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Endocrinology Vol. 142, No. 5 1710-1719
Copyright © 2001 by The Endocrine Society

ARTICLES

The Role of Phosphatidylinositol 3-Kinase and the Mitogen-Activated Protein Kinases in Insulin-Like Growth Factor-I-Mediated Effects in Vascular Endothelial Cells1

Wenli Liu, Yueqin Liu and William L. Lowe Jr.

Center for Endocrinology, Metabolism, and Molecular Medicine (W.L., W.L.L), and Robert H. Lurie Cancer Center (Y.L.), Department of Medicine, Northwestern University Medical School and Veterans Affairs Chicago Healthcare System, Lakeside Division, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15–703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite an improved understanding of the molecular mechanisms of insulin-like growth factor-I (IGF-I) signaling and the recognition that IGF-I mediates many effects in endothelial cells, some of which may be important for atherosclerosis, little is known about the signal transduction pathways that mediate the effects of IGF-I in endothelial cells. To that end, we examined the signaling pathways activated by IGF-I in endothelial cells and their contribution to IGF-I-stimulated endothelial cell migration and nuclear factor (NF)-{kappa}B-dependent transcription. Treatment of bovine pulmonary artery endothelial cells (PAEC) with IGF-I activated the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1/2 and ERK5. In contrast, IGF-I had no effect on either c-Jun amino-terminal kinase or p38 kinase activity. IGF-I also activated phosphatidylinositol (PI) 3-kinase, as reflected by increased phosphorylation of Akt. There was no evidence of cross-talk between the ERK and PI 3-kinase pathways in PAEC. In PAEC transiently transfected with pTK81-NF{kappa}B-Luc, which contained four copies of the NF-{kappa}B DNA binding site 5' to a minimal promoter and the luciferase gene, treatment with 50 ng/ml IGF-I increased luciferase activity 1.8-fold. Inhibition of ERK activity using PD98059 and PI 3-kinase activity with LY 294002 abrogated the induction of NF-{kappa}B-dependent transcription by IGF-I, suggesting that both pathways contribute to the effect of IGF-I on NF-{kappa}Bdependent transcription. In contrast to the effect of tumor necrosis factor-{alpha} on NF-{kappa}B activation, Western blot analyses demonstrated that IGF-I had no effect on I{kappa}B phosphorylation and degradation or nuclear translocation and DNA binding of NF-{kappa}B. These data suggest a direct of effect of IGF-I on nuclear NF-{kappa}B. IGF-I also increased endothelial cell migration approximately 2-fold, as demonstrated using a Boyden chamber apparatus. IGF-I-induced endothelial cell migration was inhibited, in part, by LY 294002 but not PD98059. Together, these studies demonstrate that IGF-I activates multiple signaling pathways in endothelial cells with little evidence for cross-talk between the pathways. Moreover, these pathways appear to mediate both overlapping and distinct effects in that activation of both PI 3-kinase and the ERKs contributed to the stimulation of NF-{kappa}B-dependent transcription by IGF-I, whereas only PI 3-kinase mediated IGF-I-stimulated endothelial cell migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATHEROSCLEROSIS is a complex process that occurs in response to vascular injury and results from the complex interplay among multiple cell types, including endothelial cells, vascular smooth muscle cells, and monocytes (1, 2). In the injury model of atherosclerosis, smooth muscle cell proliferation contributes to intimal hyperplasia and plaque formation, whereas endothelial cell proliferation, which promotes healing of the endothelium, is antiatherogenic (1, 2). Given the central role of cell proliferation in the pathogenesis of atherosclerosis, the role of growth factors in atherosclerosis has been studied extensively. Among the growth factors that have received attention is insulin-like growth factor-I (IGF-I). IGF-I is released by several cell types that contribute to the formation of atheroma, including vascular smooth muscle cells, macrophages, and platelets (3, 4, 5, 6). Moreover, IGF-I is expressed at sites of vascular injury, for example in the walls of arteries after balloon denudation (7, 8). Although much attention has been focused on the mechanism of IGF-I effects in smooth muscle cells, less attention has been directed toward IGF-I-stimulated signaling in endothelial cells.

The cellular events that follow IGF-I binding and account for its biological effects are still being elucidated, but signaling pathways that are activated upon IGF-I binding have been defined. Ligand binding to the receptor activates the intrinsic tyrosine kinase activity of the receptor and results in phosphorylation of members of a family of insulin receptor substrates and other molecules, including Shc, Crk, and Grb2 (9, 10). Subsequent to these initial events, two signaling pathways that mediate many of the effects of IGF-I are activated, the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) pathways (9, 10). In vascular smooth muscle cells, these pathways contribute to the regulation of IGF-I-induced DNA synthesis and cell migration (11, 12).

The purpose of the present study was to begin to define the signaling pathways important for IGF-I-mediated effects in endothelial cells. To that end, we have defined signaling pathways activated by IGF-I in endothelial cells and their contribution to two important IGF-I mediated effects in these cells: cell migration and activation of the transcription factor nuclear factor (NF)-{kappa}B. Endothelial cell migration is important for repair of the endothelium at sites of vascular injury, whereas NF-{kappa}B demonstrates increased expression at sites of high probability for atherosclerosis and, upon activation, both protects cells from apoptosis and participates in inflammatory processes (13, 14, 15, 16, 17).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Many of the previous studies examining the effect of IGF-I in endothelial cells have been performed in cells of bovine origin (reviewed in Ref. 18). Thus, for the present studies, bovine pulmonary artery endothelial cells (PAEC) were used. These cells were provided by Dr. Mark Yorek (University of Iowa, Iowa City, IA) and were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 U/ml penicillin, and 0.01 mg/ml streptomycin at 37 C in a 5% CO2 and 95% air atmosphere. Upon reaching confluence the cells were replated at a dilution of 1:4. Cells were used between passages 3 and 10.

MAPK immune complex assays
For the kinase assays, cells were plated at a density of 5 x 105 cells per 75-cm2 plate. The cells were preincubated in serum-free medium with 0.25% BSA (DMEM + 0.25% BSA) for 14–16 h before being treated with growth factors. Antibodies directed against extracellular signal-regulated kinase (ERK)-2, c-Jun amino-terminal kinase-1 (JNK1), p38 kinase, and ERK5 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The substrate protein for the JNK immune complex assay was a glutathione-S-transferase (GST)-c-Jun fusion protein, which was generated as described previously (19). The substrate for the p38 kinase assay was GST-ATF2, which was prepared using the plasmid pGST-ATF2 (kindly provided by Dr. Roger Davis, Howard Hughes Medical Institute, University of Massachusetts Medical School, Wooster, MA). The substrate for the ERK2 and ERK5 assays was myelin basic protein (Sigma, St. Louis MO). The activity of ERK2, JNK1, p38 kinase, and ERK5 was determined using immune complex assays as described previously (19, 20, 21). The final reaction products were separated by SDS-PAGE followed by autoradiography. 32P incorporation into substrate proteins was quantified using a STORM 860 Phosphor-Imager (Molecular Dynamics, Inc., Sunnyvale, CA).

Western blot analysis
Western blots were probed with polyclonal antibodies directed against phospho-ERK1/2, phospho-Akt, Akt, phospho-I{kappa}B, and I{kappa}B (New England Biolabs, Inc., Beverly, MA) at a dilution of 1:1,000 or with a polyclonal antibody directed against ERK1 and ERK2 (Santa Cruz Biotechnology, Inc.) at a dilution of 1:7,500.

Western blot analyses were performed as described previously (19, 20). Briefly, cell lysates were prepared in cell lysis buffer [150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.5), 1 µM Na orthovanadate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.7 µg/ml leupeptin], and protein content of the lysate was determined using the Coomassie blue protein assay (Bio-Rad Laboratories, Inc. Richmond, CA). Forty micrograms of protein were size separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes in a semidry apparatus. The membranes were blocked in 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween-20 (TBST) and 4% nonfat dry milk for 90 min at room temperature. Membranes were incubated for 3 h at 22 C in TBST containing nonfat milk and primary antibody, washed three times for 15 min at 22 C in TBST, and incubated for 90 min at room temperature in TBST containing nonfat dry milk and second antibody (1:7,500 dilution). Following three washes in TBST, immunoreactive bands were detected using the enhanced chemiluminescence (ECL) detection system from Amersham Pharmacia Biotech (Arlington Heights, IL), according to the manufacturer’s instructions.

Electrophoretic mobility shift assay
For the preparation of nuclear extracts, cells were harvested after the indicated treatment in 5 ml PBS containing 1 µM EDTA, 1 mM dithiothreitol (DTT), and 1 mM PMSF. The cells were resuspended in 400 µl cold buffer A [10 mM HEPES (pH 7.9), 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM p-aminobenzamindine) and incubated on ice for 15 min. Following addition of 25 µl of 10% NP-40 and vigorous vortexing for 10 sec, cell pellets were resuspended in 50 µl of cold buffer C [20 mM HEPES (pH 7.9), 0.4 M KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 20% glycerol, 1 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 mM p-aminobenzamindine] and mixed at 4 C for 15 min. The mixture was clarified, and the protein content of the supernatant was determined using the Coomassie blue protein assay. Nuclear extracts were stored at -70 C.

For the electrophoretic mobility shift assays, annealed oligonucleotides containing the consensus DNA binding sequence for NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3'; 5'-GCCTGGGAAAGTCCCCTCAACT-3') or the E-box of the adenovirus major late transcription factor promoter (5'-ATAGGTGTAGGCCACGTGACCGGGTGT-3'; 5'-ACACCCGGTCACGTGGCCTACACCTAT-3') were radiolabeled with {gamma}-32P-ATP using T4 polynucleotide kinase as described previously (22). Incubation of radiolabeled DNA with cellular nuclear extracts, separation of DNA-protein complexes using low ionic strength native PAGE, and detection of signals were performed as described previously (22). To verify the specificity of the binding reaction, a 100-fold excess of unlabeled oligonucleotide was added to the reaction before adding the labeled probe.

Transient transfection and luciferase assays
pTK81-NF{kappa}B-Luc was constructed by cloning two copies of annealed oligonucleotide containing the sequence 5'-CGCGGGGACTTTCCCgtacGGGGACTTTCCCG-CGgtac-3' in the antisense orientation 5' to the luciferase gene in the KpnI site in the vector pTK81-Luc. This sequence contained two copies (indicated in capital letters) of a NF-{kappa}B response element. For transfection assays, cells were plated onto 12-well plates at a density of 1 x 105 cells per well. Plasmid DNA was transfected into cells using the cationic lipid Lipofectin (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instruction. Cells were incubated in Opti-mem with lipofectin and plasmid DNA for 4 h and then transferred to serum-free DMEM + 0.25% BSA. For experiments using the indicated inhibitors, inhibitors were added 30 min before treating the cells with either IGF-I or tumor necrosis factor (TNF)-{alpha}. After treatment for 6 h, cells were harvested, and luciferase assays were performed as described previously (22). Light emission was measured with an AutoLumatLB953 luminometer (E.G.& G. Berthold, Bad Wildbad, Germany). The luciferase activity present in each sample was normalized using the protein content of the sample. All luciferase assays were performed in triplicate.

Cell migration assay
The migration assay was performed using a modified 48-well Boyden chamber apparatus (Neuroprobe Inc., Cabin John, MD). IGF-I was diluted to 50 ng/ml in serum-free DMEM + 0.25% BSA and loaded into the lower wells of the Boyden chamber in triplicate. The wells were subsequently covered with a polyvinylpyrrolidone-free filter with 8-µm pores (Nucleopore Corp., Pleasanton, CA) coated with type I collagen (Vitrogen, Collagen Corp., Palo Alto, CA). The cells were serum starved with serum-free DMEM + 0.1% BSA overnight and then trypsinized and resuspended at a density of 1 x 106 cells per ml. Cells (50,000 in 50 µl) were loaded into the upper wells of the Boyden chamber. For assays with inhibitors, the cells were treated with DMEM + 0.1% BSA containing the indicated inhibitor for 30 min and then trypsinized and washed. The cells were then resuspended in DMEM + 0.1% BSA containing the inhibitor and loaded into the upper well of the Boyden chamber in triplicate. The chambers were incubated for 6 h at 37 C in a 5% CO2 and 95% air atmosphere. At the end of the incubation time, the cells attached to the filter were fixed and stained in DifQuick stain ( Allegiance Healthcare Corp., Waukegan, IL). The number of cells that had migrated to the lower chamber was determined by counting the number of cells in a 6x magnification field of each well. All studies were done in triplicate.

Statistical analyses
Values are reported as the mean ± SEM. P values were calculated using the one-way repeated measures ANOVA with Tukey’s pairwise multiple comparison procedure or Kruskal-Wallis one-way ANOVA on Ranks with the Dunnett’s pairwise multiple comparison procedure, as appropriate, using SigmaStat 2.0 software (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I-stimulated signaling pathways in PAEC
As a first step in defining the signaling pathways that mediate the effects of IGF-I on endothelial cells, the ability of IGF-I to activate different signaling pathways in bovine PAEC was examined. Initial studies examined the effect of IGF-I on activation of various members of the MAPK family, including ERK1/2, JNK1, p38 kinase, and ERK5. ERK activation is dependent upon tyrosine and threonine phosphorylation by various MEKs (23). To determine whether the ERKs were activated by IGF-I, serum-starved PAEC were treated for varying periods of time with 50 ng/ml IGF-I, and Western blot analyses were performed using an antibody specific for the phosphorylated form of the ERKs (Fig. 1AGo). IGF-I-stimulated ERK phosphorylation was evident and maximal 15 min after treatment with IGF-I with a return to basal levels 120 min after IGF-I treatment (Fig. 1AGo). The ability of IGF-I to stimulate ERK phosphorylation was then compared with the effect of two well described growth factors for endothelial cells: vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) (Fig. 1BGo). As can be seen, the effect of IGF-I was similar to that of VEGF but slightly less than that of bFGF. Commensurate with the ability of IGF-I to increase ERK phosphorylation, treatment with IGF-I also increased ERK2 activity, as demonstrated using an immune complex assay (Fig. 1CGo). The IGF-I-induced increase in ERK2 activity was similar to that stimulated by bFGF and VEGF.



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  		Figure 1. A, Time course of the effect of IGF-I on ERK phosphorylation in bovine PAEC. PAEC were plated at a density of 5 x 105 cells per 75-cm2 plate, and 24 h after plating, medium was replaced with DMEM + 0.25% BSA for 16 h before treatment with 50 ng/ml IGF-I for the indicated periods of time. Proteins in cell lysates were transferred to polyvinylidene difluoride membranes. Western blot analysis was performed as described in Materials and Methods using a 1:1,000 dilution of antibody directed against phospho-ERK. After detection of phospho-ERK, the blot was stripped and reprobed with a 1:7,500 dilution of an antibody directed against ERK1 and -2. The results are representative of the results of three independent experiments performed using different cell lysates. B, The effect of growth factors on ERK phosphorylation. The cells were grown as described above and, following incubation in DMEM + 0.25% BSA for 16 h, were treated for 15 min with either DMEM + 0.25% BSA alone (Con), 5 ng/ml TNF-{alpha}, 50 ng/ml IGF-I, 20 ng/ml bFGF, or 50 ng/ml VEGF. Western blot analyses were performed as described above. The results are representative of the results of three independent experiments performed using different cell lysates. C, Autoradiogram of phosphorylated myelin basic protein. Cells were treated with either DMEM + 0.25% BSA alone (Con), 5 ng/ml TNF-{alpha}, 50 ng/ml IGF-I, 20 ng/ml bFGF, or 50 ng/ml VEGF for 15 min. ERK2 activity was measured using an immune complex assay as described in Materials and Methods. The results are representative of two independent experiments performed using different cell lysates.

 
Immune complex assays were next used to examine the effect of IGF-I on JNK1, p38 kinase, and ERK5 activity. Neither JNK1 nor p38 kinase activity was increased by treating cells for 15 min with 50 ng/ml IGF-I (Fig. 2Go, A and B). Similarly, neither VEGF nor bFGF was able to activate these MAPKs. In contrast, JNK 1 activity was markedly increased by treating cells for 15 min with 5 ng/ml TNF-{alpha}, whereas activation of p38 kinase in the PAEC was demonstrated by inducing hyperosmolarity with increasing concentrations of raffinose, a trisaccharide that is unable to cross the plasma membrane (Fig. 2Go, A and B). ERK5 activity, which is activated by oxidative stress, shear stress, and some growth factors (24, 25), was also increased by treatment of PAEC with IGF-I. In cells treated with 50 ng/ml IGF-I, maximal ERK5 activity was present within 15–30 min (Fig. 3AGo), and IGF-I increased ERK5 activity 2.5-fold (Fig. B). Epidermal growth factor (EGF), which has been shown previously to increase ERK5 activity (24), stimulated a similar increase in ERK5 activity, whereas TNF-{alpha} had no effect.



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  		Figure 2. Effect of IGF-I and other growth factors on JNK1 and p38 kinase activity in PAEC. A, Autoradiogram of phosphorylated GST-c-Jun81 substrate protein. Cells were treated with DMEM + 0.25% BSA for 16 h and then treated with either DMEM + 0.25% BSA alone (Con), 10 ng/ml EGF, 5 ng/ml TNF-{alpha}, 50 ng/ml IGF-I, 20 ng/ml bFGF, or 50 ng/ml VEGF for 15 min. JNK1 activity was measured using an immune complex assay as described in Materials and Methods. The results are representative of the results of three independent experiments performed using different cell lysates. B, Autoradiogram of phosphorylated GST-ATF2. Cells were treated as described above, and p38 kinase activity was measured using an immune complex assay. As a control, cells were also treated with either DMEM + 0.25% BSA alone or DMEM + 0.25% BSA with the indicated concentrations of raffinose. The results are representative of the results of three independent experiments performed using different cell lysates.

 


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  		Figure 3. A, Time course effect of IGF-I on ERK5 activity in PAEC. Cells were treated with DMEM + 0.25% BSA for 16 h and then treated with 50 ng/ml IGF-I for the indicated periods of time. ERK5 activity was determined using an immune complex assay with myelin basic protein as a substrate protein. Shown is a representative autoradiogram from one of three independent experiments performed using different cell lysates. B, Effect of IGF-I and other growth factors on ERK5 activity. Cells were treated with DMEM + 0.25% BSA for 16 h, and then treated with either DMEM + 0.25% BSA alone (Con), 10 ng/ml EGF, 5 ng/ml TNF-{alpha}, or 50 ng/ml IGF-I for 15 min. ERK5 activity was determined using an immune complex assay. Values represent the relative 32P incorporation into myelin basic protein compared with 32P incorporation using control lysates, which was defined as 1.0, and are the mean ± SEM of three independent experiments. *, P < 0.05 vs. the activity in control cells.

 
In addition to the ERKs, PI 3-kinase activation mediates many of the effects of IGF-I. To document IGF-I-induced activation of PI 3-kinase, Western blot analyses were performed using an antibody specific for phosphorylated Akt/protein kinase B, a kinase downstream of PI 3-kinase (Fig. 4AGo). Treatment of cells for 15 min with 50 ng/ml IGF-I stimulated a marked increase in Akt phosphorylation. VEGF and bFGF also increased Akt phosphorylation, although their effect was not as marked as that of IGF-I. In contrast, TNF-{alpha} had no effect on Akt phosphorylation. Time course studies demonstrated that Akt phosphorylation was evident within 5 min of IGF-I treatment with maximal stimulation at 15 min (Fig. 4BGo). Interestingly, the effect of IGF-I on Akt phosphorylation was sustained for up to 2 h.



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  		Figure 4. A, Effect of IGF-I and other growth factors on Akt phosphorylation in PAEC. Cells were treated with DMEM + 0.25% BSA for 16 h and then treated for 15 min with either DMEM + 0.25% BSA alone (Con), 5 ng/ml TNF-{alpha}, 50 ng/ml IGF-I, 20 ng/ml bFGF, or 50 ng/ml VEGF. Western blot analysis was performed as described in Materials and Methods using an antibody directed against phosphorylated Akt at a dilution of 1:1,000. After detection of phospho-Akt, the blot was stripped and reprobed with a 1:1,000 dilution of an antibody directed against Akt. The results are representative of the results of three independent experiments performed using different cell lysates. B, Time course effect of IGF-I on Akt phosphorylation. Cells were treated with DMEM + 0.25% BSA for 16 h and then treated for the indicated periods of time with 50 ng/ml IGF-I. Phosphorylated and total Akt levels were determined as described above. The results are representative of the results of three independent experiments performed using different cell lysates.

 
To document the efficacy of inhibitors on IGF-I-induced signaling in the PAEC and, more importantly, to determine whether cross-talk between the different signaling pathways is present, the effect of PD98059, an inhibitor of the MAPK kinase MEK1, or LY 294002, an inhibitor of PI 3-kinase, on IGF-I-induced ERK and PI 3-kinase activity was examined. Previous studies have demonstrated a role for PI 3-kinase in the activation of Ras and the ERKs (3, 4, 5, 6). As anticipated, phosphorylation of ERK1/2 was effectively inhibited by 20 µM PD98059, whereas, 20 µM LY 294002 had no effect on ERK activity (Fig. 5AGo). Similarly, 20 µM LY 294002 abrogated IGF-I-induced Akt phosphorylation, but 20 µM PD98059 had no effect on IGF-I-stimulated phosphorylation of Akt (Fig. 5Go, A and B). Finally, activation of ERK5 by IGF-I has not been previously documented, and the signaling pathway that mediates this effect of IGF-I is unknown. Treatment of cells with 20 µM LY 294002 had no effect on IGF-I-induced ERK5 activity, whereas 20 µM PD98059 partially inhibited ERK5 activation by IGF-I (Fig. 5CGo). Similarly, 100 nM Calphostin C, an inhibitor of protein kinase C, partially abrogated IGF-I-induced ERK5 activity, suggesting that protein kinase C participates in activation of ERK5 by IGF-I.



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  		Figure 5. A, Effect of PD98059 and LY 294002 on ERK phosphorylation in PAEC. Cells were treated with DMEM + 0.25% BSA for 16 h, pretreated for 30 min with either 20 µM PD98059, 20 µM LY 294002, or DMEM + 0.25% BSA alone, and then treated for 15 min with DMEM + 0.25% BSA without or with 50 ng/ml IGF-I and the indicated inhibitor. Phospho-ERK levels were detected by Western blot analysis as described, and, after detection of phospho-ERK, the blot was reprobed with an antibody directed against ERK1 and -2, as described. The results are representative of the results of three independent experiments performed using different cell lysates. B, Effect of LY 294002 and PD98059 on IGF-I-induced Akt phosphorylation in PAEC. Cells were pretreated with the indicated inhibitor as described above and then with DMEM + 0.25% BSA without or with 20 µM LY 294002, 20 µM PD98059, and/or 50 ng/ml IGF-I for 15 min as indicated. Phospho-Akt and total Akt levels were determined as described. The results are representative of the results of three independent experiments performed using different cell lysates. C, Effect of LY 294002, PD98059, and Calphostin C on IGF-I-induced ERK5 activity. Cells were treated with DMEM + 0.25% BSA for 16 h, pretreated for 30 min with either 20 µM PD98059, 20 µM LY 294002, 100 nM Calphostin C, or DMEM + 0.25% BSA alone, and then treated for 15 min with DMEM + 0.25% BSA without or with 50 ng/ml IGF-I and the indicated inhibitor. ERK5 activity was determined using an immune complex assay. Values represent the relative ERK5 activity compared with the activity in cells maintained in DMEM + 0.25% BSA (Con), which was defined as 1.0 and are the mean ± SEM of three independent experiments. *, P < 0.05 vs. level in control cells; +, P < 0.05 vs. level in cells treated with 50 ng/ml IGF-I.

 
Effect of IGF-I on NF-{kappa}B activity
Having defined signaling pathways activated by IGF-I in PAEC, the role of these pathways in IGF-I-mediated effects was examined. The first of these effects was the ability of IGF-I to increase NF-{kappa}B-dependent transcriptional activity. To address this, PAEC were transiently transfected with pTK81-NF{kappa}B-Luc, a fusion gene that contained four copies of the NF-{kappa}B DNA binding site 5' to a minimal thymidine kinase promoter and the luciferase gene in the vector pTK81. Treatment of cells for 6 h with 50 ng/ml IGF-I increased luciferase activity 1.8-fold (Fig. 6Go, top panel), whereas in cells transfected with pTK81, IGF-I increased luciferase activity only 1.1 ± 0.0-fold (mean ± SEM, n = 3). Treatment of cells with either 20 µM PD98059 or LY 294002 inhibited the IGF-I-induced increase in luciferase activity (Fig. 6Go, top panel), suggesting that both the ERK and PI 3-kinase pathways contribute to IGF-I-induced activation of NF-{kappa}B. For comparison, the effect of TNF-{alpha}, a cytokine that has been shown previously to increase NF-{kappa}B-mediated transcription, was examined. TNF-{alpha} stimulated a more robust 3.2-fold increase in NF-{kappa}B activity (Fig. 6Go, bottom panel), and, interestingly, despite the apparent lack of effect of TNF-{alpha} on ERK1/2 or PI 3-kinase activity, both LY 294002 and PD98059 partially abrogated TNF-{alpha}-induced luciferase activity (Fig. 6Go, bottom panel), suggesting that both pathways, in some manner, facilitate activation of NF-{kappa}B by TNF-{alpha}.



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  		Figure 6. A, Effect of IGF-I on NF-{kappa}B activity. pTK81-NF-{kappa}B was transfected into PAEC as described in Materials and Methods. Following transfection, the cells were treated for 16 h with DMEM + 0.25% BSA and were then preincubated for 30 min with DMEM + 0.25% BSA without or with either 20 µM LY 294002 or 20 µM PD98059. The cells were then treated for 6 h with DMEM + 0.25% without or with 50 ng/ml IGF-I and the above inhibitors. The cells were harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared with the activity in cells maintained in DMEM + 0.25% BSA alone (Con), which was defined as 1.0, and are the mean ± SEM of three independent experiments performed in triplicate. *, P < 0.05 vs. the activity in control cells; {ddagger}, P < 0.05 vs. the activity in cells treated with 50 ng/ml IGF-I. B, Effect of TNF-{alpha} on NF-{kappa}B activity. Cells were transfected and treated as described above except that treatment was with 5 ng/ml TNF-{alpha}. The values represent the relative luciferase activity compared with the activity in cells maintained in DMEM + 0.25% BSA alone (Con), which was defined as 1.0, and are the mean ± SEM of three independent experiments performed in triplicate. *, P < 0.05 vs. the activity in control cells; {ddagger}, P < 0.05 vs. the activity in cells treated with 5 ng/ml TNF-{alpha}; +, P < 0.05 compared with the activity in cells treated with either LY 294002 or PD98059 alone.

 
Subsequent studies further examined the mechanism for IGF-I-induced NF-{kappa}B activity. A key event in the activation of NF-{kappa}B is release of functional NF-{kappa}B dimers from the inhibitory protein, I{kappa}B, by phosphorylation and subsequent degradation of I{kappa}B (15). Release of the NF-{kappa}B dimers results in their translocation to the nucleus. To determine whether IGF-I stimulates I{kappa}B phosphorylation and degradation, PAEC were treated with 50 ng/ml IGF-I for varying periods of time, and Western blot analyses were performed using antibodies directed against I{kappa}B and phospho-I{kappa}B. As a control, cells were also treated with 5 ng/ml TNF-{alpha}. Interestingly, IGF-I had no effect on either I{kappa}B phosphorylation or total I{kappa}B levels (Fig. 7AGo). In contrast, TNF-{alpha} stimulated the phosphorylation of I{kappa}B, with increased phospho-I{kappa}B levels present within 5 min of TNF-{alpha} treatment, and increased I{kappa}B degradation, with a decrease in I{kappa}B levels also present within 5 min of treatment with TNF-{alpha} (Fig. 7BGo). Consistent with previous studies, recovery of I{kappa}B levels was evident within 60 min of TNF-{alpha} treatment (15).



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  		Figure 7. Effect of IGF-I and TNF-{alpha} on I{kappa}B phosphorylation in PAEC. Cells were treated for 16 h with DMEM + 0.25% BSA and then with DMEM + 0.25% BSA without or with either 50 ng/ml IGF-I (panel A) or 5 ng/ml TNF-{alpha} (panel B) for the indicated periods of time. Western blot analysis was performed as described in Materials and Methods using a 1:1,000 dilution of antibody directed against phospho-I{kappa}B. After detection, the blot was stripped and reprobed with a 1:1,000 dilution of an antibody directed against I{kappa}B. The results are representative of the results of three independent experiments using different cell lysates.

 
Given the above findings, nuclear translocation of NF-{kappa}B and NF-{kappa}B DNA binding in response to IGF-I or TNF-{alpha} treatment were next examined. To define the effect of the above ligands on nuclear translocation of NF-{kappa}B, nuclear extracts were prepared from PAEC treated for varying periods of time with either IGF-I or TNF-{alpha}, and Western blot analyses were performed using an antibody directed against the p65 subunit of NF-{kappa}B (Fig. 8AGo). Again, differing effects of IGF-I and TNF-{alpha} were observed. Consistent with its effect on I{kappa}B phosphorylation and degradation, TNF-{alpha} increased the levels of p65 in the nucleus, whereas IGF-I had no effect on nuclear p65 levels. Gel shift analyses were used to examine the effect of IGF-I and TNF-{alpha} on DNA binding of NF-{kappa}B. Treatment with IGF-I had no effect on NF-{kappa}B DNA binding activity (Fig. 8BGo). In contrast, TNF-{alpha} stimulated a marked increase in NF-{kappa}B DNA binding activity (Fig. 8CGo). As a control, protein binding to an oligonucleotide containing the E box of the adenovirus major late promoter was determined. Protein binding to this oligonucleotide was unaffected by either IGF-I or TNF-{alpha} (Fig. 8Go, B and C). Together, the above data suggest that IGF-I regulates NF-{kappa}B transcriptional activity downstream of NF-{kappa}B nuclear translocation and DNA binding, whereas TNF-{alpha} stimulates NF-{kappa}B activity via a pathway that modulates nuclear translocation and DNA binding.



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  		Figure 8. A, Western blot analysis of the p65 subunit of NF-{kappa}B levels in nuclear extracts of PAEC. Cells were treated for 16 h with DMEM + 0.25% BSA and then with DMEM + 0.25% BSA without or with 50 ng/ml IGF-I or 5 ng/ml TNF-{alpha} for the indicated periods of time. Nuclear extracts were prepared and Western blots were performed as described in Materials and Methods using a 1:1000 dilution of an antibody directed against the p65 subunit of NF-{kappa}B. The results are representative of the results of three independent experiments using different cell lysates. B and C, Effect of IGF-I and TNF-{alpha} on NF-{kappa}B DNA binding. Nuclear extracts were prepared from cells treated with either 50 ng/ml IGF-I (panel B) or 5 ng/ml TNF-{alpha} (panel C) for the indicated periods of time, and gel shift analyses were performed as described in Materials and Methods using an oligonucleotide containing either a consensus NF-{kappa}B DNA binding site or the E box of the adenovirus major late transcription factor promoter. The lane labeled 100x indicates that an 100-fold excess of unlabeled oligonucleotide was used with the extracts obtained from cells treated for 6 h with either IGF-I (panel B) or TNF-{alpha} (panel C). The unlabeled arrow indicates the band corresponding to the specific NF-{kappa}B- or E box-DNA complex. The arrow labeled N.S. indicates a nonspecific band. The results are representative of the results of three independent experiments using different cell lysates.

 
Stimulation of PAEC migration by IGF-I
To further define the role of different signaling pathways in IGF-I-induced effects in PAEC, the effect of IGF-I on PAEC migration was examined. Previous studies have documented a stimulatory effect of IGF-I on endothelial cell migration (11, 12), although the signal transduction pathways that mediate this effect have not been defined. IGF-I stimulated a 1.9-fold increase in PAEC migration (Fig. 9Go). To examine the effect of 20 µM LY 294002 and 20 µM PD98059 on IGF-I-induced cell migration, the cells were pretreated for 30 min with the respective inhibitor before initiation of the migration assay (Fig. 9Go). Inhibition of IGF-I-induced PI 3-kinase activity using LY 294002 attenuated the effect of IGF-I on endothelial cell migration; in contrast, PD98059 had no effect on endothelial cell migration. These data suggest that activation of PI 3-kinase contributes to IGF-I-stimulated endothelial cell migration but that ERK activation is not required.



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  		Figure 9. Effect of IGF-I on PAEC migration. Cell migration assays using a Boyden chamber were performed as described in Materials and Methods. For inhibitor studies, the indicated inhibitors at a concentration of 20 µM were included in the media in the upper and lower chambers. The values represent the number of cells that migrated from the upper to lower chamber and are the mean ± SEM of three independent experiments, each performed in triplicate. *, P < 0.05 vs. control; +, P < 0.05 vs. cells treated with IGF-I; #, P < 0.05 vs. cells treated with LY 294002; {ddagger}, P < 0.05 vs. cells treated with PD98059.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGF-I receptors are widespread and expressed on a variety of cell types, including endothelial cells (18). Consistent with that, a variety of effects of IGF-I on microvessel and/or large-vessel endothelial cells have been documented, including stimulation of thymidine incorporation into DNA, uptake of amino acids and glucose, proteoglycan synthesis, increased monocyte adhesion, and cell migration (18, 26, 27, 28, 29, 30). To date, however, little is known about the signal transduction pathways that mediate the effects of IGF-I in endothelial cells. In the present study, we documented that IGF-I activates ERK1 and -2 and PI 3-kinase, as reflected by increased Akt phosphorylation, in large-vessel endothelial cells from bovine pulmonary arteries. Although previous studies have documented IGF-I-induced activity of p38 kinase in cells of neural origin and JNK in breast cancer cells (19, 31, 32), IGF-I did not increase either JNK or p38 kinase activity in endothelial cells. Interestingly, IGF-I did activate a more recently described member of the MAPK family, ERK5. ERK5 activation in response to oxidative stress and shear stress in endothelial cells has been previously documented (25, 33), as has its activation by EGF and nerve growth factor in other cell types (24, 34, 35). Limited and transient activation of ERK5 by platelet-derived growth factor has also been demonstrated (34). The results reported herein are the first to demonstrate ERK5 activation by IGF-I. Additional studies sought to determine whether there was cross-talk between the different IGF-I-activated signaling pathways in endothelial cells. Interestingly, PD98059 inhibited IGF-I-induced ERK5 activity in endothelial cells, a finding consistent with a previous study that demonstrated inhibition of EGF-induced ERK5 activity in COS7 cells overexpressing ERK5 (24). Whether the findings of the previous and current study represent cross-talk between ERK1/2 and ERK5 or inhibition of MEK5, the MAPK kinase that activates ERK5, by PD98059 is not known. MEK5 is most related and 48% identical to MEK1, the target of PD98059 (36, 37), suggesting that it may be inhibited by PD98059. Despite previous reports of modulation of ERK activity by PI 3-kinase in IGF-I-treated MCF-7 cells and VEGF-induced ERK activity in endothelial cells (38, 39), inhibition of PI 3-kinase had no effect on IGF-I-induced ERK activity and vice versa in endothelial cells.

A transcription factor that may be important for atherosclerosis and has been associated with endothelial cell activation and survival as well as endothelial cell dysfunction and vascular inflammation is NF-{kappa}B (13, 14, 40, 41). NF-{kappa}B is ubiquitously expressed and exists as a dimer composed of members of the NF-{kappa}B/Rel family (15). In endothelial cells, a heterodimer of NF-{kappa}B1 (p50) and p65 (RelA) is the predominant species of NF-{kappa}B (42) In unstimulated cells, NF-{kappa}B is retained in the cytoplasm by inhibitory proteins of the I{kappa}B family (15). Upon cellular activation, I{kappa}B is phosphorylated by a multisubunit I{kappa}B kinase complex, which targets I{kappa}B for ubiquination and degradation and liberates NF-{kappa}B, resulting in nuclear translocation of NF-{kappa}B and activation of transcription (15). Previous studies have demonstrated that IGF-I increases NF-{kappa}B DNA binding in vascular smooth muscle cells and NF-{kappa}B DNA binding, nuclear p65 levels, and NF-{kappa}B-dependent gene transcription in neural cells (43, 44). The role of IGF-I in regulating NF-{kappa}B binding and activity in endothelial cells has not been previously examined, except for one study that showed a transient increase in whole-cell p65 levels 30 min following treatment with IGF-I (45). The present study demonstrated that IGF-I increases NF-{kappa}B-dependent transcription in endothelial cells, although, in contrast to the previous studies in vascular smooth muscle and neural cells, IGF-I had no effect on either nuclear p65 levels or NF-{kappa}B DNA binding. Moreover, IGF-I had no effect on I{kappa}B phosphorylation or degradation. The induction of NF-{kappa}B-dependent transcription by TNF-{alpha}, on the other hand, was associated with I{kappa}B phosphorylation and degradation, increased nuclear p65 levels, and NF-{kappa}B DNA binding. As has been described previously, subsequent to TNF-{alpha} treatment, I{kappa}B levels recovered, with full recovery evident 60 min after TNF-{alpha} treatment (15, 46). Although phospho-I{kappa}B levels also appeared to increase, I{kappa}B levels were sustained during the recovery phase. Nuclear translocation is a key event in NF-{kappa}B activation, but protein phosphorylation also appears to contribute to regulation of NF-{kappa}B activity (15). A recent study demonstrated that both PI 3-kinase and Akt were able to stimulate the transcriptional activity of NF-{kappa}B by targeting the basal levels of nuclear NF-{kappa}B as opposed to stimulating nuclear translocation (47). Thus, the effect of IGF-I in PAEC may be mediated by phosphorylation of basal nuclear NF-{kappa}B. Consistent with that possibility, the present study demonstrated that the effect of IGF-I on NF-{kappa}B transcriptional activity in endothelial cells was inhibited by LY 294002, an inhibitor of PI 3-kinase. The stimulation of NF-{kappa}B-dependent transcription by IGF-I in neural cells also was inhibited by PI 3-kinase (44).

The present studies also demonstrated that the MEK1 inhibitor PD98059 was able to attenuate IGF-I-induced NF-{kappa}B activity. These data are consistent with a role for ERK1 and -2, in addition to PI 3-kinase, in IGF-I-induced NF-{kappa}B activity, although, given the effect of PD98059 on IGF-I-induced ERK5 activity, a role for ERK5 in the stimulation of NF-{kappa}B-induced transcription by IGF-I cannot be excluded. Previous studies have suggested a role for the ERKs in NF-{kappa}B activation. In cobalt chloride-treated endothelial cells, NF-{kappa}B DNA binding was inhibited by PD98059 (44), whereas in other cell types, inhibition of ERK activation with PD98059 was associated with either decreased, no change, or increased NF-kB activity (48, 49, 50, 51, 52, 53). Interestingly, the effect of ERK inhibition is not universally dependent upon changes in NK-{kappa}B nuclear translocation or DNA binding. For example, the inhibition by PD98059 of NF-{kappa}B-dependent IL-6 production in monocytes treated with okadaic acid was independent of NF-{kappa}B DNA binding (53). Similarly, the regulation of NF-{kappa}B-dependent transcription but not nuclear translocation was dependent upon ERK activation in Cos cells treated with TNF-{alpha} (54). Together with the present studies, these data suggest that the ERKs are capable of modulating NF-{kappa}B activity via a mechanism that is independent of nuclear translocation of NF-{kappa}B.

An additional factor important in atherosclerosis is vascular cell migration. Endothelial cell migration may enhance vascular repair and, thus, attenuate the formation of atherosclerotic plaques by contributing to the repair of desquamated areas in the endothelial cell lining (1, 2). Previous studies have demonstrated the ability of IGF-I to stimulate endothelial cell migration (26, 27), although the mechanism for this effect in endothelial cells has not been examined. In the present study, we confirmed the ability of IGF-I to stimulate endothelial cell migration and have now demonstrated that this effect is dependent, in part, upon activation of PI 3-kinase and independent of ERK activation. Activation of PI 3-kinase also plays an important role in IGF-I-induced motility of Schwann cells and migration of vascular smooth muscle and colonic epithelial cells (11, 12, 55, 56). In contrast to endothelial cells, however, inhibition of ERK activity at least partially attenuates the effect of IGF-I on the migration of vascular smooth muscle and colonic epithelial cells.

In summary, despite the recognition for many years that endothelial cells express IGF-I receptors, there is little known about the signal transduction pathways that are important for IGF-I-mediated effects in these cells. We have now demonstrated that IGF-I activates multiple signaling pathways in endothelial cells, including ERK1, -2, and -5 and PI 3-kinase, with little evidence for cross-talk between these signaling pathways. The different signaling pathways appear to mediate both overlapping and distinct effects in that activation of both PI 3-kinase and the ERKs contribute to stimulation of NF-{kappa}B-dependent transcription by IGF-I, whereas only PI 3-kinase mediates IGF-I-stimulated endothelial cell migration.


   Acknowledgments
 
The authors would like to thank Drs. Mark Yorek and Robert Bar (University of Iowa, Iowa City, IA) for providing PAEC, Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School) for providing pGST-ATF2, and Dr. William Schnaper (Northwestern University) for helpful discussions.


   Footnotes
 
1 This work was supported by NIH Grant RO1 HL-58832. Back

Received November 14, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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