The Antiangiogenic Factor 16K Human Prolactin Induces Caspase-Dependent Apoptosis by a Mechanism that Requires Activation of Nuclear Factor-{kappa}B

Sébastien P. Tabruyn, Catherine M. Sorlet, Françoise Rentier-Delrue, Vincent Bours, Richard I. Weiner, Joseph A. Martial and Ingrid Struman

Laboratoire de Biologie Moléculaire et de Génie Génétique (S.P.T., C.M.S., F.R.-D., J.A.M., I.S.) and Génétique Humaine (V.B.), Université de Liège, B-4000 Liège, Belgium; and Center for Reproductive Sciences (R.I.W.), Department of Obstetrics, Gynecology and Reproductive Sciences, University of California School of Medicine, San Francisco, California 94143

Address all correspondence and requests for reprints to: Joseph Martial, Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée du 6 Aout B6A, B-4000 Liège, Belgium. E-mail: jmartial{at}ulg.ac.be.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-{kappa}B (NF-{kappa}B) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor {kappa}B-{alpha} degradation permitting translocation of NF-{kappa}B to the nucleus and reporter gene activation. Inhibition of NF-{kappa}B activation by overexpression of a nondegradable inhibitor {kappa}B-{alpha} mutant or treatment with NF-{kappa}B inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-{kappa}B dependent. These findings support the conclusion that NF-{kappa}B signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANGIOGENESIS, THE FORMATION of new capillaries from preexisting blood vessels, is necessary for many physiological processes, including embryonic vascular development and differentiation, wound healing, and organ regeneration (1). In addition, angiogenesis is associated with pathological conditions such as cancer, rheumatoid arthritis, diabetic retinopathy, and atherosclerosis (2). Angiogenesis is tightly regulated by both activators and inhibitors of endothelial cell proliferation, migration, and organization. Many growth factors and cytokines are known to stimulate angiogenesis. Among these, the best characterized are basic fibroblast growth factor (3) and vascular endothelial growth factor (VEGF) (4). On the other hand, factors with antiangiogenic activity include endostatin (5), angiostatin (6), thrombospondin (7), and the 16-kDa N-terminal fragment of human PRL (16K hPRL) (8). In addition, we have shown that recombinant 16-kDa N-terminal fragments of the hPRL family members (i.e. GH, variant GH, and placental lactogen) are also antiangiogenic (9).

The use of antiangiogenic factors as therapeutic agents is now being widely tested. Biologically active 16K hPRL was expressed and secreted from HCT116 human colon cancer cells stably transfected with an expression vector encoding 16K hPRL. 16K hPRL production by the transfected HCT116 cells inhibited tumor growth and neovascularization when implanted sc in Rag1 mice (10). Furthermore, using an adenovirus transfer vector, Kim et al. (11) have shown that expression of 16K hPRL in prostate cancer cells markedly reduced their ability to form tumors in a xenograft animal model. These new data strongly suggest that 16K hPRL has potential as an anticancer drug.

The mechanisms by which 16K hPRL inhibits angiogenesis have been partially elucidated. 16K hPRL inhibits capillary endothelial cell proliferation, migration, and organization into microvessels (8). This antiangiogenic effect appears to be mediated, in capillary endothelial cells, by a saturable high-affinity 16K hPRL binding site that differs from the PRL receptor (12). To date, the receptor mediating 16K hPRL activity remains unknown. Formation of new microvasculature requires activation of proteases, including urokinase. Lee et al. (13) have shown that 16K hPRL inhibits urokinase activation by increasing the expression of type 1 plasminogen activator inhibitor. The antiproliferative effect of 16K hPRL appears to involve inhibition of VEGF-induced Ras activation resulting in blocking of MAPK activation (14). Recently, Martini et al. (15) demonstrated that 16K hPRL induces apoptosis of bovine brain capillary endothelial (BBCE) cells and human umbilical vein endothelial cells. Signaling events associated with 16K hPRL-induced apoptosis include increased DNA fragmentation, activation of caspases-1 and -3, inactivation of two caspase-3 substrates: poly (ADP-ribose) polymerase and the inhibitor of caspase-activated DNase. Furthermore, treatment with 16K hPRL induces conversion of the antiapoptotic form of Bcl-X, Bcl-XL, to its proapoptotic form, Bcl-XS.

To better understand the mechanisms by which 16K hPRL activates programmed cell death in endothelial cells, we have analyzed the effect of 16K hPRL on NF-{kappa}B activation in bovine adrenal cortex capillary endothelial (BACE) cells. The role of nuclear factor-{kappa}B (NF-{kappa}B) in apoptotic signaling is complex. It protects many cells from cell death stimuli, but in a few cases it contributes to apoptosis (16). We show here that 16K hPRL can activate NF-{kappa}B by causing degradation of its inhibitor (I{kappa}B-{alpha}). NF-{kappa}B is essential to 16K hPRL-induced apoptosis, because inhibition of the NF-{kappa}B pathway prevents 16K hPRL-induced activation of caspase-3 and DNA fragmentation. The apoptotic cascade can be initiated either by death receptor activation, involving activation of initiator protease caspase-8, or by release of mitochondrial components, involving activation of initiator enzyme caspase-9. Once these caspases are activated, the cascade they have initiated leads to activation of the downstream effector protease caspase-3. We show that in BACE cells, both pathways are essential to 16K PRL-induced apoptosis, because inhibitors of either caspase-8 or caspase-9 prevent 16K hPRL-mediated cell death. Furthermore, we show that NF-{kappa}B plays a role in regulating this process, because NF-{kappa}B inhibitors abolish caspase-8 and -9 activation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
16K hPRL Activates NF-{kappa}B in BACE Cells
EMSAs were performed to determine whether treatment with 16K hPRL could cause NF-{kappa}B activation in BACE cells. Nuclear extracts from BACE cells stimulated by increasing concentrations of 16K hPRL were incubated with a 32P-labeled {kappa}B DNA sequence before gel electrophoresis (Fig. 1AGo). The band corresponding to the {kappa}B/NF-{kappa}B complex was found to increase with the 16K hPRL concentration from 2–10 nM (lanes 1–4) as compared with the band obtained with unstimulated cells (lane 1). The specificity of the {kappa}B/NF-{kappa}B complex detected was demonstrated by the reduced band intensity observed in the presence of a 100-fold molar excess of unlabeled {kappa}B probe (lane 8) but not with an unlabeled mutated {kappa}B probe (lane 9). Furthermore, the p50/p65 heterodimer was identified by its immunoreactivity toward p50- and p65-specific antibodies (lanes 6–7).



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1. 16K hPRL Induces NF-{kappa}B Activation in Endothelial Cells

A, EMSAs were performed with BACE cell nuclear extracts treated or not for 40 min with various concentrations of 16K hPRL or endotoxin C (0.08 ng/ml) and preincubated either without (lanes 1–5) or with addition of p50-specific antibody (lane 6) or p65-specific antibody (lane 7). In competition assays, extracts were preincubated with a 100-fold molar excess of unlabeled oligonucleotide containing either wild-type (wt) (lane 8) or mutated (mut) (lane 9) NF-{kappa}B binding sites. B–D, Luciferase activity was measured in BACE (B), ABAE (C), and BBCE (D) cells transfected with the pElam-Luc reporter gene vector after incubation for 6 h with increasing concentrations of 16K hPRL. E, Western blot detection of I{kappa}B-{alpha} in cytoplasmic extracts of BACE cells treated for 30 min with increasing concentrations of 16K hPRL. F, Transfected BACE cells were treated for 6 h with 5 nM 16K hPRL or 1 ng/ml endotoxin, these preparations being or not being boiled beforehand for 2 min or preincubated with a polyclonal antibody (no. 602) (1:250) recognizing 16K hPRL. All luciferase activities, normalized with respect to the total protein content, are expressed as enhancement factors (activity measured divided by the activity of untreated cells). Each bar represents the mean ± SD, n = 3. Endotoxin C (0.08 ng/ml) was added as a control for endotoxin contamination.

 
A luciferase reporter assay was used to confirm 16K hPRL-induced activation of NF-{kappa}B. BACE cells were transiently transfected with the pElam-Luc plasmid coding for a luciferase reporter gene under the control of the Elam-1 promoter [containing three NF-{kappa}B binding sites (17)]. 16K hPRL was found to induce luciferase activity in a concentration-dependent manner in the transfected cells (Fig. 1BGo). Induction was maximal (8.5-fold) with 10 nM 16K hPRL. The effect of 16K hPRL on adult bovine aortic endothelial (ABAE) and BBCE cells was also tested, with similar results (Fig. 1Go, C and D). Because I{kappa}B-{alpha} is the most common NF-{kappa}B inhibitor, we then examined by Western blotting the effect of 16K hPRL on the degradation of this inhibitor. When BACE cells were treated for 30 min with 16K hPRL, the level of I{kappa}B-{alpha} protein in the cytoplasm of these cells decreased progressively as the 16K PRL concentration increased (Fig. 1EGo). The lowest level of I{kappa}B-{alpha} was observed with 10 nM 16K hPRL. I{kappa}B-{alpha} degradation was first detected 20 min after 16K hPRL treatment (data not shown).

Because the 16K hPRL used for these studies was produced in Escherichia coli, it was necessary to demonstrate that the observed effects were not due to bacterial contaminants (e.g. endotoxin). The amount of endotoxin (0.08 ng/ml) present in 10 nM 16K hPRL (endotoxin C lane) was found to induce only a weak response in the various NF-{kappa}B assays. This could not account for the observed activation of NF-{kappa}B by our 16K hPRL preparation. In addition, the action of 16K hPRL was reduced when the protein was boiled for 2 min before its addition to the cells (concentration, 10 nM). Boiling for 2 min did not affect the action of endotoxin (1 ng/ml) (Fig. 1FGo). Furthermore, immunoneutralization with polyclonal antibody against 16K hPRL (1:250) significantly decreased the effect of 10 nM 16K hPRL, but not that of endotoxin (1 ng/ml). These results clearly demonstrate that 16K hPRL causes activation of NF-{kappa}B in BACE cells, independent of any effect of contaminating endotoxin.

16K hPRL Induces Apoptosis of BACE Cells
Figure 2AGo shows that an 18-h treatment of BACE cells with 16K hPRL resulted in the appearance of DNA fragments in the cytoplasm of the treated cells and that the effect was dose dependent. The highest concentration of 16K hPRL (5 nM) caused an 8.5-fold increase in DNA fragmentation as compared with untreated cells. Endotoxin C caused only a 2-fold increase in DNA fragmentation. Heat denaturation and immunoneutralization treatments significantly reduced the effect observed with the 16K hPRL preparation but not the action of 1 ng/ml endotoxin (Fig. 2BGo). These results show that the DNA fragmentation elicited by the recombinant 16K hPRL preparation is due to 16K hPRL itself and not to endotoxin contamination.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. 16K hPRL Induces Apoptosis in BACE Cells

DNA fragmentation induced by 16K hPRL. A, Cells were treated for 16 h with increasing concentrations of 16K hPRL. Endotoxin C (0.04 ng/ml) was added as a control for endotoxin contamination. B, Cells were treated for 16 h with 5 nM 16K hPRL or 1 ng/ml endotoxin. 16K hPRL and endotoxin samples were also boiled for 2 min or preincubated with a polyclonal antibody (no. 602) (final dilution 1:250) recognizing 16K hPRL. DNA fragmentation was measured using the Cell Deathplus Detection ELISA kit assay (Roche Molecular Biochemicals), and results are expressed as enhancement factors (treated vs. untreated cells). Caspase-3 activation by 16K hPRL. C, BACE cells were treated for 6 h with increasing concentrations of 16K hPRL or with 0.04 ng/ml endotoxin (endotoxin C). D, BACE cells were treated with 10 nM 16K hPRL for the indicated time. Caspase-3 activation was measured with the CaspACE Assay System Fluorometric (Promega Corp.). Each result is expressed as an enhancement factor (treated vs. untreated cells). Each bar represents the mean ± SD, n = 3.

 
To confirm that 16K hPRL induces apoptosis in BACE cells, we studied activation of the caspase cascade. Activation of the effector protease caspase-3 is one of the most common events of the apoptotic signaling pathway. When used to treat BACE cells for 6 h, 16K hPRL was found to induce caspase-3 activation in a concentration-dependent manner (Fig. 2CGo). Maximal activation (20-fold) was achieved with 10 nM 16K hPRL. Endotoxin C induced only a slight increase in activity (3-fold) as compared with untreated cells. In a time course assay with 10 nM 16K hPRL, caspase-3 activation was already visible after a 2-h incubation and maximal after 3 h (Fig. 2DGo).

A Nondegradable Mutant of I{kappa}B-{alpha} Blocks 16K hPRL-Induced Apoptosis
Given the dual effect of NF-{kappa}B on apoptosis, we further examined the role of this factor in 16K hPRL-induced apoptosis. BACE cells were cotransfected with the pElam-Luc reporter plasmid and either the empty pcDNA-3 plasmid (control) or plasmid pmutI{kappa}B, coding for a mutant I{kappa}B-{alpha} (mutI{kappa}B-{alpha}) characterized by serine-to-alanine substitutions at residue positions 32 and 36. These mutations are known to prevent I{kappa}B-{alpha} phosphorylation and subsequent degradation. In this experiment, 16K hPRL-induced enhancement of luciferase activity was reduced in cells expressing pmutI{kappa}B as compared with cells transfected with the empty vector (Fig. 3AGo). These data confirm that expression of mutI{kappa}B-{alpha} can inhibit NF-{kappa}B activation by 16K hPRL in BACE cells.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Expression of Mutant I{kappa}B-{alpha} Decreases 16K hPRL-Induced Apoptosis

A, BACE cells were cotransfected with the pElam-Luc reporter gene vector and either an expression vector for mutant I{kappa}B-{alpha} (pmutI{kappa}B-{alpha}) or an empty vector (pcDNA-3). Twenty-four hours after transfection, the cells were treated with 5 or 10 nM 16K hPRL. Luciferase activity was measured 6 h later. Luciferase activities, normalized with respect to the total protein content, are expressed as enhancement factors (treated vs. untreated cells). B, BACE cells were transfected with an expression vector for mutant I{kappa}B-{alpha} (pmutI{kappa}B-{alpha}) or with an empty vector (pcDNA-3). Twenty-four hours after transfection, the cells were treated with 5 nM or 10 nM 16K hPRL for 6 h. Caspase-3 activation was measured using the CaspACE Assay System Fluorometric (Promega Corp.). Results are expressed as enhancement factors (treated vs. untreated cells). The asterisk denotes a P value < 0.05 vs. the corresponding pcDNA-3-transfected cells. Each bar represents the mean ± SD, n = 3. The experiment was repeated three times with similar results.

 
We then asked whether overexpression of mutI{kappa}B-{alpha} would inhibit the activation of caspase-3 by treatment with 16K hPRL (5 or 10 nM 16K hPRL for 6 h). In treated cells expressing mutI{kappa}B-{alpha}, caspase-3 activity was lower than in cells transfected with the control plasmid (Fig. 3BGo). In four independent experiments overexpression of mutI{kappa}B-{alpha} inhibited 16K hPRL-induced caspase-3 activation by 29% (Fig. 3Go shows a representative experiment).

Chemical Inhibitors of NF-{kappa}B Block 16K hPRL-Induced Apoptosis
To confirm the hypothesis that NF-{kappa}B plays a role in 16K hPRL-induced programmed cell death, we used different pharmacological inhibitors of NF-{kappa}B (BAY 11–7082, BAY 11–7085, and gliotoxin) known to interfere with NF-{kappa}B activation at various levels (18, 19). In a luciferase reporter-gene expression assay, we showed that all these inhibitors block 16K hPRL-induced activation of NF-{kappa}B. Treatment for 1 h with BAY 11–7082 (1 µM), BAY 11–7085 (1 µM), or gliotoxin (300 nM) before addition of 5 nM 16K hPRL to the cells strongly reduced the increase of luciferase activity (Fig. 4AGo). For each compound, we have used the maximal nontoxic concentration. The action of gliotoxin was specific, because the inactive analog, methylgliotoxin (300 nM), had no effect (19).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. Chemical Inhibitors of NF-{kappa}B Reduce 16K hPRL-Induced Apoptosis

A, BACE cells transfected with the pElam-Luc reporter gene vector were pretreated or not for 1 h with BAY 11–7082 (1 µM), BAY 11–7085 (1 µM), gliotoxin (300 nM), or methylgliotoxin (300 nM) before stimulation for 6 h with 5 nM 16K hPRL. Luciferase activities, normalized with respect to the total protein content, are expressed as enhancement factors (treated vs. untreated cells). BACE cells were treated as above. B, DNA fragmentation as estimated with the Cell Deathplus Detection ELISA kit assay (Roche Molecular Biochemicals). Results are expressed as enhancement factors (treated vs. untreated cells). C, Caspase-3 activity measured by means of the CaspACE Assay System Fluorometric (Promega Corp.). Results are expressed as enhancement factors (treated vs. untreated cells). Each bar represents the mean ± SD, n = 3.

 
Pretreatment for 1 h with BAY 11–7082 (1 µM), BAY 11–7085 (1 µM), or gliotoxin (300 nM) significantly reduced 16K hPRL-induced DNA fragmentation in BACE cells (Fig. 4BGo). Methylgliotoxin (300 nM) failed to block 16K hPRL-induced DNA fragmentation. Likewise, a 1-h pretreatment with either of the three inhibitors strongly reduced caspase-3 activation by 5 nM 16K hPRL, whereas methylgliotoxin, again, had no effect (Fig. 4CGo).

16K hPRL Induces Caspase-8 and Caspase-9 Activation via NF-{kappa}B
To investigate the initial events leading to caspase-3 activation, we monitored caspase-8 and -9 activities. The highest concentration of 16K hPRL (10 nM) caused a significant 2-fold increase in caspase-8 activity in BACE cells, as compared with untreated cells (Fig. 5AGo). A stronger 10-fold activation of caspase-9 was observed after treatment with 16K hPRL (Fig. 5BGo). Activation of both caspases was detected after 2 h and peaked at 3 h. Endotoxin C treatment led to only slightly increased caspase-8 and caspase-9 activities.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. 16K hPRL Induces NF-{kappa}B-Dependent Caspase-8 and Caspase-9 Activation

A and B, BACE cells were treated for 3 h with 5 nM or 10 nM 16K hPRL or with 0.08 ng/ml endotoxin (endotoxin C). In the time course experiments, BACE cells were treated with 10 nM 16K hPRL for the indicated time. Caspase-8 activation was measured with the Caspase-8 Assay kit Fluorimetric (Sigma). Caspase-9 activation was measured using the Caspase-9 Fluorimetric Assay (R&D Systems). C, BACE cells were pretreated or not with Z-IETD-FMK (10 µM), Z-LEHD-FMK (10 µM), or dimethylsulfoxide (0.5% vol/vol - control) before stimulation for 3 h with 10 nM 16K hPRL. D and E, BACE cells were pretreated or not for 1 h with BAY 11–7082 (1 µM), BAY 11–7085 (1 µM), gliotoxin (300 nM), or methylgliotoxin (300 nM) before stimulation for 3 h with 10 nM 16K hPRL. Caspase-8 activation (D) and caspase-9 activation (E) were measured as described above. Results are expressed as enhancement factors (treated vs. untreated cells). Each bar represents the mean ± SD, n = 3.

 
To determine whether caspase-3 activation is dependent on caspase-8, caspase-9, or both, we examined caspase-3 activation in the presence of either a caspase-9 inhibitor (Z-LETD-FMK) or a caspase-8 inhibitor (Z-IETD-FMK). Both inhibitors were found to abolish 16K hPRL-induced caspase-3 activity almost totally in BACE cells (Fig. 5CGo). This indicates that both initiator caspases are necessary for caspase-3 activation.

To establish whether activation of caspase-8 and caspase-9 is NF-{kappa}B dependent, we then measured caspase-8 and caspase-9 activities in the presence of two NF-{kappa}B inhibitors. A 1-h pretreatment with BAY 11–7082 (1 µm) or BAY 11–7085 (1 µM) efficiently blocked caspase-8 and caspase-9 activation by 10 nM 16K hPRL (Fig. 5Go, D and E).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To better understand the signaling mechanisms involved in activation of endothelial cell apoptosis by 16K hPRL, we have investigated the effect of 16K hPRL on the caspase signaling cascade and on activation of the NF-{kappa}B signaling pathway. We show that, in endothelial cells, 16K hPRL can induce translocation to the nucleus and activation of NF-{kappa}B. In BACE cells, this activation appears to be mediated by I{kappa}B-{alpha} degradation. Degradation of I{kappa}B-{alpha} permits translocation of NF-{kappa}B to the nucleus. Recently, rat 16K PRL has been shown to induce NF-{kappa}B translocation in fibroblasts. However, in this nonendothelial cell type, this translocation requires I{kappa}B-ß but not I{kappa}B-{alpha} degradation (20).

To determine the role of NF-{kappa}B in 16K hPRL-induced apoptosis, we first used the nondegradable I{kappa}B-{alpha} serine-32 and serine-36 mutant. In BACE cells, phosphorylations on serine 32 and 36 of I{kappa}B-{alpha} are required for 16K hPRL-triggered NF-{kappa}B activation, because no activation occurs when these serines are mutated to alanine. This is consistent with the previous finding that phosphorylation of these serines triggers I{kappa}B-{alpha} degradation (21). Transfection with the pmutI{kappa}B-{alpha} (S32-S36) plasmid totally inhibits NF-{kappa}B activation. On the contrary, transfection with the pmutI{kappa}B-{alpha} (S32-S36) plasmid reduces 16K hPRL-induced caspase-3 activation by only 29%. This discrepancy can be explained by the fact that all cells that harbor the pElam-Luc plasmid also harbor the pmutI{kappa}B-{alpha} (S32-S36) plasmid, leading to a complete repression. On the other side, the caspase activity assay is performed on all cells whereas only a minor fraction of them have been transfected by the pmutI{kappa}B-{alpha} (S32-S36) plasmid. In this assay, therefore, the repression level is restricted by the transfection efficiency. According to a common transfection rate obtained with primary endothelial cells [~30% (22)], we have observed a repression of about 29%, which is the maximal we could expect. To confirm our results, we used drugs that inhibit NF-{kappa}B activation: BAY 11–7082, BAY 11–7085, or gliotoxin. Using these chemical inhibitors, the repression levels are similar in both NF-{kappa}B luciferase reporter- and caspase-3 activity-assays. These two last experiments provide evidence that 16K hPRL-induced apoptosis is fully NF-{kappa}B dependent.

The antiapoptotic action of NF-{kappa}B is well documented (23). In contrast, this study shows that NF-{kappa}B participates in a signaling cascade leading to apoptosis of BACE cells. Induction of NF-{kappa}B appears to be a general mechanism of 16K hPRL-induced endothelial cell apoptosis, because 16K hPRL can activate NF-{kappa}B in two other endothelial cell types (ABAE and BBCE). Recent reports have shown that NF-{kappa}B activation is essential for p53-induced (24) and glutamate-induced programmed cell death (25). In endothelial cells, Aoki et al. (26) have demonstrated that oxidative stress can induce apoptosis through NF-{kappa}B activation. In addition, NF-{kappa}B is reported to play a proapoptotic role in growth factor withdrawal-induced apoptosis of B lymphocytes (27). To date, the mechanisms underlying the proapoptotic action of NF-{kappa}B remain unclear. Several findings suggest that the ability of NF-{kappa}B to induce programmed cell death is due to its capacity to activate genes encoding Fas, Fas ligand, TRAIL, p53, or c-Myc (28, 29, 30, 31). The Fas/Fas ligand pathway has been shown to mediate induction of apoptosis by two other antiangiogenic factors: thrombospondin-1 and pigment-epithelium-derived factor (32). Triggering of apoptosis by activation of cell-surface death receptors involves subsequent activation of caspase-8. As NF-{kappa}B is known to activate death ligand or death receptor expression, we speculate that 16K hPRL might induce caspase-8 activation through a similar extrinsic pathway. This hypothesis is consistent with our results because NF-{kappa}B inhibition prevents caspase-8 activation and caspase-8 inhibition suppresses 16K hPRL-induced caspase-3 activation. We also show that inhibition of caspase-9, known to play a role in the mitochondria-dependent apoptosis pathway, suppresses caspase-3 activation. The requirement for both caspase-8 and caspase-9 is not contradictory, as caspase-9 might act to amplify the caspase cascade in situations where only a small amount of caspase-8 is activated (33). Indeed, this is the case here as 16K hPRL activates caspase-8 only slightly.

That an antiangiogenic factor induces apoptosis via NF-{kappa}B activation is intriguing. In all previous studies, NF-{kappa}B is described as a proangiogenic factor because it can activate expression of VEGF and IL-8, two angiogenesis-promoting molecules (34). In addition, Shono et al. (35) have demonstrated that inhibition of NF-{kappa}B abolishes induction of angiogenesis by H2O2. Here we propose an antiangiogenic role for NF-{kappa}B, linked to its ability to induce apoptosis of 16K hPRL-treated endothelial cells.

The blockade of apoptosis by the constitutive activation of NF-{kappa}B signaling pathways in cancer cells limits the efficacy of chemotherapy and radiotherapy (36). Based on this observation, treatment with NF-{kappa}B inhibitors was proposed to improve the apoptotic response to radiotherapy or chemotherapy (37). Here we show that in highly differentiated, nontransformed, capillary endothelial cells, the apoptotic process requires NF-{kappa}B signaling pathways. If similar mechanisms occur in vivo, treatment with NF-{kappa}B inhibitors might increase apoptosis of cancer cells but might also inhibit the activity of naturally occurring antiangiogenic factors. This blocking of antiangiogenic factors could result in increased angiogenesis, thus countering the proapoptotic action of the inhibitors on the tumor cells.

In conclusion, we have demonstrated that 16K hPRL induces NF-{kappa}B activation in endothelial cells. This induction leads, by the activation of two upstream caspases, caspase-8 and caspase-9, to the cell apoptosis. This finding reveals a novel important mechanism involved in the antiangiogenic action of 16K hPRL. Indeed, this article presents detailed functional evidence that NF-{kappa}B activation is fundamental for 16K hPRL-induced apoptosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Production of Recombinant Proteins and Antibodies
Recombinant 16K hPRL was produced in E. coli. Briefly, plasmid pT7L containing 23K hPRL cDNA was altered by site-directed mutagenesis: Cys 58 (TGG) of the construct was mutated to Ser (TCC), and the GAT codon corresponding to Glu 140 was mutated to TAA so as to generate a premature stop codon. The 16K hPRL coding sequences were obtained by site-directed mutagenesis performed on the cDNA encoding hPRL minus the corresponding signal peptide inserted into the pT7L expression vector (38). An ATG was genetically engineered 5' to the first codon, Cys 58 (TGC) was mutated to serine (TCC) to prevent incorrect disulfite bonds, and Glu 140 (GAA) was mutated to TAA to generate a premature stop codon. In brief, after induction of protein expression by treatment with isopropyl-ß-D-thiogalactopyranoside, the cells (E. coli BL21 DE3) were disrupted and the inclusion bodies were isolated. After washing, the inclusion bodies were solubilized in 20 mM ethanolamine-HCl (pH 9), containing 8 M deionized urea, 1% ß-mercaptoethanol, heated at 55 C for 10 min, and incubated overnight at room temperature. Denatured proteins were purified by anion exchange chromatography (Hitrap Q, Amersham Pharmacia Biotech, Arlington Heights, IL). Elution was performed in 20 mM ethanolamine-HCl (pH 9), 6 M deionized urea, with a gradient of 0–1 M NaCl. The renaturation was performed by a two-step dialysis during 72 h. For the first 6 h, a dialysis against 10 volumes of 20 mM ethanolamine-HCl (pH 9), 6 M deionized urea was performed. Then, urea from this bath was removed by buffer-exchange dialysis against 500 volumes of 20 mM ethanolamine-HCl (pH 9). Purification was then performed by molecular sieve chromatography (Sephadex G100, Amersham Pharmacia Biotech) performed in 50 mM NH4HCO3 (pH 7.5), 0.1 M NaCl. Purified proteins were dialyzed against 100 mM ethanolamine-HCl (pH 9), and stored at -20 C with 0.1 mg/ml of BSA.

The purity of the recombinant protein exceeded 95% (as estimated by Coomassie blue staining), and the endotoxin level was 0.0005 ng/ng recombinant protein, as quantified with the rapid endo test of the European endotoxin testing service (BioWhittaker, Verviers, Belgium). The anti-16K hPRL antibody used (no. 602) is a rabbit polyclonal antibody recognizing 16K hPRL. E. coli (serotype 055:B5) endotoxin was purchased from Sigma (St. Louis, MO). In experiments where the action of 16K hPRL was compared with that of endotoxin, the amount of endotoxin used was the amount present in the highest 16K hPRL concentration used in the assay.

Cell Cultures
BACE, ABAE, and BBCE cells were isolated as previously described (37). The cells were grown and serially passaged in low-glucose DMEM containing 10% fetal calf serum and 100 U/ml penicillin/streptomycin (10% fetal calf serum medium). Recombinant human basic fibroblast growth factor (Sigma) was added (1 ng/ml) to the culture every other day. Confluent cells corresponding to passages 7–12 were used in the experiments.

Preparation of Cell Extracts
BACE cells were treated or not for 40 min with various concentrations of 16K hPRL or endotoxin. The cells were scraped into PBS and centrifuged at 300 x g for 5 min, and the pellet was resuspended in 80 µl of ice-cold hypotonic buffer [10 mM HEPES (pH 7.9), 2 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 0.2% Nonidet P-40, 10 µg/ml leupeptin (Sigma), 0.7 µg/ml aprotinin (Roche, Indianapolis, IN), 0.1 µg/ml Pefabloc (Boehringer Ingelheim GmbH, Ingelheim, Germany)] for 30 sec just before centrifugation at 300 x g for 5 min. Aliquots of supernatant containing the cytoplasmic proteins were quickly frozen and stored at -80 C. The nuclear pellet was washed in PBS before being resuspended in 40 µl of extraction buffer [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM EDTA, 1 mM PMSF, 0.5 mM DTT, and 630 mM NaCl] and allowed to stand for 30 min at 4 C. After centrifugation (30 min at 2500 x g), aliquots of supernatant containing the nuclear proteins were stored at -80 C.

Protein concentrations were determined by the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).

Western Blot Analysis
Thirty micrograms of cytoplasmic cell lysates were resolved by SDS-PAGE (12%) and transferred to a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA). The blots were blocked for 1 h with 8% milk in Tris-buffered saline with 0.1% Tween 20 and were probed for 1 h with rabbit antihuman I{kappa}B-{alpha} pAb (C-21, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:600 dilution (333 ng/ml). After three washings with Tris-buffered saline with 0.1% Tween-20, the antigen-antibody complexes were detected with goat peroxidase-conjugated secondary antibody and an enhanced fluoro-chemiluminescent system (ECL-plus; Amersham Pharmacia Biotech). Finally, the blots were visualized on a Molecular Imager Fx (Bio-Rad Laboratories, Inc.).

EMSAs
The double-stranded oligonucleotide containing a palindromic variant of the {kappa}B enhancer of the IL-2 receptor {alpha}-chain binding site (39) has the following sequence: 5'-TTGGCAACGGCAGGGGAATTCCCCTCTCCTTA-3' (the core of the NF-{kappa}B binding site is underlined). This {kappa}B sequence (Eurogentec, Seraing, Belgium) was labeled using the Klenow fragment of E. coli DNA polymerase (Life Technologies, Inc., Gaithersburg, MD), [{alpha}-32P]dCTP, and [{alpha}-32P]dATP.

In the supershift assay, 1 µl antibody against p50 or p65 (Santa Cruz Biotechnology, Inc.: sc 114X, sc 109X) was added to the extract (30 min on ice) before incubation with the labeled {kappa}B probe. In the competition assay, a 100-fold molar excess of unlabeled wild-type or mutant {kappa}B was added to the binding reaction mixture 15 min before incubation with the nuclear extract.

The gel was dried for 1 h at 80 C before visualization with a Molecular Imager Fx (Bio-Rad Laboratories, Inc.).

DNA Fragmentation ELISA
BACE cells were treated or not with various concentrations of 16K hPRL or endotoxin for the indicated time. DNA fragmentation was measured with the Cell Deathplus Detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions.

Caspase Activity Assay
BACE cells were treated or not with various concentrations of 16K hPRL or endotoxin. After the indicated incubation time, caspase-3 activity was measured with the CaspACE Assay System Fluorometric (Promega Corp., Madison, WI), caspase-8 with the Caspase-8 Assay kit Fluorimetric (Sigma), and caspase-9 with the Caspase-9 Fluorimetric Assay (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Z-IETD-FMK and Z-LEHD-FMK were purchased from Calbiochem (La Jolla, CA).

Transient Transfections
BACE cells were transfected with 0.4 µg pElam-Luc reporter plasmid alone or in combination with 0.2 µg pmutI{kappa}B-{alpha}(S32-S36) plasmid or 0.2 µg of pcDNA-3 plasmid (luciferase assay). In the caspase-3 assay, cells were transfected with 0.4 µg pmutI{kappa}B-{alpha}(S32-S36) plasmid or with the same quantity of plasmid pcDNA-3. Transfections were performed with Fugene 6 liposomes (Roche) according to the manufacturer’s instruction. The pElam-Luc plasmid contains, upstream from the luciferase gene (17), the -730 to +52 region of the E-Selectin promoter, containing three copies of the NF-{kappa}B binding site. Plasmid pmutI{kappa}B-{alpha}(S32-S36) was kindly provided by Dr. C. Jobin (University of North Carolina, Chapel Hill, NC) (40).

Luciferase Activity
Cells were transfected as described above and treated or not for 6 h with various concentrations of 16K hPRL or endotoxin. They were washed once with cold PBS before addition of 200 µl lysis buffer (25 mM Tris; 8 mM MgCl2; 1 mM EDTA; 1% Triton; 15% glycerol; 1 mM DTT; 0.2 mM PMSF) and incubation for 20 min. Luciferase activity was measured in 100 µl supernatant with a 96-well plate reader (Wallac Victor, Perkin-Elmer Corp., Norwalk, CT).

Statistical Analysis
All values are expressed as means ± SD. All experiments were performed in triplicate at least three times. Comparisons between different treatments were assessed with Student’s t test. The statistical significance limit was set at P < 0.05.


    ACKNOWLEDGMENTS
 
We thank M. Lion for her excellent technical assistance and for 16K hPRL production.


    FOOTNOTES
 
This work was supported by grants from the FRIA (Fond pour la Recherche Industrielle et Agricole) (to S.T. and C.S.), FNRS (Fond National pour la Recherche Scientifique") (to I.S.), Télévie, les Services Fédéraux des Affaires Scientifiques, Techniques et Culturelles de Belgique (PAI 5/35), by Fortis Bank assurances, and by 4C Biotech (Seneffe, Belgium).

Abbreviations: ABAE, Adult bovine aortic endothelial; BACE, bovine adrenal cortex capillary endothelial; BBCE, bovine brain capillary endothelial; DTT, dithiothreitol; hPRL, human prolactin; I{kappa}B-{alpha}, inhibitor {kappa}B-{alpha}; NF-{kappa}B, nuclear factor- {kappa}B; PMSF, phenylmethylsulfonyl fluoride; VEGF, vascular endothelial growth factor.

Received for publication April 10, 2003. Accepted for publication May 23, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Carmeliet P 2000 Mechanisms of angiogenesis and arteriogenesis. Nat Med 6:389–395[CrossRef][Medline]
  2. Carmeliet P, Jain RK 2000 Angiogenesis in cancer and other diseases. Nature 407:249–257[CrossRef][Medline]
  3. Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper R, Gospodarowicz d, Bohlen P, Guillemin R 1985 Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci USA 82:6507–6511[Abstract]
  4. Ferrara N, Henzel WJ 1989 Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161:851–858[Medline]
  5. O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J 1997 Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277–285[Medline]
  6. O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Saga EH, Folkman J 1994 Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315–328[Medline]
  7. Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA, Bouck NP 1990 A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 87:6624–6628[Abstract]
  8. Clapp C, Martial JA, Guzman RC, Rentier-Delrue F, Weiner RI 1993 The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology 133:1292–1299[Abstract]
  9. Struman I, Bentzien F, Lee H, Mainfroid V, D’Angelo G, Goffin V, Weiner RI, Martial JA 1999 Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: an efficient mechanism for the regulation of angiogenesis. Proc Natl Acad Sci USA 96:1246–1251[Abstract/Free Full Text]
  10. Bentzien F, Struman I, Martini JF, Martial JA, Weiner R 2001 Expression of the antiangiogenic factor 16K hPRL in human HCT116 colon cancer cells inhibits tumor growth in Rag1(-/-) mice. Cancer Res 61:7356–7362[Abstract/Free Full Text]
  11. Kim J, Luo W, Chen DT, Earley K, Tunstead J, Yu-Lee LY, Lin SH 2003 Antitumor activity of the 16-kDa prolactin fragment in prostate cancer. Cancer Res 63:386–393[Abstract/Free Full Text]
  12. Clapp C, Weiner RI 1992 A specific, high affinity, saturable binding site for the 16-kilodalton fragment of prolactin on capillary endothelial cells. Endocrinology 130:1380–1386[Abstract]
  13. Lee H, Struman I, Clapp C, Martial JA, Weiner RI 1998 Inhibition of urokinase activity by the antiangiogenic factor 16K prolactin: activation of plasminogen activator inhibitor 1 expression. Endocrinology 139:3696–3703[Abstract/Free Full Text]
  14. D’Angelo G, Martini JF, Iiri T, Fantl WJ, Martial JA, Weiner RI 1999 16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells. Mol Endocrinol 13:692–704[Abstract/Free Full Text]
  15. Martini JF, Piot C, Humeau LM, Struman I, Martial JA, Weiner RI 2000 The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation. Mol Endocrinol 14:1536–1549[Abstract/Free Full Text]
  16. Karin M, Lin A 2002 NF-{kappa}B at the crossroads of life and death. Nat Immunol 3:221–227[CrossRef][Medline]
  17. Schindler U, Baichwal VR 1994 Three NF-{kappa}B binding sites in the human E-selectin gene required for maximal tumor necrosis factor {alpha}-induced expression. Mol Cell Biol 14:5820–5831[Abstract]
  18. Pierce JW, Schoenleber R, Jesmok G, Best J, Moore SA, Collins T, Gerritsen ME 1997 Novel inhibitors of cytokine-induced I{kappa}B{alpha} phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem 272:21096–21103[Abstract/Free Full Text]
  19. Pahl HL, Krauss B, Schulze-Osthoff K, Decker T, Traenckner EB, Vogt M, Myers C, Parks T, Warring P, Muhlbacher A, Czernilofsky AP, Baeuerle PA 1996 The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-{kappa}B. J Exp Med 183:1829–1840[Abstract]
  20. Macotela Y, Mendoza C, Corbacho AM, Cosio G, Eiserich JP, Zentella A, Martinez de la Escalera G, Clapp C 2002 16K prolactin induces NF-{kappa}B activation in pulmonary fibroblasts. J Endocrinol 175:R13–R18
  21. Ghosh S, Karin M 2002 Missing pieces in the NF-{kappa}B puzzle. Cell 109 (Suppl):S81–S96
  22. Tanner FC, Carr DP, Nabel GJ, Nabel EG 1997 Transfection of human endothelial cells. Cardiovasc Res 35:522–528[CrossRef][Medline]
  23. Bours V, Bentires-Alj M, Hellin AC, Viatour P, Robe P, Delhalle S, Benoit V, Merville MP 2000 Nuclear factor-{kappa}B, cancer, and apoptosis. Biochem Pharmacol 60:1085–1089[CrossRef][Medline]
  24. Ryan KM, Ernst MK, Rice NR, Vousden KH 2000 Role of NF-{kappa}B in p53-mediated programmed cell death. Nature 404:892–897[CrossRef][Medline]
  25. Uberti D, Grilli M, Memo M 2000 Contribution of NF-{kappa}B and p53 in the glutamate-induced apoptosis. Int J Dev Neurosci 18:447–454[CrossRef][Medline]
  26. Aoki M, Nata T, Morishita R, Matsushita H, Nakagami H, Yamamoto K, Yamazaki K, Nakabayashi M, Ogihara T, Kaneda Y 2001 Endothelial apoptosis induced by oxidative stress through activation of NF-{kappa}B: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension 38:48–55[Abstract/Free Full Text]
  27. Sohur US, Chen CL, Hicks DJ, Yull FE, Kerr LD 2000 Nuclear factor-{kappa}B/Rel is apoptogenic in cytokine withdrawal-induced programmed cell death. Cancer Res 60:1202–1205[Abstract/Free Full Text]
  28. Rivera-Walsh I, Waterfield M, Xiao G, Fong A, Sun SC 2001 NF-{kappa}B signaling pathway governs TRAIL gene expression and human T-cell leukemia virus-I Tax-induced T-cell death. J Biol Chem 276:40385–40388[Abstract/Free Full Text]
  29. Kasibhatla S, Genestier L, Green DR 1999 Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor {kappa}B. J Biol Chem 274:987–992[Abstract/Free Full Text]
  30. Qin ZH, Chen RW, Wang Y, Nakai M, Chuang DM, Chase TN 1999 Nuclear factor {kappa}B nuclear translocation upregulates c-Myc and p53 expression during NMDA receptor-mediated apoptosis in rat striatum. J Neurosci 19:4023–4033[Abstract/Free Full Text]
  31. Chan H, Bartos DP, Owen-Schaub LB 1999 Activation-dependent transcriptional regulation of the human Fas promoter requires NF-{kappa}B p50–p65 recruitment. Mol Cell Biol 19:2098–2108[Abstract/Free Full Text]
  32. Volpert OV, Zaichuk T, Zhou W, Reiher F, Ferguson TA, Stuart PM, Amin M, Bouck NP 2002 Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor. Nat Med 8:349–357[CrossRef][Medline]
  33. Scaffidi C, Fulda S, Srinivasan A, Friesen CI, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME 1998 Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17:1675–1687[Abstract/Free Full Text]
  34. Huang S, Robinson JB, Deguzman A, Bucana CD, Fidler IJ 2000 Blockade of nuclear factor-{kappa}B signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res 60:5334–5339[Abstract/Free Full Text]
  35. Shono T, Ono M, Izumi H, Jimi SI, Matsushima K, Okamoto T, Kohno K, Kuwano M 1996 Involvement of the transcription factor NF-{kappa}B in tubular morphogenesis of human microvascular endothelial cells by oxidative stress. Mol Cell Biol 16:4231–4239[Abstract]
  36. Baldwin AS 2001 Control of oncogenesis and cancer therapy resistance by the transcription factor NF-{kappa}B. J Clin Invest 107:241–246[Free Full Text]
  37. Adams J 2002 Proteasome inhibition: a novel approach to cancer therapy. Trends Mol Med 8:S49–S54
  38. Paris N, Rentier-Delrue F, Defontaine A, Goffin V, Lebrun JJ, Mercier L, Martial JA 1990 Bacterial production and purification of recombinant human prolactin. Biotechnol Appl Biochem 12:436–449[Medline]
  39. Ballard DW, Dixon EP, Peffer NJ, Bogerd H, Doerre S, Stein B, Greene WC 1992 The 65-kDa subunit of human NF-{kappa} B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc Natl Acad Sci USA 89:1875–1879[Abstract]
  40. Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U 1995 Control of I {kappa}B-{alpha} proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485–1488[Medline]