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Endocrinology, doi:10.1210/en.2006-0348
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Endocrinology Vol. 147, No. 10 4627-4637
Copyright © 2006 by The Endocrine Society

S179D Prolactin Primarily Uses the Extrinsic Pathway and Mitogen-Activated Protein Kinase Signaling to Induce Apoptosis in Human Endothelial Cells

Eric K. Ueda, Hsin-Lung Lo, Paolo Bartolini and Ameae M. Walker

Division of Biomedical Sciences (E.K.U., H.-L.L., A.M.W.), University of California, Riverside, California 92521; and Biotechnology Department (E.K.U., P.B), Instituto de Pesquisas Energéticas e Nucleares-Centro de Engenharia Nuclear, University of Sao Paulo, 2242, Cidade Universitaria, 05508-900 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Ameae M. Walker, Division of Biomedical Sciences, University of California, Riverside, California 92521. E-mail: Ameae.Walker{at}ucr.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have demonstrated that S179D prolactin (PRL) is potently antiangiogenic in vivo. Here, we examined apoptosis in human endothelial cells, using procaspase-8 and cytochrome c release as markers of the extrinsic and intrinsic pathways, respectively. Both pathways converge at caspase-3, which is responsible for cleavage of DNA fragmentation factor (DFF45). A 3-d incubation in 50 ng/ml S179D PRL quadrupled the number of early apoptotic cells; this effect was doubled at 100 ng/ml and became maximal at 500 ng/ml. DFF45 and procaspase 8 cleavage were detectable at 100 ng/ml. Cytochrome c, however, was unaffected until 500 ng/ml. The p21 increased at 24 h, whereas a change in p53 required both triple the time and higher doses. The p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). Because S179D PRL and basic fibroblast growth factor (bFGF) have both been shown to activate ERK, the effect of S179D PRL on bFGF-induced ERK signaling was examined. S179D PRL blocked ERK phosphorylation in response to bFGF, whereas continued coincubation caused a delayed and prolonged activation of ERK. PD98059 inhibited this delayed activation of ERK and effects of S179D PRL on all measures except p53 levels or activity of the Bax promoter. We conclude that S179D PRL blocks bFGF-induced ERK signaling and yet uses ERK in a different time frame to elevate p21 and activate the extrinsic pathway. Prolonged incubations and high concentrations additionally activate the intrinsic pathway using an alternate intracellular signal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
S179D PROLACTIN (PRL) IS A molecular mimic of phosphorylated human PRL (1). In previous studies, we have demonstrated that this molecule inhibits the growth of prostate tumors in vivo (2). Partly, this is brought about by blockade of the autocrine and growth-promoting unmodified PRL produced by prostate epithelial cells (2), and partly this is likely due to the antiangiogenic effects of S179D PRL (3). In terms of its antiangiogenic activity, S179D PRL has been previously shown to be very effective in the mouse corneal and chicken chorioallantoic membrane assays (3). Using human endothelial cells in vitro, S179D PRL was shown to decrease expression of a number of important growth factors/antiapoptotic molecules including vascular endothelial growth factor, basic fibroblast growth factor (bFGF), heme-oxygenase 1, angiogenin, and endogenous PRL and to result in DNA fragmentation consistent with apoptosis (3). Furthermore, because the effects of S179D PRL were seen in medium containing bFGF, it was clear that S179D PRL could overcome the substantial growth factor effects of bFGF (3) as well as affecting endogenous production. In addition, S179D PRL increased expression of two inhibitors of matrix metalloproteases, TIMPs 1 and 2 and the short PRL receptors (PRLRs) (3). Up-regulation of TIMPs 1 and 2 likely contributes to the decrease in migratory capacity of endothelial cells that results from treatment with S179D PRL (3). In other systems, up-regulation of the short PRLR has been shown to increase ERK 1/2 signaling in response to S179D PRL, and this ERK signaling is in turn responsible for up-regulation of the cell cycle regulatory protein, p21 (4).

In the current study, we have further analyzed the apoptotic response to S179D PRL, asking which pathways were used and how it is possible for both bFGF and S179D PRL to signal via ERK 1/2 and yet have bFGF treatment culminate in growth (5) and S179D PRL treatment culminate in apoptosis. A preliminary experiment concerning the latter has already been published (3).

Apoptosis can be brought about by initiation of either the intrinsic or extrinsic pathways, both of which involve caspases (cysteine proteases) as key effectors (reviewed in Ref. 6). In the intrinsic pathway, also dubbed the mitochondrial pathway, one of the major events is the release of cytochrome c from the mitochondria. In the presence of ATP, this cytochrome c can induce oligomerization of Apaf-1 (apoptosome formation), which in turn recruits pro-caspase 9 (inactive), causing its dimerization and activation. In this instance, activation of the caspase is due to a conformational change (reviewed in Ref. 7). Activated caspase 9 can then trigger apoptosis by activating other effector caspases such as pro-caspase-3 (8, 9, 10, 11, 12). This pathway plays a role in apoptosis derived from growth factor deprivation (reviewed in Ref. 8) but can also amplify a signal initiated via the extrinsic pathway (6). The extrinsic pathway involves signaling from a cell surface receptor, which then initiates formation of the death-inducing complex responsible for pro-caspase-8 sequestration and activation (13). In this instance, activation is by proteolytic cleavage (8). If cells are fully able to activate apoptosis via caspase-8, they have been dubbed type I cells (13). If mitochondrial amplification of the extrinsic signal is used, then the cells are known as type II (14). Both the extrinsic and intrinsic pathways converge at caspase-3, which is responsible for the cleavage of DNA fragmentation factor [DFF45, also known as inhibitor of caspase-activated deoxyribonuclease (ICAD)], thereby allowing the caspase-activated deoxyribonuclease to cleave DNA. DNA is cleaved internucleosomally, one of the hallmarks of apoptosis (15).

Two cell cycle regulatory proteins, p21/waf1/cip1 and p53, have been linked to the induction of apoptosis. It is very well documented that p21 inactivates cyclin-dependent kinases (reviewed in Ref. 16), a move that in the short-term can produce cell cycle arrest and the protection of cells against apoptosis (16). What moves the cell from cycle arrest toward apoptosis after elevation of p21 is less clear, but a role for elevated p21 in the induction of apoptosis is also well documented in some systems and p21 expression can be regulated by both p53-dependent and -independent mechanisms (16, 17, 18, 19). Activation of p53 can be an important initiator of the intrinsic pathway. It can also amplify apoptosis initiated by the extrinsic pathway either through pathway cross talk involving Bid cleavage by caspase 8 (6), or subsequent to DNA damage. This latter effect works via Bax, a proapototic member of the Bcl-2 family, which causes cytochrome c release from the mitochondria and thus activation of the intrinsic cascade (7, 17, 20, 21). The p53/Bid/Bcl-2/Bax pathway is triggered upon DNA damage and results in the formation of an apoptosome. This is followed by caspase 3 activation and the sequence culminates with more DNA fragmentation, thereby amplifying the apoptotic signal (7, 20, 22).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant proteins (PRL and S179D PRL)
Recombinant PRL and S179D PRL were produced in Escherichia coli as previously described (1). By expressing both PRLs in E. coli, they gain no posttranslational modifications except the formation of disulfide bonds during the in vitro refolding process. Endotoxin levels in the preparations were detected and measured with the Limulus amoebocyte lysate assay (E-Toxate kit; Sigma, St. Louis, MO).

Tissue culture
Primary human umbilical vein endothelial cells (HUVEc) were obtained from Clonetics (San Diego, CA). The cells were usually used up to passage 5, but some confirmatory experiments used cells up to passage 10. HUVEc were grown on collagen type I-coated plates in medium M 199 (Invitrogen, Carlsbad, CA) supplemented with 10 mM HEPES, 2.5 µg/ml thymidine, 140 United States Pharmacopeia units/ml heparin, 5 ng/ml human basic fibroblast growth factor (bFGF, Sigma) and 20% fetal bovine serum (FBS, Life Technologies, Inc., Grand Island, NY). For assays with a serum-deprived environment, cells were cultivated in endothelial growth medium SFM (serum-free medium) (Invitrogen) supplemented with 5 ng/ml human bFGF and 10 ng/ml epidermal growth factor (EGF; Sigma).

Flow cytometry
DNA content analysis.
HUVEc were cultured for 3 d in endothelial growth medium SFM supplemented with either PRL or S179D PRL at concentrations ranging from 10–1000 ng/ml for the dose response studies. For the cells cotreated with PD98059, they were cultured for 2 d in endothelial growth medium plus 5% FBS. Cells were harvested by trypsinization, washed with cold Dulbecco’s PBS (DPBS), and fixed with 75% ethanol in DPBS at 4 C for 30 min. Cell pellets were resuspended in 0.1% Triton X-100 (Sigma) in DPBS containing 200 µg/ml ribonuclease (Sigma) and 10 µg/ml of propidium iodide (Sigma) and incubated at room temperature for 30 min. The fluorescence of individual cells was measured with a FACScan cytofluorometer equipped with CellQuest software (Becton Dickinson and Co., Franklin Lakes, NJ).

Annexin V/propidium iodide (PI) dual staining.
Dual staining with fluorescein isothiocyanate-labeled-annexin V (FITC-annexinV) and PI was performed to detect the proportion of cells undergoing apoptosis. For this purpose, a BD Apopalert Annexin-V kit (BD Biosciences Clontech, Mountain View, CA) was employed following the manufacturer’s directions. Briefly, HUVEc were collected by trypsinization, washed, and incubated with FITC-annexin V and PI as directed. Bivariant analysis of FITC-fluorescence (FL-1) and PI-fluorescence (FL-3) gave different cell populations where FITC negative (FITC–) and PI negative (PI–) were designated viable cells; FITC positive (FITC+) and PI– as early apoptotic cells, and FITC+ and PI positive (PI+) as late apoptotic cells. The fluorescence of individual cells was measured as above.

Western blotting
Preparation of whole cell protein extracts.
To obtain whole cell lysates, cells were harvested in DPBS and lysed in 100–200 µl of lysis buffer [1% Nonidet P-40, 20 mM Tris (pH 8), 137 mM NaCl, 10% glycerol] supplemented with a protease inhibitor mix on ice for 15 min.

Preparation of cytosolic protein extracts.
To evaluate mitochondrial cytochrome c release, cytosolic protein extracts were obtained using a protocol modified from one previously described (23). Briefly, cells were washed twice, harvested with ice-cold DPBS, and cell pellets were then resuspended in 1 ml ice-cold buffer containing 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Cells were disrupted by passing the cells through a 22-gauge needle three times. Lysate was centrifuged at 10,000 x g for 10 min and the resultant supernatant was further centrifuged at 10,000 x g for 1 h.

Immunoblotting.
To detect apoptotic proteases, cell cycle regulatory proteins, and ERK 1/2, confluent HUVEc from 100-mm plates were harvested in cold DPBS and cell lysates obtained. Equal quantities of lysate (40 µg/well for all Western blots, except for the ERK 1/2, JNK, and Akt activation/phosphorylation studies where 20 µG protein/well was employed) were resolved by SDS-PAGE (8%, 10%, and 15% as appropriate) and transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Separate Western blots were performed using a variety of antibodies: rabbit antihuman caspase-8 (1:4000; BD PharMingen, Franklin Lakes, NJ), and rabbit antihuman DFF45/ICAD (1:1000), mouse antihuman p21 (1:100), rabbit antihuman p53 (1:200), and mouse antihuman phosphorylated ERK 1/2 (1:1000), all from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). To estimate the amount of cytochrome c release from the mitochondria, cytosolic extracts were obtained and analyzed as described above using rabbit antihuman cytochrome c (1:1000; BD PharMingen). To normalize lysate loading, the membranes were stripped and reprobed with mouse antihuman actin (1:100; Santa Cruz Biotechnology Inc.) for all blots except ERK 1/2 studies where rabbit antitotal ERK 1/2 was used (1:1000; Santa Cruz Biotechnology Inc.). Antigen-antibody interactions were detected using horseradish peroxidase-coupled secondary antibodies and the ECL chemiluminescent system (Amersham Biosciences, Piscataway, NJ). Controls included the use of matched nonspecific antibodies/sera and second antibodies alone. Multiple exposures ensured that the bands could be compared on a relative, semiquantitative basis.

Constructs, transient transfection experiments, and measurement of luciferase and ß-galactosidase activities
Plasmids.
To study the p21 promotor activity, a construct (pp21-luc) containing the full-length p21 promotor region was used (24). For the p53 pathway, we used a plasmid (pBax-luc) with the full-length Bax promoter (25, 26). Transactivation of the Bax gene is important for the tumor suppression function of p53, and this gene is up-regulated when levels of stable p53 are increased due to an apoptotic event. A dominant-negative, kinase inactive K52R ERK in a pCEP vector was used as a second measure of mediation through the MAPK pathway. This construct has been described by Khoo et al. (27).

Transfection.
Constructs and pSV-ß-galactosidase were transiently transfected into HUVEc. Transfection experiments were carried out on semiconfluent (80–90%), cultured (fourth passage) HUVEc in 35-mm-well tissue culture plates, in triplicate. DNA (pSV-ß-galactosidase)-lipofectamine 2000 (LF2000) complexes were preformed by incubation of DNA (2 µg construct/2 µg pSV-ß-galactosidase) and lipofectamine 2000 (8 µl) in endothelial growth SFM (without supplements) per well for 20 min. Cultures were washed twice with endothelial growth SFM and then transiently transfected with the various preformed DNA-lipofectamine complexes in the same medium for 4 h before changing medium to complete growth medium (supplemented). The transfected cells were treated with the different prolactins and PD 98059 24 h after the transfection, and treatment was carried out for 12 h or 24 h (see figure legends). ß-Galactosidase activity and total protein concentration (Bradford protein assay; Bio-Rad, Hercules, CA) were used to correct for differences in DNA uptake and cell number, respectively.

Statistical analysis
All numerical data are presented as the mean ± SD. Except where noted, all experiments used triplicates and all were conducted a minimum of three times. Statistical significance was calculated using Student’s t test to compare individual means, with Bonferroni correction where appropriate. A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In a previous study, we had demonstrated that incubation HUVEc in S179D PRL lead to DNA fragmentation, a result that was not duplicated by unmodified PRL or lipopolysaccharide (LPS) (endotoxin), the latter at 10-fold the concentration present in the recombinant proteins (3). DNA fragmentation was suggestive of apoptosis. In the current study, our aim was to use several measures of apoptosis and to determine whether the intrinsic or extrinsic pathway was used. Initial studies used high concentrations of S179D PRL and compared the result to the same concentration of unmodified PRL. Later studies focused on a more detailed concentration-response and time course analysis of the effect of S179D PRL. Because of the reported sensitivity of HUVEc to LPS, LPS was used as a control throughout.

Because both the intrinsic and extrinsic pathways converge at the caspase 3 cleavage of DFF45 (ICAD), we first assessed the effect of S179D PRL on the amount of this inhibitor present in cell lysates. As can be seen in Fig. 1AGo, a 24-h incubation in S179D PRL reduced the amount of DFF45 by about 50%, whereas unmodified PRL was without effect. Next, we moved upstream, using the cleavage of procaspase 8 as a measure of the extrinsic pathway, and the appearance of cytosolic cytochrome c as a measure of the intrinsic pathway. Figure 1BGo shows decreased amounts (about half) of procaspase 8 in response to S179D PRL, and Fig. 1CGo shows increased cytosolic cytochrome c (about double) also within the first 24 h of treatment. Once again, unmodified PRL was without effect. Cell cycle regulation via p21 and p53 is often preliminary to activation of apoptosis. Figure 2AGo shows that the amount of p21 was doubled after 24 h in S179D PRL. At 24 h, there was no effect on p53 levels (not shown), but by 72 h they too were doubled in cell lysates (Fig. 2BGo). Transient transfection of a p21 promoter-luciferase construct (p21-luc) illustrated that S179D PRL doubled the activity of the promoter in 24 h of treatment, whereas unmodified PRL and LPS were without effect (Fig. 2CGo). The Bax promoter was used as a relevant measure of p53 activity because Bax is one of the main targets of p53 during the induction of apoptosis (20). Figure 2DGo shows that S179D PRL treatment also enhanced activity at the Bax promoter.


Figure 1
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  		FIG. 1. Markers of apoptosis after treatment with S719D PRL. A, Western blot analysis of DFF 45 in whole cell lysates of HUVEc treated for 24 h with high concentration (1 µg/ml) PRL (P) or S179D PRL (S) and no treatment [control (C)] in serum-deprived medium. B, Western blot analysis of procaspase-8 cleavage in the same lysates. C, Western blot analysis of cytochrome c in cytosolic lysates of HUVEc treated equivalently. *, P value of 0.05 vs. C and P.

 

Figure 2
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  		FIG. 2. Effect of S179D PRL on cell cycle regulators p21 and p53. A, Western blot analysis of p21 in cell lysates treated for 24 h with high concentration (1 µg/ml) PRL (P), S179D PRL (S) and no treatment (C) in serum deprived medium. B, Western blot analysis of p53 in whole cell lysates treated for 72 h with high concentration PRL (P), S179D PRL (S) and no treatment (C) in serum-deprived medium. C and D, Reporter assays. Exponentially growing HUVEc were transiently cotransfected with either pSV-ß-galactosidase/pp21-luc or pSV-ß-galactosidase/pBax-luc and after the recovery period were treated with high concentration PRL (P), S179D PRL (S), LPS or no treatment (C) for 24 h. The data shown are the average from three independent experiments. Each bar represents the mean ± SD. *, P value of 0.05 vs. control (C) and P.

 
Next, we focused on several measures of apoptosis and conducted a full concentration-response to S179D PRL. Figure 3Go shows fluorescence-activated cell (FACs) analysis of DNA fragmentation. Panels A and B show representative scans without and with the highest concentration, and panel C shows quantification from four separate analyses. The percent of cells with sub G1 amounts of DNA after a 3-d incubation in S179D PRL is labeled "A" for apoptotic and the numerical value for A in each scan illustrated is given in the panels. As can be seen from panel C, S179D PRL resulted in a significant increase in DNA fragmentation at 25 ng/ml, whereas the LPS, which was 10-fold the concentration present in the S179D PRL, was without effect.


Figure 3
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  		FIG. 3. Concentration dependency of effect of S179D PRL on HUVEc DNA content. A and B, Cells were cultured in growth medium for 3 d with different concentrations of S179D PRL followed by staining with PI and flow cytometry. Events falling under the 100 (0–100) FL2 channel scale were considered apoptotic events resulting from DNA fragmentation. Percents are the proportion of gated cells undergoing apoptosis (A). C, Histogram showing percentage of cells undergoing apoptosis (sub G1 peak) after treatment with either the different concentrations of S179D PRL or 0.02 EU/ml of LPS. Each bar represents the mean ± SD (n = 4). *, P value ≤ 0.05 vs. control (concentration 0 ng/ml). Representative histograms are shown in A and B. Columns represent means ± SD of five independent experiments.

 
A second measure of apoptosis is the externalization of phosphatidyl serine and the subsequent ability to label it with fluorescently tagged annexin V. Concurrent propidium iodide (PI) staining is a measure of cell viability. Thus annexin V-positive, PI-negative cells were considered to be in the early stages of apoptosis, whereas annexin V-positive, PI-positive cells were considered to be in the late stage of apoptosis. As represented in Fig. 4Go, A and B, the lower right panels are early apoptosis, and the upper right panels are late apoptosis. Panel C quantifies early and late apoptosis together, and Table 1Go separates early and late apoptosis (data derived from five independent experiments). As can be seen from Table 1Go, a 3-d incubation in 50 ng/ml S179D PRL quadrupled the number of early apoptotic cells, with a small effect on the number of late apoptotic cells. At 100 ng/ml, the early apoptotic cells were increased to 10-fold the number in the controls, and late apoptotic cells were doubled. Levels of apoptosis became maximal at 500 ng/ml.


Figure 4
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  		FIG. 4. Annexin V/PI dual staining of HUVEc treated with different concentrations of S179D PRL. HUVEc were stained with FITC-annexinV and PI and analyzed by flow cytometry. Early apoptotic cells were defined as FITC+/PI– cells (lower right quadrant), and late apoptotic cells were defined as FITC+/PI+ (upper right quadrant). Numbers in the quadrants give the percent of FITC+ (lower) or FITV+/PI+ cells (upper) (n = 3). *, P value ≤ 0.05 vs. control (concentration 0 ng/ml). Representative dot plots are shown. Columns represent means ± SD of five independent experiments.

 

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  		TABLE 1. Percentage of cells undergoing apoptosis visualized by dual staining with Annexin V-FITC/PI

 
Concentration-response analysis of DFF45, procaspase 8, cytochrome c, p21, and p53 protein levels are shown in Fig. 5Go. All but the p53 panel (E) were the result of a 24-h incubation; p53 was measured after 72 h because there was no effect at 24 h. As can be seen, the point of convergence of the extrinsic and intrinsic pathways at DFF45 cleavage showed a definitive response at 100 ng/ml (panel A). This was also the concentration at which pro-caspase 8 cleavage was first evident (panel B). A change in cytosolic cytochrome c, however, was not evident until 500 ng/ml (panel C). Analysis of p21 showed a significant change at 24 h (panel D), whereas a change in p53 required triple the time and was not maximal until 1 µg/ml (panel E).


Figure 5
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  		FIG. 5. Concentration response analysis of apoptosis markers and cell cycle regulatory proteins, p21 and p53. Cells were treated with the different concentrations of S179D PRL for 24 h (A–D) or 72 h (E) in serum-deprived medium. Western blots: A, DFF45; B, pro-caspase 8; C, cytochrome c, D, p21; E, p53. F and G, Reporter assays. Exponentially growing HUVEc were transiently cotransfected as for Fig. 2Go and treated for either 12 or 24 h with different concentrations of S179D PRL. The data shown are the average from three independent experiments. Each bar represents the mean ± SD. *, P value ≤ 0.05 vs. control (0 ng/ml).

 
Concentration-response analysis of p21 promoter activation and Bax promoter activation are also shown in Fig. 5Go, F and G. At 24 h, p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the activity of the Bax promoter, which increased further at 1 µg/ml.

Work in other systems has demonstrated that S179D PRL primarily signals through ERK 1/2 (4, 28). This, however, is also true of bFGF (5) which is antiapoptotic and promotes the growth of endothelial cells. In a previous study, this was resolved by demonstration that S179D PRL blocked the immediate rise in ERK 1/2 phosphorylation in response to bFGF, while causing a delayed and prolonged activation of ERK in its own right (3). This finding was confirmed and extended in the present study. Figure 6Go shows blockade of early signaling from bFGF with prolonged activation of ERK at 40–240'. Furthermore, and importantly for the current study, this figure shows the ability of the MAPK pathway inhibitor, PD98059 to inhibit this delayed activation of ERK.


Figure 6
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  		FIG. 6. Blockade of bFGF ERK signaling by S179D PRL. Confluent HUVEc were starved in SFM and bFGF-depleted medium for 16 h and then treated with bFGF (25 ng/ml), bFGF plus S179D PRL (1 µg/ml), or bFGF plus S179D PRL plus PD 98059 (10 µM) for the indicated times before lysis. Each blot represents the result of a typical experiment conducted three times. Representative Western blots are shown. Columns represent means ± SD of at least three independent experiments. *, P value ≤ 0.05 vs. control (time 0 min). P-ERK, Phosphorylated ERK.

 
Because bFGF is a normal medium component, required for endothelial cell survival in vitro, analysis of the role of ERK signaling in the initiation of apoptosis in response to S179D PRL is complicated by the concurrent blockade of bFGF signaling. Tests at different time intervals, however, allowed us to settle on a 2-d incubation period in the presence of the MAPK pathway inhibitor as sufficiently short as to not result in significant cell death with the inhibitor alone, but sufficiently long to see an effect of S179D PRL on DNA fragmentation. Figure 7Go shows that in this time frame PD 98059 alone does not increase the percentage of cells with sub G1 amounts of DNA, whereas S179D PRL more than quadruples this percentage, an effect blocked by PD98059. Thus, blockade of the ERK 1/2 signaling pathway blocks apoptosis in response to S179D PRL.


Figure 7
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  		FIG. 7. Blockade of S179D-induced apoptosis by PD 98059 as determined by DNA Content. HUVEc were cultured in growth medium for 2 d and then treated with S179D PRL (1 µg/ml). Events falling under the 100 (0–100) FL2 channel scale were considered apoptotic events resulting from DNA fragmentation. Percents represent the proportion of gated cells undergoing apoptosis (A); cells in the G1/G0 phase (G1/0) and cells in the G2 phase (G2) ± SD.

 
Performance of a similar analysis of the effect of PD98059 on signaling to apoptosis is shown in Fig. 8Go. In this experiment, which was shorter in duration than the one shown in Fig. 4Go, S179D PRL increased the number of early apoptotic cells to a level more than 3-fold that in the controls, and late apoptotic cells to a level double that in the control cells. The late apoptotic effect was completely blocked by PD98059, whereas the early was mostly blocked by PD98059.


Figure 8
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  		FIG. 8. Blockade of S179D-induced apoptosis by PD98059 as determined by annexin V and PI dual staining. HUVEc were stained with FITC-annexinV and PI and analyzed by flow cytometry. Apoptotic cells were defined as early or late as described above. Numbers in the quadrants give the percent of FITC+ (lower) or FITC+/PI+ cells (upper) ± SD (n = 3). A, Untreated cells grown for 2 d in growth medium; B, cells treated with PRL (1 µg/ml) for 2 d in growth medium; C, cells treated with PD 98059 (20 µM) for 2 d in growth medium; D, cells cotreated with S179D PRL (1 µg/ml) and PD 98059 (20 µM) for 2 d in growth medium; E, cells treated with S179D PRL (1 µg/ml) for 2 d in growth medium.

 
Analysis of the role of ERK signaling in the production of elevated levels of p21 and p53 (Fig. 9Go) showed PD98059 to block the effect of S179D PRL on p21 but to have no effect on p53 levels. Cleavage of DFF45 was also blocked by PD98059. Analysis of p21 promoter activity in response to either bFGF or S179D PRL showed that bFGF was without effect and that PD98059 blocked the ability of S179D PRL to increase promoter activity. This effect of PD98059 was reproduced by dominant-negative ERK (Fig. 9DGo). Similar analyses of the Bax promoter showed no effect of PD98059 (not shown).


Figure 9
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  		FIG. 9. Blockade of effects of S179D PRL on cell cycle regulators and DFF45 by PD98059. A–C, Western blot analyses of cells treated with PRL (P) at 1 µg/ml, S179D PRL (S) at 1 µg/ml, endotoxin (L) at 0.02 EU/ml, or S179D PRL plus PD 98059 (20 µM), and no treatment [control (C)] in serum-deprived medium. Incubation for A and C was for 24 h and for B was 72 h. D, Reporter assay. Exponentially growing HUVEc were transiently cotransfected with either pSV-ß-galactosidase/pp21-luc or pSV-ß-galactosidase/pp21-luc/pERK-DN (dominant-negative ERK mutant), and after the recovery period were treated/cotreated with PRL, S179D PRL, PD 98059, and bFGF (25 ng/ml). The data shown are the average from three independent experiments. Each bar represents the mean ± SD. *, P value ≤ 0.05 vs. cotreatment with PD 98059 and S179D PRL; {dagger}, samples not different from one another, but different from C with a P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
From these experiments, it is clear that S179D PRL exerts some of its antiangiogenic effects via the promotion of endothelial cell apoptosis. Effects on markers of apoptosis could be seen in as little as 24 h with concentrations of S179D PRL between 25 and 100 ng/ml, depending on the sensitivity of the measure. An earlier study assessing cell number after a 3-d incubation required much higher concentrations for an effect to be appreciated (3). These proapoptotic effects of S179D PRL were observed despite the presence of bFGF in the culture medium. Thus, S179D PRL is not only proapoptotic, but it overcomes the antiapoptotic activities of bFGF.

The concentration response analyses show an effect on the extrinsic pathway at one fifth the concentration required to see an effect on the intrinsic pathway. Because the lower concentration was all that was required to see an effect at the point of convergence of the two pathways, this suggests that the extrinsic pathway is most important to initial apoptotic events. At higher concentrations of S179D PRL, which might be thought of as hyperactivating the extrinsic pathway, the release of cytochrome c from mitochondria could be initiated by caspase-8 cleavage of Bid, a member of the proapoptotic Bcl-2 family (6). Although elevated p53 can lead to increased cytochrome c release, it is unlikely that the initial cytochrome c release was subsequent to increased p53 because a change in cytochrome c was observed at 24 h and no change was seen in p53 until 72 h. Effects on the cell cycle regulatory protein, p21, were seen at the lower concentrations, and signaling from S179D PRL to p21 and apoptosis occur via ERK activation and hence are extrinsic. Analysis of other signaling pathways, including Akt and JNK showed no definitive effects of S179D PRL (data not shown). ERK activation, however, was not involved in the effect of S179D PRL on p53 levels or activation of the Bax promoter. This confirms the idea that p21 elevation is not a consequence of p53 activation. Most likely, later apoptotic events such as DNA damage increased p53 (7) levels and activation, which may have in turn contributed to later cytochrome c release and amplification via the intrinsic pathway. Other antiangiogenic compounds also rely on increased expression of p21 and p53 (29, 30, 31) and also activate both the extrinsic and intrinsic pathways (32, 33).

bFGF signals to growth/antiapoptosis via rapid activation of the MAPK pathway (5). In the current study, we confirm the ability of bFGF to cause a rapid phosphorylation of ERK 1/2 and show that S179D PRL blocked this rapid effect and yet caused a delayed and sustained increase in ERK 1/2 phosphorylation. Other investigators have shown that a delayed and sustained elevation of ERK 1/2 phosphorylation results in up-regulation of p21 expression (34, 35, 36, 37), and we have shown in another system that delayed and prolonged activation of ERK1/2 by S179D PRL resulted in elevated p21 (4). A delayed ERK activation has also been shown to prevent the formation of tubule-like structures in Matrigel by HUVEc (38). To verify the role of ERK 1/2 signaling in cell cycle arrest/apoptosis in these cells, we used the MEK inhibitor, PD98059 to block the cascade. The design of these experiments was complicated by the concurrent effects on signaling from bFGF, but preliminary experiments allowed us to settle on a time frame (48 h) where deprivation of bFGF signaling was not evident in terms of DNA content and annexin V/PI staining compared with control. Having demonstrated that PD98059 blocked the delayed and sustained activation of ERK 1/2 by S179D PRL, we then showed that PD98059 also blocked the effects of S179D PRL on DNA content and annexin V/PI staining. A sustained ERK phosphorylation resulting in diminished HUVEc survival has been previously observed for hederacolchiside-A1, a saponin with antiangiogenic properties (38). The effect of S179D PRL on endothelial cells is not limited to the induction of apoptosis but also includes interference with cell migration, invasion, and the ability to form tubular structures (3). In this regard, sustained ERK activation has been shown to decrease endothelial cell adhesion and cell migration (39). In a 24-h period, it was also possible to show that PD98059 blocked cleavage of DFF45, and up-regulation of p21 protein and promoter activity. There was, however, no effect of PD98059 on p53 protein even after 72 h. ERK mediation of the effect on the p21 promoter was confirmed using a dominant-negative ERK. We conclude from these experiments that low concentrations of S179D PRL use the MAPK pathway to promote apoptosis. Up-regulation of p53 protein and Bax promoter activity by higher concentrations of S179D PRL, however, was not dependent on the MAPK pathway. Because blockade of the MAPK pathway blocked apoptosis in the time frame used even at high concentrations, this argues that elevated p53/activation of p53 is insufficient to induce apoptosis. Dependence of p21 elevation on activation through the extrinsic pathway is consistent with studies in other systems (40, 41). Shin et al. have also described systems where p21 was essential for programmed cell death (42). Furthermore, our studies with S179D PRL are in agreement with the work of Maxwell (43), which showed that HUVEc were resistant to p53 induction of apoptosis. Because bFGF had no effect on either p21 or Bax promoter activity, neither the effect of the lower or higher concentrations of S179D PRL on p21 or p53 seem to be the result of blocked bFGF signaling, but rather are the result of extrinsic initiation of apoptosis and, if treated for sufficient time, subsequent amplification via the intrinsic pathway.

We conclude that S179D PRL primarily signals through the MAPK pathway to up-regulate p21 and initiate apoptosis in endothelial cells. Given that S179D PRL has been shown to dimerize both short PRLRs (44) and up-regulates expression of the short forms of the PRLR in these cells (3), it is likely that one or both of the short forms are the primary mediators of the relevant activities. Others have reported direct angiogenic effects of unmodified PRL (reviewed in Ref. 45), thought to be through an as-yet-uncloned endothelial cell-specific receptor, and there is also evidence for indirect angiogenic effects of unmodified PRL via VEGF production in other cells (46). In addition, previous work from this lab has shown up-regulation of hemeoxygenase-1(3), an angiogenic factor, by unmodified PRL. Given the presence of both short forms of the PRLR (as well as some extracellularly deleted forms of the PRLR) in these cells (3), it appears that the PRLR mediates both antiangiogenic and angiogenic effects on endothelial cells. Apart from the possible compensatory mechanisms that occur during the development of null mice, it is assumed therefore that the apparently normal angiogenic profile of the PRL null (47) and PRLR null (48) mice in most tissues is the result of the loss of both angiogenic and antiangiogenic signaling through PRLRs.

S179D PRL is a molecular mimic of phosphorylated PRL, which usually constitutes 5–30% of the prolactin released by the pituitary (49, 50). At the lower concentrations of S179D PRL, therefore, what was observed may have physiological significance because apoptosis was increased about 17-fold at 25 ng/ml, even in the presence of bFGF. There are, however, many angiogenic and antiangiogenic factors in the circulation, and so analysis in vivo will be required to determine the relative importance of physiological proportions and concentrations of phosphorylated PRL. During the induction of pituitary tumors by estrogens, phosphorylation of PRL is lost (51), and pituitary tumor cell lines do not produce phosphorylated PRL (52). Based on the current results, one might therefore propose the absence of phosphorylated PRL and its antiangiogenic effects to be a contributing factor in development of such tumors.

S179D PRL may be a useful antiangiogenic therapeutic that can use more than one pathway to promote endothelial cell apoptosis, one active at physiological concentrations and another induced by higher, but potentially therapeutic, concentrations. Antiangiogenic activity was a likely contributing factor to the antitumor activities of S179D PRL already demonstrated in vivo (2), although direct effects on epithelial cells were also important (2, 4).


   Acknowledgments
 
We thank Drs. Leonard Freedman and Robert Vogel (Merck and Co. Inc., West Point, PA), Xuan Liu (Department of Biochemistry, University of California, Riverside), and Melanie Cobb (University of Texas Southwestern Medical Center) and Katie Defea (Division of Biomedical Sciences, University of California, Riverside) who kindly provided the p21-luc, Bax-luc and dominant-negative ERK constructs, respectively.


   Footnotes
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 61005 (to A.M.W.). E.K.U. was partially supported by a split fellowship granted by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Reference No. 200719/2003-3.

E.K.U., H.L.L., P.B., and A.M.W. have nothing to disclose related to this study.

First Published Online July 13, 2006

Abbreviations: bFGF, Basic fibroblast growth factor; DFF45, DNA fragmentation factor; DPBS, Dulbecco’s PBS; FITC, fluorescein isothiocyanate; HUVEc, human umbilical vein endothelial cells; ICAD, inhibitor of caspase-activated deoxyribonuclease; LPS, lipopolysaccharide; PI, propidium iodide; PRL, prolactin; PRLR, PRL receptor; SFM, serum-free medium; TIMPs, inhibitors of matrix metalloproteases.

Received March 16, 2006.

Accepted for publication July 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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