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‡,*
§
These authors contributed equally to this study.
†
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
*
To whom correspondence should be addressed. H.-W.C.: Department of Nutrition,
China Medical University, Taichung 404, Taiwan; telephone, +886 4 22053366, ext.
7520; fax, +886 4 2206 2891; e-mail, chenhw@mail.cmu.edu.tw. K.-L.L.:
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413,
and School of Nutrition, Chung Shan Medical University, Taichung 402; telephone,
+886 4 24730022, ext. 12136; fax, +886 4 2324 8175; e-mail, kaililiu@csmu.edu.tw
ABBREVIATIONS USED
COX-2, cyclooxygenase 2; DHA, docosahexaenoic acid; ECGS, endothelial cell
growth supplement; eNOS, endothelial NOS; EPA, eicosapentaenoic acid; ERK,
extracellular signal-regulated kinase; FBS, fetal bovine serum; GPRs, G
protein-coupled receptors; HUVECs, human umbilical vein endothelial cells; MAPK,
mitogen-activated protein kinase; MMP-9, matrix metalloproteinase 9; nNOS,
neuronal NOS; NO, nitric oxide; OA, okadaic acid; PBS, phosphate-buffered saline;
PGE2, prostaglandin E2; PI3K, phosphatidyl inositol 3-kinase; PP2A, protein
phosphatase 2A; PUFAs, polyunsaturated fatty acids; SNAP,
S-nitroso-N-acetyl-DL-penicillamine; VEGF, vascular endothelial growth factor.
Running title: DHA, Cell Migration and the GPR120/PP2A/ERK1/2/eNOS
Signaling Pathway
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. In this study, we investigated the molecular
mechanism by which DHA down-regulates VEGF-induced cell migration. We used
HUVECs as the study model and the MTT assay, Western blot, wound healing assay,
and phosphatase activity assay to explore the effects of DHA on cell migration.
GPR120 is the putative receptor for DHA action. Our 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 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)
1
INTRODUCTION
2
Docosahexaenoic acid (DHA, 22:6, n-3) is enriched in fatty fish and fish oil
3
supplements and is well-known for its anti-inflammatory,1 immunomodulatory,2 and
4
anti-cancer3 properties. Cancer is among the leading causes of death in both
5
economically developed countries and developing countries.4 Epidemiological studies
6
show that a diet rich in n-3 polyunsaturated fatty acids (PUFAs) is correlated with
7
reduced risk of angiogenic diseases such as cancers.5,6 DHA has been shown to inhibit
8
vascular sprout formation in retinal microvascular endothelial cells.7 However, the
9
mechanism underlying the inhibition of cell migration by DHA, which is critical for
10
11
angiogenesis and wound repair, is not fully understood.
G protein-coupled receptors (GPRs) are important signaling molecules involved in
12
many cellular functions. Specific ligand binding to GPRs stimulates and induces a
13
variety of cellular responses via several second messenger pathways, e.g., regulation
14
of cAMP generation, the phospholipase C pathway, ion channels, and
15
mitogen-activated protein kinases.8-10 In a previous study, Oh et al.11 found that DHA
16
exerted potent anti-inflammatory effects through GPR120.
17
Angiogenesis, the process of formation of new blood vessels by sprouting of the
18
preexisting microvascular network, is involved in numerous physiological processes
19
including embryogenesis, tissue remodeling, and wound healing,12 and disease
20
development such as diabetic retinopathy, rheumatoid arthritis, tumor growth, and
21
growth of atherosclerotic plaques.13 Angiogenesis is controlled by vascular
22
endothelial growth factor (VEGF), a proangiogenic factor.14 VEGF-induced signal
23
transduction involves binding to tyrosine kinase receptors, which leads to endothelial
24
cell proliferation, migration, and new vessel formation.15 VEGF was reported to
25
induce a wide variety of signaling pathways, including protein kinase C,
26
phospholipase C-γ, extracellular signal-regulated kinase (ERK), p38 MAPK,
27
phospholipase C, and phosphatidyl inositol 3-kinase (PI3K)/Akt.16 Hence, it is critical
28
to understand the VEGF-activated signaling pathways that play an important role in
29
VEGF-mediated cell processes.
30
Nitric oxide (NO), which is synthesized from the amino acid L-arginine by the
31
NOS family of enzymes, is a gaseous molecule with a wide range of physiological
32
and pathophysiological activities, including the regulation of angiogenesis.17 The
33
NOS family is composed of three members: neuronal NOS (nNOS), endothelial NOS
34
(eNOS), and inducible NOS (iNOS). eNOS is constitutively expressed in endothelial
35
cells, but various physical and chemical stimuli affect eNOS levels in vivo and in
36
vitro.18 Moreover, eNOS is activated upon exposure to fluid shear stress and
37
numerous agonists via cellular events such as protein phosphorylation.
38
Protein phosphatase 2A (PP2A) is a heterotrimeric complex composed of three
39
subunits including a structural subunit (PP2A-A), a regulatory subunit (PP2A-B), and
40
a catalytic subunit (PP2A-C).19 PP2A is a crucial intracellular serine/threonine
41
phosphatase.20 In addition, PP2A plays a significant role in the regulation of specific
42
signal transduction cascades including ERK1/2,21 JNK,22 and p38 MAPK.23 In recent
43
years, PP2A was shown to have tumor suppressor activity in myeloid CML-BC
44
patient-derived mononuclear marrow cells.24 Thus, enhancement of PP2A tumor
45
suppressor activity may represent a potential therapeutic strategy for malignancy.
46
The anti-tumor effect of DHA has been studied in MCF-7 human breast cancer
47
cells by induction of heme oxygenase 1 and inhibition of TPA-induced matrix
48
metalloproteinase 9 (MMP-9) expression.3 Moreover, DHA was reported to have a
49
potent anti-angiogenic effect.25 The pro-angiogenic effect of VEGF and NO has also
50
been demonstrated.15 In this study, therefore, we investigated whether DHA has an
51
opposing effect on cell migration that is associated with angiogenesis and wound
52
repair and the possible mechanisms involved.
53
54
MATERIALS AND METHODS
55
56
Reagents. Medium 199 (M199), Dulbecco’s modified Eagle’s medium, 0.25%
57
trypsin-EDTA, and penicillin-streptomycin-amphotericin solution were from
58
GIBCO-BRL (Grand Island, NY); endothelial cell growth supplement (ECGS) was
59
from Upstate Biotechnology (Lake Placid, NY); fetal bovine serum (FBS) was from
60
HyClone (Logan, UT); DHA was from Cayman Chemical (Ann Arbor, MI);
61
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sodium bicarbonate,
62
heparin, gelatin, S-nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor), LY294002
63
(PI3K kinase inhibitor), GW9508 (GPR120 agonist), and antibody against β-actin
64
were from Sigma-Aldrich (St. Louis, MO); VEGF was from Peprotech (Rocky Hill,
65
NJ); okadaic acid (OA) was from Millipore (Darmstadt, Germany); PD98059
66
(ERK1/2 inhibitor) was from TOCRIS (Ellisville, MO); and antibodies against
67
ERK1/2, phospho-ERK1/2, eNOS, and phospho-eNOS were from Cell Signaling
68
Technology (Danvers, MA).
69
Cell Cultures. The human umbilical vein endothelial cell line (HUVEC; CC-2517)
70
was obtained from Clonetics (San Diego, CA) and was cultured on gelatin-coated cell
71
culture dishes in M199 supplemented with 2.2 g/L sodium bicarbonate (NaHCO3), 0.1
72
g/L heparin, 37.5 mg/L ECGS, 20% FBS, 100,000 units/L penicillin, 0.25 mg/mL
73
amphotericin, and 100 mg/L streptomycin at 37oC in a 5% CO2 humidified incubator.
74
DHA Preparation. DHA samples were prepared as described previously.1
75
Cell Viability Assay. Cell viability was assessed by the MTT assay. The MTT assay
76
measures the ability of viable cells to reduce a yellow
77
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a purple formazan
78
by mitochondrial succinate dehydrogenase. HUVECs were grown to 80% to 90%
79
confluence and then treated with various concentrations of DHA (0-200 μM) and
80
GW9508 (1-10 μM) with or without VEGF (50 ng/mL) for 8 h in M199 containing
81
2% FBS. Finally, the medium was removed and the cells were washed with
82
phosphate-buffered saline (PBS). The cells were then incubated with MTT (0.5
83
mg/mL) in M199 medium at 37oC for an additional 3 h. The medium was removed
84
and 2-propanol was added to dissolve the formazan. After centrifugation at 10000g
85
for 5 min, the supernatant of each sample was transferred to 96-well plates, and
86
absorbance was read at 595 nm in an ELISA reader. The absorbance in the control
87
group was regarded as 100% cell viability.
88
Western Blotting Analysis. After each experiment, cells were washed twice with
89
PBS and were harvested with 150 μL lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1%
90
Triton X-100, 320 mM sucrose, 5 mM EDTA, 1 mM PMSF, 1 mg/L leupeptin, 1 mg/L
91
aprotinin, and 2 mM DTT). Cell homogenates were centrifuged at 14000g for 20 min
92
at 4oC. The resulting supernatant was used as a cellular protein for Western blotting
93
analysis. The total protein was analyzed by use of the Coomassie Plus protein assay
94
reagent kit (Pierce Biotechnology Inc., Rockford, IL). Equal amounts of cellular
95
proteins were electrophoresed in a sodium dodecyl sulfate (SDS)-polyacrylamide gel,
96
and proteins were then transferred to polyvinylidene fluoride membranes (Millipore
97
Corp., Bedford, MA). Nonspecific binding sites on the membranes were blocked with
98
5% nonfat milk in 15 mM Tris/150 mM NaCl buffer (pH 7.4) at room temperature for
99
2 h. Membranes were probed with anti-ERK1/2, anti-phospho-ERK1/2, anti-eNOS,
100
anti-phospho-eNOS, and anti-β-actin. The membranes were then probed with the
101
secondary antibody labeled with horseradish peroxidase. The bands were visualized
102
by using an enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA)
103
and were scanned by use of a luminescence image analyzer (LAS-4000, FUJIFILM,
104
Japan). The bands were quantitated with ImageGauge software (FUJIFILM).
105
Cell Migration Assay. An in vitro wound healing assay was used to measure
106
directional endothelial cell migration. HUVECs were seeded onto gelatin-coated
107
6-well plates and allowed to form a confluent monolayer. Monolayers were wounded
108
by using a sterile 200-μL pipette tip, washed with PBS to remove floating cells, and
109
photographed (time 0). Cells were then cultured in M199 medium containing 2% FBS
110
and 50 ng/mL of VEGF. Meanwhile, the cells were treated with various
111
concentrations of DHA (0-100 μM) with or without SNAP (20 μM) or OA (10 nM)
112
for 8 h. In addition, the cells were treated with GW9508 (10 μM) for 8 h. Cells were
113
then photographed (100x magnification) to monitor cell migration into the wounded
114
area, and the width of the cell-free zone (distance between the edges of the injured
115
monolayer) was calculated.
116
PP2A Activity Assay. PP2A activity was determined by use of a Ser/Thr phosphatase
117
assay kit according to the instructions of the manufacturer (Upstate, Darmstadt,
118
Germany). After treatment, cells were scraped with lysis buffer (10 mM tris/Triton
119
X-100, 0.32 M sucrose, 5 mM EDTA) and protease inhibitors, sonicated for 10 s, and
120
centrifuged at 2000g for 5 min. Thereafter, phosphopeptide (K-R-pT-I-R-R) was
121
added to the cell lysate, followed by incubation at room temperature for 15 min, and
122
then Malachite Green phosphate was added and the reaction was allowed to proceed
123
at room temperature for another 15 min for color development. The relative
124
absorbance was measured at 650 nm in a microplate reader (Bio-Rad).
125
Statistical Analysis. Data were analyzed by using analysis of variance (SAS Institute,
126
Cary, NC, USA). The significance of the difference between mean values was
127
determined by one-way analysis of variance followed by Tukey’s test. p values < 0.05
128
were taken to be statistically significant.
129
130
RESULTS
131
132
Effect of DHA and GW9508 on Cell Viability in the Presence or Absence of
133
VEGF. As measured by the MTT assay, the cell viabilities of HUVECs treated with
134
VEGF alone; 50, 100, or 200 M DHA; or VEGF and 50, 100, or 200 M DHA were
135
110.5%6.6%, 92.9%1.2%, 92.4%2.7%, 12.1%2.5%, 103.6%7.4%,
136
98.5%7.8%, and 11.8%0.8%, respectively, compared with the unstimulated
137
controls (100%). Thus, there were no adverse effects on the growth of cells up to a
138
concentration of 100 M DHA in the presence or absence of 50 ng/mL of VEGF,
139
which was used to induce cell migration. The highest concentration of DHA used in
140
the present study was 100 M.
141
The cell viabilities of HUVECs treated with VEGF alone, 1 or 10 M GW9508,
142
or VEGF and 1 or 10 M GW9508 were 116.3%5.8%, 97.6%3.5%, 99.1%2.5%,
143
107.6%4.5%, and 107.0%7.3%, respectively, compared with the unstimulated
144
controls (100%). Thus, there were no adverse effects on the growth of cells up to a
145
concentration of 10 M GW9508 in the presence or absence of 50 ng/mL of VEGF.
146
DHA Inhibits VEGF-Induced Cell Migration of HUVECs. Endothelial cell
147
migration is necessary to angiogenesis and wound repair. In addition to activation by
148
chemotactic, hepatotactic, and mechanotactic stimuli, angiogenesis involves
149
degradation of the extracellular matrix by matrix metalloproteinases (MMPs) to
150
enable progression of the migrating cells.26 Moreover, at least 10 MMPs are
151
up-regulated during wound healing by epidermal, dermal, fibroblast, and blood cells
152
in mammals.27 To determine the effect of DHA on endothelial cell migration in vitro,
153
confluent monolayers of HUVECs were scratched and cultured with M199 medium
154
containing 2% FBS plus 50 ng/mL of VEGF alone or in addition to 100 M DHA. As
155
shown in Figure 1, VEGF significantly induced cell migration, whereas co-culture
156
with DHA inhibited the VEGF-induced cell migration (p < 0.05).
157
Phosphorylation of ERK1/2 and eNOS Is Involved in VEGF-Induced Cell
158
Migration. VEGF has been shown to induce a wide variety of signaling pathways
159
that transduce the physiological or pathophysiological activities of VEGF. ERK1/2 is
160
one such pathway. It has been shown that there is a relationship between ERK1/2
161
phosphorylation and eNOS activation in rat sinusoidal endothelial cells.28 Thus, we
162
determined the effect of VEGF on both ERK1/2 and eNOS phosphorylation. As
163
shown in Figure 2A, VEGF stimulated the phosphorylation of ERK1/2 and eNOS,
164
and this activation was abolished by the ERK1/2 inhibitor PD98059. However,
165
VEGF showed no effect on Akt phosphorylation (data not shown). VEGF stimulated
166
cell migration and this effect was dependent on ERK1/2 (Figure 2B). These results
167
suggested that VEGF stimulation of cell migration is mediated by the ERK1/2/eNOS
168
169
pathway.
Inhibition of VEGF-Induced Cell Migration by DHA Is Associated with the
170
Induction of PP2A Enzyme Activity and the Inhibition of ERK1/2 and eNOS
171
Phosphorylation. Tumor metastasis, the spreading of the primary tumor to distant
172
organs, is recognized to be one of the major causes of failure in the treatment of
173
cancer.29 Thus, intervention in a key step in the metastatic process, such as
174
angiogenesis, is an effective mechanism for cancer therapy. VEGF plays a critical role
175
in tumor angiogenesis and has been shown to phosphorylate eNOS on Ser1177 in
176
glomerular endothelial cells.30 NO produced by activation of eNOS regulates
177
angiogenesis. Dephosphorylation of phosphorylated eNOS is mediated by protein
178
phosphatase 2A (PP2A).31 In the present study, the role of DHA in VEGF-induced cell
179
migration was studied by using a wound healing assay. As shown in Figure 3A, DHA
180
inhibited VEGF-induced cell migration and this inhibition was abolished by treatment
181
with the PP2A inhibitor OA. These results suggested that DHA inhibition of
182
VEGF-induced cell migration is likely associated with activation of PP2A enzyme
183
activity.
184
Recently, it was shown that PP2A has tumor suppressor activity in myeloid
185
CML-BC patient-derived mononuclear marrow cells.24 We used the Ser/Thr
186
phosphatase assay to measure PP2A enzyme activity. As shown in Figure 3B, DHA
187
significantly stimulated PP2A enzyme activity compared with that in the control
188
group and this activation was attenuated by treatment with OA. We next examined
189
whether OA reversed the DHA inhibition of VEGF-induced ERK1/2 and eNOS
190
phosphorylation. As shown in Figure 3C, VEGF induced phosphorylation of ERK1/2
191
and eNOS and pretreatment with DHA attenuated this induction. Nevertheless,
192
pretreatment with OA reversed the inhibition of eNOS phosphorylation by DHA. OA
193
also reversed the attenuation of VEGF-induced ERK1/2 phosphorylation by DHA,
194
although this effect was not significant.
195
Effect of GPR120 Agonist on VEGF-Induced Phosphorylation of ERK1/2 and
196
eNOS As Well As Cell Migration. GPR120 is a physiological receptor for long-chain
197
fatty acids, in particular n-3 fatty acids, such as -linolenic acid, eicosapentaenoic
198
acid (EPA), and DHA.11,32 To determine the role of GPR120 in DHA’s inhibition of
199
cell migration, we used the GPR120 agonist GW9508. As shown in Figure 4A,
200
GW9508 had an effect similar to that of DHA on the VEGF-induced phosphorylation
201
of ERK1/2 and eNOS. Moreover, the inhibitory effect of DHA on VEGF-induced cell
202
migration was replicated by GW9508 (Figure 4B). These results suggested that the
203
effects of DHA were highly associated with binding to GPR120.
204
205
SNAP Reverses DHA Inhibition of VEGF-Induced Cell Migration. VEGF
activates eNOS and stimulates the release of NO from HUVECs.33
206
Endothelium-derived NO is critical regulator of endothelial cell migration, survival,
207
and angiogenesis.34 To determine the effect of the NO donor SNAP on endothelial
208
cell migration in vitro, confluent monolayers of HUVECs were scratched and cultured
209
in M199 medium containing 2% FBS plus vehicle, 50 ng/mL of VEGF, 100 M DHA
210
and 50 ng/mL of VEGF, or 100 M DHA, 20 M SNAP, and 50 ng/mL of VEGF,
211
respectively. As shown in Figure 5, SNAP reversed DHA inhibition of VEGF-induced
212
cell migration.
213
214
215
DISCUSSION
Cancer has attracted considerable attention in the past decades because it is among
216
the leading causes of death globally.4 Protection against cancer by DHA is attributed
217
to the anti-angiogenic activity of DHA.35 However, the mechanisms by which DHA
218
inhibits angiogenesis are still not fully clarified. In this study, we explored the
219
inhibition of HUVEC cell migration by DHA and the possible mechanisms involved.
220
We demonstrated that DHA inhibits VEGF-induced HUVEC cell migration and that
221
this suppression is likely associated with binding to GPR120, activation of PP2A
222
enzyme activity, and inhibition of the ERK1/2/eNOS signaling pathway.
223
Several mechanisms have been proposed to demonstrate the inhibitory role of
224
DHA in angiogenesis, including induction of apoptosis and reduction of cell
225
proliferation.36-38 Rose and Connolly36 proposed that the inhibition of breast cancer
226
cell proliferation by DHA was related to the reduced production of series 2
227
eicosanoids. Although DHA may prevent the occurrence of angiogenic diseases, it has
228
potential side effects on subjects receiving revascularization therapy because of its
229
inhibition of cell migration.39 In recent years, EPA and DHA have been shown to
230
inhibit angiogenesis, possibly by regulating the production or activation of various
231
pro-angiogenic factors such as eNOS, VEGF, cyclooxygenase 2 (COX-2)-derived
232
prostanoids, and MMP-9.7,40,41 Moreover, EPA and DHA dose-dependently inhibit
233
ERK1/2 phosphorylation and this is likely associated with reduced PGE2 and VEGF
234
production by EPA and DHA in HT-29 human colorectal cells.40
235
VEGF has been shown to induce eNOS phosphorylation and cell migration in
236
HUVECs, and this activation is abolished by DHA.42 Moreover, DHA has been
237
demonstrated to inhibit TPA-induced MMP-9 expression and cell migration and
238
invasion in MCF-7 breast cancer cells.3 Consistent with the results of these previous
239
studies, our data showed that DHA exerted an inhibitory effect on VEGF-induced cell
240
migration in HUVECs (Figure 1). Cell migration is essential for angiogenesis. Thus,
241
the results of the present study suggest that DHA is a potential anti-angiogenic agent.
242
ERK1/2 is known to play an important role in cell proliferation, and protein
243
phosphatases are generally identified as inhibitors of cell proliferation via
244
dephosphorylation of ERK1/2.21 Activation of the Akt/eNOS43 or ERK1/2/eNOS30
245
pathways is reported to be involved in VEGF-induced NO release. In the present
246
study, VEGF activated phosphorylation of ERK1/2 but not of Akt (data not shown).
247
Pretreatment with the ERK1/2 inhibitor PD98059 abolished VEGF-induced
248
phosphorylation of ERK1/2 and eNOS in HUVECs (Figure 2A). These results suggest
249
that eNOS is the downstream target of ERK1/2. Moreover, DHA was shown to inhibit
250
VEGF-induced cell migration via suppression of the ERK1/2/eNOS signaling
251
pathway (Figure 3A and Figure 3C). Similar to DHA treatment, the ERK1/2 inhibitor
252
PD98059 abolished cell migration (Figure 2B). Furthermore, as shown in Figure 5,
253
SNAP reversed the DHA inhibition of VEGF-induced cell migration. These results
254
strongly suggest that the DHA inhibition of VEGF-induced cell migration is through
255
the ERK1/2/eNOS-mediated signaling pathway.
256
Reversible protein phosphorylation plays an important role in many cellular
257
processes.20 In general, cells use protein phosphorylation to alter enzyme properties
258
such as activity and cell distribution of key regulatory proteins involved in specific
259
pathways. Through the use of specific PP2A inhibitors and gene silencing technique,
260
PP2A has been shown to play a key role in the regulation of cell cycle, cell
261
morphology, and development. In addition, PP2A was demonstrated to be a tumor
262
suppressor and can be a target for cancer therapy.44 For example, it is reported that
263
conjugated linoleic acid inhibits the proliferation of MCF-7 human breast cancer cells
264
and that the working mechanisms include activation of PP2A and inhibition of
265
ERK1/2 activation.21 Pretreatment with the PP2A inhibitor OA reverses the effect of
266
conjugated linoleic acid on both increased PP2A expression level and
267
dephosphorylation of ERK1/2. This result strongly suggests that dephosphorylation of
268
ERK1/2 is mediated by PP2A.
269
270
Phosphorylated eNOS is also a substrate of PP2A.31 It is reported that endostatin
inhibits endothelial cell migration and angiogenesis by down-regulating the
271
phosphorylation of eNOS at Ser1177 via PP2A, which results in a decrease in NO
272
synthesis.45,46 In the present study, we demonstrated that DHA activated PP2A
273
enzyme activity, which was inhibited by OA pretreatment (Figure 3B). Pretreatment
274
with OA reversed the inhibitory effect of DHA on VEGF-induced cell migration and
275
phosphorylation of ERK1/2 and eNOS (Figure 3A and Figure 3C). These results
276
indicate that PP2A participates in the DHA inhibition of VEGF-induced
277
ERK1/2/eNOS phosphorylation and cell migration. Activation of PP2A by DHA is by
278
an as yet unidentified signaling pathway.
279
DHA exerts anti-inflammatory effects, but the mechanisms of these effects are not
280
fully understood. It has been shown that GPR120 functions as an n-3 PUFA receptor.11
281
In an in vitro study using RAW 264.7 cells and primary intraperitoneal macrophages,
282
stimulation of GPR120 with n-3 PUFAs or the chemical agonist GW9508 causes a
283
wide range of anti-inflammatory effects. All of these effects are abolished by GPR120
284
knockdown. Therefore, we hypothesize that DHA inhibits VEGF-induced cell
285
migration through binding with GPR120. We used the GPR120 agonist GW9508 to
286
simulate the activation of GPR120 by DHA in the suppression of VEGF-induced cell
287
migration. The results showed that GW9508 significantly inhibited VEGF-induced
288
cell migration, and the inhibitory effect was similar to that of DHA (Figure 4B). In
289
addition, GW9508 significantly suppressed VEGF-induced phosphorylation of
290
291
ERK1/2 and eNOS (Figure 4A), which is critical to cell migration.
Our findings in the present study are presented schematically in Figure 6. In
292
conclusion, DHA significantly inhibits VEGF-induced phosphorylation of ERK1/2
293
and eNOS and cell migration via binding to GPR120 and induction of PP2A enzyme
294
activity in HUVECs. Treatment with the NO donor SNAP reverses the DHA
295
inhibition of VEGF-induced cell migration. Taken together, these results support an
296
opposing role of DHA in cell migration, which is implicated in its anti-cancer and
297
anti-wound repair properties.
298
299
Funding Sources
300
This study was supported by grants NSC-101-2313-B-039-007-MY3 and
301
CMU101-ASIA-11.
302
303
Conflict of interest statement
304
Che-Yi Chao, Chong-Kuei Lii, Siou-Yu Ye, Chien-Chun Li, Chia-Yang Lu, Ai-Hsuan
305
Lin, Kai-Li Liu, Haw-Wen Chen, no conflicts of interest.
306
307
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308
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Legends
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Figure 1. DHA inhibits VEGF-induced migration of HUVECs. Cells were scratched
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and then stimulated with 50 ng/mL of VEGF in the presence or absence of 100 M
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DHA for 8 h. Migration was observed by using a phase-contrast microscope, at 100x
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magnification. Values are mean ± SD, n=3. Values not sharing the same letter are
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significantly different (p＜0.05).
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Figure 2. Phosphorylation of ERK1/2 and eNOS is involved in VEGF-induced
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migration of HUVECs. (A) Cells were starved in M199 containing 2% FBS for 7 h
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and then pretreated with 20 μM of the ERK1/2 inhibitor PD98059 for 1 h followed by
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incubation with 50 ng/mL of VEGF for another 15 min. (B) Cells were scratched and
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were co-treated with 20 μM PD98059 and 50 ng/mL VEGF or treated with 50 ng/mL
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of VEGF for 8 h. Values are mean ± SD, n=3. Values not sharing the same letter are
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significantly different (p＜0.05).
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Figure 3. Protein phosphatase 2A (PP2A) is involved in DHA inhibition of
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VEGF-induced phosphorylation of ERK1/2 and eNOS and cell migration. (A)
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HUVECs were scratched and pretreated with or without 10 nM of the PP2A inhibitor
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okadaic acid (OA) for 1 h and were then treated with 100 μM DHA along with 50
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ng/mL of VEGF or 50 ng/mL of VEGF for another 8 h. (B) HUVECs were pretreated
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with or without 10 nM OA for 1 h and were then treated with 100 μM DHA for
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another 4 h. (C) HUVECs were pretreated with 10 nM OA for 1 h and were then
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treated with 100 μM DHA for 8 h before being challenged with 50 ng/mL of VEGF
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for another 15 min. Values are mean ± SD, n=3. Values not sharing the same letter are
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significantly different (p＜0.05).
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Figure 4. Effect of GPR120 agonist on phosphorylation of ERK1/2 and eNOS and
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cell migration induced by VEGF in HUVECs. (A) Cells were pretreated with 100 μM
480
DHA or 10 μM GW9508 for 8 h followed by incubation with 50 ng/mL of VEGF for
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another 15 min. (B) Cells were scratched and were co-treated with 100 μM DHA or
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10 μM GW9508 along with 50 ng/mL of VEGF for 8 h. Values are mean ± SD, n=3.
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Values not sharing the same letter are significantly different (p＜0.05).
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Figure 5. SNAP reverses DHA inhibition of VEGF-induced migration in HUVECs.
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Cells were scratched and were treated with 50 ng/mL of VEGF alone, co-treated with
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100 μM DHA and 50 ng/mL of VEGF, or co-treated with 20 μM SNAP, 100 μM DHA,
488
and 50 ng/mL of VEGF for 8 h. Values are mean ± SD, n=3. Values not sharing the
489
same letter are significantly different (p＜0.05).
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Figure 6. Model showing the pathways that mediate DHA inhibition of
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VEGF-induced cell migration in HUVECs. DHA inhibits VEGF-induced cell
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migration via binding to GPR120, activation of PP2A, and inhibition of ERK1/2 and
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eNOS phosphorylation.
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