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. 2008 Jun 13;283(24):16293-8.
doi: 10.1074/jbc.M801803200. Epub 2008 Apr 7.

Activation of p300 histone acetyltransferase activity is an early endothelial response to laminar shear stress and is essential for stimulation of endothelial nitric-oxide synthase mRNA transcription

Wei Chen  1 , Methode Bacanamwo, David G Harrison
Affiliations

Activation of p300 histone acetyltransferase activity is an early endothelial response to laminar shear stress and is essential for stimulation of endothelial nitric-oxide synthase mRNA transcription

Wei Chen et al. J Biol Chem. .

Abstract

Previous studies have shown that the acute stimulation of endothelial nitric-oxide synthase (eNOS) mRNA transcription by laminar shear stress is dependent on nuclear factor kappa B (NFkappaB) subunits p50 and p65 binding to a shear stress response element (SSRE) in the human eNOS promoter and that mutation of the SSRE abrogates the shear-stimulated increase in eNOS promoter activity. In the present study, we found that although shear markedly increased eNOS mRNA, the increase in nuclear translocation of p50 and p65 caused by shear was only 2-fold, suggesting that shear has additional effects on NFkappaB cofactor activity beyond nuclear translocation. Chromatin immunoprecipitation assays showed that virtually no p50 or p65 was bound to the eNOS promoter at base line but that shear increased the binding of these subunits to the human eNOS SSRE by 10- to 20-fold. Co-immunoprecipitation studies demonstrated during the first 30 min of shear p300 bound to p65. Shear also increased p300 histone acetyltransferase (HAT) activity by 2.5-fold and increased acetylation of p65. The increase in eNOS mRNA caused by shear was completely blocked by pharmacological inhibition of p300/HAT activity with curcumin or by p300 small interfering RNA. Chromatin immunoprecipitation assays also showed that shear stimulated acetylation of histones 3 and 4 at the region of the eNOS promoter SSRE and extended 3' toward the eNOS coding region. This was associated with opening of chromatin at the SSRE. In conclusion, these studies reveal a previously unknown role of p300/HAT activation as a very early response to shear that is essential for increasing eNOS mRNA levels.

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Figures

FIGURE 1.
FIGURE 1.
Effect of unidirectional laminar shear on nuclear levels of the NFkB subunits p50 and p65 and binding of these to the eNOS promoter. Western blots are shown of nuclear extracts for p50 (A) and p65 (B). C shows mean densitometry analysis of three to four separate experiments for each. Data are mean ± S.E. of the mean. D shows a Western blot for the cytoplasmic protein α-tubulin and α-actin (present in both the nucleus and cytoplasm) from a nuclear preparation (lane 1) and a cytoplasmic preparation (lane 2). E shows ChIP analysis of binding of p50 and p65 to the eNOS promoter. P50 and p65 were immunoprecipitated from nuclear extracts and segments of eNOS DNA amplified by PCR. Input refers to nuclear extracts before immunoprecipitation. IgG was used as an immunoprecipitation control. F shows mean values for densitometric analysis of three similar experiments. As evident, shear only increased nuclear levels of p50 and p65 by 2-fold but dramatically increased binding of these subunits to the endogenous eNOS promoter.
FIGURE 2.
FIGURE 2.
Acetylation of p50 and p65 at base line and in response to laminar shear and association of p65 with p300. The NFκB subunits p50 and p65 were immunoprecipitated and Western blots using anti-lysine antibody performed. As shown in A and B, both p50 and p65 are acetylated at base line; however, 30 min of shear increases acetylation of p65 (A and C). C shows densitometric analysis of A and B, with the density of acetylated lysines in response to shear expressed as a ratio to the average static sample on the same blot. Shear (15 dynes/cm2 × 20 min) also increases acetyltransferase activity of p300, and this increase is prevented by the histone acetyltransferase inhibitor curcumin (80 mm in D, n = 3 for each). p300 was immunoprecipitated and acetyltransferase activity measured using histone substrate as described under “Experimental Procedures.” E shows the effect of shear on the association between p65 and p300. Mean data for densitometric analysis of three separate experiments are shown in F. Data are mean ± S.E. of the mean.
FIGURE 3.
FIGURE 3.
Effect of shear on histones associated with the eNOS promoter. A–C show mean data of ChIP assays examining acetylation of histones 3 and 4 surrounding the eNOS promoter at base line and following 30 min of shear (15 dynes/cm2). Data are from three to four experiments. D shows opening of the eNOS promoter in response to shear. Nuclei were purified from sheared and unsheared cells, and 2 × 105 nuclei/treatment were digested with 10 units of BsaI. DNA was purified, and a fragment flanking the SSRE (BsaI recognition site) was amplified by quantitative PCR. DNA from undigested nuclei was used as a positive amplification control, while digested purified genomic (naked) DNA was used as a restriction digestion control and negative amplification control. In the case of closed chromatin (histone-protected promoter), BSA1 should fail to cut this site, allowing ultimate amplification of this region using PCR. As evident, shear leads to almost complete opening of the histones surrounding the shear response element. Data are mean ± S.E. of the mean. E shows localization of primers used in experiments in A–C.
FIGURE 4.
FIGURE 4.
Role of p300 in eNOS transcription in response to laminar shear. A shows effect of the p300 inhibitor curcumin on eNOS transcription in response to 6 h of shear (n = 4–5/group). B shows effect of p300 siRNA on p300 protein levels as determined by Western blot. As evident, p300 siRNA effectively reduced p300 levels whereas control (Scrambled) siRNA did not. Down-regulation of p300 with siRNA prevented acetylation of p65 as shown in the example in C and in the mean data (n = 4) presented in D. Inhibition of p300 expression with siRNA completely prevented the increase in eNOS mRNA as determined by real-time quantitative PCR (D, n = 4–5). Data are mean ± S.E. of the mean.
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References

    1. Davies, P. F. (1995) Physiol. Rev. 75 519–560 - PMC - PubMed
    1. James, N. L., Harrison, D. G., and Nerem, R. M. (1995) FASEB J. 9 968–973 - PubMed
    1. Berk, B. C., Corson, M. A., Peterson, T. E., and Tseng, H. (1995) J. Biomech. 28 1439–1450 - PubMed
    1. Malek, A. M., and Izumo, S. (1995) J. Biomech. 28 1515–1528 - PubMed
    1. Nerem, R. M., Harrison, D. G., Taylor, W. R., and Alexander, R. W. (1993) J. Cardiovasc. Pharmacol. 21 Suppl. 1, S6–S10 - PubMed

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