Background
Many growth factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in association with their receptor tyrosine kinase (RTK) receptors, play a crucial role in angiogenesis in normal and pathological settings [
1]. Essential to most RTK-mediated signaling is the activation of the extracellular-signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade. This cascade is precisely controlled by the activity of various regulatory proteins [
2], including members of the Sprouty (SPRY) protein family.
SPRY was originally described as an antagonist of Breathless FGF receptor signaling during tracheal branching in Drosophila [
3]. Four mammalian homologs (
SPRY1-4) have been described and are widely expressed in embryonic and adult tissues, except for
SPRY3 whose expression is believed to be restricted to the brain and testes in adults [
4]. All SPRY proteins share a highly conserved, cysteine-rich C-terminal domain and a more variable N-terminal domain. They are subject to tight control at multiple levels: differential localization, post-translational modification, and regulation of protein levels. SPRY specifically inhibits RTK-mediated Ras-Erk/MAPK signaling. At which stage SPRY blocks ERK/MAPK activation remains controversial, and evidence to date suggests the existence of multiple mechanisms that depend on the cell context and/or the identity of the RTK [
5‐
7]. Due to their inhibitory activity on the ERK/MAPK pathway, SPRY generally acts as a tumor suppressor. Recently, the anti-tumor potential of SPRY4 was shown to be associated with its ability to inhibit angiogenesis [
8]. Moreover, the angiostatic activity of both SPRY2 and SPRY4 has also been demonstrated
in vivo in a mouse model of ischemia [
9].
Our laboratory and others have identified 16 K prolactin (16 K hPRL), the 16-kDa N-terminal fragment of human prolactin, and its derived peptides as very potent angiostatic compounds both
in vitro and
in vivo [
10,
11]. 16 K hPRL is able to inhibit tumor growth and metastasis in various mouse models by inhibiting neovascularization [
12‐
15]. The potential therapeutic use of 16 K hPRL has also been observed in non-cancer pathological models like retinopathy [
16]. Postpartum cardiomyopathy, a disease characterized by acute heart failure in women in the late stage of pregnancy up to several months postpartum, has been shown to be a consequence of an excessive production of 16 K hPRL [
17]. To date, the mechanisms by which 16 K hPRL inhibits angiogenesis have only been partially elucidated. In bovine endothelial cells, the angiostatic activity of 16 K hPRL appears to be mediated by a saturable high-affinity binding site distinct from the PRL receptor [
18]. 16 K hPRL triggers endothelial cell apoptosis by activation of nuclear factor κB (NF-κB) [
19,
20]. In addition, 16 K hPRL induces endothelial cell cycle arrest in G
0-G
1 and G
2-M [
21], in parallel with inhibition of bFGF and VEGF stimulated MAPK activation [
22]. More recently, we identified an important link between 16 K hPRL and the immune system using a transcriptomic analysis performed on 16 K hPRL-treated endothelial cells. 16 K hPRL induces leukocyte adhesion to endothelial cells by activating NF-κB [
23].
Interestingly,
SPRY1 was amongst the targets of 16 K hPRL found in the aforementioned transcriptomic study. SPRY1 has been implicated in the inhibition of bFGF and VEGF-induced proliferation and differentiation
in vitro [
24], however the physiological role of SPRY1 in angiogenesis still remains to be elucidated. Here, after confirming upregulation of
SPRY1 expression by 16 K hPRL both
in vitro (in primary endothelial cells) and
in vivo (in a mouse xenograft tumor model), we performed
SPRY1-knockdown experiments to test the possible involvement of
SPRY1 in regulating angiogenesis. Indeed,
SPRY1 emerges as a novel endogenous angiogenesis inhibitor with potential applicability in the clinic.
Discussion
Since the emergence of angiogenesis as a crucial step in tumor growth and metastasis, great efforts have been made to discover new angiogenesis regulators. In order to identify new genes that control angiogenesis, we previously performed a transcriptomic analysis on endothelial cells after treatment with the potent angiogenesis inhibitor 16 K hPRL [
23]. In the list of 16 K hPRL upregulated genes we found
SPRY1, earlier described as a regulator of branching during trachea development in Drosophila [
3]. As angiogenesis is morphologically somewhat similar to branching of the Drosophila tracheal system,
SPRY1 appeared to be a good candidate. In addition, SPRY1 is a strong inhibitor of growth-factor-induced MAPK signaling required for angiogenesis [
27,
32] and SPRY1 was demonstrated to block endothelial cell proliferation and differentiation by inhibition of ERK/MAPK signaling induced by bFGF and VEGF [
24]. Moreover, SPRY2 and SPRY4 , two other SPRY-family members, are reported to play a role in angiogenesis [
8,
9,
33]. Based on these data, we hypothesized that SPRY1 might be an endogenous angiogenesis inhibitor and we therefore decided to study its properties in several angiogenesis models, including tumor-induced angiogenesis in mice.
The results of the present study corroborate our hypothesis. We first confirmed
in vitro that treatment with the angiostatic agent 16 K hPRL stimulates
SPRY1 expression both on transcript- and protein-levels. We further demonstrated in our xenograft tumor model that 16 K hPRL specifically enhanced the transcript-level of
SPRY1 in the (murine) vascular compartment. These data might be very useful in future cancer treatment since
SPRY1 expression is repressed during tumor development as shown in prostatic and breast cancers [
34,
35]. Therefore, the re-expression of
SPRY1 when tumor growth is abolished might be a powerful tool to monitor tumor response to angiostatic treatment or to decide on treatment strategies.
We further show that
SPRY1 silencing activates endothelial cells to proliferate, adhere to ECM proteins like fibronectin and vitronectin, to migrate, and to form complex vascular networks in a capillary-like-tube formation assay. In addition,
SPRY1 silencing protects endothelial cells from apoptosis. All these processes are highly relevant to angiogenesis. At least some of the observed effects of
SPRY1 knockdown might be linked to the previously described role of SPRY1 as an inhibitor of the MAPK pathway [
32,
36]. Effectively, some reports have already linked MAPK/ERK to cell migration. Pintucci notably highlighted the necessity of ERK1/2 activation for bFGF-induced endothelial cell migration [
37]. In line with these data, we observed an increased ERK1/2 activation and a higher migration capacity in
SPRY1-silenced cells. Moreover, SPRY2, a family member of SPRY1, has been shown to inhibit migration of tumor cells in response to serum and several growth factors [
38]. They also demonstrated that the anti-migratory effect of SPRY2 is mediated by the inhibition of Rac1 activation in epithelial cells [
39]. According to our data, SPRY1 seems to have similar effects to SPRY2 on endothelial cell migration. However, further studies are still needed to clarify whether Rac1 inhibition is also involved in the anti-migratory action of SPRY1.
The adhesion of endothelial cells to the ECM plays a major role in cell migration. To date, the potential involvement of SPRY1 in endothelial cell adhesion to ECM proteins has never been studied. According to our results, deletion of
SPRY1 potentiates adhesion of endothelial cells to fibronectin and vitronectin. The differential adhesion to vitronectin might be related to the MAPK/ERK signaling as well. Previous reports have shown in osteoblasts that inhibition of MAPK/ERK signaling decreases adhesion of these cells on different substrates, including vitronectin [
40]. This was accompanied by a reduction of α
vβ
3 integrin expression which was shown to mediate adhesion to vitronectin. Adhesion to fibronectin has also been shown to be dependent on MAPK/ERK activation [
41].
Proteins of the Sprouty family, like SPRY2, have been demonstrated to possess anti-apoptotic properties. Edwin and coworkers notably demonstrated that silencing of
SPRY2 abolishes the anti-apoptotic action of serum in adrenal cortex adenocarcinoma cells [
42]. Moreover, SPRY2 has also been implicated in the inhibition of UV radiation-induced apoptosis in HRas-transformed human fibroblasts [
43]. Here, we reported a pro-apoptotic effect for SPRY1, suggesting differential roles for SPRY1 and SPRY2 in controlling apoptosis. However, in a few cases, SPRY2 has been attributed to pro- apoptotic capacities such as in differentiated neuronal cells [
44]. On the other hand, apoptosis can also be regulated by the MAPK pathway, as demonstrated by Gupta, who showed that VEGF protects HDMECs from apoptosis by activating MAPK/ERK signaling [
45]. The pro-apoptotic role of
SPRY1 deduced from our study may thus be due to
SPRY1-mediated inhibition of MAPK signaling.
To understand how
SPRY1 regulates cell proliferation, we examined the MAPK related factors
p21 and
cyclinD1, whose products respectively downregulate and upregulate cell cycle progression [
29,
46]. The regulation of
p21 by the ERK signaling pathway however, has been under debate. In some cases, ERK signaling induces p21 accumulation, as demonstrated in chondrocyte maturation [
47]. Other studies have highlighted the importance of ERK1/2 inhibition in inducing
p21 expression. For example, Han and coworkers reported that fibronectin induces lung cancer carcinoma cell proliferation by activation of the MAPK pathway, leading to a reduction in
p21 expression [
48]. Moreover, terbinafin-induced cell-cycle arrest through an up-regulation of p21 in HUVECs was shown to be mediated by the inhibition of ERK activation [
31]. We demonstrated here that the induction of cell proliferation by
SPRY1 silencing in endothelial cells is associated with increased
cyclinD1 and reduced
p21 transcript levels. Therefore, our results reinforce the inhibitory role of ERK1/2 in the regulation of
p21.
The results we obtained here are in line with the effects we previously showed for the potent angiostatic agent 16 K hPRL which was used to identify
SPRY1. Similar to
SPRY1 which is upregulated by 16 K hPRL, Tabruyn et al. demonstrated that 16 K hPRL induces endothelial cell-cycle arrest in association with a decrease in c
yclinD1 expression and the induction of
p21 [
21]. In addition we showed that
SPRY1 expression induced by 16 K hPRL requires NF-κB activation like the angiostatic protein 16 K hPRL. Therefore we attempted to connect the effects of 16 K hPRL on endothelial cells to SPRY1. However, 16 K hPRL still induces apoptosis and inhibits proliferation after
SPRY1 silencing (data not shown). Thus, SPRY1 does not seem to be essential for the induced apoptosis or decreased proliferation by 16 K hPRL. According to the microarray data previously obtained [
23], these results are not surprising. The transcriptomic study revealed 216 transcripts differentially expressed after 2 h of 16 K hPRL treatment. So it could be predicted that suppression of only one target gene of 16 K hPRL would not be able to completely abolish the effects of 16 K hPRL. Nevertheless, the fact that endothelial cells respond opposite to treatment with
SPRY1 siRNA, regarding proliferation and apoptosis, compared to 16 K hPRL treatment indicates that
SPRY1 might be involved in the effects of 16 K hPRL.
Methods
Production of recombinant protein and chemical compounds
Recombinant 16 K hPRL was produced and purified from
E. Coli as previously described [
20]. The purity of the recombinant protein exceeded 95% (as estimated by Coomassie blue staining) and the endotoxin level was found to be 0.5 pg/ng recombinant proteins, as quantified with the "Rapid Endo Test" from the European Endotoxin Testing Service (Lonza, Verviers, Belgium). BAY 1170-82 was purchased from Calbiochem (La Jolla, CA).
Cell cultures
ABAE (Adult Bovine Aortic Endothelial) cells were isolated as previously described [
49]. The cells were grown in low-glucose DMEM containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. Recombinant bFGF (Promega) was added (1 ng/ml) to the culture every other day. Confluent cells corresponding to passages 8 to 13 were used in the experiment. HMVEC (Human Microvascular Endothelial cell) cultures were maintained in EBM2 medium (Lonza, Walkersville, Walkersville, USA) containing 0.1% hEGF, 0.04% hydrocortisone (kit EGM-2 SingleQuots, Cambrex Bio Science Walkersville, Walkersville, USA), 10% FBS, and 100 U/ml penicillin/streptomycin. HCT116 cells (human colorectal carcinoma cells) were grown in McCoy's 5a medium containing 10% FBS and 100 U/ml penicillin/streptomycin. HEK 293 (Human Embryonic Kidney) cells and adenovirus-E1-transformed HEK 293 cells (BD Biosciences, San Diego, CA) were grown in DMEM supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, 100 U/ml penicillin/streptomycin, and 2.5 μg/ml fugisone.
Adenovirus vectors
16 K-Ad is a defective recombinant E1-E3-deleted adenovirus vector encoding a secreted peptide consisting of the first 139 amino acids of PRL. This adenovirus vector was constructed as described in [
16] with the help of the Adeno-X expression system (BD Biosciences, Erembodegem, Belgium). Briefly, the 16 K hPRL complementary DNA was cloned into a pShuttle vector in an expression cassette, which was then inserted into the Adeno-X viral DNA. Recombinant adenoviruses were constructed and amplified in HEK 293 cells. The BD Adeno-X Virus Purification kit (BD Biosciences, Erembodegem, Belgium) and the Adeno-X Rapid Titer Kit (BD Biosciences, Erembodegem, Belgium) were used to perform purification and titration, respectively, of the recombinant adenoviruses (according to the manufacturer's instructions). Null-Ad is a control adenovirus carrying an empty expression cassette.
Mice
Adult female NMRI nude mice (6-8 weeks of age) purchased from Janvier Breeding (Elevage Janvier, France) were used for tumor growth experiments. The animal experiment protocol used was approved by the Institutional Ethics Committee of the University of Liege.
Mouse xenograft tumor model
Subconfluent HCT116 cells were trypsinized, washed, and resuspended in PBS. Cell suspension (3.10
6 cells in 50 μl PBS) was injected
s.c. into the right flank of NMRI nude mice 2 weeks before the first adenovirus administration. Sixteen mice were used and randomly divided into two groups of 8 mice. Mice received four intratumoral injections of 5.10
8 pfu 16 K-Ad or Null-Ad (control) starting when the HCT116 tumors reached 150 mm
3. These injections were repeated every 2 days. Ten days after the first adenoviral vector injection, the mice were euthanized and their tumors harvested. Tumor growth was assessed by measuring the length and width of each tumor every 2 or 3 days and calculating tumor volume by means of the formula: length × width
2 × 0.5 [
50].
SiRNA Transfections
Small interfering RNA (siRNA) duplexes were obtained from Integrated DNA Technologies (Integrated DNA Technologies, Coralville, USA), two targeting SPRY1 and one negative control. Cells were transfected by the CaPO4 method. Briefly, 90,000 ABAE cells were seeded into a 6-well plate and allowed to adhere overnight. One hour before transfection, the medium was replaced with fresh medium without antibiotics. SiRNA-CaCl2 complexes were made by first combining siRNA with 10 μl of 2.5 M CaCl2. One hundred microliters of HSBP (280 mM NaCl, 1.9 mM Na2HPO4; 12 mM glucose, 10 mM KCl; 50 mM Hepes, pH 7.05) were added and the mix was incubated for one minute at room temperature. Next the mix was added dropwise to the cells followed by an incubation period of 16 h. Cells were then collected and seeded for further tests.
Quantitative real time PCR (qRT-PCR) analysis
Total RNA was extracted with the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Synthesis of cDNA was performed from 1 μg total RNA, which was reverse transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Clinical Laboratories, Indianapolis, IN) according to the manufacturer's instructions. The resulting cDNA was used for quantitative real-time PCR with the one-step 2× Mastermix (Diagenode, Liège, Belgium) containing SYBR green. Thermal cycling was performed on an Applied Biosystem 7000 detection system (Applied Biosystems, Foster City, CA). No-template controls were run for all reactions, and random RNA preparations were also subjected to sham reverse transcription to check for the absence of genomic DNA amplification. The relative transcript level of each gene was obtained by the 2
-ΔΔCt method [
51] and normalized with respect to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (
in vitro assays) or cyclophilin A (PPIA) (mouse assays). Primers were designed with the Primer Express software and selected so as to span exon-exon junctions to avoid detection of genomic DNA (see Additional file
3 - List of primers used in quantitative RT-PCR). In order to verify species specificity of the PCR, PCR combining human or mouse cDNAs with human or mouse primers have been performed on cloned cDNAs for PPIA or Sprouty obtained form the German Resource Center for Genome Research (RZPD, IMAGENES, Germany). For analysis by end-point PCR, the final products of the qRT-PCR obtained after 40 cycles of PCR was loaded on agarose gel for electrophoresis.
Cells were washed twice with cold PBS and scraped into lysis buffer [25 mM HEPES (pH 7.9), 150 mM NaCl, 0.5% Triton, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride] on ice. Insoluble cell debris was removed by centrifugation at 10000 × g for 15 min. Aliquots of protein-containing supernatant were stored at -80°C. Protein concentrations were determined by the Bradford method, with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA).
Western blot analysis
Soluble cell lysate (30 μg) was resolved by SDS-PAGE (12%) and transferred to a polyvinylidene fluoride membrane (Milipore Corp., Bedford, MA). Blots were blocked overnight with 8% milk in Tris-buffered saline with 0.1% Tween 20 and probed for 1 h (or 2 h at 37°C for anti-SPRY1 antibody) with primary antibodies: anti-Prolactin A602 (home-made), anti-SPRY1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 Map Kinase (Thr202/Tyr204) antibody (Cell Signaling Technology, Beverly, MA), anti-MAP Kinase 1/2 (Millipore, Billerica, MA), polyclonal rabbit anti-beta-tubulin (Abcam plc, Cambridge CB4 OFW, UK). After three washes with Tris-buffered saline containing 0.1% Tween 20, antigen-antibody complexes were detected with peroxidase-conjugated secondary antibody and an enhanced fluoro-chemiluminescent system (ECL-plus; Amersham Biosciences, Arlington Heights, IL).
Immunostaining
ABAE cells were fixed with paraformaldehyde 1% for 30 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. The samples were blocked with 0.2% bovine serum albumin in PBS for 30 min and incubated with rabbit anti-SPRY1 over night at 4°C. This was followed by incubation with a goat anti-rabbit-Cy3 for 30 min. Fluorescence was analyzed with an Olympus fluorescence microscope and a camera linked to the Analysis software (Soft Imaging System GmbH, Münster, Germany).
Caspase-3 activity assay
Control and SPRY1-siRNA-transfected cells were plated in 24-well culture plates at a density of 20,000 cells per well in 500 μl of 10% FCS/DMEM. Caspase-3 activity was measured 48 h post-transfection with the CaspACE Assay System Fluorimetric (Promega Corp., Madison, WI) according to the manufacturer's instructions.
Analysis of cell proliferation
Transfected cells were plated in 96-well culture plates at a density of 5,000 cells per well in 10% FCS/DMEM and allowed to adhere for 6 h. Following this, complete medium was replaced with DMEM free for 24 h. The transfected cells were then incubated in 10% FBS/DMEM or DMEM containing 10 ng/ml bFGF and proliferation was analyzed 24 h later by measuring BrdU incorporation by means of the Cell Proliferation ELISA, BrdU (Colorimetric) (Roche, Clinical Laboratories, Indianapolis, IN)
The ability of SPRY1-siRNA-transfected ABAE cells to form capillary networks was evaluated in a Matrigel™angiogenesis assay. Briefly, 80,000 cells were plated in 24-well plates coated beforehand with 300 μl Matrigel. Control-siRNA- and SPRY1-siRNA-transfected cells were seeded into 200 μl of DMEM or 10% FBS/DMEM for 16 h. In order to visualize vessels under a fluorescence microscope, the cells were incubated with calcein-AM (2 μM) for 20 min. Quantitative analysis of network structures was performed by measuring the number of connections between vessels in the network. Photographs were taken with an Olympus fluorescence microscope and a camera linked to the Analysis software (Soft Imaging System GmbH, Münster, Germany)
Migration assay
Eight-micrometer 24-well Boyden chambers (Transwell; Costar Corp, Cambridge, MA) were used for cell migration assays. Both sides of the membrane were coated overnight with 0.005% gelatin. The lower chamber was filled with 600 μl DMEM containing 1% BSA and 10 ng/ml bFGF. ABAE cells transfected with siRNA duplexes, as described above, were placed in 300 μl of 0.1% BSA/DMEM in the upper chamber and allowed to migrate for 16 h at 37°C. After fixation, cells stained with 4% Giemsa were counted on the lower side of the membrane. Cell counting was performed with an ImageJ
http://rsbweb.nih.gov/ij/ macro relying on color thresholding in the RGB color space, followed by connected component labeling with the "Analyze Particles" function with size and circularity criteria. The same set of parameters was used for the experiments, and detection masks were produced and double-checked by visual examination.
Adhesion assay
Cell adhesion experiments were performed in 96-well plates coated with either vitronectin or fibronectin. Wells were coated with 50 μl vitronectin (10 μg/ml) or fibronectin (10 μg/ml) for 1 h, and then washed twice with PBS. Briefly, 50,000 siRNA-transfected cells were plated on the coated 96-well plates and allowed to adhere for 1 h. The wells were then washed twice with medium to remove non-adherent cells. The cells were fixed and stained with 0.01% crystal violet in methanol, then the wells were washed extensively with water and the dye was solubilized in methanol. Quantification was performed by reading the optical density at 550 nm with a microplate reader (Wallac Victor2; Perkin Elmer, Norwalk, Finland).
Luciferase reporter Assays
NF-κB luciferase reporter assays were performed as previously described [
20]. Luciferase activity was normalized using the β-galactosidase activity with the β-gal Reporter Gene Assay Kit (Roche).
Quantification and statistical analysis
Quantification of Western blots was performed using ImageJ software
http://rsbweb.nih.gov/ij/. All data are expressed as means ± SD unless stated differently. Analyses for statistical significance (the Mann-Whitney test) were carried out with Prism 4.0 software (GraphPad Software, San Diego, CA, USA). Statistical significance was set at
P < 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CS participated in experimental design, performed in vitro studies and statistical analysis, interpreted the data and wrote the manuscript. AC carried out the experimental design for the animal study and performed the analysis of tumor growth. LM performed quantitative RT-PCR on tumor extracts, undertook analysis of primer specificity and participated in data interpretation. ST participated in the design of the study and in revision of the manuscript. IS and JM conceived the study, and participated in experimental coordination and in manuscript revision. KC revised the manuscript. All the authors read and approved the final manuscript.