Characterization of heparin affin regulatory peptide signaling in human endothelial cells.

Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. We have previously reported that HARP is mitogenic for different types of endothelial cells and also affects cell migration and differentiation (12). In this study we examined the signaling pathways involved in the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) induced by HARP. We report for the first time that receptor-type protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), which is a receptor for HARP in neuronal cell types, is also expressed in HUVEC. We also document that HARP signaling through RPTPbeta/zeta leads to activation of Src kinase, focal adhesion kinase, phosphatidylinositol 3-kinase, and Erk1/2. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002, and U0126 inhibit HARP-mediated signaling and HUVEC migration and tube formation. In addition, RPTPbeta/zeta suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTPbeta/zeta as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.

Heparin affin regulatory peptide (HARP), 1 also known as pleiotrophin or heparin-binding growth-associated molecule, is an 18-kDa growth factor that has a high affinity for heparin. HARP is highly conserved among species and shares 50% homology with midkine and the avian analogue of midkine retinoic-induced heparin-binding protein. The above proteins constitute a relatively new family of growth factors with high affinity for heparin (1).
HARP has been originally purified from perinatal rat brain as a molecule that induces neurite outgrowth (2). HARP is also expressed in the uterus (3), cartilage (4), and bone extracts (5). Several reports have established a strong correlation between HARP expression and tumor growth and angiogenesis (6 -8). High levels of the protein were found in many human cancers and cell lines derived from human tumors (9,10). HARP has been reported to be mitogenic for different types of endothelial cells (11) and to be angiogenic in vivo and in vitro (12).
HARP exerts its biological activity through interactions with cell surface proteoglycans, such as N-syndecan (13) or through binding to more specific cell surface receptors. Receptor-type protein-tyrosine phosphatase ␤/ (RPTP␤/) and its secreted variant phosphacan (14), as well as anaplastic lymphoma kinase (15), have been recently reported to bind HARP and to be implicated in its signaling. HARP has been shown to activate both the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)-Akt signaling axes (16,17), and inhibitors of Erk1/2 or PI3K inhibit DNA synthesis stimulated by HARP on bovine epithelial lens cells. Additionally, an analysis of tyrosine-phosphorylated proteins following HARP stimulation showed induction of Shc and Erk1/2 phosphorylation (15). Nevertheless, the signals from specific receptors to PI3K or MAPK are still not well documented, although it has been hypothesized that Src is involved in the process (18,19).
In the present work we examined the effect of HARP on the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) and investigated the signaling pathway induced by HARP during this process. We report that HARP induces migration of endothelial cells through binding to RPTP␤/ leading to activation of Src, focal adhesion kinase (FAK), PI3K, and Erk1/2.
Cell Culture-HUVEC were isolated from human umbilical cords, cultured as described previously (11), and used at passages 1-2. The cells were grown as monolayers in medium M199 supplemented with 15% fetal calf serum, 150 g/ml endothelial cell growth supplement, 5 * This work was supported by grants from the Research Committee of the University of Patras (Karatheodoris) and Empeirikio Foundation, Greece. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Migration Assay-Migration assays were performed as described previously (12) in 24-well microchemotaxis chambers (Costar, Avon, France) using uncoated polycarbonate membranes with 8-m pores. Serum-starved HUVEC were harvested and resuspended at a concentration of 10 5 cells/0.1 ml in M199 containing 0.25% bovine serum albumin (BSA). The bottom chamber was filled with 0.6 ml of M199 containing 0.25% BSA and the tested substances. The upper chamber was loaded with 10 5 cells and incubated for 4 h at 37°C. After completion of the incubation, the filters were fixed and stained with 0.33% toluidine blue solution. The cells that migrated through the filter were quantified by counting the entire area of each filter, using a grid and an Optech microscope at a 20ϫ magnification.
Matrigel Tube Formation Assay-The matrigel tube formation assay was performed as described previously (12). Briefly, Matrigel TM that was growth factor-reduced was used to coat the wells of 96-well tissue culture plates (0.04 ml/well) and was left to polymerize for 1 h at 37°C. After polymerization, 15,000 cells suspended in 0.15 ml of M199 were added to each well. HARP was added directly in the medium. After 6 h of incubation at 37°C, the medium was removed, the cells were fixed and stained, and the length of the tube network was measured in the total area of the wells, as described previously (12).
RNA Interference-RNA oligonucleotide primers were obtained from Ambion, Inc. The following sequences were used as described previously (20): RPTP␤/ sense, 5Ј-AAAUGCGAAUCCUAAAGCGUU-3Ј; RPTP␤/ antisense, 5Ј-AACGCUUUAGGAUUCGCAUUU-3Ј. The annealing of the primers was achieved according to Ambion's instructions. Cells were passaged and grown to a confluence of 40% in medium without antibiotics. The transfection of cells was performed in serum-free medium for 4 h using annealed RNA at the concentration of 50 mM and jetSI-ENDO (Polyplus-Transfection, France) as transfection reagent. Cells were incubated for another 24 h in serum-containing medium and serum-starved before further experiments. The transfection efficiency was evaluated using Silencer TM ␤-actin siRNA control (Ambion). Double-stranded negative control siRNA from Ambion was also used in migration and differentiation assays.
Immunoprecipitation Assay-Cells were grown to confluence in 145-mm dishes. The medium was aspirated, cells were washed twice with ice-cold phosphate-buffered saline and lysed with 2 ml of ice-cold radioimmune precipitation assay buffer (1ϫ phosphate-buffered saline, 1% Triton X-100, 0.1% SDS, 20 nM sodium orthovanadate, 1 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA). Cells were scraped off the plate and transferred to Eppendorf tubes. Cell lysates were kept on ice for 30 min and then centrifuged at 20,000 ϫ g for 45 min at 4°C. Approximately 400 g of the supernatant were transferred to a new Eppendorf tube and incubated with 30 l of prepared suspension of protein A-agarose beads for 60 min at room temperature. Beads were collected by centrifugation, and the supernatants were transferred to new Eppendorf tubes. After this first precleaning step, supernatants were further incubated with 500 ng of primary antibody for 60 min at room temperature. At the end of this period, 80 l of prepared suspension of protein A-agarose beads were added, and samples were incubated for 18 h at 4°C. Protein A beads and bound proteins were collected by centrifugation (1,000 ϫ g, 4°C) and washed three times with ice-cold cell lysis buffer. The pellet was resuspended with 60 l of 2ϫ SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 0.1 M dithiothreitol) and kept at 4°C until use. When ready for electrophoresis, samples were heated to 95-100°C for 5 min and centrifuged, and 50 l of the supernatant were analyzed by Western blotting.
Western Blot-Proteins were analyzed by SDS-PAGE and transferred to Immobilon P membranes. Blocking was performed by incubating the membranes with Tris-buffered saline (TBS), pH 7.4, containing 3% BSA (in the case of HARP, RPTP␤/ and phosphotyrosine) or TBS containing 5% nonfat dry milk (in all other cases) for 2 h at room temperature. Membranes were further incubated in primary antibodies for 1 h at room temperature or for 18 h at 4°C under continuous agitation, as follows: PY20 at 1:5,000 dilution in TBS containing 0.1% Tween 20 (TBS-T) and 1% BSA, anti-FAK antibody at 1:600 dilution in TBS-T containing 3% nonfat dry milk, anti-RPTP␤/ antibody at 1:1,000 dilution in TBS-T containing 1% BSA, anti-HARP antibody at 1:5,000 dilution in TBS-T containing 1% BSA, anti-np-src antibody at 1:1,000 dilution in TBS-T containing 5% BSA, anti-Src antibody at 1:5,000 dilution in TBS-T containing 3% nonfat dry milk, anti-phos-phoErk1/2 antibody at 1:1,000 dilution in TBS-T containing 5% BSA, anti-Erk1/2 antibody at 1:5,000 dilution in TBS-T containing 3% nonfat dry milk, anti-PI3K p85 antibody at 1: 1,000 dilution in TBS-T containing 3% nonfat dry milk, and anti-␤-actin antibody at 1:5,000 dilution in TBS-T containing 3% nonfat dry milk. Membranes were further washed three times in TBS-T and incubated in secondary antibodies (1:12,500 dilution in TBS-T for anti-rabbit and anti-goat antibodies and 1:7,500 dilution in TBS-T containing 3% nonfat dry milk for anti-mouse antibody) for 1 h at room temperature under continuous agitation. Membranes were washed three times with TBS-T and twice with TBS. Detection of immunoreactive bands was performed using the ChemiLucent TM detection kit (Chemicon) according to the manufacturer's instructions. Blots for np-src, pFAK, and pErk1/2 were stripped and subjected to subsequent Western blotting for c-Src, FAK, and Erk1/2, respectively. The protein levels that corresponded to the immunoreactive bands were quantified using the ImagePC image analysis software (Scion Corporation, Frederick, MD).
Statistical Analysis-The significance of variability between the results from each group and the corresponding control was determined by unpaired t test. Each experiment included triplicate wells for each condition tested, and all results are expressed as mean Ϯ S.E. from at least three independent experiments.

HARP Stimulates Migration and Differentiation of Serumstarved HUVEC-HARP
is known to affect the migration and differentiation of different types of endothelial cells (12). In the present work, we studied the effect of HARP on serum-starved HUVEC. As shown in Fig. 1, human recombinant HARP induced migration of HUVEC in a concentration-dependent manner. The maximum increase was ϳ40% over the control at the concentration of 100 ng/ml of HARP and was inhibited by sodium orthovanadate and CS-C (Fig. 1), indicating that RPTP␤/ may be the phosphatase involved in this process. The significant decrease in the migration of unstimulated HUVEC by sodium orthovanadate could be because of inhibition of phosphatases other than RPTP␤/, which may influence baseline migration independent of HARP stimulation.
HARP also induced tube-like formation by HUVEC on matrigel, and this effect was abolished when cells were treated with sodium orthovanadate or CS-C (Fig. 1). In contrast to its effect on HUVEC migration, sodium orthovanadate had no effect on the tube formation of unstimulated cells on matrigel. Matrigel contains several growth factors and other undefined components involved in cell proliferation or/and differentiation that may affect the action of sodium orthovanadate, leading to a different response of the same cells cultivated under different conditions. RPTP␤/ Is Present in HUVEC and Associates with HARP-To investigate whether RPTP␤/ is found in HUVEC and acts as a receptor for HARP, we cultured cells in 145-mm plates to confluence. Cells were lysed with radioimmune precipitation assay buffer, and 400 g of total protein were subjected to immunoprecipitation against HARP or RPTP␤/. As shown in Fig. 2, RPTP␤/ was detected in all immunoprecipitates as one band of molecular mass ϳ200 kDa, which corresponds to a transmembrane spliced variant of RPTP␤/ (20 -22). These data suggest that RPTP␤/ in HUVEC interacts with HARP.
c-Src Kinase Lies Downstream of RPTP␤/ and Links the Receptor with FAK-We investigated whether c-Src kinase is involved in the signaling pathway triggered by the interaction of HARP with RPTP␤/ in HUVEC. We used cell lysates from confluent HUVEC and performed immunoprecipitation for RPTP␤/. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted against c-Src kinase and RPTP␤/. c-Src was detected in RPTP␤/ immunoprecipitates, indicating that RPTP␤/ can interact with c-Src in presence or absence of HARP. Moreover, it seems that the interaction of c-Src with RPTP␤/ is increased in the presence of HARP (Fig. 3, lanes 1  and 2).
Because c-Src is known to bind and further activate FAK (23), we tested whether c-Src binds FAK in HUVEC. In HU-VEC lysates we performed immunoprecipitation against FAK and Western blot against c-Src kinase, np-src, and FAK. As shown in Fig. 3 (lanes 3 and 4), c-Src was detected as a band of 60 kDa, indicating the existence of a c-Src-FAK complex, which was increased in the presence of HARP. Moreover, the amounts of np-src interacting with FAK were increased in the presence of HARP (Fig. 3, lanes 3 and 4).
Interestingly, FAK or RPTP␤/ was not detected in RPTP␤/ or FAK immunoprecipitates, respectively (data not shown). Taken together, the above data indicate that c-Src is an intermediate molecule in the transduction of HARP signaling to FAK.
HARP Induces the Association of FAK with PI3K-We also investigated whether PI3K is involved in the HARP signaling  pathway in endothelial cells. We used cell lysates from confluent HUVEC and performed immunoprecipitation for RPTP␤/, c-Src kinase, and FAK. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted against PI3K. PI3K was detected only in FAK immunoprecipitates, verifying a direct association of the two molecules (Fig. 4). In the same figure, it is evident that the association between FAK and PI3K is more intense after treatment of cells with HARP ( lanes 6 and 7), which was verified by immunoblotting the same membrane against FAK.
HARP Induces Activation of c-Src Kinase by C-terminal Dephosphorylation-The formation of a c-Src-FAK complex is essential for full phosphorylation and activation of FAK (24,25). Because full c-Src kinase activation and binding to FAK requires dephosphorylation of Tyr 527 , we examined whether HARP can affect the phosphorylation status of c-Src kinase at this site. We incubated serum-starved HUVEC with 100 ng/ml of HARP for different time periods and evaluated c-Src dephosphorylation by SDS-PAGE and Western blot analysis. HARP increased c-Src dephosphorylation at Tyr 527 , leading to its activation. Activation of c-Src was observed after 5 min and in most of the cases was maintained up to 30 min after the addition of HARP. Activation of c-Src by HARP was inhibited by both sodium orthovanadate and CS-C (Fig. 5).
HARP Induces FAK Phosphorylation through Activation of c-Src-Because c-Src was found to interact with FAK, we further investigated whether c-Src activation could be implicated to increased phosphorylation of FAK. Incubation of HUVEC with 100 ng/ml of HARP led to increased tyrosine phosphorylation of FAK 15 min after the addition of HARP in the medium of HUVEC. The activation remained at maximal  (lanes 3, 5, and 7) or without HARP (lanes 2, 4, and 6) for 15 min and then lysed with radioimmune precipitation assay buffer. The cell lysates were immunoprecipitated for RPTP␤/ (lanes 2 and 3), c-Src (lanes 4 and 5), or FAK (lanes 6 and 7), and the immunoprecipitates were analyzed by Western blot for the presence of the p85 subunit of PI3K. PI3K was detected only in the FAK immunoprecipitates, suggesting the direct interaction between these two molecules. Lane 1 corresponds to positive antigen control (Upstate, number 12-303). levels up to 30 min after HARP application and was inhibited by sodium orthovanadate and CS-C or the c-Src inhibitor PP1 (Fig. 6).
HARP Induces Phosphorylation and Activation of Erk1/2 through PI3K-We further tested the effect of HARP on the activation of Erk1/2. HARP induced the activation of both Erk1 and Erk2 within 5 min after the addition of HARP in the medium of HUVEC, with the maximum effect observed after 15 min. HARP-induced Erk1/2 activation was inhibited by sodium orthovanadate and CS-C and PP1, suggesting that RPTP␤/ and c-Src may be involved in this process. It was also inhibited by two PI3K inhibitors, wortmannin and LY294002, suggesting that PI3K is involved and lies upstream of Erk1/2 in this signaling pathway of HARP. Finally, the well known inhibitor of MEK U01261 also inhibited HARP-induced Erk1/2 phosphorylation (Fig. 7). same inhibitors inhibited HARP-induced tube formation on matrigel (Fig. 8).

RPTP␤/ Knockdown by RNA Interference Interrupts HARP Signaling and HARP-induced Migration and Differentiation of
HUVEC-To further establish that RPTP␤/ is the receptor of HARP in HUVEC and that the biological activity of HARP is mediated by binding to RPTP␤/, we performed the same set of experiments in HUVEC after down-regulation of RPTP␤/ by RNA interference (20). As shown in Fig. 9, 48 h following transfection, a significant reduction of both mRNA and the protein levels of RPTP␤/ was achieved in HUVEC, which led to a significant reduction of HARP-induced c-Src activation, as well as FAK and Erk1/2 phosphorylation (Fig. 10).
Finally, we examined whether RPTP␤/ knockdown inhibits HARP-induced HUVEC migration and differentiation. As shown in Fig. 11, the biological effect of HARP was abolished when RPTP␤/ expression was down-regulated. It is noteworthy that RPTP␤/ knockdown results in the reduction of unstimulated cell tube formation, which is in contrast to the lack of effect of sodium orthovanadate on unstimulated cells (Fig. 1). Methodological differences or the nonspecific actions of sodium orthovanadate could be responsible for this discrepancy. DISCUSSION HARP is an 18-kDa heparin-binding growth factor that is implicated in cell growth and differentiation and has a potent role in angiogenesis and tumor growth (1). In the present work, we studied the signaling pathway that is activated by HARP in HUVEC and leads to increased cell migration.
Our data suggest that RPTP␤/ expressed by HUVEC is involved in HARP-induced HUVEC migration and tube formation on matrigel. This is supported by the co-immunoprecipitation of the two molecules, as well as the inhibition of HARP activity by sodium orthovanadate or CS-C. These results are in line with previous reports that linked RPTP␤/ to cell migration induced by HARP and midkine in neuronal cells (18,20,26), where RPTP␤/ is considered to be a HARP receptor (19,  22). This is the first time however that RPTP␤/ is reported to be a HARP receptor in non-neuronal cells and to be expressed by human endothelial cells. It is also the first time that a signaling pathway of RPTP␤/ is being characterized.
c-Src kinase is co-immunoprecipitated with RPTP␤/ in HU-VEC lysates. This is the first time that a direct association of these two molecules is shown, although a potential role of c-Src kinase as a downstream molecule of RPTP␤/ signaling has been previously implied as a potential event in the signal cascade induced by midkine (18). Activation of c-Src was inhibited by sodium orthovanadate or CS-C but not by genistein (data not shown), which suggests that binding of HARP to RPTP␤/ leads to the induction of phosphatase activity and dephosphorylation of c-Src kinase at Tyr 527 (Fig. 5B), which leads to its activation (27). The inhibition of HARP signaling by sodium orthovanadate has been reported previously (16,17), indicating a role of phosphatases in this process without, however, clarifying the phosphatase(s) involved. The lack of effect of sodium orthovanadate on the ratio of activated to inactive c-Src in unstimulated cells presented in Fig. 5 is consistent with its inhibition of phosphatases other than RPTP␤/, which may contribute to normal basal migration of HUVEC.
It is well known that HARP binding to RPTP␤/ is mediated by both the protein core and the chondroitin sulfate chains of the receptor, and this interaction is inhibited by exogenously supplied chondroitin sulfates (26). The fact that in the present study HARP binding to RPTP␤/ was inhibited by free CS-C chains further supports the notion that RPTP␤/ is the receptor involved in activation of c-Src kinase and the subsequent signaling pathway that finally leads to increased cell migration.
It has been suggested in neuronal cells that binding of HARP to RPTP␤/ leads to receptor dimerization, resulting in the inactivation of the phosphatase activity and increased phosphorylation of ␤-catenin (22). However, neither dimerization of RPTP␤/ nor inactivation of phosphatase activity has directly been shown. Moreover, increased phosphorylation of ␤-catenin can also be the result of c-Src activation (28), which seems to be the case based on our data (see above). c-Src has also been shown to be a substrate of another receptor protein-tyrosine phosphatase, RPTP␣. An interaction of the SH2 domain of Src with the C-terminal Tyr 789 of RPTP␣ results in dephosphorylation of the inhibitory phosphorylation site of c-Src (Tyr 527 ), leading to c-Src activation (29). A similar interaction with the SH2 domain of Src has been shown for RPTP⑀ (30), which suggests that it may be a common mechanism for many, if not all, receptor protein-tyrosine phosphatases.
c-Src was also found to co-immunoprecipitate with FAK, in accordance with several reports describing a FAK-Src complex (27). Because FAK was not detected in RPTP␤/ immunoprecipitates of HUVEC lysates (data not shown) we assume that c-Src kinase links FAK to RPTP␤/ and HARP signaling. The SH2 domain of c-Src kinase is known to recognize and bind phosphorylated Tyr 397 of FAK (31), leading to elevated FAK phosphorylation (27). This seems to be the case in our experiments too, because HARP-induced tyrosine phosphorylation of FAK was inhibited by CS-C, sodium orthovanadate, and PP1.
FAK interaction with c-Src results to enhanced phosphorylation of FAK and creates docking sites for several other molecules, such as PI3K and Shc (27). Several reports have implicated the PI3K-MAPK pathway in HARP signaling (16,17), however, without any data on the upstream signaling molecules involved. In the present work we indicate for the first time that activation of c-Src after the binding of HARP to RPTP␤/ can lead to activation of FAK and subsequently of the PI3K and MAPK signaling pathway, which finally leads to HARP-induced HUVEC migration and differentiation. Inter-estingly, no Akt activation was observed after treatment of HUVEC with HARP (data not shown), although PI3K was found to be involved in HUVEC signaling. Akt activation was observed in HUVEC after stimulation with midkine (32) suggesting that there are distinct pathways of HARP and midkine in these cells. Alternatively, Akt activation may not be possible to detect in our experiments because of increased basal levels (data not shown), possibly as a result of serum starvation of HUVEC.
The role of RPTP␤/ in signaling triggered by HARP in HUVEC is supported further by our results obtained after RPTP␤/ knockdown by RNA interference. Our results clearly demonstrate that RPTP␤/ down-regulation interrupts HARP signaling (Fig. 10) in HUVEC and abolishes its biological activity on cell migration and differentiation (Fig. 11). We also saw a decrease in unstimulated HUVEC migration and differentiation in these RPTP␤/ knockdown studies. This effect would be consistent with interference of endogenously expressed HARP-mediated processes in these cells (1). Moreover, RPTP␤/ binds various cell adhesion and extracellular matrix molecules (33)(34)(35)(36), whereas it is not known whether this binding affects its signaling and whether activation of its signaling is necessary for the function of RPTP␤/ after binding to such molecules. At least in the development of the nervous system, although phosphatase activity is required for many RPTPmediated developmental events, some developmental functions of RPTPs do not require tyrosine phosphatase activity (37). Finally, it is becoming increasingly recognized that receptor protein-tyrosine phosphatases are implicated in the regulation of integrin-mediated events (38), which seem to play significant role in HUVEC migration and tube formation on matrigel (39). RPTP␣-dependent differences in cell spreading on fibronectin and vitronectin substrates are mediated by the ␣ ␤ 3 integrin, and although RPTP␣ does not modulate integrin-ligand interactions, it seems to regulate the strength of integrin-cytoskeleton bonds (40). Integrin ␣ 3 ␤ 1 uses a complex involving protein kinase C␤II, RACK1, and PTP to regulate cadherin-mediated cell-cell adhesion, possibly by modulating tyrosine phosphorylation of ␤-catenin (41). RPTP␤/ seems to participate in a multimolecular complex involving the low density lipoprotein receptor-related protein 6 ectodomain, ␣ 6 ␤ 1 -integrin, and ␣ 4 ␤ 1integrin (42). Whether such a complex is formed in HUVEC and what its possible role might be is not known and is under further investigation. Taken together, our results implicate RPTP␤/ in HARP-induced migration and differentiation of human endothelial cells and identify the signaling molecules involved.