1. Lysophosphatidic Acid Dependent-Redox Signaling
in Proliferation and Atherosclerosis
Keren Pereda†
, LeAnn Rogers*, Chananat Klomsiri*, Leslie Poole*, and Larry W. Daniel*
Excellence In Cardiovascular Science Summer Research Program†
and Department of Biochemistry*
Wake Forest University
Introduction
Lysophosphatidic acid (LPA) is a small signaling lipid which is
able to promote a variety of cellular responses including growth,
differentiation, survival and motility.1,2
LPA binds to its
receptor, and is internalized as an endosome containing the
NADPH oxidase complex. NADPH oxidase produces ROS in
response to LPA, which also signals the recruitment of specific
proteins to the endosome. ROS is release from the endosome
and oxidizes these proteins.3
Many proteins need to be oxidized
in order for them to be phosphorylated leading to the
corresponding pathway activations.4
Akt and ERK are two of
many specific proteins activated by LPA that may be
phosphorylated/oxidized in response to ROS, other proteins may
also be phosphorylated/oxidized downstream or upstream of Akt
and ERK. The Akt and ERK pathways are important because
they promote cell proliferation and activation of molecules that
contribute to atherosclerosis. We examined how LPA-dependent
ROS production affects the Akt and ERK pathways in HUVEC
and SKOV3 cells.
Methods
ROS Production
HUVEC cells were cultured and plated in EBM-2 media from
Lonza containing 2% FBS. SKOV3 cells were cultured and
plated with RPMI-1640 media containing 10% FBS. All cells
were maintained at 37˚C and 5% CO2
. For imaging, cells were
treated with 1 µM LPA for 30 minutes. Cells were incubated
with H2DCF-DA (DCF) for the final 10 minutes of LPA
stimulation, then washed three times with media before
visualizing with an Olympus inverted epi-fluorescence
microscope with FITC filters. Cells were pretreated with DPI
for 10 minutes where indicated. Lines on the graph (Fig.2B)
represent individual cells monitored over time for DCF
fluorescence.
Western Blot
SKOV3 cells were cultured, plated, and maintained as
described. Where indicated, cells were challenged with DPI or
EUK-134 30 min prior to LPA stimulation. The cells were
harvested by detergent lysis, sonicated, and centrifuged to
remove cellular debris. Protein concentration was determined
using Pierce BSA Protein Assay Reagent in order to control for
protein loading amounts, and 40 µg of protein was resolved by
SDS-PAGE. The proteins were then transferred onto
nitrocellulose, and blocked in 5% milk in TBS-T. The blot was
incubated overnight at 4˚C with primary antibodies specific for
phospho- or total-MAPK p44/42(ERK) or phospho(Ser473)- or
total-Akt, followed by secondary HRP-linked antibodies for 2
hrs at room temperature. The blot was developed using Perkin
Elmer Western Lightning Chemiluminescence Reagent.
References
1. Rivera-Lopez, C.M., Tucker, A.L. & Lynch, K.R. Lysophosphatidic acid (LPA) and
angiogenesis. Angiogenesis 11, 301-10 (2008).
2. Teo, S.T., Yung, Y.C., Herr, D.R. & Chun, J. Lysophosphatidic acid in vascular
development and disease. IUBMB Life 61, 791-9 (2009).
3. Daniel, et.al. unpublished.
4. Poole LB, Nelson KJ. Discovering mechanisms of signaling-mediated cysteine
oxidation. Curr Opin Chem Biol. 1, 1218-24 (2008).
Results
A. Control B. +LPA
Figure1. ROS production in HUVEC Cells. Control
(left panel) and LPA treated (right panel).
0 10 120 240 36030 60Time (min)
LPA - - + - + - + - + - + - +
p-ERK
p-Akt
Figure 3. LPA stimulates the phosphorylation of Akt
and ERK.
Objectives
•To evaluate the production of ROS in response to LPA in
HUVEC and SKOV3 cells
•To determine the effect of “blocking” the ROS component of
LPA signaling on the phosphorylation of Akt and ERK
Conclusions
•LPA stimulates ROS production in both HUVEC and SKOV3 cells
•LPA stimulates phosphorylation of proteins in pathways
important in cancer cell proliferation and atherosclerosis
•Phosphorylation of Akt and ERK in response to LPA is dependent
on ROS production and protein oxidation
Acknowlegments
Research supported by NHLBI R25 HL 092618
Figure 4. DPI, an inhibitor of NADPH oxidase an dflavin
containing proteins, and EUK-134, a SOD and catalase mimic,
inhibit LPA stimulated phorphorylation of Akt and ERK
p-Akt
p-ERK
Akt
ERK
50 500 100 0 100
10 60Time (min.)
Euk-134(µM)10 µM DPI
Time (min.)
+ + + + +- - - - -
5 10 30 60 120
A. B.
A. 0 min 10
min
20 min 30 min
ControlLPA
Figure 2. A. ROS production in SKOV3 cells. B. DPI
inhibits LPA-dependent ROS production.
B.
Time(min)