Transactivation of Fetal Liver Kinase-1/Kinase-Insert Domain-Containing Receptor by Lysophosphatidylcholine Induces Vascular Endot
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《内分泌学杂志》
Department of Pharmacology (Y.F., M.Y., Y.I., N.A., H.O., Y.K., K.I., T.T.), Graduate School of Medical Sciences
Department of Clinical Pharmacology (K.T.), Graduate School of Pharmaceutical Sciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan
Abstract
Lysophosphatidylcholine (LPC), a major lipid component of oxidized low-density lipoprotein, is a bioactive lipid molecule involved in numerous biological processes including the progression of atherosclerosis. Recently orphan G protein-coupled receptors were identified as high-affinity receptors for LPC. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, LPC-stimulated transactivation of receptor tyrosine kinase has not yet been reported. Here we observed for the first time that LPC treatment of human umbilical vein endothelial cells (HUVECs) induces tyrosyl phosphorylation of vascular endothelial growth factor receptor 2 [fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)]. Flk-1/KDR transactivation by LPC was inhibited by vascular endothelial growth factor receptor tyrosine kinase inhibitors, SU1498 and 4-[(4'-chrolo-2'-fluoro) phenylamino]6,7-dimethoxyquinazoline (VTKi) in immunoprecipitation. Furthermore, we examined the effects of the Src family kinases inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), on LPC-induced Flk-1/KDR transactivation. Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation because herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from Western blots, ERK1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also activated by LPC, and this was inhibited by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in HUVECs. LPC-induced stimulation of HUVEC proliferation was shown to be secondary to transactivation because it was suppressed by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in dimethylthiazoldiphenyltetra-zoliumbromide assay. These findings suggest that LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the progression of atherosclerosis.
Introduction
LYSOPHOSPHATIDYLCHOLINE (LPC) is known to induce a variety of vascular endothelial responses ranging from the up-regulation of adhesion molecules and growth factors to the secretion of chemokines and superoxide anion radicals (1, 2). As a component of oxidized low-density lipoprotein (oxLDL), LPC is locally generated and accumulates at the sites of wounds, inflammation, and atherosclerosis (3, 4). Atherosclerosis can be characterized as a chronic inflammatory disease in which both cell proliferation and apoptotic/necrotic cell death occur within the vascular wall. In atherosclerotic lesions, pathological examination reveals the presence of angiogenesis (5, 6). It has previously been shown that LPC induces growth factor gene expression in cultured human endothelial cells (7). It has also been shown that oxLDL and LPC induces endothelial proliferation (8).
Recently orphan G protein-coupled receptors (GPCRs) were identified as high-affinity receptors for LPC. LPC binds to G2A and GPR4, Gi-protein-coupled receptors, specifically and regulates both cell growth and immunologic responses (9, 10). Gs-protein-coupled receptor GPR119 is reported to be a novel LPC receptor involved in insulin secretion (11). LPC, interacting with its receptor, induces an elevation of Ca2+ concentration activates serum-responsive transcription factors via the MAPK kinase pathway (10, 12). Despite the continued accumulation of evidence, however, the exact mechanisms by which LPC exerts biological function in endothelial cells (ECs) have not yet been elucidated.
Transactivation of receptor tyrosine kinases (RTKs) by the binding of ligands to GPCRs has been shown to have important physiological consequences. GPCR-mediated RTK transactivation has been implicated to have a crucial role in diseases such as cardiac hypertrophy and cancer and may have an important role in vascular diseases as well (13, 14). The role of Ca2+, reactive oxygen species, cSrc, Pyk2, protein kinase C, and membrane-bound metalloproteases has been reported in GPCR-mediated transactivation of RTKs (15, 16, 17, 18). Among these intracellular and extracellular molecules, a role for c-Src tyrosine kinase in GPCR-mediated RTK transactivation has been the focus (16, 19). LPC has been reported as a regulator of tyrosine kinase activity (20, 21). In these investigations, c-Src is suggested to have an important role in LPC signaling. It has been reported that transactivation of RTK in response to stimulation of GPCRs induces MAPK and Akt activation (15, 19). Vascular endothelial growth factor (VEGF) receptor-2 [fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR)] is a major receptor tyrosine kinase transducing the effects of VEGF into ECs. The signaling of Flk-1/KDR is necessary for the execution of VEGF-stimulated proliferation as well as the survival of cultured ECs and has been shown to be involved in atherosclerosis. Therefore, we hypothesized that LPC may transactivate Flk-1/KDR and investigated the specific role of LPC-induced Flk-1/KDR activation in ECs.
In the present study, we examined whether LPC transactivates Flk-1/KDR in human umbilical vein endothelial cells (HUVECs). Thereafter we investigated the involvement of c-Src tyrosine kinase in Flk-1/KDR transactivation by LPC. The findings of the present study strongly suggest that the transactivation of Flk-1/KDR by LPC is mediated by c-Src in HUVECs. The influence of Flk-1/KDR transactivation on the activation of ERK 1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also examined. In addition, it was observed that LPC caused HUVEC proliferation, which may be related to the progression of atherosclerosis.
Materials and Methods
Chemicals
LPC (C18:0) and wortmannin were purchased from Sigma (St. Louis, MO). Recombinant human VEGF was from PepRo Tech EC, Ltd. (London, UK). VEGF receptor tyrosine kinase inhibitors, VTKi [(4-[4'-chrolo-2'-fluoro]phenylamino)6,7-dimethoxyquinazoline] and SU1498 [(E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl) amino-carbonyl] acrylonitrile], and Src family tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) and herbimycin A were from Calbiochem (San Diego, CA). MAPK kinase (MEK) 1/2 inhibitors PD98059 was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and U0126 was from Promega (Madison, WI). Anti-Flk-1/KDR polyclonal antibody, protein A/G PLUS-agarose beads, and anti-ERK1/2 antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphosphotyrosine, clone 4G10, and anti-Src antibody were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-phospho-ERK1/2 (Thr 202/Tyr 204) antibody, anti-phospho-Akt (Ser 473) antibody, and anti-Akt antibody were from Cell Signaling Technology Inc. (Beverly, MA). Anti-Src phospho-specific antibody (Tyr 418), which recognizes the activated form of c-Src, was from Biosource (Camarillo, CA). All other chemicals were of reagent grade, obtained from commercial sources, and used without further purification.
Cell culture and transient transfection
HUVECs and endothelial cell basal medium (EBM)-2 were purchased from Clonetics (San Diego, CA). HUVECs were cultured in EBM-2 supplemented with 10% fetal bovine serum (FBS), gentamicin sulfate (50 μg/ml), and amphotericin-B (50 ng/ml) in addition to human fibroblast growth factor-B (10 ng/ml), epidermal growth factor human recombinant in a buffered BSA saline solution (20 ng/ml), VEGF human recombinant (1 ng/ml), IGF-I in aqueous solution cell culture tested (1 ng/ml), ascorbic acid (1 μg/ml), heparin (3 ng/ml), and hydrocortisone (0.4 μg/ml) at 37 C and 5% CO2. For transfection of wild-type (WT) or kinase-inactive (KI) Src, commercially available pUSE mammalian expression vectors encoding pp60c-Src or catalytically inactive Src (K297R) were used (Upstate Biotechnology). For the transient expression experiments, HUVECs were transfected with CytoPure-huv transfection reagent (polyplus-transfection, QBIOGENE Inc., Carlsbad, CA) according to the manufacture’s instructions. Transfection efficiency was determined with pcDNA3.1-GFP transfection as about 30% in HUVECs.
Immunoblot analysis
HUVECs in 0.2% FBS-containing EBM-2 medium were treated with or without inhibitors for various times and then incubated with LPC. After treatment with reagents, the cells were washed twice with cold PBS. Thereafter the cells were harvested using 0.5 ml of lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride] and incubated on ice. After thawing, cells were harvested and cell lysates were sonicated (Handy Sonic UR-20 P, Tomy Seiko Co. Ltd., Tokyo, Japan) on ice for 15 sec and then centrifuged at 25,000 x g for 20 min at 4 C to precipitate cell debris. The supernatant of lysates was analyzed for protein concentration by the Bradford methods (Bio-Rad Laboratories, Hercules, CA), and equal amounts of cellular proteins were separated by SDS-PAGE. After transfer to nitrocellulose membrane, the activation levels of ERK1/2, Akt, and Src were examined using antiphosphospecific antibodies, as described previously (22, 23).
Immunoprecipitation
Lysates containing equal amounts of protein were incubated with anti-Flk-1/KDR antibody overnight and then incubated with protein A/G PLUS-agarose beads for 2 h on a roller system at 4 C. After immunoprecipitation, samples were evaluated with immunoblot assay using antiphosphotyrosine antibody (clone 4G10). Based on its 230-kDa molecular mass and tyrosyl phosphorylation, it was considered that the bands represent activated state of Flk-1/KDR as described previously (17).
Cell viability assay
When HUVECs reached 40–50% confluence in 35-mm collagen-coated dishes (IWAKI, Osaka, Japan), growth was arrested using EBM-2 medium with 0.2% FBS for 24 h. SU1498, VTKi, PD98059, U0126, and herbimycin A were added and incubated for 30 min. PP2 was added and incubated for 2 h. After 24 h incubation with LPC (20 μM), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) was added at a final concentration of 0.5 mg/ml, and after a further 2 h incubation, HUVECs were lysed with isopropanol containing 0.04 M HCl. MTT reduction was read at 550 nm by a spectrophotometer.
Statistical analysis
Values are presented as means ± SD for five separate experiments. One-way ANOVA was used to determine significance among groups, after which post hoc test with the Bonferroni correction were used for comparison between individual groups. A value at P < 0.05 was considered to be significant.
Results
LPC stimulates transactivation of Flk-1/KDR in HUVECs
We first examined whether Flk-1/KDR is activated by LPC in HUVECs. HUVECs were treated at various times and with various concentrations of LPC. Activation of Flk-1/KDR in the cell lysates was determined by tyrosyl phosphorylation as described in Materials and Methods. As shown in Fig. 1A, LPC rapidly activated Flk-1/KDR (with a peak at 10 min) and then sustained activation for at least 60 min. Figure 1B shows the dose response for the activation of Flk-1/KDR by LPC in HUVECs. Flk-1/KDR activation was determined by a 10-min incubation period. Flk-1/KDR activity was increased in a dose-dependent manner by LPC (1–20 μM), and maximal activation occurred at 20 μM of LPC. To confirm that these are transactivation events, we examined the effects of VEGF receptor tyrosine kinase inhibitors, SU1498 (30 μM) and VTKi (5 μM) on LPC-induced Flk-1/KDR activation in HUVECs. The cells were pretreated with SU1498 and VTKi for 30 min before the addition of LPC (20 μM) and VEGF (10 ng/ml) for 10 min. As shown in Fig. 2A, both compounds significantly inhibited phosphorylation of Flk-1/KDR in response to 20 μM LPC. Figure 2B shows that both compounds also inhibited 10 ng/ml VEGF-induced phosphorylation of Flk-1/KDR. There were no differences in the total amounts of Flk-1/KDR observed on Western blot analysis with anti-Flk-1/KDR antibodies. These findings suggest that LPC significantly transactivates Flk-1/KDR in HUVECs.
c-Src tyrosine kinase is involved in the LPC-induced Flk-1/KDR transactivation
Many tyrosine kinases, including those of both the receptor and nonreceptor type, are important for the activation of survival and/or proliferative pathways in various cell types. Several c-Src-mediated signal transduction pathways from GPCRs to receptor tyrosine kinases have been reported (17, 18). We hypothesized that c-Src might be involved in the LPC-induced transactivation of Flk-1/KDR. To elucidate whether LPC activates c-Src in HUVECs, an examination of the effect of LPC on c-Src tyrosine kinase activity in HUVECs was undertaken. As shown in Fig. 3A, an application of 20 μM LPC caused rapid and significant phosphorylation of c-Src (peak at 5 min) at tyrosine 418, an autophosphorylation cite that leads to full catalytic activity of the kinase. c-Src activity was increased by LPC in a dose-dependent manner (1–20 μM), and maximal activation occurred at 20 μM of LPC (Fig. 3B). A tyrosine kinase inhibitor, herbimycin A (1 μM), and a Src kinase family inhibitor, PP2 (10 μM), both inhibited LPC-induced Src kinase activation (data not shown). In addition, herbimycin A (0.1–10 μM) and PP2 (1–100 μM) both inhibited LPC-induced Flk-1/KDR activation in a dose-dependent manner (Fig. 4, A and B). The cells were transfected with KI Src for 24 h and pretreated with PP2 for 2 h before the addition of LPC (20 μM) for 10 min. As shown in Fig. 4C, KI Src transfection and PP2 (100 μM) treatment did not affect VEGF-induced Flk-1/KDR activation. Furthermore, we examined the effect of KI Src transfection on LPC-induced Flk-1/KDR activation. As shown in Fig. 5A, transfection of KI Src almost abolished LPC-induced Flk-1/KDR activation, whereas WT Src transfection caused Flk-1/KDR activation with or without LPC treatment. Transfection of KI Src also inhibited LPC-induced c-Src kinase activation in HUVECs (Fig. 5B). There were no differences in the total amounts of Flk-1/KDR observed on Western blot analysis with anti-Flk-1/KDR antibody. In WT Src and KI Src cDNA-transfected samples, c-Src protein expression was different from vector-alone-transfected sample. These findings strongly suggest that c-Src is involved in LPC-induced Flk-1/KDR transactivation in HUVECs.
LPC stimulates downstream of Flk-1/KDR and c-Src
It was reported that ERK1/2 and Akt exist in the downstream region of Flk-1/KDR (24). It has also been reported that LPC stimulates ERK1/2 and Akt activation, which results in the activation of cell survival pathways (25). Thus, we examined whether LPC activates ERK1/2 and Akt through transactivation of Flk-1/KDR in HUVECs. Activation of ERK1/2 and Akt in the cell lysates were determined as described in Experimental procedures. Application of 20 μM LPC caused significant activation of ERK1/2, which peaked at 60 min, and also of Akt, which peaked at 90 min (data not shown). First, we investigated whether LPC-induced activation of ERK1/2 and Akt was inhibited by SU1498 (30 μM) or VTKi (5 μM) in HUVECs. The cells were pretreated with SU1498 and VTKi for 30 min before the addition of LPC (20 μM) to examine their effects on ERK1/2 or Akt activation, respectively. Both compounds significantly inhibited ERK1/2 and Akt activation in response to 20 μM LPC stimulation (Fig. 6). These findings suggest that LPC-induced transactivation of Flk-1/KDR is involved in the activation of ERK1/2 and Akt downstream. Furthermore, we examined the effects of herbimycin A, PP2, and KI Src transfection on ERK1/2 and Akt activation by LPC to investigate whether c-Src is involved in these phenomena. The cells were pretreated with herbimycin A (1 μM) for 30 min and PP2 (10 μM) for 2 h before the addition of LPC (20 μM) to examine their respective effects on ERK1/2 or Akt activation. Herbimycin A and PP2 both inhibited LPC-induced ERK1/2 and Akt phosphorylation (Fig. 7, A and B). Transfection of KI Src also inhibited LPC-induced ERK1/2 and Akt phosphorylation in HUVECs (Fig. 7, C and D). These findings suggest that ERK1/2 and Akt are downstream effector molecules of c-Src, which is suggested to be involved in the LPC induction of Flk-1/KDR transactivation.
LPC-induced Flk-1/KDR transactivation stimulates HUVEC proliferation
Flk-1/KDR activation is reported to induce cell growth and differentiation (24). Therefore, to investigate pathophysiological implications of LPC-induced Flk-1/KDR transactivation, we examined the effect of LPC on HUVEC proliferation. As shown in Fig. 8A, MTT assay revealed that stimulation with LPC increased HUVEC viability over a 24-h incubation period in a dose-dependent manner (1–20 μM). In addition, we examined whether LPC-induced HUVEC proliferation was mediated by Flk-1/KDR transactivation as well as activation of ERK1/2 and Akt. The cells were pretreated with SU1498 (30 μM), VTKi (5 μM), the MEK1/2 inhibitors PD98059 (10 μM) and U0126 (10 μM), and the phosphatidylinositol 3-phosphate kinase (PI3K) inhibitor LY294002 (10 μM) and wortmannin (10 nM) for 30 min before the addition of LPC (20 μM). SU1498, VTKi, PD98059, U0126, LY294002, and wortmannin inhibited LPC-induced HUVEC proliferation (Fig. 8B). These results suggest that ERK1/2 and Akt are involved in LPC-induced proliferation of HUVECs. To characterize the role of c-Src in LPC-induced HUVEC proliferation, we then examined the effect of herbimycin A and PP2 on LPC-induced HUVEC proliferation. The cells were pretreated with herbimycin A (1 μM) for 30 min and PP2 (10 μM) for 2 h before the addition of LPC (20 μM). Both herbimycin A and PP2 significantly inhibited the HUVEC proliferation induced by LPC (Fig. 8C). Transfection of KI Src also inhibited LPC-induced HUVEC proliferation, whereas WT Src transfection induced HUVEC proliferation in the absence of LPC stimulation (Fig. 8D). These findings strongly suggest that c-Src is involved in LPC-induced HUVEC proliferation, which is mediated through Flk-1/KDR transactivation.
Discussion
The major finding of the present study is that LPC transactivates VEGF receptor Flk-1/KDR in HUVECs via c-Src tyrosine kinase activation. SU1498 and VTKi, which have been shown to inhibit Flk-1/KDR, inhibited LPC-induced Flk-1/KDR activation. Herbimycin A and PP2, specific inhibitors for Src family kinases, both inhibited LPC-induced Flk-1/KDR activation in a concentration-dependent manner. Transfection of KI Src also inhibited LPC-induced Flk-1/KDR activation. From these findings, it was suggested that c-Src is involved in Flk-1/KDR transactivation in HUVECs. Moreover, LPC-induced ERK1/2 and Akt activation, which are both reported to be downstream of Flk-1/KDR (17), were also inhibited by SU1498, VTKi, herbimycin A, PP2, and transfection with KI Src. It was also found that LPC-induced Flk-1/KDR transactivation resulted in a stimulation of HUVEC proliferation. VEGF receptor inhibitors, MEK1/2 inhibitors, PI3K inhibitors, Src family inhibitors, and transfection with KI Src all inhibited LPC-induced HUVEC proliferation.
The transactivation of RTK in response to the stimulation of a number of GPCRs, such as transactivation of the epithelial growth factor receptor by GPCR ligands such as thrombin, angiotensin II, lysophosphatidic acid, and endothelin-1 has been reported (18, 26). However, LPC-stimulated transactivation of RTK has not yet been reported or elucidated. Nevertheless, the role of LPC in the pathogenesis of atherosclerosis and systemic autoimmune diseases is well documented (27, 28), even though its specific cell surface receptor was not identified until quite recently. To date, a subfamily of GPCRs consisting of G2A and GPR4 have been identified as the receptors for LPC (9, 10). It is reported that G2A is expressed in atherosclerotic lesions (29). On the other hand, it is reported that G2A and GPR4 are proton-sensing GPCRs (30, 31). However, it is also reported that GPR119 is a newly identified receptor for LPC (11). Although it still remains to be clarified what GPCRs are responsible for LPC action, we found that LPC caused Flk-1/KDR transactivation in HUVECs. LPC has been implicated in the pathological states of vascular ECs that may be related to the progressive pathological events of atherosclerosis. It has been reported that the plasma concentration of LPC is elevated in patients with coronary artery diseases, compared with that of normal subjects (32). However, the precise intracellular mechanisms mediated by LPC stimulation in ECs have not yet been clarified. In the present study, we found for the first time that LPC induces Flk-1/KDR transactivation in HUVECs (Figs. 1 and 2). Because Flk-1/KDR activation is reported to be an important factor for atherogenesis (33, 34), LPC-induced Flk-1/KDR transactivation is taken to be one of the pathological developments of atherosclerosis.
Several mechanisms for RTK transactivation have been proposed. For example, Pyk2, reactive oxygen species, intracellular-free Ca2+, and matrix metalloproteases have been identified as factors that activate RTK in many types of cells (17, 18). We and others previously reported that c-Src is involved in the downstream signaling events after stimulation of GPCR and RTK (15, 16, 19). Therefore, we examined whether c-Src mediates LPC-induced Flk-1/KDR transactivation in the present study. As shown in Fig. 3, c-Src was rapidly and significantly activated by LPC in HUVECs. Furthermore, herbimycin A, PP2, and transfection of KI Src significantly inhibited LPC-induced Flk-1/KDR transactivation (Figs. 4 and 5). From these findings, it is strongly suggested that c-Src is involved in LPC-induced Flk-1/KDR transactivation, and c-Src may be localized near the receptor and plasma membrane. However, because we did not determine whether c-Src directly regulates Flk-1/KDR activity in this study, further studies are required to define the precise role and the mechanisms of Src kinase in Flk-1/KDR transactivation.
One previous report suggested that a PI3K-Akt- endothelial nitric oxide synthase pathway exists downstream of the Flk-1/KDR transactivation by sphingosine-1 phosphate because the sphingosine-1 phosphate-induced Akt and endothelial nitric oxide synthase activation were inhibited by SU1498 and VTKi (17). In the present study, we found similarly that LPC-induced ERK1/2 and Akt activation were inhibited by SU1498 and VTKi (Fig. 6). It is reported that ERK1/2 and Akt are activated by VEGF as early as 5–10 min after administration (35). In the present study, we found that LPC-induced ERK1/2 and Akt activation were observed. Both the ERK1/2 and Akt activation after transactivation are likely to have an important role in EC function.
The proliferation of ECs is a prominent feature of atherosclerotic lesions because it is an important step toward angiogenesis as well as contributing to the intimal thickening that develops after endothelial injury (8, 36). LPC is closely related to atherosclerosis because LPC is a component of the oxLDL, which is widely considered to be a major risk factor for atherosclerosis (3, 4). Therefore, we further examined the effect of LPC on HUVEC proliferation. LPC-induced HUVEC proliferation in a concentration-dependent manner with the maximal effect of LPC at 20 μM (Fig. 8A). This result is consistent with the findings of Wolfram et al. (8), who reported that 20 μM LPC induces vascular endothelial proliferation. However, we also observed that LPC at higher concentrations than 25 μM brought about HUVEC death rather than proliferation (data not shown). At higher concentration than 25 μM, LPC has been shown to cause vascular smooth muscle cell apoptosis (37). It is also reported that LPC can induce GPCR-mediated apoptosis (38). However, in contrast with these findings, it is reported that LPC at 10–20 μM facilitates cell growth, differentiation, and proliferation (8, 39). In agreement with these later findings, our results show that 20 μM LPC significantly induced HUVEC proliferation (Fig. 8A). As shown by the results of pretreatment with SU1498, VTKi, PD98059, U0126, LY294002, wortmannin, herbimycin A, and PP2 and transfection with KI Src, LPC-induced HUVEC proliferation was inhibited (Fig. 8, B–D). On the other hand, we measured lysophospholipase D that produces physiologically active lysophosphatidic acid from LPC (40, 41). In this study, LPC did not activate lysophospholipase D in HUVEC (data not shown). From these findings, it is suggested that LPC-induced Flk-1/KDR transactivation via c-Src is implicated as a contributor to the development of atherosclerosis, especially those steps in which EC proliferation plays a pivotal role.
In summary, we show for the first time that VEGF receptor Flk-1/KDR is transactivated by LPC in HUVECs. c-Src activation is suggested to be involved in LPC-induced Flk-1/KDR transactivation. In addition, LPC-induced Flk-1/KDR transactivation caused ERK1/2 and Akt activation in HUVECs. Although the specific mechanistic steps of the pathophysiological role of LPC remain to be elucidated, LPC-induced Flk-1/KDR transactivation and the resultant proliferation of vascular ECs very likely have a role in cardiovascular diseases such as atherosclerosis.
Acknowledgments
We thank Dr. A. Tokumura (Faculty of Pharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima Graduate School) for excellent advisement and technical support.
Footnotes
First Published Online December 1, 2005
Abbreviations: EBM, Endothelial cell basal medium; EC, endothelial cell; FBS, fetal bovine serum; Flk-1, fetal liver kinase-1; GPCR, G protein-coupled receptor; HUVEC, human umbilical vein endothelial cell; KDR, kinase-insert domain-containing receptor; KI, kinase inactive; LPC, lysophosphatidylcholine; MEK, MAPK kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; oxLDL, oxidized low-density lipoprotein; PI3K, phosphatidylinositol 3-phosphate kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine; RTK, receptor tyrosine kinase; SU1498, (E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl) amino-carbonyl; VEGF, vascular endothelial growth factor; VTKi, (4-[4'-chrolo-2'-fluoro]phenylamino)6,7-dimethoxyquinazoline; WT, wild type.
Accepted for publication November 21, 2005.
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Department of Clinical Pharmacology (K.T.), Graduate School of Pharmaceutical Sciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan
Abstract
Lysophosphatidylcholine (LPC), a major lipid component of oxidized low-density lipoprotein, is a bioactive lipid molecule involved in numerous biological processes including the progression of atherosclerosis. Recently orphan G protein-coupled receptors were identified as high-affinity receptors for LPC. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, LPC-stimulated transactivation of receptor tyrosine kinase has not yet been reported. Here we observed for the first time that LPC treatment of human umbilical vein endothelial cells (HUVECs) induces tyrosyl phosphorylation of vascular endothelial growth factor receptor 2 [fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)]. Flk-1/KDR transactivation by LPC was inhibited by vascular endothelial growth factor receptor tyrosine kinase inhibitors, SU1498 and 4-[(4'-chrolo-2'-fluoro) phenylamino]6,7-dimethoxyquinazoline (VTKi) in immunoprecipitation. Furthermore, we examined the effects of the Src family kinases inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), on LPC-induced Flk-1/KDR transactivation. Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation because herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from Western blots, ERK1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also activated by LPC, and this was inhibited by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in HUVECs. LPC-induced stimulation of HUVEC proliferation was shown to be secondary to transactivation because it was suppressed by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in dimethylthiazoldiphenyltetra-zoliumbromide assay. These findings suggest that LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the progression of atherosclerosis.
Introduction
LYSOPHOSPHATIDYLCHOLINE (LPC) is known to induce a variety of vascular endothelial responses ranging from the up-regulation of adhesion molecules and growth factors to the secretion of chemokines and superoxide anion radicals (1, 2). As a component of oxidized low-density lipoprotein (oxLDL), LPC is locally generated and accumulates at the sites of wounds, inflammation, and atherosclerosis (3, 4). Atherosclerosis can be characterized as a chronic inflammatory disease in which both cell proliferation and apoptotic/necrotic cell death occur within the vascular wall. In atherosclerotic lesions, pathological examination reveals the presence of angiogenesis (5, 6). It has previously been shown that LPC induces growth factor gene expression in cultured human endothelial cells (7). It has also been shown that oxLDL and LPC induces endothelial proliferation (8).
Recently orphan G protein-coupled receptors (GPCRs) were identified as high-affinity receptors for LPC. LPC binds to G2A and GPR4, Gi-protein-coupled receptors, specifically and regulates both cell growth and immunologic responses (9, 10). Gs-protein-coupled receptor GPR119 is reported to be a novel LPC receptor involved in insulin secretion (11). LPC, interacting with its receptor, induces an elevation of Ca2+ concentration activates serum-responsive transcription factors via the MAPK kinase pathway (10, 12). Despite the continued accumulation of evidence, however, the exact mechanisms by which LPC exerts biological function in endothelial cells (ECs) have not yet been elucidated.
Transactivation of receptor tyrosine kinases (RTKs) by the binding of ligands to GPCRs has been shown to have important physiological consequences. GPCR-mediated RTK transactivation has been implicated to have a crucial role in diseases such as cardiac hypertrophy and cancer and may have an important role in vascular diseases as well (13, 14). The role of Ca2+, reactive oxygen species, cSrc, Pyk2, protein kinase C, and membrane-bound metalloproteases has been reported in GPCR-mediated transactivation of RTKs (15, 16, 17, 18). Among these intracellular and extracellular molecules, a role for c-Src tyrosine kinase in GPCR-mediated RTK transactivation has been the focus (16, 19). LPC has been reported as a regulator of tyrosine kinase activity (20, 21). In these investigations, c-Src is suggested to have an important role in LPC signaling. It has been reported that transactivation of RTK in response to stimulation of GPCRs induces MAPK and Akt activation (15, 19). Vascular endothelial growth factor (VEGF) receptor-2 [fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR)] is a major receptor tyrosine kinase transducing the effects of VEGF into ECs. The signaling of Flk-1/KDR is necessary for the execution of VEGF-stimulated proliferation as well as the survival of cultured ECs and has been shown to be involved in atherosclerosis. Therefore, we hypothesized that LPC may transactivate Flk-1/KDR and investigated the specific role of LPC-induced Flk-1/KDR activation in ECs.
In the present study, we examined whether LPC transactivates Flk-1/KDR in human umbilical vein endothelial cells (HUVECs). Thereafter we investigated the involvement of c-Src tyrosine kinase in Flk-1/KDR transactivation by LPC. The findings of the present study strongly suggest that the transactivation of Flk-1/KDR by LPC is mediated by c-Src in HUVECs. The influence of Flk-1/KDR transactivation on the activation of ERK 1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also examined. In addition, it was observed that LPC caused HUVEC proliferation, which may be related to the progression of atherosclerosis.
Materials and Methods
Chemicals
LPC (C18:0) and wortmannin were purchased from Sigma (St. Louis, MO). Recombinant human VEGF was from PepRo Tech EC, Ltd. (London, UK). VEGF receptor tyrosine kinase inhibitors, VTKi [(4-[4'-chrolo-2'-fluoro]phenylamino)6,7-dimethoxyquinazoline] and SU1498 [(E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl) amino-carbonyl] acrylonitrile], and Src family tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) and herbimycin A were from Calbiochem (San Diego, CA). MAPK kinase (MEK) 1/2 inhibitors PD98059 was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and U0126 was from Promega (Madison, WI). Anti-Flk-1/KDR polyclonal antibody, protein A/G PLUS-agarose beads, and anti-ERK1/2 antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphosphotyrosine, clone 4G10, and anti-Src antibody were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-phospho-ERK1/2 (Thr 202/Tyr 204) antibody, anti-phospho-Akt (Ser 473) antibody, and anti-Akt antibody were from Cell Signaling Technology Inc. (Beverly, MA). Anti-Src phospho-specific antibody (Tyr 418), which recognizes the activated form of c-Src, was from Biosource (Camarillo, CA). All other chemicals were of reagent grade, obtained from commercial sources, and used without further purification.
Cell culture and transient transfection
HUVECs and endothelial cell basal medium (EBM)-2 were purchased from Clonetics (San Diego, CA). HUVECs were cultured in EBM-2 supplemented with 10% fetal bovine serum (FBS), gentamicin sulfate (50 μg/ml), and amphotericin-B (50 ng/ml) in addition to human fibroblast growth factor-B (10 ng/ml), epidermal growth factor human recombinant in a buffered BSA saline solution (20 ng/ml), VEGF human recombinant (1 ng/ml), IGF-I in aqueous solution cell culture tested (1 ng/ml), ascorbic acid (1 μg/ml), heparin (3 ng/ml), and hydrocortisone (0.4 μg/ml) at 37 C and 5% CO2. For transfection of wild-type (WT) or kinase-inactive (KI) Src, commercially available pUSE mammalian expression vectors encoding pp60c-Src or catalytically inactive Src (K297R) were used (Upstate Biotechnology). For the transient expression experiments, HUVECs were transfected with CytoPure-huv transfection reagent (polyplus-transfection, QBIOGENE Inc., Carlsbad, CA) according to the manufacture’s instructions. Transfection efficiency was determined with pcDNA3.1-GFP transfection as about 30% in HUVECs.
Immunoblot analysis
HUVECs in 0.2% FBS-containing EBM-2 medium were treated with or without inhibitors for various times and then incubated with LPC. After treatment with reagents, the cells were washed twice with cold PBS. Thereafter the cells were harvested using 0.5 ml of lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride] and incubated on ice. After thawing, cells were harvested and cell lysates were sonicated (Handy Sonic UR-20 P, Tomy Seiko Co. Ltd., Tokyo, Japan) on ice for 15 sec and then centrifuged at 25,000 x g for 20 min at 4 C to precipitate cell debris. The supernatant of lysates was analyzed for protein concentration by the Bradford methods (Bio-Rad Laboratories, Hercules, CA), and equal amounts of cellular proteins were separated by SDS-PAGE. After transfer to nitrocellulose membrane, the activation levels of ERK1/2, Akt, and Src were examined using antiphosphospecific antibodies, as described previously (22, 23).
Immunoprecipitation
Lysates containing equal amounts of protein were incubated with anti-Flk-1/KDR antibody overnight and then incubated with protein A/G PLUS-agarose beads for 2 h on a roller system at 4 C. After immunoprecipitation, samples were evaluated with immunoblot assay using antiphosphotyrosine antibody (clone 4G10). Based on its 230-kDa molecular mass and tyrosyl phosphorylation, it was considered that the bands represent activated state of Flk-1/KDR as described previously (17).
Cell viability assay
When HUVECs reached 40–50% confluence in 35-mm collagen-coated dishes (IWAKI, Osaka, Japan), growth was arrested using EBM-2 medium with 0.2% FBS for 24 h. SU1498, VTKi, PD98059, U0126, and herbimycin A were added and incubated for 30 min. PP2 was added and incubated for 2 h. After 24 h incubation with LPC (20 μM), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) was added at a final concentration of 0.5 mg/ml, and after a further 2 h incubation, HUVECs were lysed with isopropanol containing 0.04 M HCl. MTT reduction was read at 550 nm by a spectrophotometer.
Statistical analysis
Values are presented as means ± SD for five separate experiments. One-way ANOVA was used to determine significance among groups, after which post hoc test with the Bonferroni correction were used for comparison between individual groups. A value at P < 0.05 was considered to be significant.
Results
LPC stimulates transactivation of Flk-1/KDR in HUVECs
We first examined whether Flk-1/KDR is activated by LPC in HUVECs. HUVECs were treated at various times and with various concentrations of LPC. Activation of Flk-1/KDR in the cell lysates was determined by tyrosyl phosphorylation as described in Materials and Methods. As shown in Fig. 1A, LPC rapidly activated Flk-1/KDR (with a peak at 10 min) and then sustained activation for at least 60 min. Figure 1B shows the dose response for the activation of Flk-1/KDR by LPC in HUVECs. Flk-1/KDR activation was determined by a 10-min incubation period. Flk-1/KDR activity was increased in a dose-dependent manner by LPC (1–20 μM), and maximal activation occurred at 20 μM of LPC. To confirm that these are transactivation events, we examined the effects of VEGF receptor tyrosine kinase inhibitors, SU1498 (30 μM) and VTKi (5 μM) on LPC-induced Flk-1/KDR activation in HUVECs. The cells were pretreated with SU1498 and VTKi for 30 min before the addition of LPC (20 μM) and VEGF (10 ng/ml) for 10 min. As shown in Fig. 2A, both compounds significantly inhibited phosphorylation of Flk-1/KDR in response to 20 μM LPC. Figure 2B shows that both compounds also inhibited 10 ng/ml VEGF-induced phosphorylation of Flk-1/KDR. There were no differences in the total amounts of Flk-1/KDR observed on Western blot analysis with anti-Flk-1/KDR antibodies. These findings suggest that LPC significantly transactivates Flk-1/KDR in HUVECs.
c-Src tyrosine kinase is involved in the LPC-induced Flk-1/KDR transactivation
Many tyrosine kinases, including those of both the receptor and nonreceptor type, are important for the activation of survival and/or proliferative pathways in various cell types. Several c-Src-mediated signal transduction pathways from GPCRs to receptor tyrosine kinases have been reported (17, 18). We hypothesized that c-Src might be involved in the LPC-induced transactivation of Flk-1/KDR. To elucidate whether LPC activates c-Src in HUVECs, an examination of the effect of LPC on c-Src tyrosine kinase activity in HUVECs was undertaken. As shown in Fig. 3A, an application of 20 μM LPC caused rapid and significant phosphorylation of c-Src (peak at 5 min) at tyrosine 418, an autophosphorylation cite that leads to full catalytic activity of the kinase. c-Src activity was increased by LPC in a dose-dependent manner (1–20 μM), and maximal activation occurred at 20 μM of LPC (Fig. 3B). A tyrosine kinase inhibitor, herbimycin A (1 μM), and a Src kinase family inhibitor, PP2 (10 μM), both inhibited LPC-induced Src kinase activation (data not shown). In addition, herbimycin A (0.1–10 μM) and PP2 (1–100 μM) both inhibited LPC-induced Flk-1/KDR activation in a dose-dependent manner (Fig. 4, A and B). The cells were transfected with KI Src for 24 h and pretreated with PP2 for 2 h before the addition of LPC (20 μM) for 10 min. As shown in Fig. 4C, KI Src transfection and PP2 (100 μM) treatment did not affect VEGF-induced Flk-1/KDR activation. Furthermore, we examined the effect of KI Src transfection on LPC-induced Flk-1/KDR activation. As shown in Fig. 5A, transfection of KI Src almost abolished LPC-induced Flk-1/KDR activation, whereas WT Src transfection caused Flk-1/KDR activation with or without LPC treatment. Transfection of KI Src also inhibited LPC-induced c-Src kinase activation in HUVECs (Fig. 5B). There were no differences in the total amounts of Flk-1/KDR observed on Western blot analysis with anti-Flk-1/KDR antibody. In WT Src and KI Src cDNA-transfected samples, c-Src protein expression was different from vector-alone-transfected sample. These findings strongly suggest that c-Src is involved in LPC-induced Flk-1/KDR transactivation in HUVECs.
LPC stimulates downstream of Flk-1/KDR and c-Src
It was reported that ERK1/2 and Akt exist in the downstream region of Flk-1/KDR (24). It has also been reported that LPC stimulates ERK1/2 and Akt activation, which results in the activation of cell survival pathways (25). Thus, we examined whether LPC activates ERK1/2 and Akt through transactivation of Flk-1/KDR in HUVECs. Activation of ERK1/2 and Akt in the cell lysates were determined as described in Experimental procedures. Application of 20 μM LPC caused significant activation of ERK1/2, which peaked at 60 min, and also of Akt, which peaked at 90 min (data not shown). First, we investigated whether LPC-induced activation of ERK1/2 and Akt was inhibited by SU1498 (30 μM) or VTKi (5 μM) in HUVECs. The cells were pretreated with SU1498 and VTKi for 30 min before the addition of LPC (20 μM) to examine their effects on ERK1/2 or Akt activation, respectively. Both compounds significantly inhibited ERK1/2 and Akt activation in response to 20 μM LPC stimulation (Fig. 6). These findings suggest that LPC-induced transactivation of Flk-1/KDR is involved in the activation of ERK1/2 and Akt downstream. Furthermore, we examined the effects of herbimycin A, PP2, and KI Src transfection on ERK1/2 and Akt activation by LPC to investigate whether c-Src is involved in these phenomena. The cells were pretreated with herbimycin A (1 μM) for 30 min and PP2 (10 μM) for 2 h before the addition of LPC (20 μM) to examine their respective effects on ERK1/2 or Akt activation. Herbimycin A and PP2 both inhibited LPC-induced ERK1/2 and Akt phosphorylation (Fig. 7, A and B). Transfection of KI Src also inhibited LPC-induced ERK1/2 and Akt phosphorylation in HUVECs (Fig. 7, C and D). These findings suggest that ERK1/2 and Akt are downstream effector molecules of c-Src, which is suggested to be involved in the LPC induction of Flk-1/KDR transactivation.
LPC-induced Flk-1/KDR transactivation stimulates HUVEC proliferation
Flk-1/KDR activation is reported to induce cell growth and differentiation (24). Therefore, to investigate pathophysiological implications of LPC-induced Flk-1/KDR transactivation, we examined the effect of LPC on HUVEC proliferation. As shown in Fig. 8A, MTT assay revealed that stimulation with LPC increased HUVEC viability over a 24-h incubation period in a dose-dependent manner (1–20 μM). In addition, we examined whether LPC-induced HUVEC proliferation was mediated by Flk-1/KDR transactivation as well as activation of ERK1/2 and Akt. The cells were pretreated with SU1498 (30 μM), VTKi (5 μM), the MEK1/2 inhibitors PD98059 (10 μM) and U0126 (10 μM), and the phosphatidylinositol 3-phosphate kinase (PI3K) inhibitor LY294002 (10 μM) and wortmannin (10 nM) for 30 min before the addition of LPC (20 μM). SU1498, VTKi, PD98059, U0126, LY294002, and wortmannin inhibited LPC-induced HUVEC proliferation (Fig. 8B). These results suggest that ERK1/2 and Akt are involved in LPC-induced proliferation of HUVECs. To characterize the role of c-Src in LPC-induced HUVEC proliferation, we then examined the effect of herbimycin A and PP2 on LPC-induced HUVEC proliferation. The cells were pretreated with herbimycin A (1 μM) for 30 min and PP2 (10 μM) for 2 h before the addition of LPC (20 μM). Both herbimycin A and PP2 significantly inhibited the HUVEC proliferation induced by LPC (Fig. 8C). Transfection of KI Src also inhibited LPC-induced HUVEC proliferation, whereas WT Src transfection induced HUVEC proliferation in the absence of LPC stimulation (Fig. 8D). These findings strongly suggest that c-Src is involved in LPC-induced HUVEC proliferation, which is mediated through Flk-1/KDR transactivation.
Discussion
The major finding of the present study is that LPC transactivates VEGF receptor Flk-1/KDR in HUVECs via c-Src tyrosine kinase activation. SU1498 and VTKi, which have been shown to inhibit Flk-1/KDR, inhibited LPC-induced Flk-1/KDR activation. Herbimycin A and PP2, specific inhibitors for Src family kinases, both inhibited LPC-induced Flk-1/KDR activation in a concentration-dependent manner. Transfection of KI Src also inhibited LPC-induced Flk-1/KDR activation. From these findings, it was suggested that c-Src is involved in Flk-1/KDR transactivation in HUVECs. Moreover, LPC-induced ERK1/2 and Akt activation, which are both reported to be downstream of Flk-1/KDR (17), were also inhibited by SU1498, VTKi, herbimycin A, PP2, and transfection with KI Src. It was also found that LPC-induced Flk-1/KDR transactivation resulted in a stimulation of HUVEC proliferation. VEGF receptor inhibitors, MEK1/2 inhibitors, PI3K inhibitors, Src family inhibitors, and transfection with KI Src all inhibited LPC-induced HUVEC proliferation.
The transactivation of RTK in response to the stimulation of a number of GPCRs, such as transactivation of the epithelial growth factor receptor by GPCR ligands such as thrombin, angiotensin II, lysophosphatidic acid, and endothelin-1 has been reported (18, 26). However, LPC-stimulated transactivation of RTK has not yet been reported or elucidated. Nevertheless, the role of LPC in the pathogenesis of atherosclerosis and systemic autoimmune diseases is well documented (27, 28), even though its specific cell surface receptor was not identified until quite recently. To date, a subfamily of GPCRs consisting of G2A and GPR4 have been identified as the receptors for LPC (9, 10). It is reported that G2A is expressed in atherosclerotic lesions (29). On the other hand, it is reported that G2A and GPR4 are proton-sensing GPCRs (30, 31). However, it is also reported that GPR119 is a newly identified receptor for LPC (11). Although it still remains to be clarified what GPCRs are responsible for LPC action, we found that LPC caused Flk-1/KDR transactivation in HUVECs. LPC has been implicated in the pathological states of vascular ECs that may be related to the progressive pathological events of atherosclerosis. It has been reported that the plasma concentration of LPC is elevated in patients with coronary artery diseases, compared with that of normal subjects (32). However, the precise intracellular mechanisms mediated by LPC stimulation in ECs have not yet been clarified. In the present study, we found for the first time that LPC induces Flk-1/KDR transactivation in HUVECs (Figs. 1 and 2). Because Flk-1/KDR activation is reported to be an important factor for atherogenesis (33, 34), LPC-induced Flk-1/KDR transactivation is taken to be one of the pathological developments of atherosclerosis.
Several mechanisms for RTK transactivation have been proposed. For example, Pyk2, reactive oxygen species, intracellular-free Ca2+, and matrix metalloproteases have been identified as factors that activate RTK in many types of cells (17, 18). We and others previously reported that c-Src is involved in the downstream signaling events after stimulation of GPCR and RTK (15, 16, 19). Therefore, we examined whether c-Src mediates LPC-induced Flk-1/KDR transactivation in the present study. As shown in Fig. 3, c-Src was rapidly and significantly activated by LPC in HUVECs. Furthermore, herbimycin A, PP2, and transfection of KI Src significantly inhibited LPC-induced Flk-1/KDR transactivation (Figs. 4 and 5). From these findings, it is strongly suggested that c-Src is involved in LPC-induced Flk-1/KDR transactivation, and c-Src may be localized near the receptor and plasma membrane. However, because we did not determine whether c-Src directly regulates Flk-1/KDR activity in this study, further studies are required to define the precise role and the mechanisms of Src kinase in Flk-1/KDR transactivation.
One previous report suggested that a PI3K-Akt- endothelial nitric oxide synthase pathway exists downstream of the Flk-1/KDR transactivation by sphingosine-1 phosphate because the sphingosine-1 phosphate-induced Akt and endothelial nitric oxide synthase activation were inhibited by SU1498 and VTKi (17). In the present study, we found similarly that LPC-induced ERK1/2 and Akt activation were inhibited by SU1498 and VTKi (Fig. 6). It is reported that ERK1/2 and Akt are activated by VEGF as early as 5–10 min after administration (35). In the present study, we found that LPC-induced ERK1/2 and Akt activation were observed. Both the ERK1/2 and Akt activation after transactivation are likely to have an important role in EC function.
The proliferation of ECs is a prominent feature of atherosclerotic lesions because it is an important step toward angiogenesis as well as contributing to the intimal thickening that develops after endothelial injury (8, 36). LPC is closely related to atherosclerosis because LPC is a component of the oxLDL, which is widely considered to be a major risk factor for atherosclerosis (3, 4). Therefore, we further examined the effect of LPC on HUVEC proliferation. LPC-induced HUVEC proliferation in a concentration-dependent manner with the maximal effect of LPC at 20 μM (Fig. 8A). This result is consistent with the findings of Wolfram et al. (8), who reported that 20 μM LPC induces vascular endothelial proliferation. However, we also observed that LPC at higher concentrations than 25 μM brought about HUVEC death rather than proliferation (data not shown). At higher concentration than 25 μM, LPC has been shown to cause vascular smooth muscle cell apoptosis (37). It is also reported that LPC can induce GPCR-mediated apoptosis (38). However, in contrast with these findings, it is reported that LPC at 10–20 μM facilitates cell growth, differentiation, and proliferation (8, 39). In agreement with these later findings, our results show that 20 μM LPC significantly induced HUVEC proliferation (Fig. 8A). As shown by the results of pretreatment with SU1498, VTKi, PD98059, U0126, LY294002, wortmannin, herbimycin A, and PP2 and transfection with KI Src, LPC-induced HUVEC proliferation was inhibited (Fig. 8, B–D). On the other hand, we measured lysophospholipase D that produces physiologically active lysophosphatidic acid from LPC (40, 41). In this study, LPC did not activate lysophospholipase D in HUVEC (data not shown). From these findings, it is suggested that LPC-induced Flk-1/KDR transactivation via c-Src is implicated as a contributor to the development of atherosclerosis, especially those steps in which EC proliferation plays a pivotal role.
In summary, we show for the first time that VEGF receptor Flk-1/KDR is transactivated by LPC in HUVECs. c-Src activation is suggested to be involved in LPC-induced Flk-1/KDR transactivation. In addition, LPC-induced Flk-1/KDR transactivation caused ERK1/2 and Akt activation in HUVECs. Although the specific mechanistic steps of the pathophysiological role of LPC remain to be elucidated, LPC-induced Flk-1/KDR transactivation and the resultant proliferation of vascular ECs very likely have a role in cardiovascular diseases such as atherosclerosis.
Acknowledgments
We thank Dr. A. Tokumura (Faculty of Pharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima Graduate School) for excellent advisement and technical support.
Footnotes
First Published Online December 1, 2005
Abbreviations: EBM, Endothelial cell basal medium; EC, endothelial cell; FBS, fetal bovine serum; Flk-1, fetal liver kinase-1; GPCR, G protein-coupled receptor; HUVEC, human umbilical vein endothelial cell; KDR, kinase-insert domain-containing receptor; KI, kinase inactive; LPC, lysophosphatidylcholine; MEK, MAPK kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; oxLDL, oxidized low-density lipoprotein; PI3K, phosphatidylinositol 3-phosphate kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine; RTK, receptor tyrosine kinase; SU1498, (E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl) amino-carbonyl; VEGF, vascular endothelial growth factor; VTKi, (4-[4'-chrolo-2'-fluoro]phenylamino)6,7-dimethoxyquinazoline; WT, wild type.
Accepted for publication November 21, 2005.
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