • 2.Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519–560. doi: 10.1152/physrev.1995.75.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Corson MA, James NL, Latta SE, Nerem RM, Berk BC, Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res. 1996;79:984–991. doi: 10.1161/01.res.79.5.984. [DOI] [PubMed] [Google Scholar]
  • 4.Kuchan MJ, Frangos JA. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am J Physiol. 1994;266:C628–636. doi: 10.1152/ajpcell.1994.266.3.C628. [DOI] [PubMed] [Google Scholar]
  • 5.Harrison DG, Venema RC, Arnal JF, Inoue N, Ohara Y, Sayegh H, Murphy TJ. The endothelial cell nitric oxide synthase: is it really constitutively expressed? Agents Actions Suppl. 1995;45:107–117. doi: 10.1007/978-3-0348-7346-8_16. [DOI] [PubMed] [Google Scholar]
  • 6.Davis ME, Cai H, Drummond GR, Harrison DG. Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways. Circ Res. 2001;89:1073–1080. doi: 10.1161/hh2301.100806. [DOI] [PubMed] [Google Scholar]
  • 7.Weber M, Hagedorn CH, Harrison DG, Searles CD. Laminar shear stress and 3′ polyadenylation of eNOS mRNA. Circ Res. 2005;96:1161–1168. doi: 10.1161/01.RES.0000170651.72198.fa. [DOI] [PubMed] [Google Scholar]
  • 8.Hirose Y, Manley JL. RNA polymerase II and the integration of nuclear events. Genes Dev. 2000;14:1415–1429. [PubMed] [Google Scholar]
  • 9.Proudfoot N, O’Sullivan J. Polyadenylation: a tail of two complexes. Curr Biol. 2002;12:R855–857. doi: 10.1016/s0960-9822(02)01353-2. [DOI] [PubMed] [Google Scholar]
  • 10.Corden JL, Cadena DL, Ahearn JM, Jr., Dahmus ME. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc Natl Acad Sci U S A. 1985;82:7934–7938. doi: 10.1073/pnas.82.23.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Dahmus ME. Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem. 1996;271:19009–19012. doi: 10.1074/jbc.271.32.19009. [DOI] [PubMed] [Google Scholar]
  • 12.Proudfoot N. New perspectives on connecting messenger RNA 3′ end formation to transcription. Curr Opin Cell Biol. 2004;16:272–278. doi: 10.1016/j.ceb.2004.03.007. [DOI] [PubMed] [Google Scholar]
  • 13.Komarnitsky P, Cho EJ, Buratowski S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 2000;14:2452–2460. doi: 10.1101/gad.824700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sims RJ, 3rd, Belotserkovskaya R, Reinberg D. Elongation by RNA polymerase II: the short and long of it. Genes Dev. 2004;18:2437–2468. doi: 10.1101/gad.1235904. [DOI] [PubMed] [Google Scholar]
  • 15.Fujita T, Ryser S, Tortola S, Piuz I, Schlegel W. Gene-specific recruitment of positive and negative elongation factors during stimulated transcription of the MKP-1 gene in neuroendocrine cells. Nucleic Acids Res. 2007;35:1007–1017. doi: 10.1093/nar/gkl1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ryser S, Fujita T, Tortola S, Piuz I, Schlegel W. The rate of c-fos transcription in vivo is continuously regulated at the level of elongation by dynamic stimulus-coupled recruitment of positive transcription elongation factor b. J Biol Chem. 2007;282:5075–5084. doi: 10.1074/jbc.M607847200. [DOI] [PubMed] [Google Scholar]
  • 17.Dewey CF, Jr., Bussolari SR, Gimbrone MA, Jr., Davies PF. The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng. 1981;103:177–185. doi: 10.1115/1.3138276. [DOI] [PubMed] [Google Scholar]
  • 18.Ramasamy S, Parthasarathy S, H DG. Regulation of endothelial nitric oxide synthase gene expression by oxidized linoleic acid. J Lipid Res. 1998;39:268–276. [PubMed] [Google Scholar]
  • 19.Szocs K, Lassegue B, Sorescu D, Hilenski LL, Valppu L, Couse TL, Wilcox JN, Quinn MT, Lambeth JD, Griendling KK. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vasc Biol. 2002;22:21–27. doi: 10.1161/hq0102.102189. [DOI] [PubMed] [Google Scholar]
  • 20.Price DH. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol. 2000;20:2629–2634. doi: 10.1128/mcb.20.8.2629-2634.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wada T, Takagi T, Yamaguchi Y, Watanabe D, Handa H. Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro. Embo J. 1998;17:7395–7403. doi: 10.1093/emboj/17.24.7395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yamaguchi Y, Takagi T, Wada T, Yano K, Furuya A, Sugimoto S, Hasegawa J, Handa H. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell. 1999;97:41–51. doi: 10.1016/s0092-8674(00)80713-8. [DOI] [PubMed] [Google Scholar]
  • 23.Kosmidou I, Moore JP, Weber M, Searles CD. Statin treatment and 3′ polyadenylation of eNOS mRNA. Arterioscler Thromb Vasc Biol. 2007;27:2642–2649. doi: 10.1161/ATVBAHA.107.154492. [DOI] [PubMed] [Google Scholar]
  • 24.Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui DS, Yang SX, Simoncini T, Yamada M, Rabkin E, Allen PG, Huang PL, Bohm M, Schoen FJ, Moskowitz MA, Liao JK. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest. 2000;106:15–24. doi: 10.1172/JCI9639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gimbrone MA, Jr., Resnick N, Nagel T, Khachigian LM, Collins T, Topper JN. Hemodynamics, endothelial gene expression, and atherogenesis. Ann N Y Acad Sci. 1997;811:1–10. doi: 10.1111/j.1749-6632.1997.tb51983.x. [DOI] [PubMed] [Google Scholar]
  • 26.Proudfoot NJ, Furger A, Dye MJ. Integrating mRNA processing with transcription. Cell. 2002;108:501–512. doi: 10.1016/s0092-8674(02)00617-7. [DOI] [PubMed] [Google Scholar]
  • 27.Karantzoulis-Fegaras F, Antoniou H, Lai SL, Kulkarni G, D’Abreo C, Wong GK, Miller TL, Chan Y, Atkins J, Wang Y, Marsden PA. Characterization of the human endothelial nitric-oxide synthase promoter. J Biol Chem. 1999;274:3076–3093. doi: 10.1074/jbc.274.5.3076. [DOI] [PubMed] [Google Scholar]
  • 28.Chan Y, Fish JE, D’Abreo C, Lin S, Robb GB, Teichert AM, Karantzoulis-Fegaras F, Keightley A, Steer BM, Marsden PA. The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation. J Biol Chem. 2004;279:35087–35100. doi: 10.1074/jbc.M405063200. [DOI] [PubMed] [Google Scholar]
  • 29.Fish JE, Matouk CC, Rachlis A, Lin S, Tai SC, D’Abreo C, Marsden PA. The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code. J Biol Chem. 2005;280:24824–24838. doi: 10.1074/jbc.M502115200. [DOI] [PubMed] [Google Scholar]
  • 30.Ryser S, Tortola S, van Haasteren G, Muda M, Li S, Schlegel W. MAP kinase phosphatase-1 gene transcription in rat neuroendocrine cells is modulated by a calcium-sensitive block to elongation in the first exon. J Biol Chem. 2001;276:33319–33327. doi: 10.1074/jbc.M102326200. [DOI] [PubMed] [Google Scholar]
  • 31.Sano M, Schneider MD. Cyclin-dependent kinase-9: an RNAPII kinase at the nexus of cardiac growth and death cascades. Circ Res. 2004;95:867–876. doi: 10.1161/01.RES.0000146675.88354.04. [DOI] [PubMed] [Google Scholar]
  • 32.Chao SH, Price DH. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J Biol Chem. 2001;276:31793–31799. doi: 10.1074/jbc.M102306200. [DOI] [PubMed] [Google Scholar]
  • 33.Robb GB, Carson AR, Tai SC, Fish JE, Singh S, Yamada T, Scherer SW, Nakabayashi K, Marsden PA. Post-transcriptional regulation of endothelial nitric-oxide synthase by an overlapping antisense mRNA transcript. J Biol Chem. 2004;279:37982–37996. doi: 10.1074/jbc.M400271200. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp1
NIHMS172806-supplement-Supp1.pdf (289.6KB, pdf)

RESOURCES