Article pubs.acs.org/JAFC
Docosahexaenoic Acid Inhibits Vascular Endothelial Growth Factor (VEGF)-Induced Cell Migration via the GPR120/PP2A/ERK1/2/eNOS Signaling Pathway in Human Umbilical Vein Endothelial Cells Che-Yi Chao,†,∥ Chong-Kuei Lii,†,‡,∥ Siou-Yu Ye,‡ Chien-Chun Li,#,⊥ Chia-Yang Lu,‡ Ai-Hsuan Lin,‡ Kai-Li Liu,*,#,⊥ and Haw-Wen Chen*,‡ †
Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan Department of Nutrition, China Medical University, Taichung, Taiwan # School of Nutrition, Chung Shan Medical University, Taichung, Taiwan ⊥ Department of Nutrition, Chung Shan Medical University Hospital, Taichung, Taiwan ‡
ABSTRACT: Cell migration plays an important role in angiogenesis and wound repair. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is essential for endothelial cell survival, proliferation, and migration. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, shows both anti-inflammatory and antioxidant activities in vitro and in vivo. This study investigated the molecular mechanism by which DHA down-regulates VEGF-induced cell migration. HUVECs were used as the study model, and the MTT assay, Western blot, wound-healing assay, and phosphatase activity assay were used to explore the effects of DHA on cell migration. GPR120 is the putative receptor for DHA action. The results showed that DHA, PD98059 (an ERK1/2 inhibitor), and GW9508 (a GPR120 agonist) inhibited VEGF-induced cell migration. In contrast, pretreatment with okadaic acid (OA, a PP2A inhibitor) and S-nitroso-N-acetyl-DL-penicillamine (an NO donor) reversed the inhibition of cell migration by DHA. VEGF-induced cell migration was accompanied by phosphorylation of ERK1/2 and eNOS. Treatment of HUVECs with DHA increased PP2A enzyme activity and decreased VEGF-induced phosphorylation of ERK1/2 and eNOS. However, pretreatment with OA significantly decreased DHA-induced PP2A enzyme activity and reversed the DHA inhibition of VEGF-induced ERK1/2 and eNOS phosphorylation. These results suggest that stimulation of PP2A activity and inhibition of the VEGF-induced ERK1/2/eNOS signaling pathway may be involved in the DHA suppression of VEGF-induced cell migration. Thus, the effect of DHA on angiogenesis and wound repair is at least partly by virtue of its attenuation of cell migration. KEYWORDS: cell migration, docosahexaenoic acid (DHA), human umbilical vein endothelial cells (HUVECs), nitric oxide (NO), vascular endothelial growth factor (VEGF)
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INTRODUCTION Docosahexaenoic acid (DHA, 22:6, n-3) is enriched in fatty fish and fish oil supplements and is well-known for its antiinflammatory,1 immunomodulatory,2 and anticancer3 properties. Cancer is among the leading causes of death in both economically developed countries and developing countries.4 Epidemiological studies show that a diet rich in n-3 polyunsaturated fatty acids (PUFAs) is correlated with reduced risk of angiogenic diseases such as cancers.5,6 DHA has been shown to inhibit vascular sprout formation in retinal microvascular endothelial cells.7 However, the mechanism underlying the inhibition of cell migration by DHA, which is critical for angiogenesis and wound repair, is not fully understood. G protein-coupled receptors (GPRs) are important signaling molecules involved in many cellular functions. Specific ligand binding to GPRs stimulates and induces a variety of cellular responses via several second messenger pathways, for example, regulation of cAMP generation, the phospholipase C pathway, ion channels, and mitogen-activated protein kinases.8−10 In a previous study, Oh et al.11 found that DHA exerted potent antiinflammatory effects through GPR120. Angiogenesis, the process of the formation of new blood vessels by sprouting of the preexisting microvascular network, is © 2014 American Chemical Society
involved in numerous physiological processes including embryogenesis, tissue remodeling, wound healing,12 and disease development such as diabetic retinopathy, rheumatoid arthritis, tumor growth, and growth of atherosclerotic plaques.13 Angiogenesis is controlled by vascular endothelial growth factor (VEGF), a pro-angiogenic factor.14 VEGF-induced signal transduction involves binding to tyrosine kinase receptors, which leads to endothelial cell proliferation, migration, and new vessel formation.15 VEGF was reported to induce a wide variety of signaling pathways, including protein kinase C, phospholipase C-γ, extracellular signal-regulated kinase (ERK), p38 MAPK, phospholipase C, and phosphatidyl inositol 3-kinase (PI3K)/Akt.16 Hence, it is critical to understand the VEGFactivated signaling pathways that play an important role in VEGF-mediated cell processes. Nitric oxide (NO), which is synthesized from the amino acid L-arginine by the NOS family of enzymes, is a gaseous molecule Received: Revised: Accepted: Published: 4152
February 12, 2014 April 1, 2014 April 15, 2014 April 15, 2014 dx.doi.org/10.1021/jf5007165 | J. Agric. Food Chem. 2014, 62, 4152−4158
Journal of Agricultural and Food Chemistry
Article
The medium was removed, and 2-propanol was added to dissolve the formazan. After centrifugation at 10000g for 5 min, the supernatant of each sample was transferred to 96-well plates, and absorbance was read at 595 nm in an ELISA reader. The absorbance in the control group was regarded as 100% cell viability. Western Blotting Analysis. After each experiment, cells were washed twice with PBS and were harvested with 150 μL of lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 320 mM sucrose, 5 mM EDTA, 1 mM PMSF, 1 mg/L leupeptin, 1 mg/L aprotinin, and 2 mM DTT). Cell homogenates were centrifuged at 14000g for 20 min at 4 °C. The resulting supernatant was used as a cellular protein for Western blotting analysis. The total protein was analyzed by use of the Coomassie Plus protein assay reagent kit (Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of cellular proteins were electrophoresed in a sodium dodecyl sulfate (SDS)−polyacrylamide gel, and proteins were then transferred to polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). Nonspecific binding sites on the membranes were blocked with 5% nonfat milk in 15 mM Tris/150 mM NaCl buffer (pH 7.4) at room temperature for 2 h. Membranes were probed with anti-ERK1/2, antiphosphoERK1/2, anti-eNOS, antiphospho-eNOS, and anti-β-actin. The membranes were then probed with the secondary antibody labeled with horseradish peroxidase. The bands were visualized by using an enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA, USA) and were scanned by use of a luminescence image analyzer (LAS-4000, FUJIFILM, Japan). The bands were quantitated with ImageGauge software (FUJIFILM). Cell Migration Assay. An in vitro wound-healing assay was used to measure directional endothelial cell migration. HUVECs were seeded onto gelatin-coated 6-well plates and allowed to form a confluent monolayer. Monolayers were wounded by using a sterile 200 μL pipet tip, washed with PBS to remove floating cells, and photographed (time 0). Cells were then cultured in M199 medium containing 2% FBS and 50 ng/mL VEGF. Meanwhile, the cells were treated with various concentrations of DHA (0−100 μM) with or without SNAP (20 μM) or OA (10 nM) for 8 h. In addition, the cells were treated with GW9508 (10 μM) for 8 h. Cells were then photographed (100× magnification) to monitor cell migration into the wounded area, and the width of the cell-free zone (distance between the edges of the injured monolayer) was calculated. PP2A Activity Assay. PP2A activity was determined by use of a Ser/Thr phosphatase assay kit according to the instructions of the manufacturer (Upstate, Darmstadt, Germany). After treatment, cells were scraped with lysis buffer (10 mM tris/Triton X-100, 0.32 M sucrose, 5 mM EDTA) and protease inhibitors, sonicated for 10 s, and centrifuged at 2000g for 5 min. Thereafter, phosphopeptide (K-R-pTI-R-R) was added to the cell lysate, followed by incubation at room temperature for 15 min, and then Malachite Green phosphate was added and the reaction was allowed to proceed at room temperature for another 15 min for color development. The relative absorbance was measured at 650 nm in a microplate reader (Bio-Rad). Statistical Analysis. Data were analyzed by using analysis of variance (SAS Institute, Cary, NC, USA). The significance of the difference between mean values was determined by one-way analysis of variance followed by Tukey’s test. p values
