Reciprocal Regulation of Syndecan-2 and Notch Signaling in Vascular Smooth Muscle Cells*

Background: The interaction of endothelial and smooth muscle cells is critical for blood vessel formation. Results: Endothelial cells induce syndecan-2 expression in smooth muscle cells through Notch signaling, and syndecan-2 acts as a Notch coreceptor. Conclusion: Notch signaling and syndecan-2 cooperate to govern smooth muscle cell differentiation. Significance: Interaction of syndecan-2 and Notch signaling is a novel strategy for the regulation of smooth muscle cell differentiation. Vascular cell interactions mediated through cell surface receptors play a critical role in the assembly and maintenance of blood vessels. These signaling interactions transmit important information that alters cell function through changes in protein dynamics and gene expression. Here, we identify syndecan-2 (SDC2) as a gene whose expression is induced in smooth muscle cells upon physical contact with endothelial cells. Syndecan-2 is a heparan sulfate proteoglycan that is known to be important for developmental processes, including angiogenesis. Our results show that endothelial cells induce mRNA expression of syndecan-2 in smooth muscle cells by activating Notch receptor signaling. Both NOTCH2 and NOTCH3 contribute to the increased expression of syndecan-2 and are themselves sufficient to promote its expression independent of endothelial cells. Syndecan family members serve as coreceptors for signaling molecules, and interestingly, our data show that syndecan-2 regulates Notch signaling and physically interacts with NOTCH3. Notch activity is attenuated in smooth muscle cells made deficient in syndecan-2, and this specifically prevents expression of the differentiation marker smooth muscle α-actin. These results show a novel mechanism in which Notch receptors control their own activity by inducing the expression of syndecan-2, which then acts to propagate Notch signaling by direct receptor interaction.

. Syndecan-2 knockdown in zebrafish leads to defective angiogenic sprouting (16), and syndecan-2 inactivation in microvascular endothelial cells causes impairments in capillary tube-like structures (17,18). In addition to regulating angiogenesis, functional studies have demonstrated a role in left-right axis formation (19) and promotion of membrane protrusions and migration (20 -22). Syndecan-2 is a heparan sulfate proteoglycan with glycosaminoglycan chains attached to the extracellular domain (ectodomain) of the protein (23). The glycosaminoglycan ectodomains of syndecan family members are known to control both cell-matrix and cell-cell interactions and serve as a coreceptor for growth factor PDGF, FGF, and VEGF signaling (13,14) and TGF␤ signaling (24). In this work, we describe the interaction of two cell surface signaling mediators, NOTCH3 and syndecan-2. We show that syndecan-2 expression is induced in smooth muscle cells by coculturing with endothelial cells, and this induction relies on Notch signaling. Furthermore, we demonstrate that syndecan-2 augments Notch activity and directly binds to the NOTCH3 receptor. These data highlight the importance of crosstalk between individual signaling pathways in governing cell communication within the vasculature.

EXPERIMENTAL PROCEDURES
Cell Culture-Primary cultures of human aortic smooth muscle cells (HAoSMCs) and human coronary artery smooth muscle cells were purchased from Lonza and grown in DMEM (Mediatech, Inc.) supplemented with 10% FBS (HyClone), 2 mM glutamine, 1 mM sodium pyruvate, and 100 units/ml penicillin/ streptomycin. Human dermal neonatal fibroblasts (HDFNs) were purchased from Cascade Biological and cultured in DMEM supplemented as described above with 5% FBS. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and grown in EBM-2 supplemented with the Bul-letKit components as recommended by the manufacturer. Primary cells between passages 6 and 9 were used for all experiments. Human hepatoblastoma (HepG2) cells and human adenocarcinoma (HeLa) cells were purchased from American Type Culture Collection and cultured in DMEM supplemented as indicated with 10% FBS. For virus production, TN-293 cells were purchased from Stratagene and cultured in 10% DMEM as described above. All cultures were maintained in humidified 5% CO 2 at 37°C. For coculture, 6 ϫ 10 4 mural cells were plated in 12-well plates, and after adhesion, 6 ϫ 10 4 HUVECs were added. To separate endothelial cells from fibroblasts and smooth muscle cells, anti-PECAM1conjugated Dynabeads (Invitrogen) were used according to the manufacturer's instructions. All cell coculture experiments, unless indicated, were performed in medium consisting of EBM-2 supplemented with all BulletKit components except FBS, VEGF, and basic FGF. This medium was supplemented with 1% FBS and 30 ng/ml VEGF-A 165 (PeproTech). N-[ (3,acetyl]-L-alanyl-2-phenylglycine 1,1dimethylethyl ester (DAPT; Calbiochem) was added to specified wells at the time of plating. Transwell experiments were performed as described previously (12). Transwell inserts (12well type, Corning Costar) with 0.4-m pores were coated with 50 g/ml rat tail collagen I (BD Biosciences). 2 ϫ 10 4 HDFNs or HUVECs were first plated on the outside of the polycarbonate membrane of the Transwell inserts. After cell adherence, the Transwell inserts were inverted and reinserted onto 12-well plates, and 2 ϫ 10 4 HDFNs or HUVECs were plated on the top surface of the insert and cultured in a final volume of 1.3 ml of medium (0.3 ml in the insert and 1 ml in the well). Following incubation for 48 h, cells grown on the top side of the inserts were harvested by trypsinization and processed for quantitative real-time PCR (qPCR).

Syndecan-2 and Notch Signaling
UUC CAG CUC A; and syndecan-2 siRNA sequence, 5Ј-GCU GAC AUC UGA UAA AGA CAU. All siRNAs were transfected at 100 nM. Following transfection, cells were cocultured with HUVECs for 48 h, separated, and collected for qPCR analysis and Western blotting.
Lentivirus Expression-Human NOTCH2 intracellular domain (NICD2) cDNA (a gift from Dr. Igor Prudovsky) was cloned with a HA-tag attached to the 3Ј-end into pCDF1-MCS2-EF1-copGFP (System Biosciences) using BamHI and EcoRI sites. NOTCH3 intracellular domain (NICD3) and dominant-negative MAML (mastermind-like 1) constructs were made as described previously (12). The human syndecan-2 open reading frame (American Type Culture Collection) was amplified by PCR and cloned into pCDF1-MCS2-EF1-copGFP using XbaI and BglII sites. A HA tag was conjugated to the 3Ј-end of syndecan-2 by PCR and cloned using XbaI and EcoRI sites. The lentivirus plasmids were transfected into TN-293 cells using Lipofectamine 2000 (Invitrogen), and the viral particles were amplified and purified as described (12). For HDFN and HAoSMC infection, equal volumes of viral particles were diluted in 10% FBS in DMEM and were incubated with cells for 24 h. The efficiency of infection was evaluated using GFP expression and qPCR. Viral particles were titrated to achieve 90 -100% infection. Expression of NICD2, NICD3, and syndecan-2 cDNAs was confirmed by qPCR and Western blot analysis.
Plasmid Transfection and Luciferase Assays-A 5ϫCBF1-luciferase plasmid was generated as described (12). To measure the transcriptional activity, HDFNs or HAoSMCs at 80% confluency were sequentially transfected with siRNA followed by plasmids using Lipofectamine 2000. Cells were then cocultured with an equal number of HDFNs or HAoSMCs (as a control) or HUVECs for an additional 48 h. Cells were collected, and promoter activity was measured by luciferase assays using Steady-Glo reagent (Promega). To normalize the transfection efficiency, Hsp-␤-galactosidase (LacZ) was cotransfected, and luciferase activities were normalized based on an equivalent amount of LacZ activity. Luciferase and LacZ activities were measured as described (27) and quantified using a Molecular Devices SpectraMax luminometer. All experiments were performed in duplicate and repeated a minimum of three times.
Statistical Analysis-Data analyses were performed using GraphPad Prism, and comparisons between data sets were made using Student's t test. Differences were considered significant if p Ͻ 0.05, and data are presented as means Ϯ S.E. Data shown are representative of at least three independent experiments.

Syndecan-2 Is Regulated by Endothelial Cells through Notch
Signaling-Using a screen to find genes regulated by endothelial-mural cell interaction, we identified an array of genes whose

Coculture of endothelial cells increases syndecan-2 expression in mural cells.
A, mural cells (HDFNs), HAoSMCs, and human coronary artery smooth muscle cells (HCASMCs) were cultured in the presence or absence of endothelial cells (HUVECs) for 48 h; separated using anti-PECAM1-conjugated Dynabeads; and subjected to qPCR analysis for syndecan-2 transcript expression. B, HepG2 and HeLa cells were cultured alone or cocultured as described for mural cells, and the expression of syndecan-2 was measured by qPCR. C, HUVECs and HDFNs were plated on either side of a Transwell insert as indicated and cultured for 48 h, and the top chamber was harvested for qPCR to examine syndecan-2 mRNA expression. *, p Ͻ 0.05; **, p Ͻ 0.01 relative to the control. n.s., not significant. MAY 11, 2012 • VOLUME 287 • NUMBER 20 expression was altered when cells of the vasculature were cocultured (28). One of these genes was the heparan sulfate proteoglycan syndecan-2 (SDC2) (15). Coculture of HUVECs with HDFNs, HAoSMCs, or human coronary artery smooth muscle cells led to a significant induction of syndecan-2 mRNA expression in mural cells as assessed by qPCR (Fig. 1A). The induction in the different mural cell subtypes ranged from ϳ10-fold in fibroblasts to 4-fold in coronary artery smooth muscle cells, which exhibited a higher level of basal syndecan-2 expression. In contrast, two non-mural cell types, HepG2 and HeLa cells, showed no significant induction of syndecan-2 when cultured with HUVECs (Fig. 1B). To determine whether the induction of syndecan-2 requires cell-cell contact between neighboring endothelial and mural cells, we performed coculture experiments using Transwell inserts to physically separate the cells. Compared with control wells that had endothelial and mural cells cultured together, when endothelial cells were separated by a 0.4-m pore membrane, the level of syndecan-2 expression remained low in the mural cells, similar to that seen in noncocultures (Fig. 1C). These data indicate that endothelial cells induce syndecan-2 expression in mural cells and that this is dependent upon cell-cell contact.

Syndecan-2 and Notch Signaling
Our laboratory previously showed the importance of Notch signaling for the communication of endothelial and mural cells (12,28). Given that syndecan-2 induction requires cell-cell contact and Notch receptor activation relies on contact from an adjacent ligand-presenting cell, we tested the involvement of the Notch pathway. To do so, we used the ␥-secretase inhibitor DAPT, which prevents Notch receptor cleavage. In the presence of endothelial cells, 3 M DAPT blocked the up-regulation of syndecan-2 in both fibroblasts and smooth muscle cells (Fig.  2, A and B). We further confirmed the role of Notch signaling by using the dominant-negative MAML construct, which blocks Notch transcriptional activity (29). Similar to DAPT inhibition, syndecan-2 transcript induction by endothelial cells was inhibited in the presence of dominant-negative MAML (Fig. 2, C  and D).
NOTCH2 and NOTCH3 are prominently expressed in vascular smooth muscle cells (30 -32). To determine whether one or both of these Notch receptors is responsible for the transactivation of syndecan-2, we performed knockdown experiments with NOTCH2 and NOTCH3 utilizing siRNA ( Fig. 3 and supplemental Fig. 1). Under coculture conditions, knockdown of NOTCH2 significantly blocked syndecan-2 induction by endothelial cells, whereas knockdown of NOTCH3 had little effect (Fig. 3, A and B). siRNA inhibition of both the NOTCH2 and NOTCH3 receptors resulted in a complete loss of the inductive effects of endothelial cells on syndecan-2 expression. To determine whether the Notch receptors are sufficient for syndecan-2 gene expression, we overexpressed the intracellular domains of NOTCH2 (NICD2) and NOTCH3 (NICD3) by lentivirus transduction in smooth muscle cells and measured syndecan-2 expression (Fig. 3, C and D). The data show that both NOTCH2 and NOTCH3 can promote syndecan-2 expression in smooth muscle cells. Together, these results show that Notch signaling is both sufficient and necessary for the endothelial cell-dependent expression of syndecan-2.
To determine whether syndecan-2 expression might be regulated by Notch2 and Notch3 in vivo, we examined the highly vascularized yolk sacs of embryos deficient in Notch2 (25) and Notch3 (26). Syndecan-2 expression was significantly attenuated in yolk sacs of mouse embryos deficient in both Notch2 and Notch3, with the most pronounced decrease occurring in the absence of both Notch family members (Fig. 4). These data show a dependence of syndecan-2 gene expression on Notch signaling within the vasculature.
Syndecan-2 Regulates Notch Signaling-Previously, we showed that endothelial cells activate Notch signaling in neighboring mural cells to promote an autoregulatory loop resulting in NOTCH3 induction, followed by NOTCH3-dependent differentiation of smooth muscle cells (12). Because syndecan-2 is a target of Notch signaling, we asked if the up-regulation of syndecan-2 might affect Notch signaling in smooth muscle cells. To address this, we first examined endothelial cell-activated Notch signaling using a CBF1-luciferase reporter construct, which serves as a general Notch signaling sensor. HAoSMCs were cotransfected with the CBF1-luciferase or control luciferase construct along with siRNA to knock down syndecan-2 expression (Fig. 5, A and B). Following coculture with endothelial cells, Notch transcriptional activity was measured by luciferase assays. As published previously (12), endothelial cells promoted robust activity of the CBF1-reporter (Fig.  5A) and caused an increase in NOTCH3 RNA and protein (Fig.  5, C and D). However, when syndecan-2 was knocked down in smooth muscle cells, Notch signaling was greatly attenuated (Fig. 5A), and furthermore, NOTCH3 expression was decreased, as a likely consequence of its inability to autoactivate its own expression (Fig. 5, C and D).   MAY 11, 2012 • VOLUME 287 • NUMBER 20

JOURNAL OF BIOLOGICAL CHEMISTRY 16115
To more precisely examine the downstream effect that the loss of syndecan-2 has on Notch signaling, we measured the expression of known targets of NOTCH3 in smooth muscle cells cocultured with endothelial cells. Consistent with our previous findings, expression of HEYL/HRT3, PDGF receptor-␤, smooth muscle ␣-actin, SM22␣, and CNN1 (calponin-h1) was up-regulated in smooth muscle cells by endothelial cell coculture (Fig. 6, A-F). In the absence of syndecan-2, however, the expression of most of these genes was abrogated. CNN1 showed a slight but not significant decrease, suggesting that it is regulated differently. These data indicate that syndecan-2 facilitates Notch signaling in smooth muscle cells and is an important mediator of smooth muscle differentiation by regulating the expression of some smooth muscle genes.
To assess whether syndecan-2 is sufficient to activate Notch signaling and smooth muscle gene expression, we overexpressed syndecan-2 cDNA by lentivirus transduction in smooth muscle cells and measured gene expression by qPCR and Western blotting. Syndecan-2 was overexpressed by Ͼ50-fold (Fig.  7A). In smooth muscle cells cultured alone, the overexpression of syndecan-2 could not induce NOTCH3 expression or the expression of Notch signaling targets HRT3, PDGF receptor-␤, and smooth muscle ␣-actin (Fig. 7). Moreover, in cells cocultured with HUVECs, in which Notch signaling is activated, overexpression of syndecan-2 could not further induce any of the tested Notch targets (Fig. 7).
Syndecan-2 and NOTCH3 Physically Interact-Our data indicated that syndecan-2 modulates Notch signaling, and because both proteins are localized within the cell membrane, we speculated that syndecan-2 was facilitating Notch signaling through direct binding to Notch receptors. Because our attempts to use commercial antibodies to detect human syndecan-2 were unsuccessful, we performed co-immunoprecipitation experiments with a HA-tagged full-length syndecan-2 (HA-SDC2) protein, followed by immunoblotting to detect endogenous NOTCH3. HA-SDC2 was lentivirally transduced into cells, followed by immunoprecipitations with IgG or anti-HA antibodies. NOTCH3-specific immunoblots demonstrated that NOTCH3 protein was pulled down with syndecan-2 (Fig. 8). The reverse experiment of immunoprecipitating endogenous NOTCH3, followed by probing for HA-tagged syndecan-2 by Western blotting, showed a similar result (Fig. 8). Thus, our data show that these two proteins physically associate in cultured cells, providing a mechanism by which syndecan-2 modulates Notch signaling.

DISCUSSION
The signaling events that govern the interaction of vascular cells are critical for proper formation and function of blood vessels. The data presented here provide mechanistic insight into how cells within the vasculature communicate to control the function of each other. Previously, we demonstrated a role for NOTCH3 in endothelial cell/smooth muscle cell communication (12). Our data showed that Notch signaling is important for endothelial cell-induced differentiation of smooth muscle cells, but the mechanisms down-FIGURE 5. Syndecan-2 regulates Notch signaling. A, luciferase reporter assays were used to assess Notch signaling activity. HAoSMCs were transiently transfected with control (siCon) or syndecan-2 (siSDC2) siRNA along with the pGL3-promoter-luciferase (pGL3-LUC) plasmid as control or with a plasmid with five CBF1-binding elements upstream of the promoter (CBF-LUC). 24 h later, HUVECs were cocultured with a subset of cells, and luciferase activity was measured after 48 h. B, efficient siRNA knockdown of syndecan-2 was determined by qPCR. C, qPCR was used to assess NOTCH3 expression (autoregulation) after syndecan-2 siRNA (siSYNDECAN-2) knockdown. D, Western blotting using anti-NOTCH3 antibody indicated that loss of syndecan-2 attenuated NOTCH3 protein expression. *, p Ͻ 0.05; **, p Ͻ 0.01.

Syndecan-2 and Notch Signaling
stream of NOTCH3 were not defined. Here, we have shown that syndecan-2 is also induced by endothelial cells and is dependent upon NOTCH2 and NOTCH3 for this up-regulation. Moreover, in the absence of syndecan-2, the expression of downstream targets of Notch signaling is attenuated, indicating that syndecan-2 acts as a facilitator of Notch receptor activity. Consistent with this, in the absence of activated Notch signaling, syndecan-2 is not sufficient to induce expression of Notch target genes.
Notch signaling, particularly Notch3, has been shown to be important for the regulation of smooth muscle differentiation (12,30,33). Our data suggest that syndecan-2 acts to promote smooth muscle differentiation via modulation of Notch signaling. A precise role for syndecan-2 in smooth muscle has not been reported; however, studies with syndecan family members suggest a potential function in controlling the switch between proliferation and differentiation. Deletion of syndecan-1 causes increased intimal hyperplasia and smooth muscle proliferation particularly in response to PDGF-B (34). Loss of syndecan-4 limits neointimal hyperplasia and reduces smooth muscle proliferation (35). An earlier report showed a requirement for syndecan-4 in thrombin-induced proliferation (36). Unpublished in vitro results from our laboratory indicate that syndecan-2 inhibits smooth muscle proliferation, consistent with a role in governing differentiation in collaboration with Notch signaling. 3 Whether syndecan-2 has functions independent of the Notch pathway in smooth muscle cells remains to be determined.
Very little is known about the regulation of syndecan-2 gene expression. Expression levels have been reported in many cell types and linked to certain cancers (13)(14)(15). One report showed that syndecan-2 levels increase upon treatment with tumor necrosis factor-␣ (37). In vascular smooth muscle cells,   MAY 11, 2012 • VOLUME 287 • NUMBER 20 was shown to induce the expression of syndecan-4, but not syndecan-2 (38). Our results show that Notch signaling regulates syndecan-2 gene expression in vitro and in vivo. We have shown that both NOTCH2 and NOTCH3 are necessary and sufficient for syndecan-2 induction in smooth muscle cells. Currently, we do not know if the NICD and cofactor CBF (RPB-J) bind directly to the syndecan-2 gene to activate its transcription. Like syndecan-2, Notch receptors are widely expressed and are known to be important for development and tumor progression (8). Our results examined Notch-dependent activation of syndecan-2 only in dermal fibroblasts and coronary artery and aortic smooth muscle cells. Given the potential overlap of Notch receptors and syndecan-2 expression in other cell types, it is interesting to speculate that Notch receptors regulate syndecan-2 expression in other tissues, and in turn, syndecan-2 acts to govern Notch signaling.

Syndecan-2 and Notch Signaling
One of the most interesting findings from our study is that syndecan-2 acts to reinforce Notch signaling in smooth muscle cells. Syndecan-2 appears to do this through direct proteinprotein interaction. The syndecan family has been reported to interact with growth factors and their receptors (13,14), and specifically syndecan-2 has been shown to directly bind to the TGF␤ type III receptor, betaglycan (24). More interestingly, Pisconti et al. (39) demonstrated a direct link between syndecan-3 and Notch1 in skeletal muscle satellite cells. These authors showed that Notch1 and syndecan-3 directly interact and that syndecan-3 regulates Notch1 cleavage by ADAM17/ tumor necrosis factor-␣-converting enzyme. Thus, like growth factor receptors, Notch family members may be common targets for syndecan regulation. Our results show for the first time that syndecan-2 is a target of Notch signaling in smooth muscle cells, and syndecan-2 acts in a feedforward loop to enhance the actions of Notch signaling through direct contact with the NOTCH3 receptor (Fig. 9). Taken together, these results provide new information for the role of syndecan-2 in smooth muscle biology.