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Ephrin-B2 controls PDGFRβ internalization and signaling

Ephrin-B2 is essential for supporting mural cells; namely, pericytes and vascular smooth muscle cells (VSMCs). Nakayama et al. find that ephrin-B2 controls platelet-derived growth factor receptor β (PDGFRβ) distribution in the VSMC plasma membrane, endocytosis, and signaling. VSMCs lacking ephrin-B2 exhibited a redistribution of PDGFRβ from caveolin-positive to clathrin-associated membrane fractions and enhanced PDGF-B-induced PDGFRβ internalization. Mice lacking ephrin-B2 in vascular smooth muscle developed vessel wall defects and aortic aneurysms. These results suggest that ephrin-B2 is an important regulator of PDGFRβ endocytosis in mural cells.

Cell surface receptors integrate numerous signals from the tissue environment and can thereby induce fundamental changes in cellular behavior, which are the basis of numerous growth, migration, and tissue patterning processes. Increasing evidence indicates that endocytosis not only regulates receptor levels and bioavailability in the plasma membrane but can also influence the strength and identity of downstream signal transduction events (Abella and Park 2009;Hansen and Nichols 2009;Kumari et al. 2010). The concept that receptor internalization and trafficking can regulate signal transduction events has been initially proposed for the epidermal growth factor receptor (Vieira et al. 1996), but, subsequently, similar findings have been reported for the receptors binding insulin growth factor, transforming growth factor b, Wnt, or vascular endothelial growth factor (VEGF) (Chow et al. 1998;Di Guglielmo et al. 2003;Yu et al. 2007;Finger et al. 2008;Lanahan et al. 2010;Sawamiphak et al. 2010;Wang et al. 2010;Morcavallo et al. 2012).
Platelet-derived growth factor receptor b (PDGFRb), a receptor tyrosine kinase (RTK) activated by PDGF, suppresses the differentiation and promotes the proliferation of vascular smooth muscle cells (VSMCs) and other mesenchymal cell types (Andrae et al. 2008;Olson and Soriano 2011). Signals induced by PDGF binding to PDGFRb include the activation of Ras-mitogen-activated protein (MAP) kinase, phosphoinositide 3-kinase, the small GTPase Rac1, and c-Jun N-terminal kinase (JNK) (Andrae et al. 2008). Different membrane domains and endocytic pathways have been proposed to regulate PDGFRb function (Liu et al. 1996;Sundberg et al. 2009). Treatment of cultured human fibroblasts with dynamin inhibitors, which block clathrin-dependent endocytosis, argues for both dynamin-dependent and -independent PDGFRb internalization processes (Sadowski et al. 2013). Caveolin-1 can interfere with PDGF-induced signal transduction (Fujita et al. 2004;Yamamoto et al. 2008), which suggests that lipid rafts or caveolae might harbor a passive RTK pool devoid of signaling activity. Recently, it also has been proposed that PDGFRb endocytosis in cultured cells can be mediated by macropinocytosis involving dorsal, clathrin-containing membrane ruffles (Moes et al. 2012). However, the precise roles of different membrane domains for PDGFRb function, the underlying molecular processes, and the regulation of downstream signal transduction responses remain incompletely understood.
Ephrins, membrane-anchored ligands for Eph family RTKs, are emerging as key regulators of endocytosis and trafficking processes (Bethani et al. 2010;Pitulescu and Adams 2010). In the developing cardiovascular system, ephrin-B2, a transmembrane protein belonging to the B-class ephrin subfamily, is an important regulator of endothelial cell behavior and blood vessel growth. This function was recently linked to the regulation of ligandinduced VEGF receptor (VEGFR) internalization from the plasma membrane, which enhances certain downstream signaling events such as MAP kinase activation (Lanahan et al. 2010;Sawamiphak et al. 2010;Wang et al. 2010). Ephrin-B2, like other ephrins, not only activates Eph RTKs (termed ''forward'' signaling) on adjacent cells but also has receptor-like (''reverse'') signal transduction capacity that contributes to its role in VEGFR endocytosis (Sawamiphak et al. 2010;Wang et al. 2010;Nakayama et al. 2013). In addition to its functions in the endothelial monolayer of blood vessels, ephrin-B2 also controls the behavior of pericytes and VSMCs in the blood vessel wall. Targeting of ephrin-B2 (encoded by the gene Efnb2) in these perivascular cell types led to the formation of unstable blood vessels, hemorrhaging, and perinatal lethality (Foo et al. 2006). Many of these features resembled macroscopic defects observed in mutant mice lacking PDGF-B or PDGFRb (Lindahl et al. 1997;Tallquist et al. 2003). However, the exact mechanistic role of ephrin-B2 in perivascular cells and possible links to PDGF signaling have not been explored so far.

Vessel wall and signaling defects in VSMC-specific ephrin-B2 mutants
To circumvent the previously reported perinatal lethality of general mural cell-specific ephrin-B2 mutants (Foo et al. 2006), probably a consequence of pericyte defects, and inactivate the ligand specifically in VSMCs, we interbred mice carrying a loxP-flanked version of the Efnb2 gene (Efnb2 lox/lox ) (Grunwald et al. 2004) and SM22a-Cre transgenics (Lepore et al. 2005). While a fraction of the resulting Efnb2 DSMC mutants were viable, reached adulthood, and were fertile, only 42% of the expected number (i.e., 10.7% instead of 25%) was obtained at weaning age (Supplemental Fig. 1A). The absence of ephrin-B2 protein in mutant VSMCs was confirmed by immunostaining of Efnb2 DSMC aorta sections (Fig. 1A). The body weight of 30-to 60-wk-old mutants was reduced compared with age-matched littermates (Supplemental Fig. 1B). In addition, adult Efnb2 DSMC mice showed significant dilation of the aorta accompanied by reduced thickness of the aortic VSMC layer, which was most obvious for the aortic arch region (Fig. 1B,C; Supplemental Fig. 1C,D). The mutant tunica media appeared flattened, and the elastic lamella failed to show the wavy morphology observed in control aortae (Supplemental Fig. 1E). Consistent with the reduced thickness of the adult Efnb2 DSMC vessel wall, VSMC number and proliferation were reduced in mutants at postnatal day 8 (P8) (Fig. 1D-F), whereas the number of apoptotic cells was not significantly changed (data not shown). Our previous characterization of mural cellspecific Efnb2 mutants had shown that loss of ephrin-B2 resulted in impaired association of pericytes and VSMCs with perinatal blood vessels (Foo et al. 2006). In addition to the aorta, VSMC-specific Cre activity in SM22a-Cre transgenic mice is detectable in the embryonic dermis and postnatal retinal vasculature (Supplemental Fig. 1F; data not shown). Accordingly, arterial smooth muscle cell coverage was reduced and irregular in Efnb2 DSMC embryonic day 17.5 (E17.5) skin and P8 retina (Supplemental Fig. 1G,H; data not shown). In contrast, the (untargeted) pericytes in Efnb2 DSMC mutants showed no overt differences from control littermates (Supplemental Fig. 1H; data not shown).
To gain insight into the molecular changes associated with the vessel wall defects in Efnb2 DSMC mutants in vivo, we investigated the activation status of key signaling pathways in adult mutant and littermate control aorta lysates. Activation of MAP kinase Erk1/2, JNK, and PDGFRb was significantly increased in Efnb2 DSMC aorta samples (Fig. 1G,J). In line with the reduced VSMC proliferation, Efnb2 DSMC lysates contained elevated levels of p27 kip1 , an inhibitor of G1-to-S-phase transition in the cell cycle (Bond et al. 2008), whereas the active, GTP-associated form of the small GTPase Rac1 was strongly reduced ( Fig. 1H-J). Thus, loss of ephrin-B2 in vascular smooth muscle led to vessel wall defects and altered activation of multiple key signaling pathways in vivo.

Regulation of Tiam1-Rac1 signaling by ephrin-B2
To gain further insight into the functional role of ephrin-B2 in VSMCs, we compared the gene expression profiles of previously generated control and Efnb2 knockout VSMCs (Foo et al. 2006) by Affymetrix microarray analysis. This revealed that loss of ephrin-B2 led to pronounced changes in gene expression (Supplemental Fig. 2A,B). One of the top down-regulated genes (49-fold reduction compared with control) was Tiam1, which encodes T-cell lymphoma invasion and metastasis-inducing protein 1, a key regulator of cell morphology and polarity (Mertens et al. 2006). As Tiam1 is guanine nucleotide exchange factor (GEF) and thereby an activator of Rac1, we reasoned and EdU-labeled VSMCs (F). P-values were calculated using two-tailed Student's t-test (n = 3). Error bars indicate SD. (G) Western blot analysis of total and phosphorylated JNK (p-JNK), Erk1/2 (p-Erk1/2), and PDGFRb (p-PDFGRb) in control and Efnb2 DSMC aorta lysate, as indicated. Tubulin is shown as a loading control. Molecular weight markers (in kilodaltons) are indicated. (H,I) Strongly decreased levels of active Rac1 (GTPÁRac1) in Efnb2 DSMC aorta lysate relative to control (shown in H). (I) Tiam1 protein was nearly undetectable in mutant samples, whereas amounts of p27 kip1 were elevated. Tubulin is shown as a loading control. Molecular weight markers are indicated. (J) Quantitative analysis of band intensities in the Western blots shown in H and I and replicates. P-values were calculated using two-tailed Student's t-test (n = 3). Error bars indicate SD. that its down-regulation might be causally linked to the reduced Rac1 activity in Efnb2 DSMC aortas (Fig. 1H). Western blot analysis confirmed reduced Tiam1 protein levels in Efnb2 DSMC aorta lysate (Fig. 1I) as well as in cultured murine Efnb2 knockout VSMCs ( Fig. 2A).
As both MAP kinase and Rac1 signaling play important roles in the regulation of smooth muscle cell proliferation (Zou et al. 1998;Bond et al. 2008), we next investigated the interplay between these two pathways. Inhibition of Rac1 by administration of the small compound NSC23766 significantly reduced proliferation of cultured VSMCs and led to up-regulation of p27 kip1 (Fig. 2B,C). VSMC proliferation was also slightly reduced and p27 kip1 proliferation was increased after overactivation of MAP kinase (Fig. 2B,C), which was achieved by expressing a Raf/ estrogen receptor (ER) fusion protein (DRaf1-ER) with tamoxifen-inducible kinase activity (Thiel et al. 2009). Further reduction of VSMC mitosis was obtained after combined NSC23766 administration and DRaf1-ER activation ( Fig. 2B), which resembled the low Rac1 activity but elevated phospho-Erk1/2 observed in Efnb2 DSMC aorta lysates (Fig. 1G,H). We reported previously that cultured Efnb2 knockout VSMCs display spreading defects (Foo et al. 2006), which can be mimicked by treatment of VSMCs with the Rac1 inhibitor NSC23766 (Fig. 2D,E; Supplemental Fig. 2C). Defective spreading of Efnb2 knockout cells was rescued by re-expression of full-length Tiam1 (Fig. 2D,E), which also significantly restored the proliferation of ephrin-B2-deficient VSMCs (Fig. 2F). Thus, ephrin-B2 is a critical regulator of Tiam1/Rac1 and thereby controls VSMC spreading and mitosis.
Next, we investigated the regulation of Tiam1 expression by upstream signals. When control or Efnb2 knockout cells were treated with the Erk1/2 inhibitor U0126, Tiam1 expression was significantly increased at both the mRNA and protein levels ( Fig. 2G,H). Conversely, Erk1/2 activation with tamoxifen-inducible DRaf1-ER reduced Tiam1 mRNA and protein in murine VSMCs (Fig. 2I,J). The regulation of Tiam1 by ephrin-B2 does not appear to be regulated by acute reverse signal transduction. While the stimulation of VSMCs with recombinant EphB4/Fc fusion protein led to detectable phosphorylation of B-class ephrins after 15 and 30 min, there was no appreciable change in Tiam1 protein levels under the same conditions (Supplemental Fig. 2D). We showed previously that prolonged EphB4/Fc stimulation triggers pronounced internalization and degradation of ephrin-B2 after 2.5 and 6 h (Foo et al. 2006). The strong reduction of ephrin-B2 at these time points was accompanied by down-regulation of Tiam1 protein (Fig. 2K). This reduction of Tiam1 was prevented by the addition of the proteasome inhibitor MG132 (Fig.  2K,L), which indicates a role of protein degradation in this process.
Together, these data strongly argue for positive regulation of Tiam1 in smooth muscle cells by ephrin-B2, whereas MAP kinase activity reduces Tiam1 expression. Accordingly, the combination of absent ephrin-B2 expression and elevated phospho-Erk1/2, as observed in mutant aorta lysates, can explain the lost expression of Tiam1 and low Rac1 activity in Efnb2 DSMC VSMCs.

Signaling defects in ephrin-B2-deficient VSMCs are linked to PDGFRb
An increasing body of evidence connects the Eph/ephrin system to the modulation of other cell surface receptors, such as VEGFRs in endothelial cells (Bethani et al. 2010;Pitulescu and Adams 2010). Given the prominent role of PDGF signaling in VSMCs and other cell types of the mesenchymal lineage (Andrae et al. 2008;Olson and Soriano 2011), we next investigated the role of ephrin-B2 in the regulation of PDGFRb. Stimulation of murine control VSMCs with PDGF-B led to transient autophosphorylation of PDGFRb, which was accompanied by JNK and Erk1/2 activation (Fig. 3A). In line with findings in Efnb2 DSMC aorta lysates (Fig. 1G), PDGFRb tyrosine phosphorylation as well as phospho-JNK and phospho-Erk1/2 levels were substantially increased in Efnb2 knockout VSMCs at 5 and 15 min after PDGF-B treatment (Fig. 3A). In contrast, other factors triggering MAP kinase activation in VSMCs, such as insulin-like growth factor 1 (IGF-1) and tumor necrosis factor a (TNF-a) (Hayashi et al. 1999;Yoshimura et al. 2005), led to comparable Erk1/2 phosphorylation in control and Efnb2 knockout VSMCs (Supplemental Fig. 3A). Thus, the modulatory role of ephrin-B2 is confined to certain growth factors but not others. Consistent with the strongly reduced Tiam1 expression in Efnb2 knockout cells, PDGF-B-induced activation of Rac1 was impaired in comparison with control VSMCs (Fig. 3B). However, NSC23766 treatment of cultured VSMCs did not result in appreciable alterations in PDGF-B-induced JNK and Erk1/2 phosphorylation, suggesting that the up-regulation of these signals in Efnb2 knockout cells is not a direct consequence of Tiam1/Rac1 defects (Supplemental Fig. 3B).
Since ephrin-B2 positively regulates VEGFR internalization and thereby promotes the downstream activation of MAP kinase and Rac1 in endothelial cells (Sawamiphak et al. 2010;Wang et al. 2010;Nakayama et al. 2013), we next investigated whether PDGFRb function is modulated by ephrin-B2. Immunostaining of surface PDGFRb in nonpermeablized murine VSMCs and surface biotinylation experiments indicated that PDGF-B-induced uptake of the receptor was accelerated in Efnb2 knockout cells relative to controls ( Fig. 3C-F). The faster internalization of PDGFRb in ephrin-B2-deficient cells was accompanied by more rapid degradation of the RTK (Fig.  3E). Moreover, strongly enhanced association of PDGFRb with the clathrin heavy chain (CHC) suggested that clathrinmediated endocytosis of this receptor might be enhanced in Efnb2 knockout VSMCs as well as in Efnb2 DSMC aorta lysate ( Fig. 3G-I).

Ephrin-B2 controls PDGFRb distribution in the plasma membrane
PDGFRb distribution and endocytosis have been assigned to a number of different membrane domains, including clathrin-positive membrane ruffles, macropinosomes, and caveolae (Liu et al. 1996;Sundberg et al. 2009;Moes et al. 2012). To study the association of PDGFRb with caveolin-1-positive or clathrin-containing membrane ). P-values were calculated using ANOVA with Tukey's post-hoc test (n = 3). Error bars indicate SD. (C) Levels of the cell cycle inhibitor p27 kip1 were increased by Rac1 inhibition and were not restored by Erk1/2 activation (DRaf1:ER+TMX) in cultured murine VSMCs. Tubulin is shown as a loading control. Densiometric readings for p27 kip1 bands and molecular weight markers (in kilodaltons) are indicated. (D) Images of automatic cell shape analysis. Phalloidin-stained VSMCs (blue) and DAPI-stained nuclei (green) were segmented. Arrows mark Efnb2 knockout cells that express pEGFP-Tiam1 (yellow); arrowheads in the images at the right indicate rejected cells due to undetectable GFP expression. (E) Box-andwhiskers diagram of shape factor data of D. A dot marks the median, the box spans 30% of the values, and whiskers span 50% of the values. P-values were calculated using ANOVA and Tukey's post-hoc test (control, n = 263; knockout [KO], n = 263; KO+pEGFP Tiam1, n = 232). Smaller shape factor of Efnb2 cells was rescued by re-expression of Tiam1. (F) Re-expression of Tiam1 restored cell proliferation defects (measured by 2 h of EdU incorporation) in Efnb2 knockout smooth muscle cells. P-values were calculated using ANOVA (n = 3). Error bars indicate SD. (G) Western blot showing increased Tiam1 protein after Erk1/2 inhibition (U0126) for 24 h. Total Erk1/2 and p-Erk1/2 bands, which were strongly reduced by U0126, are shown below. (H) Quantitative PCR (qPCR) of Tiam1 expression in control or Efnb2 knockout VSMCs incubated with U0126 for 24 h. P-values were calculated using two-tailed Student's t-test (n = 3). Error bars indicate SD. (I,J) Western blot (I) and qPCR results (J) showing reduced Tiam1 levels after Erk1/2 activation (DRaf1:ER+TMX) for the indicated times. P-values were calculated using two-tailed Student's t-test (n = 3). Error bars indicate SD. (K) Tiam1 and ephrin-B2 protein levels were decreased after stimulation of VSMCs with EphB4/Fc for the indicated times (left), which was strongly reduced after administration of MG132 (right). (L) Densiometric analysis of data shown in K.
fractions, cultured murine VSMCs were analyzed by sucrose density gradient centrifugation. In the 11 fractions collected from control cells, ephrin-B2 and caveolin-1 were found together in fractions 3-6, which also contained the vast majority of PDGFRb (Fig. 4A). In Efnb2 knockout VSMCs, the distribution of caveolin-1 appeared unaltered, while a significant portion of PDGFRb had shifted to the bottom fractions (8-11) containing the CHC (Fig. 4A,B). Indicating association of ephrin-B2 and PDGFRb, both proteins were coimmunoprecipitated from the pooled sucrose gradient fractions 3-6 of cultured control but not Efnb2 knockout VSMCs (Fig. 4C). Arguing further for an ephrin-B2-dependent sequestering of PDGFRb into caveolin-1-containing membrane fractions, immunofluorescence showed that signals for ephrin-B2 (detected by binding of EphB4/Fc), PDGFRb, and caveolin-1 overlapped in spot-like structures in cultured control VSMCs (Fig. 4D). In contrast, EphB4/Fc binding was absent in ephrin-B2-deficient cells, and little or no overlap was seen between PDGFRb and caveolin-1 signals (Supplemental Fig. 4A).
PDGF-B-induced tyrosine phosphorylation and therefore activation of PDGFRb was almost exclusively associated with the CHC-containing fractions 8-11, while the vast majority of total PDGFRb protein remained in fractions 4-6 and showed only weak tyrosine phosphorylation (Fig. 4E). In Efnb2 knockout cells, a significantly larger fraction of PDGFRb was located in fractions 8-11, and only this pool showed substantial and, compared with the control, enhanced tyrosine phosphorylation (Fig.  4E). Likewise, phospho-Erk1/2 and JNK were found in fractions 8-11 after sucrose gradient centrifugation (Supplemental Fig. 4B). The notion that PDGFRb signaling is associated with clathrin-mediated endocytosis was further supported by nystatin treatment of murine VSMCs. This inhibitor, which binds to cholesterol and disrupts caveolae-mediated endocytosis (Sakane et al. 2010), did not compromise PDGFRb internalization but enhanced phosphorylation of PDGFRb, JNK, and Erk1/2 in response to PDGF-B (Supplemental Fig. 4C,D).
Taken together, the above results argue that PDGFRb signaling activity is primarily linked to the clathrin pathway in VSMCs. Ephrin-B2 interacts with PDGFRb and directs the receptor into caveolin-1-containing membrane domains, which negatively control clathrin-mediated PDGFRb endocytosis as well as downstream activation of JNK and MAP kinase. Further supporting that ephrin-B2 indeed counteracts clathrin-mediated PDGFRb internalization and degradation not only in vitro (Fig. 3E,G), PDGFRb immunostaining was significantly reduced in Efnb2 DSMC retinal arterial smooth muscle cells in vivo (Fig. 4F,G). Providing an internal control, anti-PDGFRb signals in the untargeted pericytes were comparable in control and mutant retinas (Fig. 4F,G).
Given the important role of ephrin-B2 in internalization processes in endothelial cells, we tested whether the ligand might also direct the distribution of VEGF receptors to specific membrane domains. However, sucrose density gradient centrifugation of cultured murine endothelial cells failed to reveal overt alterations in VEGFR3 distribution in the absence of ephrin-B2 (Supplemental Fig. 4E,F).

Role of Eph-ephrin interactions
To investigate whether the loss of ephrin-B2 reverse signaling or the lack of ligand-induced (forward) EphB acti-vation was responsible for the observed smooth muscle cell defects, we stimulated cultured VSMCs with ephrin-B2/Fc or EphB4/Fc fusion proteins. Remarkably, the stimulation of ephrin-B2 with EphB4/Fc not only triggered the internalization of ephrin-B2 but also led to pronounced clustering and endocytosis of surface PDGFRb (Fig. 5A,B;  Supplemental Fig. 5A). In contrast, the stimulation of EphB receptors with recombinant ephrin-B2/Fc had no overt effect on PDGFRb ( Fig. 5A; data not shown). The EphB4/ Fc-induced internalization of PDGFRb was not accompanied by appreciable tyrosine phosphorylation of the PDGFR and was largely unaffected by nystatin treatment (Fig. 5B,C). Nystatin also had no substantial effect on EphB4/Fc-induced ephrin-B2 internalization, which argues against a crucial role of caveolae in this process (Fig. 5B,C). PDGFRb showed a substantial level of colocalization with EphB4/Fc fusion protein in VSMCs at different time points after stimulation ( Fig. 5D; Supplemental Fig. 5B). Further supporting a role in PDGFRb internalization, a fraction of the EphB4/Fc-positive (i.e., ephrin-B2) and PDGFRbpositive spots overlapped with EEA1, a marker of early endosomes (Fig. 5D). Likewise, sucrose density gradient centrifugation confirmed that PDGFRb protein was shifted into CHC-containing fractions (8-11) at 30 min after EphB4/Fc stimulation (Fig. 5E). Following EphB4/Fc treatment and concomitant with the removal of ephrin-B2 from the cell surface, PDGF-B-mediated phosphorylation of PDGFRb and Erk1/2 was gradually increased ( Fig. 5F; Supplemental Fig. 5C), which resembled the enhanced activation of these molecules in Efnb2 knockout VSMCs (Fig. 3A). In contrast, no appreciable alteration in Erk1/2 activation was seen in cultured cells treated with ephrin-B2/Fc (Supplemental Fig. 5D,E).
We showed previously that cultured and freshly isolated VSMCs express the receptors EphB2, EphB3, and EphB4, all of which can interact with ephrin-B2 (Foo et al. 2006). The data above indicate that interactions with the local microenvironment, such as neighboring EphB receptor-presenting smooth muscle cells, can alter ephrin-B2 surface presentation and thereby strongly influence PDGFRb internalization and signaling in VSMCs. Specifically, ephrin-B2-expressing cells that encounter high levels of EphBs in their direct environment would down-regulate Tiam1 and thereby Rac1 signaling (Fig. 2K), whereas PDGFRb endocytosis as well as MAP kinase and JNK activation are concomitantly enhanced. We therefore propose that both the nature and quantity of signal transduction processes downstream from PDGFRb are critically controlled by ephrin-B2 and its interactions with EphB receptors.

Ephrin-B2 modulates PDGFRb activity
RTKs can activate a variety of downstream signal transduction processes, leading to very different cellular behaviors, such as proliferation, migration, differentiation, or cell shape modulation. Previous work has shown that coreceptors, which interact with RTKs at the cell surface, can strongly influence their activity and integrate different signals from the local tissue environment. For example, low-density lipoprotein (LDL) receptor-related protein 1 (LRP1)-a transmembrane protein that can bind ligands as diverse as lipoproteins, proteases, growth factors, cytokines, and matrix proteins-associates with PDGFRb and modulates its expression, internalization, and signaling (Boucher et al. 2003;Lehti et al. 2009;Muratoglu et al. 2010). Some controversy surrounds the exact role of LRP1 in PDGFRb function, and it has been proposed that it can suppress (Boucher et al. 2003;Zhou et al. 2009) or support (Muratoglu et al. 2010) MAP kinase activation. LRP6, another member of the LDL receptor-related protein family, can also interact with PDGFRb, promote its degradation, and reduce PDGF-induced VSMC proliferation (Keramati et al. 2011). Neuropilin-1, a coreceptor for VEGFs in endothelial cells, has been found in VSMCs, where it promotes PDGF-induced smooth muscle cell migration. This is thought to involve an interaction with PDGFRa, the second member of the PDGFR family (Pellet-Many et al. 2011). Here, we identified ephrin-B2 as a novel interaction partner of PDGFRb, which modulates the membrane distribution, internalization from the cell surface, and signaling activity of this RTK (Fig. 6). Our data suggest that PDGFRb-expressing cells can have very different signaling outputs in response to PDGF depending on the presence and expression levels of ephrin-B2. In particular, the absence of ephrin-B2 strongly enhances PDGF-Binduced MAP kinase and JNK activation, whereas Tiam1/ Rac1 signaling, a pathway critical for cell migration, proliferation, and spreading, gets diminished. Thus, ephrin-B2 can act as a molecular switch for different downstream signals induced by PDGF-B/PDGFRb.

Ephrin-B2 and surface receptor internalization
Members of the large Eph/ephrin gene families appear to be expressed in virtually all cell types and tissues, which raises the question of how a large but nevertheless limited set of ligand-receptor interactions can be translated into highly diverse biological responses. An increasing body of evidence indicates that the functional versatility of Eph/ ephrin molecules is achieved by molecular cross-talk with receptors from other families and, in particular, the modulation of their surface availability, clustering, trafficking, and endocytosis (Bethani et al. 2010;Pitulescu and Adams 2010). Among the surface molecules that are controlled by ephrin-B2, the VEGFRs VEGFR2 and VEGFR3 are most similar to PDGFRb because all three RTKs share a similar organization of several extracellular immunoglobulin domains and a single cytoplasmic kinase domain and are evolutionarily closely related to Drosophila PVR (PDGF/ VEGF receptor). While previous work has revealed that ephrin-B2 controls VEGFR2 and VEGFR3 internalization and signaling activity in vitro and in vivo, it is remarkable that these processes are positively regulated by the ephrin, which is the opposite of what we observed for PDGFRb. It is currently unclear whether these differences reflect distinct molecular properties of PDGFRs and VEGFRs, cell type-specific features distinguishing endothelial cells and VSMCs, or the influence of ephrin-associated or independently acting additional regulators. Therefore, it will be important to identify the relevant molecular interaction partners of ephrin-B2 and PDGFRb in caveolin-positive membrane domains as well as in the clathrin uptake machinery. Our findings also highlight that the precise role of ephrin-B2 in the modulation of other surface receptors can be complex and follow distinct mechanistic principles.

Potential disease relevance
Our data indicate that ephrin-B2 is a critical regulator of PDGF-B-induced signaling responses in vascular smooth muscle. While the phosphorylation of JNK and MAP kinase were strongly enhanced in Efnb2 mutant aortae and cultured cells, the expression of the GEF Tiam1 and activation of Rac1 were diminished. These changes were associated with diminished VSMC proliferation and vessel wall defects in Efnb2 DSMC mutants. While future work will have to address whether ephrin-B2 might be relevant in the context of VSMC-associated human diseases such as aortic aneurysms, our results suggest that the levels or availability of ephrin-B2 at the cell surface could potentially affect a variety of pathobiological processes involving PDGFs and their receptors (Ostman and Heldin 2007;Andrae et al. 2008). Examples include the autocrine and cell-autonomous PDGF-B/PDGFRb signaling that promotes glioma growth and invasiveness in the brain (Uhrbom et al. 1998) or the PDGF-B overexpression in experimental cancer models, which induces the recruitment of vessels and stromal fibroblasts (Forsberg et al. 1993). High levels of PDGF expression and PDGFR signaling have been also associated with atherosclerotic lesions, and the administration of neutralizing antibodies and Figure 6. Schematic summary of findings. Ephrin-B2 sequesters PDGFRb into caveolin-1-positive membrane domains and thereby counteracts clathrin-mediated endocytosis and excessive activation of the RTK. In particular, this process limits PDGF-Binduced Erk1/2 and JNK activation. Accordingly, down-regulation of ephrin-B2 levels (for example, via EphB-induced internalization through interactions with other VSMCs) enhances Erk1/2 and JNK signaling in response to PDGF-B. At the same time, ephrin-B2 positively regulates Tiam1 expression and thereby PDGF-Binduced Rac1 activation. MAP kinase overactivation reduces Tiam1 transcript levels and protein, which leads to impaired smooth muscle cell spreading and proliferation. aptamers can reduce smooth muscle cell proliferation and neointima formation in mice (Raines 2004;Andrae et al. 2008). PDGF-B/PDGFRb signaling in hepatic stellate cells, renal fibroblasts, and myofibroblasts has been linked to fibrosis in the liver, kidney, and skin, respectively (Andrae et al. 2008). As these examples highlight the great clinical relevance of the PDGF pathway, it should be worthwhile to investigate whether the presence or absence of ephrin-B2, related B-class ephrins, or the corresponding EphB receptors might alter PDGFRb surface availability, internalization, and signaling in pathological settings. Likewise, it remains to be resolved whether therapeutic modulation of ephrin-B2 expression might be beneficial in disease processes involving dysregulated PDGF-B/PDGFRb activity.
All animal experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by local animal ethics committees.
For staining of paraffin sections of aorta, sections were deparaffinized and incubated in 1% hydrogen peroxide for 10 min after antigen unmasking. Then, sections were blocked with 5% goat serum in PBS (30 min at room temperature) prior to incubation with Cy3-conjugated anti-a-SMA antibody (1:400; Sigma, A2547) diluted in the blocking solution in combination with TO-PRO3 (1:1000; Invitrogen). Elastin was detected by autofluorescence on paraffin after staining with Cy3-conjugated anti-aSMA antibody. Detection of ephrin-B2 on paraffin sections was performed with goat anti-ephrin-B2 antibodies (1:200; R&D Systems, AF496) as previously described (Batlle et al. 2002) in combination with Cy3-conjugated anti-a-SMA antibody (1:400; Sigma, A2547). For the labeling of proliferating cells, P8 pups received intraperitoneal injections with 5-ethynyl-29-deoxyuridine (EdU; dissolved 2 mg/mL in PBS; Invitrogen). After 2 h, pups were humanely sacrificed, and aortas were dissected and fixed for 1 h on ice and paraffin-embedded. After anti-a-SMA-Cy3 staining, EdU was detected with a Click-iT EdU Alexa Fluor 647 imaging kit (Invitrogen) according to the manufacturer's instructions and counterstained with DAPI.

Immunofluorescence
Control and Efnb2 knockout cells were plated on glass coverslips, washed in PBS, fixed in 4% PFA for 10 min on ice, washed in PBS, permeabilized with blocking buffer containing 0.1% Triton X-100 for 10 min, and incubated in blocking buffer (1% BSA in PBS) for 1 h at room temperature. Primary antibodies in blocking buffer were incubated overnight. Antibodies used were directed against PDGFRb (1:50 dilution; R&D Systems, AF1042), caveolin-1 (1:250; Becton Dickinson, 610493), or EEA1 (1:250; Abcam, ab2900). The cells were then washed three times for 10 min with PBS and incubated with appropriate secondary antibodies covalently linked to Alexa Fluor 488 or Alexa Fluor 546 (1:500) for 1 h at room temperature in PBS and counterstained with Alexa Fluor 546-conjugated anti-phalloidin antibody and/or DAPI (1:1000; Sigma). To detect PDGFRb on the surface of cultured VSMCs, cells were incubated with anti-PDGFRb antibody without permeabilization. The number of PDGFRb signals in 1 mm 2 was calculated from five cells from each group; three independent experiments were performed. To visualize ephrin-B2 in cultured VSMCs, 5 mg/mL EphB4/Fc (R&D Systems, 466-B4) was incubated with blocking solution. After washing with PBS, cells were incubated with Cy3-conjugated anti-human IgG/Fc (1:500; Jackson Laboratories, 109-165-008). For the labeling of proliferating cells, cultures were incubated with EdU (dissolved 10 mM in culture medium; Invitrogen) for 2 h before staining. EdU was detected with a Click-iT EdU Alexa Fluor 647 imaging kit (Invitrogen) according to the manufacturer's instructions.
Images were recorded with a Leica SP5 confocal microscope and Volocity software (Improvision).

Cell stimulation experiments
Control (Efnb2 lox/lox) and Efnb2 knockout cells were seeded at a density of 3.0 3 10 5 in 6-cm dishes and incubated for 24 h. After overnight serum starvation, cells were then incubated in serumfree growth medium containing 10 ng/mL PDGF-B (R&D Systems, catalog no. 520-BB), 2 ng/mL IGF (Peprotech, 100-11), or 10 ng/mL TNF-a (R&D Systems, 2279-BT) at 37°C for the indicated times; collected with 300 mL of 13 SDS sample buffer; and subjected to immunoblotting with antibodies binding phospho- Cell proliferation was measured by using the Premix WST-1 cell proliferation assay system (Takara, MK400) according to the manufacturer's instructions. Briefly, 0.1 3 10 4 cells per well were cultured in microtiter plates (tissue culture grade, 96 wells, flat bottom) in a final volume of 100 mL of culture medium per well. After the incubation period (48 h), 10 mL of Premix WST-1 was added per well, and cells were incubated for 1-4 h. The absorbance of the samples against a background control (the same volume of culture medium and Premix WST-1) was measured by using a microtiter plate (ELISA) reader.

Automatic cell shape analysis
Electroporation was performed by using the Amaxa Nucleofection system. Nonconfluent cells were trypsinized, washed in medium, and resuspended at a concentration of 1 3 10 6 cells per 100 mL in an Amaxa Basic Nucleofector kit. Two micrograms of plasmid DNA was then mixed with 100 mL of cells and placed in a cuvette. After electroporation by using setting T-030, cells were directly transferred to warm medium. Control or pEGFPtagged full-length Tiam1 transfected cells were seeded on sixwell dishes, fixed, and stained with Alexa Fluor 546 phalloidin (Invitrogen, 1:500), Alexa Fluor488-coupled anti-GFP antibody (Invitrogen, 1:500), and DAPI. The images were automatically analyzed by a custom-developed image-processing journal detecting the DAPI-stained nuclei followed by detection of cell outlines (using the Alexa Fluor 488 image) separating touching cells with watershed lines. Cells touching the border were rejected. A shape factor (4 3 p 3 area/perimeter 2 ) was calculated for each cell.

Microarray and quantitative PCR analysis
RNA was isolated from control and Efnb2 knockout cells after 3 d of culturing in IFN-g-free cell medium using the RNAeasy kit (Qiagen) following the manufacturer's guidelines. Generation of gene expression profiles for control and Efnb2 knockout cells was carried out by the GeneChip microarray service at the Paterson Institute for Cancer Research using Affymetrix moe430A 2.0 chips.
For real-time PCR, cDNA synthesis was achieved from 500 ng of total RNA by using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's guidelines. Then, TaqMan gene expression assays (Applied Biosystems) for murine GAPDH and Tiam1 were used in combination with TaqMan gene expression master mix.

Quantification and image processing
Volocity (Improvision), Photoshop CS, and Illustrator CS (Adobe) software was used for image processing without distorting or misrepresenting results. Data were based on at least three independent experiments or three mutant and control animals for each stage and result shown.