Accepted Manuscript Gastrin-releasing peptide induces monocyte adhesion to vascular endothelium by upregulating endothelial adhesion molecules Mi-Kyoung Kim, Hyun-Joo Park, Yeon Kim, Hyung Joon Kim, Soo-Kyung Bae, MoonKyoung Bae PII:
S0006-291X(17)30092-X
DOI:
10.1016/j.bbrc.2017.01.058
Reference:
YBBRC 37120
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 4 January 2017 Accepted Date: 12 January 2017
Please cite this article as: M.-K. Kim, H.-J. Park, Y. Kim, H.J. Kim, S.-K. Bae, M.-K. Bae, Gastrinreleasing peptide induces monocyte adhesion to vascular endothelium by upregulating endothelial adhesion molecules, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.01.058. 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 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.
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Gastrin-releasing peptide induces monocyte adhesion to vascular endothelium by upregulating endothelial adhesion
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molecules
Mi-Kyoung Kim1§, Hyun-Joo Park1,2§, Yeon Kim1, Hyung Joon Kim1, Soo-Kyung
Department of Oral Physiology, 2 Department of Dental Pharmacology, BK21 PLUS
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Bae2, Moon-Kyoung Bae1*
Project, School of Dentistry, Pusan National University, Yangsan 626-870, South
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Korea
Denotes co-first authors
*
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Running Title: Effect of GRP on monocyte adhesion to endothelial cells
Corresponding Author: Moon-Kyoung Bae, Department of Oral Physiology, School
of Dentistry, Pusan National University, Yangsan 626-770, South Korea; Tel. 82-51-510-8239; Fax. 82-51-510-8238; E-mail:
[email protected]
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ABSTRACT
Gastrin-releasing peptide (GRP) is a neuropeptide that plays roles in various
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pathophysiological conditions including inflammatory diseases in peripheral tissues; however, little is known about whether GRP can directly regulate endothelial inflammatory processes. In this study, we showed that GRP promotes the adhesion of
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leukocytes to human umbilical vein endothelial cells (HUVECs) and the aortic endothelium. GRP increased the expression of intercellular adhesion molecule-1
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(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by activating nuclear factor-κB (NF-κB) in endothelial cells. In addition, GRP activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38MAPK, and AKT, and the inhibition of these signaling pathways significantly reduced GRP-induced monocyte adhesion to the
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endothelium. Overall, our results suggested that GRP may cause endothelial dysfunction, which could be of particular relevance in the development of vascular inflammatory
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disorders.
KEYWORDS Gastrin-releasing peptide; endothelial adhesion molecules; NF-kappaB; vascular endothelial cells 2
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INTRODUCTION
The inflammatory reaction requires multifaceted interactions between the
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vascular endothelium and inflammatory cells (monocytes, neutrophils, and lymphocytes) [1]. In an inflammatory environment, activated vascular endothelial cells express adhesion molecules such as selectins, intercellular adhesion molecule-1
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(ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) to enable the trafficking and adherence of monocytes or lymphocytes to the endothelium [2]. The expression
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of endothelial adhesion molecules [3] is significantly induced in response to several inflammatory stimuli including lipopolysaccharide, interleukin-1, tumor necrosis factor-α (TNF-α), and neuropeptides such as substance P [4]. Gastrin-releasing
peptide
(GRP),
a
primary
member
of
mammalian
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bombesin-like peptides, is a small regulatory neuropeptide [5], which is a homolog of the amphibian bombesin peptide. GRP has been implicated in different physiological processes including exocrine and endocrine secretions, gastrointestinal motility, pain
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perception, and stress responses [6]. In addition, GRP acts as a growth factor,
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morphogen [7], and pro-angiogenic factor [8] in some types of cancers [9]. GRP also promotes neutrophil migration and recruitment [10], indicating that GRP is an endogenous inflammatory mediator. Biological effects of GRP are mediated by GRP receptor (GRP-R, BB2), a member of the G protein–coupled receptor (GPCR) family that is expressed in various cell types [11]. It has been reported that GRP receptors are present in human vascular endothelial cells and mediate the pro-angiogenic effects of GRP [8]. 3
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In this study, we examined the effects of GRP on the adhesion of monocytes to vascular endothelial cells and the expression of endothelial adhesion molecules in GRP-stimulated HUVECs. We demonstrated that GRP increased monocyte adhesion to
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HUVECs at least in part through the activation of the NF-κB and MAPK/AKT signaling
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pathways.
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MATERIALS AND METHODS
Reagents and antibodies
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GRP and RC-3095 were purchased from Sigma. Calcein-AM was obtained from Molecular Probes. LY294002 and SB203580 were purchased from Calbiochem. PD98059 and pyrrolidine dithiocarbamate (PDTC) were supplied by A.G. Scientific
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and Sigma, respectively. The antibodies for ICAM-1, VCAM-1, phospho-IKK, IKK,
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phospho-IκB, IκBα, and NF-κB p65 were supplied by Santa Cruz Biotechnology. The antibodies for phospho-ERK, ERK, phospho-p38, p38, phosphor-Akt, and Akt were obtained from Cell Signaling. Human α-tubulin and β-actin antibodies were purchased from Bioworld Technology. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Thermo Fisher Scientific. Alexa 488-conjugated
Cell culture
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goat anti-mouse IgG was purchased from Life Technologies.
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Primary human umbilical vein endothelial cells (HUVECs) (passage 5-8) were
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purchase from CLONETICS. HUVECs were plated onto 0.2% gelatin-coated dishes and grown in sterile endothelial growth medium (EGM-2, CLONETICS). EGM-2 was composed of endothelial basal medium (EBM-2, CLONETICS) and trace elements, growth factors, and antibiotics. The U937 monocytic cells were grown in RPMI-1640 (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotics. The cells were grown at 37°C in a humidified 95% air/5% CO2 environment.
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In vitro monocyte adhesion assay HUVECs were plated on 24-well plates at 5×104 cells/well and incubated with GRP alone or in combination with inhibitors for 2 hours. The U937 cells were then
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added (5×105 cells/mL, 200 µL/well) to the confluent monolayers of HUVECs and incubated for 1 h. The non-adherent monocytes were removed by washing twice with PBS. The adherent cells were fixed and washed twice with PBS. The adherent
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monocytes were counted in 3 consistent fields using a fluorescence microscope
for each set of four wells.
Ex vivo monocyte adhesion assay
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(Nikon). The average number of adherent monocytes was calculated in the three fields
Male Sprague-Dawley rats (SD, 6 weeks of age), weighing 210−230 g were
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obtained from Samtako, Osan, Korea. The aortas were opened longitudinally, and incubated with GRP alone or in combination with inhibitors for 4 hours. The aortas were then incubated for 1 h with 1×106 fluorescence-labeled (using Calcein-AM)
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monocytes. After incubation, the unbound monocytes were washed away. The
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adherent monocytes were counted in 3 consistent fields using a fluorescence microscope (Nikon).
Western blot analysis
Harvested cells were lysed in a lysis buffer (40 mM Tris-Cl, 10 mM EDTA, 120 mM NaCl, and 0.1% NP-40 with protease inhibitor cocktail (Sigma)). A constant protein concentration (30 µg/lane) was used. Proteins were separated by SDS/PAGE 6
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and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% skim milk in PBS containing 0.1% Tween-20 for 1 h at room temperature and probed with the appropriate antibodies. This signal was
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developed with the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech).
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RT-PCR analysis
Total RNA was isolated from HUVECs with a TRIzol reagent kit (Invitrogen). cDNA
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synthesis was performed on 2 µg of total RNA with a reverse transcription kit (Promega). The oligonucleotide primers for PCR were designed as follows: β-actin, 5′-GACTACCTCATGAAGATC-3′ and 5′-GATCCACATCTGCTGGAA-3′; ICAM-1, 5′-CAGTGACCATCTACAGCTTT-3′ and 5′- GCTGCTACCACAGTGATGAT-3′;
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TTC-3′.
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VCAM-1, 5′-GATACAACCGTCTTGGTCAG-3′ and 5′- CATATACTCCCGCATCC
Real-time PCR analysis
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Real-time PCR quantification was performed using a SYBR Green method (Light Cycler; Roche Applied Science). Cycling parameters included 1 cycle at 95°C for 10 min, followed by amplification for 30 cycles at 95°C for 10 s, 57°C for 5 s, and 72°C for 7 s. A melting curve program was subsequently applied with continuous fluorescence measurements. The entire cycling process, including data analysis, took less than 1 h and was monitored using Light Cycler software (version 4.0). The oligonucleotide
primers
for
real-time 7
PCR
were
as
follows:
β-actin,
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5′-ACTCTTCCAGCCTTCCTTCC-3′
and
5′-TGTTGGCGTACAGGTCTTTG-3′;
ICAM-1, 5′- CCCCACCATGAGGACATACA-3′ and 5′- GTGTGGGCCTTTGTGT TTTG-3′; VCAM-1, 5′- GGCTGTGAATCCCCATCTTT-3′ and 5′- TCCACCTGGA
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TTCCCTTTTC-3′.
Transfection by Amaxa Nucleofector II
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For electroporation with Amaxa Nucleofector II (Lonza), cells were washed with PBS
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and detached with trypsin (Invitrogen). Approximately 1 × 106 cells were transferred into a new tube, centrifuged at 200 g for 10 min at room temperature and the pellet was suspended in 100 µl of transfection solution. After addition of 3 µg of plasmid DNA, the cells were transferred into the electroporation cuvette without introducing air bubbles and then pulsed. Afterwards, 400 µl of supplemented full medium warmed to
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37°C were added to the pulsed cells, followed by a 15 min incubation at 37°C in an atmosphere of 5% (v/v) CO2. The cells were dispensed into 4 wells of a 24-well plate.
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After 24 h, the cells were analyzed by luciferase assay.
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Luciferase assay
Cells were harvested 24 h after transfection, rinsed with PBS, resuspended in reporter cell lysis buffer (Promega), and incubated for 10 minutes at room temperature. The lysate was centrifuged at 12,000 × g for 5 min to pellet the cell debris. The supernatants were transferred to a fresh tube. A 10 µL-aliquot of the extract was added to 25 µL of the luciferase assay substrate (Promega) and the luminescence of samples was read immediately on a 20/20 Luminometer (Tuner Biosystems). β-galactosidase 8
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activity was used for normalizing the transfection efficiency and protein input. A 20 µL-aliquot
of
the
cell
extract
was
mixed
with
20
µL
of
O-nitrophenyl-β-D-galactopyranoside (ONPG) solution. The absorbance of the
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mixture was determined at 420 nm after 30 min of incubation at 37°C. Each extract was assayed at least three times and the relative luciferase activity was calculated as RLU/β-galactosidase. The ICAM-1 and VCAM-1 luciferase reporter constructs with
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the full-length (ICAM-1: -1350 to +45 bp, VCAM-1: -1716 to +119 bp) and truncated forms (ICAM-1: -485 to +45 bp, VCAM-1: -213 to +119 bp) were used as previously
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described [12].
Immunocytochemistry
Cells cultured on coverslips were fixed in 4% paraformaldehyde for 10 min,
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blocked with 0.5% Triton X-100/PBS for 5 min, and then labeled with the appropriate primary and FITC-conjugated secondary antibodies. Coverslips were mounted in Vectastain containing DAPI (Vector Laboratories). The cells were analyzed by
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fluorescence microscopy (Nikon).
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Statistical analysis
Data shown are the mean ± standard deviation (S.D.) obtained for at least three
independent experiments. Statistical comparisons between groups were made by one-way analysis of variance (ANOVA) followed by the Student’s t-test.
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RESULTS
GRP promotes monocyte adhesion to vascular endothelial cells
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Adhesion of leukocytes to the vascular endothelial cells is a critical step in vascular inflammation [2]. To determine the role of GRP in vascular inflammation, we first tested whether GRP promotes the adhesion of leukocytes to endothelial cells. HUVECs
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were exposed to various concentrations of GRP for 2 h and co-cultured with U937 monocytes for an additional 1 h. As shown in Figure 1A, the adhesion of
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fluorescent-labeled U937 cells onto endothelial cells was augmented by GRP stimulation in a dose-dependent manner. Next, an ex vivo monocyte adhesion assay was further conducted in a co-culture system of fluorescent-labeled U937 monocytes and an aorta isolated from a SD rat. As shown in Figure 1B, fluorescent-labeled monocytes
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were observed on the surface of the aortic endothelium, and GRP treatment increased the number of adherent cells compared to the number observed on the control aorta.
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HUVECs
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GRP increases the mRNA and protein expression of ICAM-1 and VCAM-1 in
ICAM-1 [13] and VCAM-1 [3] are both well-known to mediate monocyte
adhesion to the endothelium during inflammation. To examine the effect of GRP on the expression of ICAM-1 and VCAM-1 in endothelial cells, HUVECs were treated with GRP for the indicated times, and then levels of ICAM-1 and VCAM-1 protein were measured by western blot analysis. As shown in Figure 2A, GRP significantly increased ICAM-1 and VCAM-1 protein levels in a dose-dependent manner, with a peak at 4 10
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hours, followed by a slight decline by 8 hours. The levels of ICAM-1 and VCAM-1 mRNA in GRP-treated HUVECs were further analyzed by RT-PCR. Consistent with the western blot analysis, GRP treatment significantly induced the expression of ICAM-1
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and VCAM-1 mRNA in HUVECs (Fig. 2B). Using real-time RT-PCR, we also quantified the expression levels of ICAM-1 and VCAM-1 mRNA enhanced by GRP in a time- and dose-dependent manner (Fig. 2 C and D). The action of GRP is mediated by
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GRP receptor, which is expressed on many types of cells including endothelial cells [8]. The presence of GRP receptor mRNA and protein in endothelial cells was confirmed by
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RT-PCR analysis and western blot analysis, respectively (Fig. 2E). Next, we tested the effect of RC-3095, a selective GRP receptor antagonist, on the GRP-mediated increase expression of ICAM-1 and VCAM-1 mRNA. As shown in Figure 2F, GRP-induced expression of ICAM-1 and VCAM-1 mRNA was suppressed by pretreatment with
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RC-3095.
GRP increases transcriptional activity of ICAM-1 and VCAM-1 genes via NF-κB
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activation in HUVECs
To determine whether GRP enhanced ICAM-1 and VCAM-1 transcriptional
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activity, we performed promoter assays using luciferase reporter constructs. The full-length ICAM-1 promoter (1.3 kb) contains binding sites for several transcription factors, including NF-κB, AP1, and STAT, while the full-length VCAM-1 promoter (1.8 kb) has binding sites for NF-κB, TRE, and GATA [12]. Both the ICAM-1 and VCAM-1 truncated forms contain proximal NF-κB binding sites. The transcriptional activities of these constructs were determined following transient transfection into HUVECs. As 11
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shown in Figure 3A and B, GRP treatment increased promoter activity of the truncated ICAM-1 and VCAM-1 promoters, and a similar increase was also observed in the reporter activities of full-length ICAM-1 and VCAM-1 genes. The pGL3 control vector
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without ICAM-1 and VCAM-1 promoter sequences was not affected by GRP. These results indicated that NF-κB binding sites were necessary for the GRP-induced transcriptional activity of ICAM-1 and VCAM-1 genes.
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NF-κB activation mainly occurs via phosphorylation, ubiquitination, and proteolytic degradation of IκB-α, which releases the p65 subunit of NF-κB for
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translocation to the nucleus [14]. Therefore, we determined whether GRP stimulated the phosphorylation and degradation of IκB-α in HUVECs. HUVECs were exposed to GRP, and IκB-α activation was analyzed by western blot analysis using a specific antibody against the phosphorylated form of IκB-α. As shown in Figure 3C, GRP treatment
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resulted in IκB-α phosphorylation with maximal effect after 30 min of GRP stimulation. Some degradation of IκB-α was observed by 30 min. Next, we wanted to confirm the translocation
of
NF-κB
by
GRP
using
immunofluorescence
microscopy.
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Immunocytochemistry assay showed that in untreated cells, p65 was localized primarily
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in the cytoplasm, whereas GRP treatment induced p65 translocation into the nucleus (Fig. 3D).
Involvement of the ERK1/2, p38MAPK, and PI3K/Akt pathways in GRP-induced vascular inflammation To elucidate the underlying signaling pathway by which GRP exerted its endothelial inflammatory activity, the effect of GRP treatment on the phosphorylation of 12
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MAP kinases (ERK1/2 and p38) and Akt in HUVECs was examined. HUVECs were treated with GRP for the indicated times, and then the phosphorylation of ERK, p38MAPK, and Akt was measured by western blot analysis. As shown in Figure 4A,
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GRP treatment significantly increased phosphorylation of ERK1/2, p38MAPK, and Akt in a time-dependent manner, with a peak at 10 min, followed by a slight decline by 30 min. Next, to determine the effect of pharmacological kinase inhibitors of MEK
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(PD98059), p38MAPK (SB203580), and PI3-kinase (LY294002) on GRP-induced monocyte binding to the ex vivo aortas, an ex vivo adhesion assay was performed using
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fluorescent-labeled U937 monocytes and an aorta isolated from a SD. As shown in Figure 4B, pretreatment with PD98059, SB203580, and LY294002 decreased GRP-induced adhesion of monocytes to aortic endothelium; especially, SB203580 markedly suppressed GRP-induced adhesion between the monocytes and the
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endothelium.
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DISCUSSION
GRP was first characterized as a neuropeptide that stimulates gastrin secretion
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in the gastrointestinal tract [15]. GRP acts by interacting with the GRP receptor, a member of the G-protein coupled receptor superfamily expressed in the digestive, endocrine, and nervous systems [16]. The GRP receptor mediates gastrointestinal
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motility and secretion of pancreatic enzymes, hormones in the gastrointestinal tract, and neurotransmitters in the central nervous system [15]. In addition, GRP and the
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GRP receptor are overexpressed in many kinds of cancer cells, and the presence of both GRP and the GRP receptor results in tumor cell proliferation, growth, invasion, and metastasis [17]. Specific GRP receptor antagonists have been developed as candidate anticancer compounds, such as RC-3095, which shows attractive antitumor
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activity in vitro and in vivo [18]. Recent investigations have unveiled a potential role for GRP in various inflammatory responses through pharmacological inhibition with GRP receptor antagonists [19]. It has been reported that GRP receptor antagonism has
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anti-inflammatory effects in experimental arthritis [20], an animal model of sepsis
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[21], and an acute inflammation model in rats [22]. RC-3095 inhibits NF-κB and AP-1 signaling pathways and the production of inflammatory mediators, monocyte chemoattractant protein-1 (MCP-1) and IL-6, in macrophages stimulated with LPS [23]. GRP levels are correlated with the production and release of proinflammatory cytokines in patients with rheumatoid arthritis [24]. In addition, GRP directly induces GPR receptor-mediated neutrophil chemotaxis [10]. In this study, we demonstrated that GRP directly activated vascular endothelial cells and subsequently increased 14
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monocyte-endothelial adhesion through the GRP receptor. Therefore, it is possible that blocking the GRP receptor with antagonists, antisense oligonucleotides, and monoclonal antibodies may reduce the vascular inflammatory response by
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downregulating the expression of adhesion molecules on endothelial cells as well as by inhibiting transendothelial neutrophil migration.
The NF-κB transcription factor plays a critical role in the development of
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inflammation through regulating the expression of pro-inflammatory genes in response to various inflammatory stimuli [14, 25]. Activating the phosphoinositide 3-kinase
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(PI3K)/Akt pathway stimulates NF-κB p65/RelA transactivation [26]. In addition, MAPK pathways are required for p65/RelA transactivation [27]. We demonstrated in this study that GRP induced monocyte adhesion to endothelial cells through parallel pathways; an NF-κB-dependent pathway and ERK1/2, p38MAPK, and Akt signaling
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pathways. However, we cannot exclude the possibility that GRP may regulate NF-κB activity via activating signal transduction pathways such as MAPK and PI3K/Akt pathways. This possibility is currently under investigation.
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Our data in this study and other reports showed that the GRP receptor is expressed on
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vascular endothelial cells [8]. GRP directly stimulates endothelial migration and tubule formation via endothelial GRP receptor, and GRP blockade reduces GRP-induced endothelial angiogenesis [8, 28]. Additionally, GRP treatment enhances endothelial cell proliferation and decreases the expression of key autophagy proteins [29]. In this study, we demonstrated that GRP directly stimulated endothelial dysfunction. There is increasing evidence that autophagy can modulate the inflammatory response during pathophysiological conditions [30, 31]. Further studies will be needed to examine 15
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whether GRP mediates crosstalk between inflammation and autophagy in resting or activated endothelium. In conclusion, these results provided the first evidence that GRP increases
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expression of ICAM-1 and VCAM-1 through NF-κB activation in endothelial cells, and that GRP stimulates monocyte adhesion onto vascular endothelial cells. Our findings suggest a potential role for GRP in the pathogenesis of vascular diseases associated with
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vascular inflammatory reaction.
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ACKNOWLEDGEMENTS
This research was supported by a grant from Basic Science Research Program
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through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015R1A2A2A01002980) and by the Financial Supporting Project of Long-term Overseas Dispatch of PNU's Tenure-track Faculty,
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2014.
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Cell Biol. 91 (2013) 250-258. [31] P. Lapaquette, J. Guzzo, L. Bretillon, M.A. Bringer. Cellular and molecular connections between autophagy and inflammation, Mediators Inflamm. 2015 (2015)
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FIGURE LEGENDS
Figure 1. GRP increases monocytes adhesion to the endothelium
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(A) For the in vitro monocyte adhesion assay, HUVECs were incubated with GRP at the indicated concentrations for 2 h, and the calcein-exposed monocytes were co-incubated with HUVECs for 1 h. The number of adherent monocytes was counted. (B) An ex vivo
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monocyte adhesion assay was performed using rat aorta. The rat aortas were treated GRP for 4 h. Adherent monocytes to endothelium were observed under fluorescence
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microscope and photographed. The number of adherent monocytes was counted. These data represent the mean ± SE of three experiments. *P<0.05 vs. control.
Figure 2. GRP increases expression of ICAM-1 and VCAM-1 in HUVECs
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HUVECs were incubated with GRP (100 nM) for the indicated times (A, B, and C) or stimulated with various concentrations of GRP for 4 h (D). (A) ICAM-1 and VCAM-1 protein levels were examined by western blotting using anti-ICAM-1 and anti-VCAM-1
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antibodies. β-actin served as the loading control. (B) Total RNA were isolated and then
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analyzed by RT-PCR using specific primers to human ICAM-1 and VCAM-1. β-actin served as an internal control. (C-D) Using real-time PCR, the levels of ICAM-1 and VCAM-1 mRNA were also quantified. *P<0.05; **P<0.01 vs. control. (E) Protein expression level of NMB-R was confirmed by western blotting using an anti-GRP-R antibody. β-actin served as the loading control. The total RNA was isolated and then analyzed by RT-PCR using primers specific to human GRP receptor (GRP-R). β-actin served as an internal control. HMVECs, human dermal microvascular endothelial cells. 23
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(F) HUVECs were incubated with GRP (100 nM) alone or in combination with RC-3095 (100 nM) for 4 h. Using real-time PCR, the expression levels of ICAM-1 and VCAM-1 mRNA were also quantified. The expression level of the control (untreated)
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was set to 1, and the values were normalized to the β-actin mRNA levels. *P<0.01 vs. control; #P<0.05 vs. GRP alone.
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Figure 3. GRP stimulates transcriptional activity of ICAM-1 and VCAM-1 genes via NF-κB in HUVECS
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(A and B) Top, schematic representation of full-length and truncated (containing NF-κB motif) promoter of ICAM-1 (A) and VCAM-1 (B) genes. HUVECs were transiently transfected with ICAM-1 or VCAM-1 luciferase plasmids that contain full length ICAM-1 (1.3 kbp) or truncated ICAM-1 (-200 bp) promoter regions, respectively (A),
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or the VCAM-1 (1.8 kbp) or truncated VCAM-1 (65 and 75 bp) promoter regions, respectively (B), together with a β-galactosidase plasmid, and exposed to GRP (100 nM) for 4 h. Luciferase activity was normalized to β-galactosidase activity. Data are
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means ± SE of luciferase light units from triplicate experiments with the activity of
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untreated cells set at 100%. *P<0.05 vs. control. (C) HUVECs were incubated with GRP (100 nM) for the indicated times. Western blots were probed with anti-phospho-IκB-α and anti-IκB-α antibodies. β-actin served as the loading control. (D) HUVECs were incubated with GRP (100 nM) for 1 h. Immunocytochemical analysis of NF-κB p65 was performed. The cells were stained with NF-κB p65 (green) and nuclei were stained with DAPI (blue). Representative images are shown from at least two independent experiments. 24
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Figure 4. ERK1/2, p38MAPK, and PI3K/Akt pathways are involved in GRP-induced leukocyte adhesiveness to endothelium
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(A) HUVECs were treated with GRP (1 µM) for the various times shown. Levels of total and phosphorylated ERK1/2, p38MAPK, and Akt were determined by western blotting.
These data represent the mean ± SE of three experiments. (B) Rat aortas
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were incubated with GRP (1 µM) for 4 h in the presence or absence of LY294002 (LY, 20 µM), PD98059 (PD, 50 µM), or SB203580 (SB, 50 µM). Then, fluorescent-labeled
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monocytes were co-incubated with aortas. Aortas were observed under a fluorescent microscope, and the number of adherent cells on endothelium was counted. Three independent experiments were performed. *P<0.05 vs. control; #, P<0.05 vs. GRP
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Research Highlights
● Gastrin-releasing peptide (GRP) induces adhesion of monocytes to human umbilical vein
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endothelial cells (HUVECs) and aortic endothelium.
● GRP markedly increases the expression of ICAM-1 and VCAM-1 through the activation of
ERK1/2, p38MAPK, and PI3K/Akt pathways are involved in the GRP-induced leukocyte
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adhesiveness to endothelium
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●
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NF-κB in vascular endothelial cells.