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Cell Biology International (2010) 34, 1155–1161 (Printed in Great Britain)
Down-regulation of CREB-binding protein expression inhibits thrombin-induced proliferation of endothelial cells: possible relevance to PDGF-B
Hong Jiang12, Jing Chen1, Lang Wang, Li‑hua Zhu and Hua‑zhi Wen
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, Peoples Republic of China

Thrombin acts as a potent mitogenic factor for ECs (endothelial cells) by the release of several growth factors, including PDGF-B (platelet-derived growth factor-B). CBP (CREB-binding protein), which functions as a transcriptional coactivator, links the changes in the extracellular stimuli with alterations in gene expression. Therefore, we hypothesized that CBP could mediate thrombin-induced proliferation of ECs via PDGF-B-dependent way. Short hairpin RNA was used to down-regulate the expression of CBP in ECs. CBP and PDGF-B levels were analysed by real-time RT-PCR and Western blot. To evaluate ECs proliferation, cell cycle and DNA synthesis were analysed by flow cytometry and BrdU (bromodeoxyuridine) incorporation assay, respectively. PDGF-B was involved in the mitogenic effect of thrombin on ECs. Down-regulation of CBP attenuated ECs proliferation and inhibited cell cycle progression induced by thrombin. Silencing CBP expression also suppressed thrombin-induced PDGF-B expression in ECs. Mitogenic activity of thrombin was impaired by silencing CBP expression in ECs. This inhibitory effect was, in part, related to the inability to up-regulate PDGF-B expression in ECs. CBP could be regarded as a potential therapeutic target for vascular injury.

Key words: cell cycle, CREB-binding protein, endothelial cell, growth, transcription coactivator

Abbreviations: BrdU, bromodeoxyuridine, CBP, CREB-binding protein, CREB, cAMP-response-element-binding protein, EBM, endothelial basal medium, ECs, endothelial cells, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HAT, histone acetyltransferase, MOI, multiplicities of infection, MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, PDGF-B, platelet-derived growth factor-B, shRNA, short hairpin RNA, VSMCs, vascular smooth muscle cells, VEGF, vascular endothelial growth factor

1Hong Jiang and Jing Chen contributed equally to this work.

2To whom correspondence should be addressed (email

1. Introduction

Thrombin is generated when perturbation of ECs (endothelial cells) activates coagulation cascade during vascular injury, and high plasmatic level of thrombin persists during vascular lesion formation (Coughlin, 2000; Martorell et al., 2008a). In addition to its roles in blood coagulation, growing evidence points to the fact that thrombin can be a potent mitogenic factor for ECs (Borrelli et al., 2001; Dupuy et al., 2003; Zania et al., 2008). Many cellular effects of thrombin on ECs contribute to the mechanism of thrombin-induced proliferation. Several studies have provided evidence that thrombin can induce DNA synthesis through direct activation of protein kinase C, mitogen-activated protein kinase (Olivot et al., 2001) and downstream up-regulation of NOR-1 in ECs (Martorell et al., 2007). On the other hand, some researches have showed that mitogenic activity of thrombin is mediated indirectly by the release of several growth factors, including VEGF (vascular endothelial growth factor) (Dupuy et al., 2003), angiopoietin-2 (Huang et al., 2002), PDGF-B (platelet-derived growth factor-B) (Minami et al., 2004) and epidermal growth factor (Igura et al., 1996). Although the precise cellular mitogenic mechanism of thrombin has not been elucidated in ECs, it is known that these events are associated with vascular gene expression through key transcription factors, such as Ets-1 (Wu et al., 2002), NF-κB (Martin et al., 2009), HIF-1 (hypoxia-inducible factor 1) (Yamakawa et al., 2003) and CREB (cAMP-response-element-binding protein) (Martorell et al., 2008b). Besides the participation of transcription factors, extracellular and intracellular signalling pathways require additional transcriptional coactivators to achieve the desired and specific response. Thus, more work is needed to understand the role of transcriptional coactivators in regulating thrombin-induced ECs proliferation.

CBP (CREB-binding protein) functions as a transcriptional coactivator that mediates communication between transcription factors and transcriptional machinery and appears to be important for gene transcription (Chan and La Thangue, 2001). CBP is thought to be activated among numerous signal transduction pathways, thus the availability of CBP may be a limiting factor for the appropriate execution of many biological processes stimulated with various cytokines (Chan and La Thangue, 2001), including thrombin. The gene expression program regulated by CBP includes a myriad of cellular functions, such as DNA repair, inflammation, cell growth, differentiation and apoptosis (Goodman and Smolik, 2000). Further findings in knockout mice indicate that lack of CBP results in embryonic lethality and severe defects in both vasculogenesis and angiogenesis (Yao et al., 1998; Oike et al., 1999). Therefore, we hypothesize that CBP is involved in the ECs proliferation induced by thrombin.

Here, to uncover functions of CBP in endothelial biology, we investigated whether CBP knockdown could inhibit ECs proliferation induced by thrombin and determined the underlying mechanism. In the present study, we found that mitogenic activity of thrombin in ECs was impaired by silencing CBP expression. This inhibitory effect was, in part, related to the inability to up-regulate PDGF-B expression in ECs.

2. Materials and methods

2.1. Primary ECs culture and adenovirus transfection

All procedures followed were in accordance with the Animal Ethical Commission of Wuhan University. ECs were isolated from the thoracic aortas of rats and cultured in EBM (endothelial basal medium) medium (Cambrex) with 20% FBS (fetal calf serum; Invitrogen) and ECs growth supplements (Cambrex), as described previously (Suschek et al., 1997; Hoffmann et al., 2001). CD31 (BD Pharmingen™) of rat ECs was detected with flow cytometric analysis for cell purity examination. The total percentage of CD31-positive cells between passages four and six is approximately 85–90%.

Recombinant adenovirus CBP–shRNA/Ad containing shRNAs (short hairpin RNAs) against CBP was prepared as described previously (Chen et al., 2008). CBP–shRNA/Ad is a replication-deficient vector based on human adenovirus serotype 5 that contains the expression cassettes for green fluorescent protein and shRNAs against CBP. Ad (N), which has a non-homologous shRNA sequence, was used as the control adenovirus. The specific target sequences of CBP are as follows:





The non-homologous shRNA sequence is 5′-AAGCTTCATAAGGCGCATAGC-3′. For adenovirus transfection, subconfluent ECs were incubated with CBP–shRNA/Ad and Ad (N) at 25 or 50 MOI (multiplicity of infection) in serum-free media for about 4–6 h. Then, the media was removed, and cells were incubated with complete EBM (EBM containing 20% FBS and growth supplements) for the indicated time courses. Cell viability was assessed by Trypan Blue exclusion. No significant decrease of cell viability was detected in all investigated groups.

2.2. RT-PCR

Isolation of total cellular RNA was carried out by Trizol reagent (Invitrogen). One microgram of total RNA was reverse transcribed with oligo(dT). Then cDNA was amplified by real-time PCR with SYBR Green PCR master mix (Invitrogen) according to manufacturer's instructions. Thermal cycling conditions comprised an initial denaturation step at 94°C for 10 min, followed by 40 cycles (94°C for 30 s; 60°C for 30 s; 72°C for 60 s). Levels of CBP and PDGF-B mRNA were calculated based on the method of $2^{\hyphen \rDelta \rDelta {{\rm C}_{\rm T}}} $ between the investigated group and control group. The primers were as follows: CBP, forward primer 5′-ACTGGCAGACCTCGGAAAGA-3′, reverse primer 5′-TCTGGCGCCGCAAAAA-3′. PDGF-B, forward primer 5′-AGGTGTTCCAGATCTCGC-3′, reverse primer 5′-GTCACTGTGGCCTTCTTG-3′. GAPDH (glyceraldehyde-3-phosphate dehydrogenase), forward primer 5′-TCAACGGCACAGTCAAGG-3′, reverse primer 5′-TGAGCCTTCCACGATG-3′.

2.3. Immunoprecipitation and immunoblotting

ECs, 2×106, were harvested and lysed in 500 μl lysis buffer. Protein concentration was determined by the Bipec (bicinchoninic acid protein assay). Whole cell extracts (0.5 mg protein) were incubated with 2 μg anti-CBP antibody (Santa Cruz) overnight at 4°C and further incubated with Protein A/G-conjugated agarose beads (Santa Cruz) for 2 h at 4°C. The beads were washed five times with lysis buffer. Total cell lysates and CBP immunoprecipitates were boiled for 3 min and electrophoresed. Protein was transferred from the gels to nitrocellulose membranes and blocked with 5% non-fat dry milk for 2 h. Blots were probed with polyclonal antibodies against CBP, PDGF-B and GAPDH (Santa Cruz) and then probed with secondary antibodies (Pierce). Proteins were detected by the ECL Plus detection kit (Pierce). The expression level of CBP and PDGF-B were indicated as a ratio to GAPDH, respectively.

2.4. PDGF-B ELISA assay

ECs, seeded at 4×105/ml into six-well plates, were cultured for 24 h in standard medium and then stimulated for 72 h with thrombin concentrations ranging from 0.01 to 10 units/ml in EBM with 2% FBS. The conditioned medium was collected, centrifuged and stored at −80°C. The PDGF-B immunoassays (R&D Systems) were performed using supernatants according to the manufacturer.

2.5. Cell proliferation assay

Cells (25000 cells/well) were seeded in 24-well culture plates for 24 h. After 72 h adenovirus infection, different treatments were performed, and cells underwent evaluation for proliferation by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and BrdU (bromodeoxyuridine) assay.

For MTT assay, cells were washed twice with PBS, and l ml of serum-free medium containing 100 μg/ml MTT was added to each well. After incubation at 37°C for 4 h, MTT-containing medium was removed; cells were washed with PBS, 0.5 ml DMSO was added to dissolve MTT crystals. One hundred microlitres of solution from each well was transferred into 96-well microplate and measured for absorbance at 570 nm.

For BrdU incorporation assay, BrdU, final concentration 10 μmol/l, was introduced into the culture medium for the cells labelling during the final 24 h of incubation. Then, the culture plate was washed, and a colorimetric BrdU cell proliferation assay was performed according to the manufacturer's instruction (Calbiochem). Cells incubated without BrdU served as the negative control. The absorbance was read at a wavelength of 450 nm with a spectrophotometric plate reader. The mean absorbance for each group was determined after subtracting the mean value of the negative control.

2.6. Cell cycle analysis

After treatment, cells were harvested and fixed with 70% ethanol at 4°C overnight, followed by incubation with 100 μl RNase (Sigma–Aldrich) at 37°C for 30 min. The samples were stained with propidium iodide (Sigma–Aldrich) at 4°C for 2 h and analysed with a FACSscan (Becton–Dickinson).

2.7. Data analysis

Data are presented as means±S.E.M. All values were analysed using one-way ANOVA (analysis of variance) and the Newman–Keuls–Student t test. A P-value <0.05 was considered significant.

3. Results

3.1. Inhibition of CBP gene expression by shRNA-mediated silencing

As shown in Figure 1, CBP expression level was dramatically reduced in ECs transfected with CBP–shRNA/Ad, but not in ECs transfected with Ad (N). Compared with the control group, 45% and 64.3% reduction of CBP mRNA were observed in ECs transfected with CBP–shRNA/Ad at the MOI of 25, 50 in the presence of 0.1 unit/ml thrombin, respectively. Western blot analysis also verified that expression of CBP protein was abolished by 33.1 and 60.6%, respectively (P<0.05 compared with thrombin group).

3.2. PDGF-B is involved in the mitogenic effect of thrombin on ECs

Thrombin induced PDGF-B expression in a dose-dependent manner (Figure 2A). Levels of PDGF-B in the medium reached a peak value after treatment with 0.1 unit/ml thrombin. ECs stimulated with thrombin at all concentrations release more PDGF-B than the control. In addition, thrombin significantly caused dose-dependent increase of ECs proliferation (Figure 2B), assayed by BrdU incorporation assay, concomitant with an increase in PDGF-B release.

To assess whether the proliferative activity of thrombin depends on the release of PDGF-B from ECs, the inhibitory effect of anti-PDGF-B antibody was evaluated. The anti-PDGF-B antibody at concentrations of 10, 20 and 40 μg/ml deceased the BrdU incorporation by 23.8, 39.0 and 49.9% (P<0.05 compared with thrombin group) (Figure 2C), respectively. Non-specific IgG antibody did not affect the mitogenic effect of thrombin on ECs. Furthermore, anti-PDGF-B antibody could also inhibit cell cycle progression induced by thrombin in a dose-dependent manner (Figures 2D and 2E).

3.3. Down-regulation of CBP attenuates ECs proliferation induced by thrombin

Thrombin, 0.1 unit/ml, induced an ∼200% increase in MTT assay and a 180% increase in BrdU incorporation assay compared with the control (P<0.05 compared with control group). Down-regulation of CBP inhibited thrombin-induced proliferation of ECs both in MTT assay and BrdU incorporation assay (Figure 3). The antiproliferative effect of CBP down-regulation was dose dependent, with maximal inhibition by CBP–shRNA/Ad at the MOI of 50, the highest CBP gene-silence tested (P<0.05 compared with thrombin group). These findings were in good agreement with the results obtained with MTT assay.

3.4. Silencing CBP expression induces cell cycle arrest in thrombin-stimulated ECs

Cells in control group were mainly distributed in the G0-/G1-phase (79.4±2.90%). After 24 h treatment with 0.1 unit/ml thrombin, FACS analysis revealed approximately 63.7% and 23.9% of cells in G0-/G1- and S-phases, respectively. Down-regulation of CBP increased the percentage of cells in G0-/G1-phase and decreased the fraction in S-phase (Figure 4). ECs transfected with CBP–shRNA/Ad at the MOI of 25, 50 remained mostly in the G0-/G1-phase with only approximately 15.3% and 9.6% of cells entering S-phase, respectively (P<0.05 compared with thrombin group).

3.5. Down-regulation of CBP inhibited PDGF-B expression induced by thrombin in ECs

As shown in Figure 5, in non-transfected cells, thrombin significantly enhanced PDGF-B expression. In comparison, thrombin failed to promote PDGF-B expression in ECs transfected with CBP–shRNA/Ad. Western blot analysis indicated that total PDGF-B was blunt by 18.1% and 49.0% in ECs transfected with CBP–shRNA/Ad at the MOI of 25 and 50, respectively. A similar effect on PDGF-B mRNA was also observed.

4. Discussion

Several studies have shown that CBP is activated by different mitogens for vascular cell, such as transforming growth factor-β (Topper et al., 1998) and VEGF (Zippo et al., 2004), indicating that CBP may be involved in ECs proliferation. Our results demonstrated that direct inhibition of CBP expression prevented the mitogenic effect of thrombin on ECs, suggesting that CBP could be one of the key transcription coactivators to regulate ECs proliferation in response to thrombin. This conclusion is based on several evidence in our study: (i) the growth inhibition of endothelial cell by silencing CBP expression was not the result of cytotoxic mechanism, since no significant decrease of cell viability was detected in ECs transfected with CBP–shRNA/Ad assessed by Trypan Blue exclusion, (ii) the antiproliferative effect of CBP knockdown is due to the inability of DNA synthesis and S-phase progression.

One major question we asked was how CBP silencing inhibited ECs proliferation induced by thrombin. Since some evidence has implicated PDGF-B as an important downstream mediator of the angiogenic effect of thrombin (Borrelli et al., 2001), we found that thrombin-induced PDGF-B expression in ECs was suppressed by CBP down-regulation. Therefore, we suspect that ECs with CBP knockdown are generally defective in PDGF-B transcription stimulated with thrombin. Because CBP possesses HAT (histone acetyltransferase) activity and is capable of multiple interactions with transcription regulators (Vo and Goodman, 2001), it may function as a signal integrator and co-ordinate complex signal transduction events at the level of gene transcription. Transcription factor Egr-1 is involved in overexpression of PDGF-B induced by multiple physiological stimuli in ECs (Khachigian et al., 1996, 1997). Moreover, CBP acts as a transcription coactivator for Egr-1 (Silverman et al., 1998; Silverman and Collins, 1999), which is expressed at high levels in ECs induced by thrombin (Wu et al., 2002; Minami et al., 2004) or in human atherosclerosis (McCaffrey et al., 2000; Harja et al., 2004). Thus, CBP silencing could decrease the functional interaction with Egr-1 and inhibit the Egr-1-dependent gene transcription, including PDGF-B.

Although our previous study indicated that thrombin could induce CBP expression in VSMCs (vascular smooth muscle cells) (Chen et al., 2008), there was no such effect observed in ECs. This discordance in thrombin response between ECs and VSMCs is not unique to CBP. For example, thrombin induces expression on VEGF in fibroblasts (Ollivier et al., 2000) and VSMCs (Bassus et al., 2001), whereas there is no effect of thrombin on stimulation of VEGF in ECs (Minami et al., 2004). These results suggest that thrombin signalling varies between cell types, and these differences may be regulated by differential expression and/or activity of its receptors, since other studies have suggested that levels of thrombin-binding receptors are differentially regulated in ECs and VSMCs (Minami et al., 2004). Although we found that thrombin did not affect CBP expression, CBP knockdown had inhibitory effect on ECs proliferation induced by thrombin. Previously, it has been reported that MAP kinase, which is activated by thrombin in ECs, can phosphorylate CBP, and this aids CBP-mediated transcriptional activation (Janknecht and Nordheim, 1996). We propose that down-regulation of CBP attenuated its potential activation in response to thrombin and thus failed to activate some transcription factors, which are important for cell proliferation. Further experiments are needed to prove this hypothesis.

Because PDGF-B works as a mitogenic factor for ECs, decreased PDGF-B level due to CBP knockdown could inhibit cell cycle progression. However, we could not exclude the possibility that CBP may also play a direct role in cell cycle. One characteristic of CBP involving its HAT activity (Chan and La Thangue, 2001), which can influence chromatin remoulding by modulating histones, controls the G1/S transition during cell cycle (Ait-Si-Ali et al., 2000). CBP HAT activity is maximal at a time point preceding the G1/S transition (Ait-Si-Ali et al., 2000). Some studies suggest that, near the G1/S transition, CBP is recruited to transcription factor E2F during G1/S transition (Wang et al., 2007), leading to increased gene expression either in DNA synthesis or in cell cycle controls (Trouche et al., 1996; Lee et al., 1998; Morris et al., 2000). Therefore, the possibility could not be excluded that down-regulation of CBP could, in part, impair ECs proliferation in response to some stimuli directly via cell cycle pathway.

In summary, CBP has emerged as an important player in thrombin-regulated ECs proliferation. Since CBP is regarded as the transcriptional coactivator to play a central role in integrating multiple signal-dependent events, it is possible that CBP is also involved in the process of proliferation induced by other external stimuli, such as VEGF, basic fibroblast growth factor and epidermal growth factor. Therefore, further elucidation of the precise roles of CBP in response to these factors will increase our understanding of the important effect of CBP on ECs proliferation during vascular injury.

Author contribution

Hong Jiang and Jing Chen contributed to the study design and most of the experiments. Lang Wang and Li-hua Zhu contributed to the cell culture. Hua-zhi Wen contributed to the ELISA assay.


This work was supported by the National Science Foundation of China NSDC [grant no. 30770849].


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Received 16 October 2009/12 July 2010; accepted 19 August 2010

Published as Cell Biology International Immediate Publication 19 August 2010, doi:10.1042/CBI20090304

© The Author(s) Journal compilation © 2010 Portland Press Limited

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)