The Syndecan-4/Protein Kinase Cα Pathway Mediates Prostaglandin E2-induced Extracellular Regulated Kinase (ERK) Activation in Endothelial Cells and Angiogenesis in Vivo*

Background: Prostaglandin E2 (PGE2) induces tumor growth and angiogenesis. Results: PGE2-induced ERK activation in endothelial cells and angiogenesis are driven by syndecan-4-dependent PKCα activation. Conclusion: The syndecan-4/PKCα/ERK pathway is important for PGE2-induced angiogenesis in vitro and in vivo. Significance: Sdc4/PKCα activation is a novel finding in PGE2 signaling and may represent a pharmacological target. Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4−/− mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4−/− endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα−/− mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.

Prostaglandins (PGs) 2 and thromboxane A 2 (TXA 2 ), termed together prostanoids, are a class of lipid mediators generated by cyclooxygenase enzymes and terminal synthase enzymes starting from arachidonic acid. Among prostanoids, prostaglandin E 2 (PGE 2 ) production has been linked with acute inflammatory symptoms (redness, swelling, fever, and pain). However a large body of evidence indicates that uncontrolled PGE 2 synthesis plays a major role in sustaining chronic inflammation (1) and promoting angiogenesis and tumor growth (2). PGE 2 exerts its effects via four related G-protein coupled receptors, termed E-prostanoid receptors (EP1-4). EP receptors couple to a striking variety of signaling pathways. EP2 and EP4 couple to G s , which increases intracellular cAMP and activates protein kinase A. EP1 activates G q , which initiates the Phospholipase C/IP3 pathway. EP3 can induce G i activation, which leads to inhibition of cAMP synthesis. Furthermore, the complexity of PGE 2 -induced signaling is increased by the existence of cross-talk between EPs and tyrosine kinase receptors such FGFR1 (3) or EGFR (4). Among PGE 2 receptors, EP4 is particularly important in the vasculature. A specific endothelial deletion of EP4 decreases reperfusion after ischemia and increases infarct size in the brain (5). EP4 has also been identified as the major EP receptor mediating effects of PGE 2 -induced tumor angiogenesis and lymphangiogenesis (6).
In this study, we investigated the role of syndecan-4 (Sdc4), a transmembrane proteoglycan that belongs to the four-member syndecan family (Sdc1-4) in PGE 2 signaling. Sdc4 can act as a FGFR1 coreceptor and modulate its signaling (7), or it can also signal in an independent manner (8). Sdc4 signaling can lead to either activation of ERK and cell proliferation (8,9) or of Rac1, leading to cell migration (10). Both are dependent on activation of PKC␣, an event that requires dephosphorylation of a Ser-183 site in its cytoplasmic domain (11).
Sdc4 null mice are viable but show a number of defects in inflammatory settings, such as delayed wound healing (12), decreased myocardial infarction recovery (13), and impaired response to LPS injection (14).
We find that in Sdc4 Ϫ/Ϫ mice and Sdc4 Ϫ/Ϫ endothelial cells, PGE 2 -induced ERK activation is decreased. We further show that PGE 2 induces ERK activation by activating PKC␣ in an Sdc4-dependent manner and that activation of Sdc4-PKC␣-ERK is involved in modulation of PGE 2 -dependent proangiogenic effects (i.e. migration and endothelial cord formation). In agreement with these results, Sdc4 Ϫ/Ϫ and PKC␣ Ϫ/Ϫ mice show a reduction in PGE 2 -induced angiogenesis in vivo. Thus, Sdc4 plays a key role in mediation of PGE 2 angiogenic signaling.
Primary mouse EC were isolated as described previously (15). Briefly, organs (heart, lung, vein, and artery) of both wild-type and knockout mice were harvested, finely minced with scissors, and digested with 25 ml collagenase (2 mg/ml) at 37°C for 45 min under gentle agitation. The crude preparation was triturated, passing it 12 times through a cannula needle, and then filtered on a 70-M sterile cell strainer. The filtered preparation was spun at 400 ϫ g, and the pellet was resuspended in 2 ml of 0.1% BSA. For EC selection, magnetic beads (Invitrogen) coated with anti-mouse CD31 (BD Biosciences) were added to the cell suspension and incubated with rotation at room temperature for 15 min. The bead-bound cells were recovered with a magnetic separator and washed with DMEM containing 20% FBS. Cells were suspended in 10 ml of complete DMEM and seeded on 10-cm plates. Lenti-X TM HEK 293T (Clontech) and HEK 293A (Invitrogen) cells were cultured in 10% FBS DMEM (Invitrogen) containing penicillin/streptomycin at the same concentration as indicated above.
Virus Generation-Lentivirus for stable shRNA integration into the host genome were generated as described previously (16). Briefly, packaging plasmids (Addgene) were mixed with shRNA plasmid (Mission shRNA, Sigma-Aldrich) in Optimem medium (Invitrogen) and Lipofectamine 2000 (Invitrogen) with the following ratios: 5 g pMDL/pRRE, 2.5 g pRSV-Rev, 2.5 g pVCMV-VSG, and 10 g shRNA. The mixture was transferred to 90% confluent 293T cells in 10-cm dishes for 6 h. The medium was replaced with regular DMEM 10% FBS and collected after 48 h. Medium containing virus was filtered through a 0.45-M filter and used immediately for HUVEC transduction. The target sequence for human Sdc4 was 5Ј-CCGGGCCAGGTTCTTCTTGAGCTTTCTCGAG-AAAGCTCAAGAAGAACCTGGCTTTTTG-3Ј.
Site-directed mutagenesis of Sdc4 was conducted using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the instructions of the manufacturer. PCR reactions were assembled and performed under the following conditions: annealing, 60°C, 50 s; extension, 68°C, 7 min; and denaturation, 95°C 50 s, 18 cycles. Then the DNA resulting from the PCR was digested to remove the template DNA, and the remaining plasmids were transformed into One Shot Top 10 Escherichia coli (Invitrogen). The mutated clones were selected and confirmed by sequencing. Adenoviruses expressing a rat full-length or mutated rat Sdc4 sequence were then generated as reported previously (16). Briefly, HA-tagged full-length or mutated Sdc4 coding sequences were subcloned into pENTR/D (Invitrogen) and then transferred into the adenovirus vector pAD/CMV/V5-DEST (Invitrogen). The adenovirus was generated by transfection of this plasmid into HEK 293A (Invitrogen) according to the instructions of the manufacturer.
Stable Knockdown and Silencing Experiments-To achieve a stable knockdown, HUVEC P2 were seeded on 10-cm plates and transduced at 70% confluence with freshly produced lentivirus carrying a Scrambled or Sdc4 shRNA sequence and expressing the puromycin-resistant gene. Cells were kept with virus-rich medium for 6 h, and then the medium was replaced with complete M199 medium (same ingredients reported in cell cultures paragraph). Forty-eight hours post-infection, puromycin (0.8 g/ml) was added to cells, and selection was allowed for 3 days. Cells were used in the experiment or split for propagation. Selected cells were maintained in complete M199 medium with puromycin (0.4 g/ml) and used for a maximum of two more passages after initial selection.
For PKC␣ silencing, HUVEC were seeded on 6-well plates and transfected at 70% confluence. PKC␣ or Scrambled siRNA (Origene) were resuspended in the provided buffer, and transfection was done using Lipofectamine RNAiMAX (Invitrogen) according to the instructions of the manufacturer. Cells were used for experiments 72 h post-transfection.
Western Blot Analysis-HUVEC or primary mouse EC were seeded onto 6-cm plates. Confluent cells were starved overnight (HUVEC) or 48 h (mouse EC) in 0.5% FBS and then stimulated with the indicated agent. For inhibition experiments, the PI3K inhibitor LY290042 (50 M) and EP4 antagonist AH23848 (10 M) (17) were preincubated for 30 min prior PGE 2 treatment. Rescue experiments were carried out by infecting HUVEC with adenovirus (multiplicity of infection ϭ 10) for 6 h and then starved for 18 h in 0.5% FBS. For cell stimulation, the PGE 2 concentration (100 nM) was the same except were indicated. Following stimulation, cells were rapidly washed twice with ice-cold PBS and lysed with 200 l of 0.1% TritonX-100 lysis buffer (Cell Signaling Technology, Inc.) containing protease inhibitor (Roche) and phosphatase inhibitor (Roche) mixtures. Total lysates were cleared with a 15,000 ϫ g spin, and protein concentration was determined using the BCA method (Thermo-Scientific). The protein concentration of each lysate was adjusted accordingly, added to 1ϫ reducing loading buffer, and boiled for 5 min. Samples were loaded on 4 -15% gels for SDS-PAGE separation and then transferred to an Immobilon-P membrane (Millipore). Membranes were blocked 1 h with 5% fat dry milk in Tris-buffered saline containing 0.05% Tween20 (TBS-T) and then incubated overnight at 4°C with primary antibody. Protein bands were visualized using HRP-conjugated secondary antibodies associated to enhanced chemiluminescence (Immobilon TM Western, Millipore).
Densitometric Quantification-The signal from the chemiluminescence reaction was recorded in a digital acquisition system (G-Box by Syngene) equipped with a 1.4-megapixel charge-coupled device (CCD) camera with a "true" 1.4-megapixel resolution. The linear range is automatically calculated by the software and is displayed as a histogram with each acquired image. Multiple images of the same blot were acquired with incremental 1-min exposure. Images without band saturation were used for densitometric quantification. The total intensity of each band was determined with ImageJ software (18) as described, following published guidelines for background correction (19). For determination of phosphorylation levels, controls were always repeated in each experiment and loaded sideby-side with treated samples in the same gel. This allows each experiment to develop all sample signals in the same acquired image. Samples were probed with an antibody that recognizes the phosphorylated form (e.g. pERK) and with another one that recognizes both phosphorylated and non-phosphorylated form (e.g. tERK). After quantification (see above), the band intensity of the phosphorylated protein was normalized to the intensity of total protein in the same sample. These normalized values were used for calculation of the phosphorylation fold change in treated versus control samples. Fold change values were collected from replicated independent experiments (n ϭ at least 3) and used for statistical analysis.
Scratch Assay-Cells were seeded on 6-well plates, allowed to reach confluence, and then starved overnight in 0.5% FBS. Each well was marked below the plate surface by drawing a vertical line. This allowed identification of the same scratched area to take consistent pictures. After overnight starving, five different scratches intercepting the marked line were done in each well using a 200-l sterile tip. Picture of scratches were taken just before stimulation (time 0) and after 8 h (time 8). Except where indicated, the same concentration of PGE 2 (100 nM) was used for the scratch assay. Migration was calculated using the scratch area at time 0 (A t0 ) and the correspondent scratch area at time 8 (A t8 ). The scratch area was measured using ImageJ software. Migration was expressed as the percentage of scratch closure after 8 h compared with the initial area according to the following formula: % closure ϭ [(A t0 -A t8 )/A t0 ]ϫ100. Migration in each well is defined by the mean closure of five different scratches.
In Vitro EC Cord Formation-Cells were starved overnight in 0.5% FBS, detached with trypsin, and seeded again in 12-well plates coated previously with 300 l of reduced-growth factor Matrigel (BD Biosciences). 80,000 cells/well were seeded in 0.5% FBS with or without PGE 2 (100 nM) or VEGF-A as a positive control. 7 h after cell seeding, five random pictures (ϫ40 magnification) were taken in each well, and the total length of cord formation was quantified in each field. The mean of five total lengths/well represents an independent experimental point. Cord length in each random field was assessed using the NeuronJ plug-in (20) for ImageJ.
In Vivo Angiogenesis-In vivo angiogenesis was evaluated by Matrigel plug assay as reported previously (21) with modifications. Briefly, 8-week-old male mice were injected subcutaneously with reduced-growth factor Matrigel (BD Biosciences) in the flank area. Matrigel plugs containing either vehicle, PGE 2 (10 M), or VEGF-A (100 ng/ml ϩ 5 units of heparin) were removed 7 days after injection, embedded in Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek), and allowed to solidify on a dry ice bed. Embedded plugs were cryosectioned (10-m thickness) and processed by fixation and staining. Sections were stained with CD31 primary antibody (BD Biosciences) followed by fluorescence-conjugated secondary antibody. Four random images from each section were acquired and used for quantification of angiogenesis (see Fig. 5 for details).
Statistics-All statistical analyses were done using GraphPad Prism (GraphPad). Student's t test comparison was used in two-

Syndecan-4 Mediates PGE 2 Signaling in EC
group data sets. One-way analysis of variance followed by Bonferroni or Dunnet post-tests was used in more than two-group data sets.

Sdc4
Regulates PGE 2 -induced ERK Activation-In agreement with previous studies that have shown that PGE 2 induces ERK activation in EC, HUVEC displayed ERK activation as early as 2 min after PGE 2 administration (Fig. 1A). Although in nonendothelial cell types PGE 2 -induced ERK activation has been shown to involve a PI3K-dependent mechanism (22,23), in agreement with previous publications (24) we could not detect PGE 2 -induced AKT activation in HUVEC (Fig. 1A). Furthermore, PGE 2 -induced ERK activation could not be blocked with the PI3K inhibitor LY292000 (50 M) (Fig. 1B).
Because PGE 2 -induced ERK activation in EC has been shown to require FGFR1 transactivation (3), we next examined whether Sdc4, which can interact with and modulate FGFR signaling, plays a role in PGE 2 -induced ERK activation. To investigate this possibility, primary EC were isolated from WT or Sdc4 Ϫ/Ϫ mice and treated with PGE 2. Western blot analysis of vein EC stimulated with PGE 2 demonstrated prompt activation in EC isolated from WT but not Sdc4 Ϫ/Ϫ mice (Fig. 1C). Quantitative analysis of this blot as well as of blots of EC from hearts and lungs of wild-type and Sdc4 Ϫ/Ϫ mice showed a 1.5to 2-fold increase in ERK activation after 5-min stimulation with PGE 2 in wild-type but not knockout EC (Fig. 1D).
EP4 is thought to be the principal prostanoid receptor responsible for PGE 2 -induced ERK activation (24). In agreement with this, EP4 was the highest-expressed receptor in HUVEC (Fig. 2B) 2C). One potential reason for decreased PGE 2 -induced ERK activation in Sdc4 Ϫ/Ϫ EC could be reduced EP4 expression. However, both quantitative real-time PCR and Western blot analysis did not show differences in EP4 expression level between Sdc4 Ϫ/Ϫ and WT EC (Fig. 2D).
We then decided to use HUVEC to test whether human EC showed a similar effect as mouse EC with regard to the role of Sdc4 in PGE 2 signaling. To this end, we knocked down Sdc4 in HUVEC using lentivirus carrying shRNA followed by stable selection with puromycin, achieving more that 80% Sdc4 knockdown, as shown by Western blot analysis (Fig. 2E). Knockdown of Sdc4 in HUVEC (Sdc4 sh) led to a similar decrease in PGE 2 -induced ERK activation (Fig. 2, E and F), as was observed in mouse Sdc4 Ϫ/Ϫ cells. In contrast, ERK activation induced by VEGF-A was not affected by the knockdown of Sdc4 (Fig. 2, E and F).
PKC␣ Mediates PGE 2 -induced ERK Activation via Sdc4-We next addressed the mechanism involved in Sdc4-dependent activation of ERK by PGE 2 . A key signaling event attributed to Sdc4 is recruitment and activation of PKC␣ in response to ligand-induced oligomerization (25,26). To check whether PKC␣ is involved in PGE 2 -induced ERK activation, we first used mouse EC isolated from PKC␣ Ϫ/Ϫ mouse hearts. Compared with WT EC, PGE 2 -induced ERK activation was reduced significantly in PKC␣ Ϫ/Ϫ EC (Fig. 3, A and B). To verify this, we next knocked down PKC␣ in HUVEC (PKC␣ siRNA) and also observed a significant reduction in PGE 2 -induced ERK activation (Fig. 3, C and D). ERK activation induced by VEGF-A was not affected by the knockdown of PKC␣ in HUVEC (Fig. 3, C  and D).
Prior studies have established that dephosphorylation of Ser-183 in Sdc4 cytoplasm is required for PKC␣ activation (27,28). PGE 2 treatment induced Ser-183 dephosphorylation in HUVEC (Fig. 3E) that is similar but not as extensive as dephosphorylation induced by FGF2, a known activator of this pathway (27). To verify that Sdc4-dependent activation is indeed central to PGE 2 -induced ERK activation, we mutated Ser-183 site to augment or inhibit Sdc4-dependent PKC␣ activation. Two mutants were used: a Ser-to-Ala mutation (S183A), which would be expected to favor PKC␣ activation, and S183E, which would be expected to inhibit it. Introduction of these mutant constructs in adenoviral vectors into Sdc4 sh HUVEC demonstrated that S183A (Ser/Ala) but not S183E (Ser/Glu) mutant FIGURE 1. Sdc4 regulates PGE 2 -induced ERK activation. A, confluent HUVEC were serum-starved and then treated with PGE 2 for the indicated times to assess ERK activation (pERK) and AKT activation (pAKT). Ctr, control. B, HUVEC were preincubated with the PI3K inhibitor LY294002 (50 M) for 30 min before PGE 2 stimulation for 5 min. C, primary mouse EC were isolated from WT or Sdc4 Ϫ/Ϫ mice as described under "Experimental Procedures." Confluent endothelial cells derived from the vein were serum-starved and then treated with PGE 2 for the indicated times. Shown is one representative blot of three with similar results. D, pERK quantification in WT versus Sdc4 Ϫ/Ϫ following treatment with PGE 2 in EC isolated from different tissues (vein, lung, and heart). Each diagram is derived from three independent experiments (n ϭ 3). Bars represent mean Ϯ S.E. **, p Ͻ 0.01; ***, p Ͻ 0.001. MAY 3, 2013 • VOLUME 288 • NUMBER 18 rescued ERK activation by PGE 2 (Fig. 3, F and G). Re-expression of full-length Sdc4 protein (WT) in Sdc4 sh HUVEC fully restored PGE 2 -dependent induction of ERK activation (Fig. 3, F  and G). HA-tagged Sdc4 constructs used for rescue experiments achieved a similar protein expression after adenoviral infection (Fig. 3F). As a control for nonspecific infection effects, we used an adenovirus expressing GFP at an equal multiplicity of infection as the Sdc4 virus.

Syndecan-4 Mediates PGE 2 Signaling in EC
Sdc4 Regulates PGE 2 -induced the Angiogenic Phenotype in Vitro and in Vivo-To demonstrate the biological relevance of Sdc4-dependent regulation of PGE 2 -induced ERK activation, we examined several aspects of EC behavior in vitro and in vivo. First, the effect of PGE 2 on EC migration was examined using an in vitro scratch assay. PGE 2 induced HUVEC migration in a concentration-dependent manner (Fig. 4, A and B). Next, the same assay was used to test the migration response of EC after knockdown of Sdc4. Although HUVEC infected with a scrambled shRNA increased their migration in response to PGE 2 , no such increase was observed in Sdc4 shRNA HUVEC (Fig. 4C). Sdc4 sh HUVEC were still able to respond to a general promigratory stimulus such 20% FBS (Fig. 4C). FBS was used as a positive control since it contains many factors that induce a strong promigratory response in HUVEC (29).
We then used an in vitro two-dimensional Matrigel assay to assess the effect of PGE 2 on EC cord formation (Fig. 4D). Quantification of this assay showed that Sdc4 or PKC␣ knockdown in HUVEC inhibited their ability to form cords in response to PGE 2 but not VEGF-A (Fig. 4, F and E). We next examined whether PGE 2 -induced angiogenesis is affected in Sdc4 or PKC␣ null mice. To investigate this we employed the Matrigel plug assay that has been used previously to test in vivo proangiogenic properties of PGE 2 (21). Analysis of Matrigel plugs impregnated with PGE 2 and implanted into WT, Sdc4 Ϫ/Ϫ , and PKC␣ Ϫ/Ϫ mice after 7 days showed a significant increase in the CD31-positive area in WT mouse compared with WT control mouse, whereas no such increase was observed in either Sdc4 Ϫ/Ϫ or PKC␣ Ϫ/Ϫ mice (Fig. 5,  A and B). Of note, VEGF-induced in vivo angiogenesis was not affected in Sdc4 Ϫ/Ϫ or PKC␣ Ϫ/Ϫ mice.

DISCUSSION
The proangiogenic properties of PGE 2 have been widely investigated both in vivo and vitro (2). Administration of PGE 2 to EC in vitro results in a series of "proangiogenic" events such as up-regulation of VEGF and FGF2 (30), increased migration and spreading via ␣V␤3 integrin-dependent Rac activation (31), and up-regulation of chemokine receptor CXCR4 (32). The proangiogenic phenotype promoted by PGE 2 in EC has been associated with the activation of PGE 2 -EPs downstream signaling such cAMP/protein kinase A (33) and PI3K/AKT (34) pathways. However, given the complexity of PGE 2 signaling, other pathways are likely to be involved. PGE 2 is able to induce FGFR1 transactivation in EC via a cross-talk mechanism, promoting the proangiogenic phenotype in vitro (3).
ERK activation is a key event in the proangiogenic phenotype promoted by PGE 2 in vitro (24), but the mechanism of this

Syndecan-4 Mediates PGE 2 Signaling in EC
activation remains elusive. The results of this study show that PGE 2 induces ERK activation in a Sdc4-dependent manner that, in turn, requires Sdc4-driven activation of PKC␣. Furthermore, Sdc4 plays a critical role in the regulation of angiogenic activity of PGE 2 both in vitro and in vivo. PGE 2 -induced migration and endothelial cord formation has been shown to be ERKdependent in vitro (24). Our findings show that silencing of Sdc4 in HUVEC leads to reduced migration and cord formation in response to PGE 2 . In line with our in vitro data, we also observed impairment of the angiogenesis response in Sdc4 Ϫ/Ϫ mice, demonstrating that failing to fully respond to PGE 2 , observed in vitro, has in vivo relevance. PGE 2 dependence on the Sdc4/PKC␣ pathway is demonstrated by a number of observations. First, PGE 2 treatment of EC leads to dephosphorylation of the Ser-183 site in Sdc4 cytoplasmic tail, an event known to be required for PIP2-mediated PKC␣ binding to Sdc4 and its activation (27). Because the phosphatase responsible for this event is not known, we have not been able to determine how PGE 2 achieves this effect. Second, PGE 2 signaling is reduced similarly in Sdc4 Ϫ/Ϫ and PKC␣ Ϫ/Ϫ mouse EC in vitro. Finally, PGE 2 -dependent ERK activation can be restored in Sdc4 Ϫ/Ϫ EC by a WT Sdc4 construct but not by a construct unable to activate PKC␣.
It is noteworthy that in cancer cells (23) and EP4-transfected HEK 293 cells (22), ERK activation mediated by EP4 requires PI3K involvement, but in EC EP4-induced ERK activation has been reported to be PI3K-independent (24). This study provides a mechanism for this observation, demonstrating that Sdc4/PKC␣ can activate ERK in response to PGE 2 downstream of EP4. Furthermore, in agreement with a previous finding, we did not observe PGE 2 -induced AKT activation at early time points (2-15 min). This is also supported by the fact that ERK activation was unaffected by the PI3K inhibitor.
PKC␣ can promote ERK activation in a number of ways, including direct activation of RAF (36,37) and RAS (38). ERK has been shown to be a key signal for PGE 2 -induced angiogenesis (3,24). PKC␣ ability to stimulate angiogenesis both in vivo and in vitro has been well documented. Activation of PKC␣ but not other PKC isoforms is responsible for enhancing in vitro cord formation in HUVEC (39). This is mediated by up-regula-tion of VEGF that is evident after 6 h of PKC␣ activation (39). Interestingly, VEGF up-regulation induced by PMA (non selective PKC activator) can be abolished by a specific MEK inhibitor U0126 (40). Thus, PKC␣-induced VEGF up-regulation appears to be ERK-dependent. In agreement with this, inhibition of Sdc4/PKC␣ pathway induced by overexpression of PKC␦ leads to impaired in vitro EC cord formation (28). In vivo, PKC␣ knock-out mice have impaired neovascularization following myocardial infarction (35). PGE 2 -induced VEGF up-regulation requires ERK-dependent activation of JNK in EC (30) therefore PGE 2 /Sdc4/PKC␣ could represent the missing link in this pathway.
Sdc4 Ϫ/Ϫ mice are viable and fertile but show an impaired response to insults that evoke inflammatory response such as wound healing (12) and myocardial infarction (16). Wound healing is a tightly regulated process driven and orchestrated by a plethora of inflammatory mediators (36) including PGE 2 which among other effects can induce epithelial cell proliferation (37) and promote angiogenesis in the healing tissue (38). Our data showing that Sdc4 Ϫ/Ϫ EC are unable to achieve a full angiogenic response following PGE 2 administration can potentially explain some of the findings in Sdc4 Ϫ/Ϫ mice including delayed angiogenesis in healing tissues leading to impaired wound healing (15) and larger infarct size (16). Furthermore, because Sdc4 is ubiquitously express in many cell types other than EC, including myofibroblasts, smooth muscle cells and epithelial cells among others, this pathway might be relevant in a number of other disease processes where inflammation and inflammatory response play an important role including atherosclerosis, certain chronic infections and cancer. PGE 2 is generally considered to have a potent tumor-promoting activity associated with a wide spectrum of effects which include cancer cell proliferation (39), angiogenesis (22) and metastasis (40). In tumor microenvironment the cyclooxygenase-2/PGE 2 axis is highly up-regulated (5) and a direct role of PGE 2 in tumorigenesis has been extensively showed in animal models (see (41) for Review). Finally, recent epidemiological studies (42)(43)(44) reported a marked reduction in cancer incidence/mortality and prevention of cancer onset in patients treated with low-dose aspirin or other non-steroidal anti-inflammatory drugs. This effect is believed to be associated with nonselective cyclooxygenase enzyme inhibition and reduction in prostanoid metabolites, including PGE 2 levels. Our study unveiled a novel PGE 2 signaling pathway that may provide a therapeutic opportunity to control tumor growth.