Proliferation of CECs Requires Dual Signaling through Both MAPK/ERK and PI 3-K/Akt Pathways

Abstract

purpose. To analyze the intracellular signaling involved in the proliferation of choroidal endothelial cells (CECs) in vitro.

methods. Bovine CECs were cultured in endothelial growth medium (EGM) containing 2% fetal calf serum (FCS), 10 μg/ml bovine brain extract (BBE), and 10 ng/ml epidermal growth factor (EGF) in fibronectin-coated plates. Cells were treated with various specific pharmacologic inhibitors of the mitogen-activated protein kinase (MAPK) and of the phosphatidylinositol 3-kinase (PI 3-K) pathways to analyze signaling involved in CEC proliferation. Activation of the MAPK and PI 3-K was detected by Western blot analysis, using specific antiphosphosignaling protein antibodies.

results. FCS, EGF, and BBE were all necessary to induce optimal CEC proliferation. Individually, these three components were not mitogenic. EGM-stimulated CEC proliferation involved the activation of the Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90^RSK cascade. Inhibition of Ras resulted in a 92% reduction of CEC proliferation, whereas inhibition of ERK1/2 activity reduced it by only 46%. The PI 3-K/p70^S6K/Akt pathway was also stimulated during CEC proliferation, and inhibition of PI 3-K activity resulted in a 94% reduction in CEC proliferation. Inhibition of PI 3-K/p70^S6K activities also unexpectedly inhibited ERK activity, whereas the converse was not observed, suggesting that PI 3-K acted upstream from ERK and controlled this pathway for CEC proliferation.

conclusions. CEC proliferation involves both ERK and PI 3-K. That PI 3-K signaling is a key component in cell proliferation can be demonstrated by controlling ERK activity. These data on the molecular mechanism and signaling of CEC proliferation may have major implications for developing more selective methods for antiangiogenic and antitumoral therapy.

Choroidal endothelial cells (CECs) are central to the maintenance of retinal function and the progression of many retinal diseases. ^{1 2} Choroidal neovascularization (CNV) which involves CECs, is implicated in various blinding diseases, including the exudative form of age-related macular degeneration and choroidal melanoma. ^{3 4 5} Pathologic and morphologic studies have documented the steps involved in CNV. ^{6 7} During angiogenesis, endothelial cells are stimulated to migrate, proliferate, and invade surrounding tissues to form capillaries, resulting in part from the action of soluble endothelial-cell–directed growth factors. During neovascularization, matrix metalloproteinase (MMP) expression and activation are induced, allowing the breakdown of the vascular endothelial cell (VEC) basement membrane and invasion of VECs through the interstitial tissue. ⁸ However, little is known about the molecular mechanisms and the signaling underlying these processes. In particular, the intracellular signaling that mediates CEC proliferation has not been studied. This is partly due to the difficulty of isolating and purifying large numbers of CECs. Recently, a rapid and simplified method for the isolation of bovine CECs using microdissection and Lycopersicon esculentum–coated paramagnetic beads has been described. ⁹  

Cell proliferation is regulated by a complex array of signaling pathways and the integration of these different pathways, resulting in the generation of a net signaling input. In many instances, the signal that induces cell proliferation is mediated by a series of sequentially activated protein kinases. One of these is the Ras guanosine triphosphate (GTP)–binding protein, which transmits cell proliferation signals by activating the Ras/Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade. ¹⁰ In this pathway, Ras activates the serine-threonine protein kinases Raf1, MEK1 and ERK1/2. ¹¹ In addition to controlling Raf kinases, Ras may also directly regulate a second signaling pathway involving the phosphatidylinositol 3 kinase (PI 3-K). ¹² PI 3-K activates the protein serine-threonine kinase Akt, activation of which is involved in protection from apoptosis. ¹³ PI 3-K also regulates another signaling pathway, indirectly by controlling the activity of the protein serine-threonine kinase p70 small 6 kinase (p70^S6K). ¹⁴ p70^S6K is involved in cell proliferation, malignant cell growth, ¹⁵ and prevention of apoptosis, ¹⁶ depending on the cell type. The generation of multiple signal inputs through an intentional cross talk between different signaling molecules is a mechanism by which cells commit themselves to critical responses only if the required complement of signals is present. 

We determined the culture conditions for optimal CEC proliferation and then investigated several aspects of proliferating CEC-induced intracellular signaling. Molecular aspects of the signaling were studied by pharmacologically inhibiting specific kinase pathways and analyzing the levels of activation of the signaling pathways implicated in cell proliferation. We examined whether one or several signal transduction pathways are involved in CEC proliferation and assessed possible cross talk between them to determine how proliferation of endothelial cells in CNV may be controlled. 

Methods

Materials

Culture medium, fetal calf serum (FCS), and growth factors were purchased from Bio Whittaker (Emerainville, France). Specific intracellular signaling inhibitors (PD98059, rapamycin, the Ras FPT III inhibitor LY294200, and apigenin) were the products of Calbiochem (Meudon, France). Polyclonal antibody directed against phospho-ERK1/2 (thr²⁰² and tyr²⁰⁴) was purchased from Promega (Madison, WI), and polyclonal antibodies directed against phospho-Raf1 (ser²⁵⁹), -MEK (ser²¹⁷ and ser²²¹) -p90^RSK (ser³⁸¹), -p70^S6K (thr⁴²¹ and ser⁴²⁴), and -Akt (ser⁴⁷³) were obtained from New England Biolabs/Cell Signaling Technology (Beverly, MA). Anti-von Willebrand factor polyclonal antibody and anti-CD31 monoclonal antibody were the products of Dako (Trappes, France). 

CEC Culture and Treatment of Cells

CECs were isolated and purified after microdissection of bovine choroid and using L. esculentum (LEA)–coated paramagnetic beads (Dynabeads; Dynal Biotech, Oslo, Norway), as previously described. ⁹ CECs were cultured in endothelial growth medium (EGM) containing endothelium basal medium (EBM, based on MCDB-131 medium; Bio Whittaker), 2% FCS, and bovine brain extract (BBE; 10 μg/ml), epidermal growth factor (EGF; 10 ng/ml), and hydrocortisone (1 μg/ml) in fibronectin-coated plates. Cells were used from the first to the fifth passages (split ratio 1:3). All experiments were run in triplicate and were performed at least three times. The proliferation of CECs was assessed daily by counting the number of cells, by using a Malassez chamber, and by determining[ ³H] thymidine (SA:0.92 TBq/millimole; Amersham, Orsay, France) incorporation, as previously described. ¹⁶ The number of dead cells was determined by two methods: counting the cells remaining in the culture dish after staining with trypan blue and by using 3(4,5-dimethylthiazol-, yl)2,5-diphenyltetrazolium bromide (MTT). ¹⁷  

Specific inhibitors of Ras processing (FPT inhibitor III) or of MEK1 (PD098059), PI 3-K (LY294200), p70^S6K (rapamycin), or ERK1/2 (PD98059) phosphorylation, or of both ERK1/2 and PI 3-K phosphorylation (apigenin; Calbiochem) were added, as appropriate, 12 hours before induction of cell proliferation and on the first day of the cell proliferation assay. Each inhibitor was dissolved in dimethyl sulfoxide (DMSO) and diluted in EBM so that the final concentration of DMSO in test solutions would not exceed 0.1% (0.1% DMSO has no effect on EGM-stimulated CEC proliferation). The specificity of the different signaling inhibitors is presented in Table 1 . 

Immunohistochemistry

Cells were confirmed to be endothelial cells by immunostaining for von Willebrand factor and for CD31. CECs plated on glass coverslips precoated with fibronectin were fixed with 4% paraformaldehyde (PAF) for 15 minutes at room temperature, extensively washed with cold phosphate-buffered saline (PBS), and then incubated with anti-von Willebrand factor polyclonal antibody (1:100, Dako). CECs were also fixed in cold acetone for 10 minutes on ice and incubated with anti-CD31 monoclonal antibody (1:100, Dako), or nonimmune serum (1:100) as a negative control. The antigen–antibody complexes were detected with fluorescein isothiocyanate anti-rabbit antibody (1:100), or rhodamine isothiocyanate anti-mouse antibody (1:100; Biosys, Compiegne, France), as previously described. ¹⁸ After a final wash with PBS, slides were mounted in glycerol-PBS (1:1). Components of the basement membrane were analyzed by immunochemistry. Monolayers of CECs grown on glass coverslips were fixed with 4% PAF, mounted in optimal cutting temperature compound (OCT; Tissue TeK; Miles–Bayer Diagnostics, Puteaux, France), and then rapidly frozen in liquid nitrogen. Seven-micrometer-thick sections were cut at −20°C in a cryostat and were incubated for 1 hour at room temperature with polyclonal antibodies against two basement membrane components: laminin and nidogen-entactin (1:100). The antigen-antibody complexes were detected with fluorescein isothiocyanate anti-rabbit antibody (1:100), or rhodamine isothiocyanate anti-mouse antibody, as described. Slides were observed and photographed under a fluorescence microscope (Aristoplan; Leica, Rueil-Malmaison, France, with HP5 film; Ilford, Basildon, UK). 

Western Blot Analysis

CECs were washed twice in PBS, lysed in ice-cold lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Igepal ([Schibley, Elyria, OH]), 0.5% natrium deoxycholate [5Na-DOC], 50 mM NaF, 5 mM EDTA, 40 mM β-glycerophosphate, 0.2 mM sodium orthovanadate, 1μ g/ml leupeptin, and 1 μM pepstatin) and centrifuged at 4°C for 10 minutes at 10,000g. Monoclonal antibody directed againstβ -actin was used as an internal standard for checking protein loading. Cell lysate was mixed with 3× Laemmli buffer and heated for 5 minutes at 95°C. The soluble proteins of the cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 12%–15% polyacrylamide gel), transferred onto nitrocellulose filters by electroblot and probed with polyclonal antibodies directed against Raf1, MEK1, ERK2, p90^RSK, p70^S6K, and Akt (dilution 1:100, Santa Cruz Biotech, Santa Cruz, CA) to verify the constant production of these kinases over the proliferation period. A polyclonal antibody directed against phospho-ERK1/2 (thr²⁰² and tyr²⁰⁴, 1:1000) and polyclonal antibodies directed against phospho-Raf1 (ser²⁵⁹) -MEK (ser²¹⁷ and ser²²¹), -p90^RSK (ser³⁸¹), -p70^S6K (thr⁴²¹ and ser⁴²⁴), and -Akt (ser⁴⁷³; dilution 1:1000) were used to analyze the activation of intracellular signaling during CEC proliferation. The primary antibodies were detected with a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody. Enhanced chemiluminescence (ECL) substrates were used to detect positive bands, according to the manufacturer’s instructions, and the membrane was placed against film (Hyperfilm ECL; Amersham). The protein bands detected on the fluorograph were quantified using a laser densitometer (LKB Ultrascan XL; Pharmacia, Saclay, France). 

Statistics

Each figure shows the results of experiments repeated at least three times. All data are expressed as the mean ± SEM. Two-tailed Student’s t-test normal distributions with equal variances) and the Wilcoxon or Mann–Whitney tests (nonparametric tests) were used for statistical analysis. 

Results

Determination of the Stimulating Effect of Various Mitogens on CEC Proliferation

Bovine CEC cultures were established from choroid. CECs from primary cultures collected using LEA-coated beads (Dynabeads; Dynal) and cultured in EGM formed typical cobblestone monolayer colonies, as previously observed ⁹ (Fig. 1A ). The CECs maintained this morphology until the fifth passage (Figs. 1B 1C) . The cell cultures were confirmed to be pure VECs by immunostaining for von Willebrand factor (Figs. 1D 1I) and CD-31 (data not shown). Interestingly, the basement membrane of the CECs were intensely stained by anti-laminin (Figs. 1E 1F) and nidogen-entactin (Figs. 1G 1H) antibodies, revealing two major factors of vascular endothelial cell attachment and migration. No immunostaining was observed with nonimmune serum and when the primary antibody was omitted (Figs. 1J 1K) . The effects of various mitogens on CECs cultured in EBM are presented in Figure 2 . CECs did not survive when cultured with EBM alone. The cells degenerated and began to detach from the well after 48 hours. The number of cells after 4 days of culture in EBM was 51% (P < 0.05) of that on day 1 of the culture, and only 23% (P < 0.05) of that cultured in EGM (Fig. 2A) . Addition of 2% serum to EBM did not induce cell proliferation, and the CECs cultured died after 3 days in these culture conditions: the number of CECs after 4 days of culture was 62% (P < 0.05) of that on day 1 (Fig. 2A) . EGF at 10 ng/ml had no stimulating effect on the growth but allowed cell survival, with the number of cells remaining constant for 4 days (Fig. 2A) . Similarly, BBE (10 μg/ml) alone did not stimulate growth but was essential for CEC survival (Fig. 2A) . In contrast, simultaneous addition of the FCS, EGF, and BBE to EBM induced CEC proliferation, with a doubling time of 4 days (P < 0. 01; Fig. 2A ). 

The dose-effects of each of these three components in association with a combination of the two others (at the previously reported maximal effective dose ⁹ was analyzed after 4 days of culture (Fig. 2B) . CECs were cultured with EGF and BBE at 10 ng/ml and 10 μg/ml, respectively, in the presence of various concentrations of serum (Fig. 2B , EGF + BBE). The effects of serum on growth was dose dependent. A combination of EGF plus BBE, without serum, only allowed cell survival, whereas addition of 2% and 5% of FCS resulted in a two- and threefold increase in cell number, respectively. The maximal growth effect was observed with 5% of FCS (cell number increased by 265%, P < 0.01) and higher concentration had no further effect. Note that 2% serum in the absence of the other factors did not stimulate growth (compare Fig. 2A , EBM + FCS; 2B, EGF + BBE). CECs were cultured with 5% FCS plus 10 μg/ml BBE, in the presence of various concentrations of EGF. The association of FCS and BBE was sufficient to stimulate cell proliferation by a factor of 1.8 (P < 0.01; Fig. 2B , FCS + BBE). The effect of the addition of EGF on cell proliferation was dose dependent, with a maximal effect at 10 ng/ml (P < 0.05, in comparison with baseline observed on day 0). When cultured with a combination of 10 ng/ml EGF and 5% serum, CECs did not proliferate (Fig. 2B , EGF + FCS). Addition of BBE to this combination induced cell growth which was concentration dependent and biphasic, with an optimal concentration of 10 μg/ml (P < 0.01). Concentrations higher than 10 μg/ml BBE elicited a smaller increase in CEC number (Fig. 2B , EGF + FCS). 

In conclusion, EGF was essential for CEC survival in culture, and the combination of serum and BBE induced CEC proliferation. The association of the three components—serum, BBE, and EGF—was required for optimal CEC proliferation. 

ERK2 Activation Is Stimulated and Is Required for CEC Proliferation

ERKs are key components of the transduction of signals leading to proliferation of various macrovascular endothelial cell lines ^{19 20} and nonneuronal retinal cells (retinal pigment epithelial [RPE ] and Müller cells) in vitro. ²¹ Little is known about activation of ERKs in proliferating microvascular endothelial cells. CECs were cultured in complete EGM to induce cell proliferation and the state of ERK2 phosphorylation was investigated by Western blot analysis, using an antibody that specifically recognizes active ERK1 and 2. High levels of ERK1 and 2 activation were observed within 10 minutes of the addition of EGM (Fig. 3A ). The levels of ERK phosphorylation remained high for the following 30 minutes and decreased thereafter, but phosphorylation remained detectable over the 24-hour culture period. The production of ERK2 was constant over this period of culture, showing that the increase in ERK2 activation was not due to an increase in ERK2 production by CECs (Fig. 3B) . We investigated the role of ERK1/2 in the proliferation of CECs. ERK1 and 2 (ERK1/2) are the only known substrates for MEK1 and 2 (MEK1/2). Inhibition of MEK1/2 by the pharmacologic compound PD98059 (10 μM) considerably reduced the activation of ERK1/2 throughout the 24-hour period of culture (Fig. 3B) , but resulted in only a 46% reduction in CEC proliferation (P < 0.05; Fig. 3C ). This suggests that CEC proliferation was partially mediated by ERK1/2 activation. CEC treatment with up to 40 μM PD98059 did not result in a greater reduction in ERK1/2 activation (data not shown) or inhibition of cell proliferation (Fig. 3C) , suggesting that the partial effect of ERKs’ inhibition on CEC proliferation was not due to a partial inhibition of ERKs or to a dose-limited effect of the inhibitor. 

We analyzed the upstream cascade for protein kinases responsible for the ERK1/2 activation, leading to the stimulation of CEC proliferation. The Ras pathway consists of a linear cascade of protein kinases, Raf, MEK, and ERKs. The fine-tuning of the signal strength and duration is a determinant for cell activation. We therefore studied this cascade over a 5-day culture period. The states of Raf-1, MEK1, and ERK1/2 activation were investigated by studying their phosphorylation levels by Western blot analysis, using antibodies that specifically recognize the active forms of Raf-1, ²² MEK1, ²³ and ERK1/2 ²⁴ (Fig. 4A ). Phosphorylation of Raf-1 was undetectable in the basal state. Stimulation of CEC proliferation by EGM induced a sustained and constant activation of Raf-1 over the 5-day period of culture. In contrast, MEK1 activation was biphasic. MEK1 phosphorylation was optimal after 24 hours of culture and thereafter decreased slowly but was still detectable on day 5 of the culture period. Similarly, ERK1/2 phosphorylation was biphasic, with maximum phosphorylation after 24 hours of culture and a slow decrease thereafter. The production of Raf-1, MEK1, and ERK2 was constant throughout the 5 days, showing that the increase in the levels of activation of each of these three components of the Ras cascade was not due to an increase in the production of the three kinases (data not shown). 

It has been reported that ERK1 and 2 also activate the p90 ribosomal S6 kinase (p90^RSK) through phosphorylation, ²⁵ in addition to inducing transcription factors to mediate various biologic effects. ¹¹ Thus, we investigated whether p90^RSK was activated during CEC proliferation. The state of phosphorylation of the p90^RSK was studied by Western blot, using an antibody that specifically recognizes active p90^RSK. Phosphorylation of p90^RSK was undetectable in the basal state, stimulation of CEC proliferation by EGM resulted in p90^RSK activation after 2 hours of stimulation. Thereafter, its phosphorylation decreased rapidly and was not detectable after 3 days of culture (Fig. 4A) . 

Finally, we investigated the role of Ras signaling in CEC proliferation. CEC treatment with the inhibitor of Ras processing, FPT inhibitor III at 10 μM ²⁶ resulted in a 92% reduction of CEC proliferation after 4 days of culture as assessed by cell counting (P < 0.01; Fig. 4B ). Measurement of the cell proliferation by [³H] thymidine incorporation gave a similar result after 4 days of culture (P < 0.01; Fig. 4C ), showing that Ras plays a pivotal role in transmitting signals for CEC proliferation. This observation contrasts with the finding for the inhibition of MEK1 by PD98059 which reduced CEC proliferation by only 46% (compare Figs. 3D 4B 4C ). 

PI 3-Kinase Also Mediates the Signaling Involved in CEC Proliferation and Controls ERK1/2 Activation

Inhibition of Ras processing abolished CEC proliferation, whereas inhibition of MEK1 activation only partially inhibited CEC proliferation. A possible explanation is that there is another signaling pathway also mediated by Ras that controls CEC proliferation, in addition to the Raf-1/MEK/ERK/p90^RSK pathway. In addition to controlling Raf-1, Ras may also directly regulate a number of other important proteins including PI 3-K. PI 3-K has been implicated in the control of cell proliferation and cell survival by activating the p70^S6K/Akt pathway. ^{15 27} Thus, we investigated the role of the PI 3-K in CEC proliferation. Treatment of CECs with 10 μM of the highly selective inhibitor of PI 3-K, LY294002, blocked 94% of the proliferation of CECs after 4 days of culture (P < 0.01). 

This is similar to the growth reduction obtained with the inhibition of Ras processing (compare Figs. 4B 4C 5A ), suggesting that Ras/PI 3-K is the major pathway mediating CEC proliferation. Thus, we investigated the downstream cascade of protein kinases induced by the activation of PI 3-K. The activation of the major PI 3-K downstream signaling pathway, the p70^S6K/Akt cascade, was analyzed by Western blot, using antibodies that specifically recognize active forms of p70^S6Kand Akt. ^{28 29} Phosphorylation of p90^RSK was very weak in the basal state, whereas p70^S6K was greatly and constantly activated over the 24-hour culture period after EGM stimulation (Fig. 5B) . The production of the p70^S6K was constant over the 24-hour culture period, demonstrating that p70^S6K activation was not due to stimulation of its production (Fig. 5B) . Akt, the kinase upstream from p70^S6K was also activated over the 24-hour culture period after EGM stimulation and, similarly, the production of Akt was constant over the 24-hour culture period (Fig. 5B) . 

To confirm the role of p70^S6K in CEC proliferation, we treated cells with 10 nM rapamycin, which binds to the FKBP12 complex leading to rapid inactivation of p70^S6K. ³⁰ Rapamycin was unexpectedly less potent (68% reduction of cell proliferation, P < 0.05) than the PI 3-K inhibitor in blocking CEC proliferation (compare Figs. 5B 5C ), although it abolished p70^S6K activation. A similar partial inhibition of CEC proliferation was obtained with rapamycin concentrations up to 40 nM (Fig. 5C) , suggesting that the partial effect of p70^S6K inhibition on CEC proliferation was not due to a dose-limited effect of the inhibitor. These data suggest that the activation of p70^S6K is only a part of the complete signaling mediating CEC proliferation. They also confirm that Ras/PI 3-K signaling is the major pathway mediating CEC proliferation, but suggest that PI 3-K controls another signaling pathway responsible for CEC proliferation, in addition to that mediated by p70^S6K/Akt. 

In conclusion, we have demonstrated that CEC proliferation was mediated by at least two signaling pathways involving the activation of both the Raf/MEK/ERK/p90^RSK cascade and PI 3-K/p70^S6K/Akt signaling. Inhibition of cell proliferation was complete when CECs were treated with the PI 3-K inhibitor LY294002, despite the presence of the Raf/MEK/ERK/p90^RSK pathway, which was also implicated in CEC proliferation. A possible explanation of these paradoxical results is that PI 3-K signaling controls the Raf/MEK/ERK/p90^RSK pathway. Thus, we investigated the effects of the PI 3-K inhibitor LY294002 on the activation of ERK1/2. As suspected, treatment of EGM-stimulated cells with 10 μM LY294002 reduced ERK1/2 activation greatly within 10 minutes (Fig. 6A ). This effect was complete after 2 hours of treatment. A higher concentration of PI 3-K inhibitor (40 μM) completely inhibited ERK activation in EGM-stimulated CECs after 30 minutes of treatment (Fig. 6B) , demonstrating that PI 3-K effectively controlled ERK1/2 activation. We checked that PD98059 also inhibited ERK1/2 activation. At a concentration of 40 μM, both the specific ERK1/2 inhibitor and the PI 3-K inhibitor were equally effective in ERK1/2 activation. 

If our hypothesis is correct, the reverse is not true, and ERK1/2 does not control the PI 3-K pathway. Indeed treatment of EGM-stimulated CECs with the ERK1/2 inhibitor PD98059 did not inhibit p70^S6K, whereas LY294002 greatly reduced the phosphorylation of p70^S6K (Fig. 6C) . Similarly, PD98059 did not inhibit Akt, whereas LY294002 completely abolished the activation of Akt (Fig. 6D) . 

To confirm the hypothesis that two different pathways (ERK1/2 and PI 3-K) are involved in the signaling that controls CEC proliferation, we treated EGM-stimulated CECs with apigenin, an inhibitor of both ERK1/2 and PI 3-K activation. ^{31 32} Apigenin (25 μM) completely suppressed CEC proliferation induced by EGM (P < 0.01; Fig. 6E ). To rule out the possibility that this inhibitory effect of apigenin on cell proliferation is due to its action on another unknown pathway, CECs were treated with a mix of the specific ERK and PI 3-K inhibitors PD98059 and LY294002, both at 10 μM (Fig. 6E) . Specific blockade of ERK1/2 and PI 3-K caused a complete inhibition of CEC proliferation (P < 0.01), similar to that observed with apigenin and with the inhibitor of Ras processing, the central upstream activator of these two kinases. Therefore, it is unlikely that CEC proliferation is mediated by a third pathway. 

These data show that the proliferation of the microvascular endothelial cells of the choroid was mediated by two different signaling pathways, one involving the Raf/MEK/ERK/p90^RSK cascade, the other involving the PI 3-K/p70^S6K/Akt cascade. The pathways converge at the level of ERK1/2 through the control of ERKs by PI 3-K. A proposed scheme for the cross talk between the activated MAPK/ERK and the PI 3-K/p70^S6K pathways during CEC proliferation is presented in Figure 7 . 

Discussion

ERK1/2 Signaling Plays a Major Role in the Control of CEC Proliferation

Angiogenesis is involved in pathologic processes including diabetic retinopathies and age-related macular degeneration, and tumor growth such as choroidal melanoma. We report that Ras is a pivotal component of the signaling that mediates the proliferation of the microvascular endothelial cells of the choroid. One of the primary roles of Ras is to participate in the activation of Raf-1, which belongs to the linear Raf/MEK/ERK signaling cascade that mediates cell proliferation, differentiation, migration, and survival. ³³ In the present study, we showed that this cascade of signaling leading to ERK1/2 phosphorylation was activated and was required for CEC proliferation. In previous studies, we reported that the proliferation of RPE cells was also mediated by the phosphorylation of ERK1/2. ²¹ But, ERK1/2 activation was more transient in RPE cells than in CECs, lasting only 2 hours after FGFs stimulation. These data contrast with ERK1/2 activation in CECs which is sustained, peaking after 24 hours of stimulation, and still detectable after 5 days. It has been suggested that the kinetics of ERK1/2 activation is in many cases dependent on the cell type. ³⁴ This may explain the difference between RPE cells and CECs in the kinetics of ERK1/2 activation for the same biologic event. 

A wide variety of extracellular stimuli induce activation of ERK1/2 to transduce proliferation, differentiation, migration, and survival activity. The fine regulation of the ERK1/2 signal duration is crucial to cell fate determination. The activation of MEK1, the signaling kinase directly upstream from ERK1/2, and that of p90^RSK, the signaling kinase directly downstream from ERK1/2 had very similar kinetics, suggesting a very fine and coordinated regulation of these three kinases during CEC proliferation. The difference in the duration of ERK1/2 activation may lead to the activation of different kinases and the induction of different transcription factors that are implicated in cell fate determination. For example, recent data show that overlong and sustained activation of ERK1/2 is necessary for the synthesis of the anti-apoptotic protein BcL-xl during survival of RPE cells. ³⁴ In contrast, in PC12 lines, a transient ER1/2 signal induces cell proliferation, whereas a sustained activation causes these cells to differentiate, stop growing, and survive. ^{35 36}  

More recently, it has been demonstrated that constitutively active MEK1-ERK2 signaling induces cell migration ³⁷ and that the activation of the MEK1-ERK1/2 pathway regulates the aorta endothelial cell fate between apoptosis and angiogenesis. ³⁸ This confirms the importance of the fine-tuning of the activation of the ERK signaling for determining cell fate. Thus, it would be of value to analyze whether the duration of ERK1/2 activation also determines CEC fate, by studying the kinetics of ERK1/2 phosphorylation during CEC migration and cell survival, in comparison with ERK1/2 activation during CEC proliferation. 

Is ERK1/2 the Major Signaling Pathway Controlling Vascular Endothelial Cell Fate?

It has been reported very recently that ERK1/2 is activated in models in vitro and in vivo of retinal vascular endothelial cell proliferation. ³⁹ In addition, inhibition of ERK1/2 by PD98059 partially inhibits in vitro retinal endothelial cell proliferation and in vivo neovascularization, suggesting that the ERK pathway plays an important role in the control of retinal angiogenesis. ³⁹ More recently, it has been reported that hepatocyte growth factor (HGF)-induced bovine retinal endothelial cell (BREC) growth is also ERK1/2 dependent, because inhibition of MEK 1 with PD98059 abolishes cell proliferation. ⁴⁰ HGF-induced migration of BREC is also dependent on the ERK1/2 pathway. ⁴⁰ However, the investigators did not compared the kinetics of ERK1/2 activation during cell proliferation and migration to analyze the possibility of the control of cell fate determination by ERK1/2 signal duration. In addition, it has been shown that HGF stimulates cell migration through stimulation of MMP induction and sustained activation of ERK1/2 in epidermal cells ^{41 42} and keratinocytes. ⁴³  

Thus, it is tempting to speculate that ERK1/2 may control CEC and BREC migration through the induction of MMPs, allowing the breakdown of the basement membrane of VECs. However, HGF-induced BREC proliferation and migration also involves activation of PI 3-K. ⁴⁰ Thus, it would be of value to analyze whether combined ERK1/2 and PI 3-K inhibition is additive and whether PI 3K or other signaling pathways control the ERK1/2 pathway for BREC proliferation. 

PI 3-K Cross-Talk with ERK1/2: a Complex and Specific Regulation to Control CEC Proliferation

Although the Ras/Raf/ERK pathway is essentially linear, it suggests cross-talk with other signaling pathways. A novel ERK-to-JNK cross-activation was recently proposed for VEGF-induced aortic endothelial cell proliferation, ⁴⁴ whereas parallel ERK1/2 and p38 MAP kinase signaling was shown to be implicated in a FGF2-stimulated murine macrovascular endothelial cell line. ⁴⁵ Cross-activation seems to be a recent and perhaps cell-context–specific concept that does not involve a direct action of ERK, but rather that of other kinase pathways. In addition to controlling Raf kinases, Ras may also directly regulate a number of other important proteins. Ras interacts with and stimulates the activity of the PI 3-K, which in turn activates Akt. The Ras/PI 3-K/Akt pathway has been strongly linked to protection from apoptosis and migration, ⁴⁶ whereas the Ras/Raf/MEK/ERK pathway is mainly associated with cell proliferation. 

In addition, PI 3-K also indirectly controls the activity of the serine-threonine kinase p70^S6K. ²⁷ The function of p70^S6K is essential for G1 progression. ⁴⁷ In our study ERK1/2 signaling was only a part of the pathway that mediated CEC proliferation. In contrast, PI 3-K was the major pathway, because specific inhibition of PI 3-K completely abolished CEC proliferation. PI 3-K activated p70^S6K in CECs during cell proliferation, and its pharmacologic inactivation reduced cell proliferation by half, suggesting that CEC proliferation was in part also mediated by p70^S6K. This is consistent with recent data in a study that showed that p70^S6K-mediated protein synthesis is essential for human umbilical vein endothelial cell (HUVEC) proliferation induced by serum. ⁴⁸ However, the authors did not analyze the possibility of other signaling pathways or signal cross-activation in this macrovascular endothelial cell line. In contrast, in another study it was reported that ERK1/2, PI 3-K, and p70^S6K were activated during the proliferation of VEGF-stimulated HUVEC, ⁴⁹ whereas Wu et al., ²⁰ showed that VEGF activated PI 3-K, PLCγ, and PKCε independently of one other. In their study, the PLCγ and PKCε are in the pathway through which VEGF activated ERK for cell proliferation. Thus, if the various published data on cell proliferation are not to be considered conflicting, the roles of the different signaling pathways in cell proliferation are unclear, even for a particular endothelial cell type. 

We report the first evidence of the involvement of both the ERK and the PI 3-K pathways in the proliferation of CECs. In addition, we demonstrated cross-talk between these two pathways, with PI 3-K controlling ERK signaling. The relevance of PI 3-K for activation of ERKs is controversial. Several studies in non-VECs have found that pharmacologic inhibition of PI 3-K effectively inhibits activation of the MAPK/ERK cascade. ^{50 51} Other studies have found that inhibition of PI 3-K had no effect on the activation of ERKs. ^{52 53} This suggests that the control by PI 3-K of ERK activation is in many cases dependent on cell type and cell line. VECs from different sites show antigenic and phenotypic heterogeneity. HUVECs do not exhibit tight junctions, whereas retinal endothelial cells do, and CECs are joined by gap junctions and are highly fenestrated. 

Thus, the differences in signaling that mediate the proliferation of the VECs may depend on the specificity of these cells—for example, macrovascular versus microvascular—and may be a function of the organ of origin. In addition, the differences in signaling mediating the proliferation of cells may also vary within a single cell line, depending on the kind and concentration of the stimulus used to induce cell proliferation. Duckworth et al., ⁵⁴ have demonstrated that stimulation of PI 3-K in NIH 3T3 cells provide an efficient pathway for Raf/ERK activation at low PDGF levels, whereas stimulation of these cells at higher PDGF levels provided a redundant signal through activation of a direct activation of Raf/ERK pathway by PKC. More recently, a similar control of ERK signaling by both the direct upstream linear pathway and an adjacent PI 3-K pathway was demonstrated to be EGF-concentration dependent in COS-7 cells. ¹³  

Our study demonstrates for the first time the central role of PI 3-K signaling in generating a maximal mitogenic response through both ERK1/2 and p70^S6K. No data have been reported for the signaling involved in the proliferation of microvascular cells, or branch points, or multicomponent signaling complexes. Cross-talk and synergism between adjacent pathways may be of great physiological significance. It is possible that these different signaling pathways may regulate diverse responses by integrating signals from each other. Cells thereby ensure that the required signals are present before critical responses. The identification of the complete kinase signaling system, the substrates, the branch points, and the regulatory feedback loops may provide more selective methods and strategies for the treatment of angiogenic diseases and cancers. 

 Supported by a Grant 9697 from Association de la Recherche contre le Cancer (FM) and Association Claude Bernard (AZ).

 Submitted for publication June 23, 2000; revised September 19, 2000; accepted October 6, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Summary of Data on the Kinases Studied and Kinase Inhibitors Used

Signaling Inhibitors	Selectivity
FPT inhibitor III	Ras
PD98059	MEK1
Apigenin	ERK1/2 and PI 3-K
LY294200	PI 3-K
Rapamycin	p70S6K

Figure 1.

 

Morphology and characterization of CECs. Scale bar, 15 μm. (A) Primary cultures of CECs selected using LEA-coated beads after 4 days of culture showed a large number of proliferating CECs. (B) Sparse and (C) confluent culture of CECs at the fifth passage shows cells retaining their typical cobblestone morphology. Analysis of the immunoreactivity for von Willebrand factor (D) on CECs at the first passage (scale bar, 70 μm) and analysis of the immunoreactivity for anti-laminin (E) and anti-nidogen entactin (G) in the basement membrane of CECs at the third passage. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; F, H, and K). Nonimmune serum does not label the CECs (I) and the basement membrane (J) Scale bar, 40 μm. The ribbon-like patterns of laminin and nidogen staining of the basement membrane is due to the use of the 7-μm-thick sections of the CEC monolayers deposited in the embedding medium. *P < 0. 05, **P < 0. 01.

Figure 2.

 

Effects and dose response of various mitogens on the proliferation of CECs. Cells were seeded at 1.5 × 10 ⁴ cells per well in six-well plates and cultured in EGM for 3 days. Cells were then stimulated with the three mitogens (2% FCS, 10 ng/ml EGF, and 10μ g/ml BBE) in EBM (A) or with various concentrations of one mitogen in EBM with a combination of the two other mitogens at the optimal concentration (as determined in preliminary experiments ⁹ ), and cell proliferation was measured over a 4-day period of culture (B). Similar results were obtained in four independent experiments, each performed with triplicate wells per point. Values are means ± SE, and differences between means were analyzed by the Mann–Whitney test. *P < 0. 05,** P < 0. 01.

Figure 3.

 

Analysis of ERK1/2 activation and effects of ERK1/2 inhibition on cell proliferation in EGM-stimulated CECs. (A, B) CECs were cultured in EGM for 24 hours in the absence (A) or the presence (B) of MEK1 inhibitor PD98059 at 10 μM. Cells were lysed, and equal amounts of protein were reduced and subjected to SDS-PAGE and Western blot analysis using an anti-active ERK1/2 antibody (A, B) and an anti-ERK1/2 antibody (A). (C) Proliferation of CECs treated with 10μ M or 40 μM PD98059 and stimulated by EGM was studied on days 1, 2, and 4. Similar results were obtained in three independent experiments, with triplicate wells per time point. Values are means ± SE, and differences between means were analyzed by the Mann–Whitney test.* P < 0. 05.

Figure 4.

 

Analysis of long-term activation of Raf-1, MEK1, ERK1/2, and p90^RSK and effects of the inhibition of Ras processing on cell proliferation in EGM-stimulated CECs. (A) CECs were cultured in EGM for 5 days, and phosphorylation of Raf-1, MEK1, ERK1/2, and p90^RSK was analyzed. (B) Proliferation of untreated CECs and CECs treated with 10 μM of the Ras processing inhibitor FPTIII and stimulated by EGM was studied on days 1, 2, and 4 by cell counting and [³H] thymidine incorporation. Similar results were obtained in four independent experiments, with triplicate wells per time point. Values are means ± SE, and differences between means were analyzed by the Mann–Whitney test. **P < 0. 01.

Figure 5.

 

Analysis of activation of p70^S6K and Akt and effects of the inhibition of PI 3-K and p70^S6K activities on cell proliferation in EGM-stimulated CECs. Proliferation of untreated CECs and CECs treated with 10 μM of the PI 3-K inhibitor LY294002 (A) and either 10 nM or 40 nM of the p70^S6K inhibitor rapamycin (C) and then stimulated by EGM was measured on days 1, 2, and 4. (B) EGM-stimulated cells were lysed at the indicated time, and equal amounts of protein were reduced and subjected to SDS-PAGE. Western blot analysis was performed using anti-active p70^S6K and Akt antibodies and anti-p70^S6K and Akt. Similar results were obtained in three independent experiments, each performed with triplicate wells per time point. Values are means ± SE, and differences between means were analyzed by the Mann–Whitney test. *P < 0. 05, **P < 0. 01.

Figure 6.

 

Effects of the inhibition of ERK1/2 and PI 3-K activities on ERK1/2, p70^S6K and Akt in EGM-stimulated CECs. (A through D) EGM-stimulated cells cultured in the presence of 10 μM (A) or 40 μM (B) PD98059 and of 10 μM LY294002 (A, B) were lysed at the indicated time, and equal amounts of protein were reduced and subjected to SDS-PAGE. Western blot analysis was performed using the indicated antibodies. (E) Proliferation of untreated CECs and CECs treated with 25μ M of apigenin and a mix of the PI 3-K inhibitor LY294002 and the ERK1/2 inhibitor PD98059, both at 10 μM, and then stimulated by EGM was studied on days 1, 2, and 4. Similar results were obtained in three independent experiments, with triplicate wells per time point. Values are means ± SE. Differences between means were analyzed by the Mann–Whitney test. **P < 0. 01.

Figure 7.

 

Model for MAP kinase/ERK and PI 3-K/p70^S6K signaling pathways involved in proliferation of EGM-stimulated CECs. After Ras activation, two signaling pathways are activated: the linear Raf-1/MEK1/ERK1/2/p90^RSK pathway, which accounts for half the signaling, and the PI 3-K/p70^S6K/Akt pathway. The p70^S6K/Akt pathway also accounts for half of signaling, whereas PI 3-K directly controls p70^S6K/Akt signaling and activates ERK1/2 signaling and is thus the hub of the regulation of CEC proliferation.

While the manuscript was in progress, another study of signal transduction by ERK1 and 2 in retinal angiogenesis was published (Bullard LE, Penn JS. Signal transduction proteins ERK1 and 2 are therapeutic targets for the inhibitors of retinal angiogenesis [ARVO Abstract]. Invest Ophthalmol Vis Sci. 2000;41(4):140. Abstract nr 718.)