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(The FASEB Journal. 2006;20:2150-2152.)
© 2006 FASEB

Semaphorin 3C regulates endothelial cell function by increasing integrin activity

Nazifa Banu*, Jason Teichman{dagger}, Marya Dunlap-Brown§, Guillermo Villegas{dagger} and Alda Tufro{dagger},{ddagger},1

Division of Nephrology, Departments of
* Internal Medicine and

{dagger} Pediatrics, and

{ddagger} Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, USA; and

§ Department of Microbiology, University of Virginia, Charlottesville, Virginia, USA

1Correspondence: Department of Pediatrics/Nephrology, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Forchheimer Bldg, room 708, Bronx, NY 10461 USA. E-mail: atufro{at}aecom.yu.edu

ABSTRACT

Class 3 semaphorins (sema 3) are secreted guidance proteins. Sema 3A expressed by endothelial cells controls vascular morphogenesis through integrin inhibition. Sema 3C is required for normal cardiovascular patterning. Here we examined the potential role of sema 3C as regulator of endothelial cell function in vitro using mouse glomerular endothelial cells (MGEC). We determined that MGEC express sema 3C mRNA and protein and its receptors mRNA. Recombinant sema 3C induced MGEC proliferation 18 ± 2% above control, as assessed by bromodeoxyuridine (BrdU) incorporation, and reduced starvation-induced apoptosis by 46 ± 3%, as indicated by an in situ marker of activated caspase 3. Sema 3C increased MGEC adhesion to fibronectin 79 ± 13% and to collagen 55 ± 12% as compared with control. Sema 3C-induced MGEC adhesion was prevented by integrin blocking antibodies and involved ß1 integrin serine phosphorylation. Sema 3C-induced MGEC adhesion and proliferation were similar to those induced by vascular endothelial growth factor (VEGF)-A. Sema 3C induced a 44 ± 11% increase in MGEC directional migration and stimulated MGEC capillary-like network formation on collagen I gels. Collectively, our data indicate that sema 3C promotes glomerular endothelial cell proliferation, adhesion, directional migration, and tube formation in vitro by stimulating integrin phosphorylation and VEGF120 secretion, functions that are similar to VEGF-A and opposite to sema 3A.—Banu, N., Teichman, J., Dunlap-Brown, M., Villegas, G., Tufro, A. Semaphorin 3C regulates endothelial cell function by increasing integrin activity.


Key Words: class 3 semaphorins • endothelial cell adhesion • migration • tube formation • VEGF120 secretion

CLASS 3 SEMAPHORINS are a family of secreted proteins initially characterized during nervous system development that provide guidance cues to axons, neural crest cells, and endothelial cells (1 , 2) . Semaphorins generate chemorepellent signals by inducing growth cone collapse of migrating axons (3) , inhibiting endothelial cell migration, or establishing zones of exclusion for cell migration (4 5 6 7) or induce chemoattraction by stimulating migration and survival of axons, neural crest, epithelial or tumoral cells (8 9 10) .

Class 3 semaphorins bind to a receptor complex consisting of a binding component, neuropilins 1 or 2, and a signaling component, plexins A1, A2, A3, or D1 (11 12 13) . The specificity of the semaphorin signaling depends on the combination of neuropilin/plexin present in dimeric or multimeric receptor complexes in each cell type, e.g., plexin D1 is endothelial cell specific and associates to plexin A1/NP1 to transduce sema 3A signals and to plexin A2/NP2 to transduce semaphorin (sema) 3C signals (5 , 12 13 14 15) . Semaphorin signaling engages integrins and small GTPases to regulate the organization of actin cytoskeleton and thereby mediate cell migration and remodeling (12 , 16 17 18) .

Sema 3A, the prototypical class sema 3 protein, is considered a chemorepellent with proapoptotic functions that induces growth cone collapse and endothelial cell repulsion in vitro and neural pathfinding defects and vascular patterning abnormalities in vivo, resulting in neonatal lethality (6 , 19 , 20) . Sema 3A signals inhibit integrin activity and induce F-actin depolymerization (6 , 20) . Sema 3C provides chemorepulsive guidance to sympathetic neurons and chemoattractive guidance to GABAergic neurons (8 , 10 , 21) , is a chemoattractant for neural crest cells (22) , and positively regulates branching of the lung epithelium during development (23 , 24) . Ablation of the sema 3C gene in mice resulted in severe outflow tract abnormalities, i.e., persistent troncus arteriousus, aortic arch interruption, and mispatterning of intersomitic vessels (25) . Although genetic studies clearly indicate that sema 3C plays a distinct role in endothelial cell guidance and vascular morphogenesis, the mechanisms mediating these effects remain unknown. Similarly, the function of sema 3C in the kidney is unknown.

In the present studies, we tested the hypothesis that sema 3C regulates endothelial cell function using mouse glomerular endothelial cells (MGEC). We determined that sema 3C stimulates glomerular endothelial cell proliferation and survival and enhances their adhesion, directional migration, and tube formation in vitro by stimulating integrin activity and VEGF120 secretion. Data indicate that sema 3C functions in glomerular endothelial cells are similar to VEGF-A and opposite to sema 3A.

MATERIALS AND METHODS

Cell culture
MGEC, isolated and characterized previously (26) , were grown in MCDB 131 medium (Sigma) supplemented with 15% FBS, 150 µg/ml endomitogen, 1 ng/ml epidermal growth factor (EGF), 50 U/ml heparin-Na, 1 µg/ml hydrocortisone, and 50 U/ml pen/strep (MCDB 131 complete medium). Cells were maintained at 37°C in 5% CO2 and were starved 12 h in serum free MCDB medium before each experiment unless otherwise indicated.

Additives and production of recombinant sema 3C
Recombinant human VEGF165 (R&D Systems) and recombinant sema 3C, 3A, and 3F, generated in our laboratory (27) , were used for MGEC stimulation. Mouse sema 3C cDNA (NM_013657) was cloned into EcoRI/NotI of pHIS.Parallel vector (28) . E. coli BL21 DE3 Codon Plus (Stratagene) were transformed with pHis.Parallel/sema 3C and induced with 1 mM IPTG to produce the recombinant protein. The protein was then extracted from inclusion bodies in denaturing conditions (8 M Urea, 100 mM NaH2PO4, and 10 mM Tris-HCl) and refolded with a reducing buffer (6 M Urea, 100 mM NaCl, 50 mM Tris-HCl, 10 mM DTT, 1 mM ß-mercaptoethanol, and 1 mM EDTA) followed by an oxidation buffer (100 mM ClNa, 50 mM Tris-HCl, 10 mM cysteine, 2 mM cysteine, and 1 M L-arginine). Then, the protein was dialyzed against decreasing urea concentration (100 mM NaCl, 50 mM Tris-HCl, 192 mM glycine, and 7 mM urea) and concentrated using Amicon Ultra filters (Millipore).

Reverse-transcription and semiquantitative polymerase chain reaction
Total RNA was isolated from MGEC in control conditions and after 24 h exposure to sema 3C (360 ng/ml) using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA). cDNAs were generated using 2 µg total RNA and Superscript II reverse transcriptase (Invitrogen). Semiquantitative polymerase chain reaction (PCR) was performed at 94°C for 3 min once, followed by 25–30 cycles of 94°C for 45 s, 60°C for 30 s, and 72°C for 45 s using the following specific primers: sema 3C: 5'-GCA AAA TGG CTG GCA AAG ATC C-3' and 5'-CCC ATG AAA TCT ATA TAC ATT CC-3'; plexin A1: 5'-CCT CGA GAA CAA GAA CCA CCC CAA-3' and 5'-CCC TTC ACC GGC ACC TCA GGT GCA TT-3'; plexin A2: 5'-CCT CGA GAA CAA GAA CCA CCC CAA-3' and 5'-AAC ACC TTC ACT GGG ATC TCT GGA CTG TTC-3'; neuropilin 1: 5'-GAA GGC AAC AAC AAC TAT GA-3' and 5'-ATG CTC CCA GTG GCA GAA TG-3'; neuropilin 2: 5'-AAG TGG GGG AAG GAG ACT GT-3' and 5'- GTC CAC CTC CCA TCA GAG AA-3'. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) cDNA fragment was amplified as a loading control using the following primers 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3'. PCR products were separated on 1% agarose gels.

Cell proliferation assay
MGEC resuspended in MCDB 131 complete medium were plated on fibronectin coated (1 µg/ml) 96-well microtiter plates (1.5x104 cells/well) and allowed to attach at 37°C, 5% CO2 for 4 h. After adherent cells were washed three times with PBS, they were incubated for 24 h in the presence of BrdU (1:10,000) in serum free MCDB medium with or without appropriate stimuli (sema 3C or VEGF165) at 37°C, 5% CO2. MGEC proliferation was determined by measuring the amount of incorporated BrdU in the cells using a BrdU cell proliferation assay kit (Calbiochem, San Diego, CA).

Apoptosis assay
MGEC apoptosis was assessed using a FITC-conjugated marker (CaspACE FITC-VAD-FMK In Situ Marker; Promega) that binds to activated caspase 3, an in situ marker for apoptosis. Briefly, MGEC were plated on fibronectin coated (1 µg/ml) chamber glass slides (3x104 cells/chamber) and were grown in MCDB 131 complete medium for 24 h. At 40% confluency, cells were starved for 48 h in serum free MCDB medium either in the presence or in the absence of sema 3C (360 ng/ml) or for 24 h in the presence of cycloheximide (10 µg/ml) at 37°C, 5% CO2. At the end of this time period, CaspACE FITC-VAD-FMK In Situ Marker (10 µM) was added and cells were incubated for 20 min at 37°C protected from light. After being washed three times with PBS, cells were fixed in 10% buffered formalin for 30 min at room temperature, washed, and mounted with Fluromount G (Southern Biotech). Apoptotic cells were analyzed by fluorescence microscopy (Fluoview 300, Olympus). The percentages of apoptotic cells were calculated by counting at least 300 cells from two randomly selected fields for each condition in triplicate samples.

Cell adhesion assay
MGEC resuspended in serum free MCDB 131 medium with or without sema 3C or VEGF165 were plated (1.0x104 cells/0.1 ml/well) on 96-well tissue culture plates coated with either fibronectin or collagen I or gelatin (1 µg/ml). Cells were then allowed to attach for 30 min at 37°C, 5% CO2. After being washed three times with PBS, adherent MGEC were fixed in 10% buffered formalin for 20 min at room temperature, washed, and stained with 0.1% crystal violet. The number of attached cells per well was counted under light microscopy. In some experiments, MGEC resuspended in serum free medium were incubated for 20 min at 37°C, 5% CO2 in the presence or absence of anti-{alpha}5ß1 (BMA5; Chemicon) and anti-{alpha}Vß3 (LM609; Chemicon) function blocking antibodies (20 µg/ml).

Cell migration assay
MGEC were seeded on the top of 8 µm pore membrane of tissue culture inserts (Nunc; 3x103 cells/insert) in MCDB 131 complete medium and allowed to adhere for 1 h, and the adherent cells were starved in serum free MCDB medium for 2 h. Then, the medium outside the insert was switched to serum free medium with or without sema 3C (500 ng/ml), and cells were incubated for 3 h at 37°C, 5% CO2. The inserts were washed, and cells were fixed in 10% buffered formalin for 20 min and stained with 0.1% crystal violet. MGEC from the topside of inserts were removed using cotton swabs, and the cells that had migrated to the bottom side of inserts were counted under light microscopy.

Capillary-like network formation
MGEC (2x105 cells/gel) were plated on collagen I gels (1.2 mg/ml; Collaborative Research) with complete MCDB131 medium and allowed to attach for 3 h. Then, the medium was replaced with serum free medium +10 µM Cell-Tracker (Molecular Probes) + recombinant sema 3C (360 ng/ml) or medium +10 µM Cell-Tracker + vehicle (100 mM NaCl, 50 mM Tris-HCl, 192 mM glycine, 7 mM urea). Cells were imaged using phase and confocal microscopy (Olympus Fluoview FV300) at 12, 24, or 48 h.

Western blot analysis and immunoprecipitation
At 70% confluency, MGEC were starved for 12 h in serum free MCDB medium and then stimulated with sema 3C (360 ng/ml) and harvested at different time points using cell dissociation buffer (Life Technologies). Cells were lysed in modified radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.5 mM MgCl2, 5 mM NaP2O7, 20 mM HEPES, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 50 mM NaF, and 2 mM Na3VO4) with complete EDTA-free protease inhibitor (Roche Diagnostics, Indianapolis, IN). Protein concentration was determined using the bicinchoninic acid (BCA) assay (Sigma) following the manufacturer’s instructions. 100–150 µg protein per sample were resolved in 7% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), and blotted with anti-sema 3C (MAB#1728, R&D Systems), phosphospecific anti-ß1 integrin (pS785; BioSource, Camarillo, CA), anti-focal adhesion kinase F-actin (FAK; #05–537, Upstate), and antiphosphorylated tyr397 FAK (#44–624G, Biosource) antibodies. HRP-conjugated secondary antibodies and the enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ) were used to visualize protein signals. To demonstrate equal protein loading, blots were reprobed with antiactin antibody (Sigma). In additional experiments, MGEC resuspended in serum free medium were exposed to anti-{alpha}5ß1 (BMA5; Chemicon) or anti-{alpha}Vß3 (LM609; Chemicon) function blocking antibodies (20 µg/ml), anti-VEGF neutralizing Ab (1 µg/ml; #AF393, R&D Systems), anti-mouse VEGFR2 neutralizing Ab (1 µg/ml; MAB#4431, R&D Systems), anti-NP-1 neutralizing Ab (5 µg/ml; AF#566, R&D Systems), or anti-NP2 neutralizing Ab (5 µg/ml; AF#567, R&D Systems) for 45 min at 37°C, 5% CO2 prior to stimulation with sema 3C (360 ng/ml) for various periods of time (10 min to 24 h). To assess sema 3C and VEGF-A secretion, cell supernatants were collected at the end of the experiments, concentrated (x20–240) using Amicon Ultra filters (Millipore), and 20–50 µl samples were resolved by SDS-PAGE and immunoblotted with anti-sema 3C (MAB#1728, R&D Systems) or anti-VEGF (sc#507, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies as described above. For Immunoprecipitation, the concentrated supernatant (240x) was incubated with 5 µg/ml of antisema 3C monoclonal antibody for 4 h at 4°C, and then 50 µl of protein G-agarose (#1719416, Roche) were added and incubated overnight at 4°C in a tube rotator. Beads with the antigen-antibody complex were pelleted by centrifugation and washed three times with PBS. To release antigen-antibody complex, beads were resuspended in 2 x protein sample buffer and boiled 5 min at 100°C. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot.

RESULTS

We determined that our previously characterized mouse glomerular endothelial cells (26) express sema 3C mRNA and its signaling receptors, plexins A1, A2, and D1 mRNAs (Fig. 1 A), suggesting that sema 3C may have autocrine functions. We observed that sema 3C receptors’ mRNA expression is not regulated by ligand availability (Fig. 1A ). Sema 3C protein was detected in MGEC cell lysates and in their supernatant by Western blotting and immunoprecipitation, respectively. These data indicate that sema 3C is expressed and secreted by glomerular endothelial cells (Fig. 1B ). We documented the specificity of the sema 3C antibody by immunoblotting that detected recombinant sema 3C as a single band of the expected Mr ({approx}83 kDa) and no cross-reactivity with recombinant sema 3A and sema 3F (Fig. 1C ). We previously showed that MGEC also express the binding coreceptors neuropilin 1 and neuropilin 2 (29) .


Figure 1
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  		Figure 1. MGEC express sema 3C and its receptors: plexins and neuropilins. A) RT-PCR analysis of RNA isolated from untreated (control) or sema 3C-treated MGEC. cDNAs were amplified using specific primers for sema 3C, plexins, neuropilins, or GAPDH. 25 PCR cycles were used for sema 3C and NP1, and 30 cycles were used for NP2 and plexins. Amplified products were separated on TAE/agarose gels. B) Western blots (WB) showing sema 3C protein in MGEC lysates and supernatant; detection of secreted sema 3C required immunoprecipitation before immunoblotting (IP:WB). C) Western blot showing sema 3C Ab specificity: 800 ng of recombinant sema 3A, sema 3C, and sema 3F were resolved in 8% SDS gel and immunoblotted. Mr (kDa) is shown on right-hand side of immunoblots.

Cell adhesion assays were performed to determine the role of sema 3C. Sema 3C significantly increased glomerular endothelial cell adhesion to fibronectin and collagen I substrates, whereas it did not alter adhesion to gelatin, suggesting that integrins could mediate this effect. Sema 3C-induced adhesion was similar to that induced by VEGF165 (Fig. 2 A-C). Sema 3C dose-response experiments revealed maximal cell adhesion at 360 ng/ml (Fig. 2D ). To test whether integrins play a role in sema 3C-induced increased adhesion, cell adhesion experiments were performed in the presence or absence of ß1 and ß3 integrin function blocking antibodies (BMA5 and LM609, respectively). We observed that ß1 and ß3 integrin blocking antibodies prevented sema 3C-induced adhesion, indicating that integrins mediate this effect (Fig. 2E ). We determined that sema 3C induces a significant increase in ß1 integrin serine phosphorylation, as indicated by Western analysis with a phosphoserine-specific pS785 ß1 integrin antibody (Fig. 2F ). Sema 3C-induced ß1 integrin serine phosphorylation was not altered by prior exposure to anti-VEGFR2 neutralizing antibodies but was abolished by anti-NP1 and anti-NP2 neutralizing Ab (Fig. 2G ), suggesting that sema 3C effects on ß1 integrin phosphorylation are independent of VEGF-A and are likely mediated by NP/plexin signaling. We examined whether sema 3C signaling induced focal-adhesion kinase (FAK) tyrosine phosphorylation and found that neither FAK expression level nor its Tyr397 phosphorylation were altered by sema 3C (Fig. 2H ).


Figure 2
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  		Figure 2. Sema 3C stimulates MGEC adhesion and induces ß1 integrin phosphorylation. Adhesion of MGEC to fibronectin (A), collagen (B), and gelatin (C), at each substrate concentration of 1 µg/ml, was assessed in the absence (control) or in the presence of sema 3C (180 ng/ml) or VEGF (30 ng/ml). D) Dose response to sema 3C (180–480 ng/ml) showing that sema 3C regulates MGEC adhesion to fibronectin, *P < 0.05 control vs. sema 3C, **P < 0.05 sema 3C 480 vs. 180 ng/ml; E) MGEC adhesion to fibronectin (1 µg/ml) in the absence (control=gray bars) or in the presence of sema 3C (360 ng/ml; red bars) with or without antibodies blocking {alpha}5 ß1, {alpha}V ß3, or both {alpha}5 ß1 and {alpha}V ß3 integrins. Data are mean ± SE of 3 separate experiments, carried out in quadruplicate, *P < 0.05 control vs. {alpha}5 ß1 or {alpha}V ß3 blocking antibodies, #P < 0.05 sema 3C vs. {alpha}5 ß1 or {alpha}V ß3 blocking antibodies. F) Western blot showing a 2-fold increase in phosphorylated Ser-785ß1 integrin at 30 min, reprobed with an antiactin Ab to document equal loading. G) Western blot showing that NP1 and NP2 neutralizing antibodies prevent sema 3C-induced Ser-785ß1 integrin phosphorylation whereas VEGF and VEGFR2 neutralizing antibodies do not. H) Western blot showing phosphorylated Y397 FAK and total FAK showing that sema 3C did not alter FAK tyrosine phosphorylation. Western blots are representative of ≥3 separate experiments.

Next, we examined the effect of the sema 3C on endothelial cell proliferation and survival. Sema 3C induced glomerular endothelial cell proliferation in a dose-response manner (Fig. 3 A). Sema 3C-induced increase in endothelial cell proliferation was similar to that induced by VEGF-A (Fig. 3B ). Sema 3C reduced starvation-induced apoptosis by 46 ± 3% compared with control, as indicated by an in situ marker of activated caspase 3 (Fig. 4 A-D). In contrast, cycloheximide, used as a positive control, increased MGEC apoptosis by 192 ± 31%.


Figure 3
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  		Figure 3. Sema 3C induces MGEC proliferation. Subconfluent cultures of MGEC on fibronectin were incubated in serum free medium with or without sema 3C or VEGF-A in the presence of BrdU for 24 h. A) Dose-response curve to sema 3C (180–480 ng/ml); B) Sema 3C (360 ng/ml) and VEGF (30 ng/ml) stimulate MGEC proliferation similarly. Data are expressed as mean ± SD optical density (OD) at 450/550 nm (A), and mean ± SD percent changes above control BrdU incorporation (B), n = 3 independent experiments, quadruplicates were carried out for each condition, *P < 0.05 compared with control.


Figure 4
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  		Figure 4. Sema 3C reduces MGEC apoptosis. Subconfluent cultures of MGEC on fibronectin were incubated in serum free medium in the absence (control) or in the presence of sema 3C (360 ng/ml) for 48 h or cycloheximide (10 µg/ml) for 24 h; Apoptotic cells were detected by fluorescence microscopy using a FITC-conjugated in situ marker for activated caspase 3. Bar graph is mean ± SE percent change in the number of apoptotic cells/field sema 3C vs. control; n = 3 independent experiments.

We next assessed sema 3C effect on directional endothelial cell migration and tube formation. Sema 3C increased migration of endothelial cells by 44 ± 11% at 3 h, as compared with control (Fig. 5 A). Sema 3C significantly stimulated endothelial cell network and tube formation at 48 h (Fig. 5B,C ). Since sema 3C functions described herein are similar to VEGF-A functions, we asked whether sema 3C regulates VEGF-A expression and secretion by endothelial cells. We determined that sema 3C induced a >13-fold increase in VEGF120 secretion to the MGEC supernatant, whereas VEGF164 expression in MGEC lysates remained unchanged (Fig. 5D ). VEGF120 secretion was maximal after 30 min exposure to sema 3C, suggesting that it does not require protein synthesis (Fig. 5E ). Sema 3C-induced VEGF120 secretion was not prevented by ß1 or ß3 integrin function blocking antibodies (Fig. 5F ), indicating that integrins do not mediate VEGF120 secretion. Sema 3C-induced VEGF120 secretion was decreased by NP-1 and NP-2 neutralizing antibodies (Fig. 5G ) and was not altered by VEGFR2 neutralizing Ab (data not shown), suggesting that sema 3C regulates VEGF120 secretion via NP/ plexin signaling.


Figure 5
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  		Figure 5. Sema 3C induces MGEC directional migration, capillary-like network formation and VEGF-A secretion. A) Sema 3C (500 ng/ml) induced a 44 ± 11% increase in MGEC migration at 3 h. Data are mean ± SE cell counts from three independent experiments performed in duplicate. *P < 0.05 sema 3C vs. control; B) MEGC plated on collagen I gels in serum free medium for 48 h showing minimal network formation; C) MEGC plated on collagen I gels and exposed to sema 3C (360 ng/ml) for 48 h developed extensive capillary-like network. Images are representative of 3 independent experiments performed in triplicate. D) Western blot showing VEGF164 expression in control and sema 3C-treated MGEC cell lysates and VEGF120 secretion to the supernatant, demonstrating that sema 3C induces significant VEGF120 secretion. E) Time course of sema 3C-induced VEGF120 secretion. F) Western blot showing that VEGF120 secretion is not mediated by {alpha}5 ß1 or {alpha}V ß3 integrins. G) Western blot showing that VEGF120 secretion is mediated by NP2 signaling. All Western blots are representative of ≥3 separate experiments.

Taken together, the data indicate that sema 3C stimulates glomerular endothelial cell survival and proliferation and promotes its adhesion, migration, and vascular morphogenesis in vitro by inducing ß1 integrin phosphorylation and VEGF120 secretion via NP/plexin signaling.

DISCUSSION

In this study, we present evidence indicating that sema 3C is produced and secreted by glomerular endothelial cells and that sema 3C promotes endothelial cell survival and proliferation and stimulates cell adhesion, migration, and tube formation in vitro by inducing ß1 integrin serine phosphorylation and VEGF120 secretion via NP/plexin signaling.

Class 3 semaphorins modulate cell migration and provide site-specific guidance cues to neurons, neural crest, and endothelial cells by modulating integrin function and cytoskeletal dynamics (2 , 12) . The specificity of sema 3 signals results from the plexin-neuropilin present in the receptor complex (12) . Sema 3C binds to plexin A2 /NP-1 and to plexin A2/NP-2 complexes (13) . In addition, sema 3C binding increases significantly when NP-1 or NP-2 dimerize with plexin D1 (5) .

Expression studies reported sema 3C transcripts in the conotruncal area of the developing heart and in the aorta and pulmonary vessels, suggesting that sema 3C may provide attractive signals to migrating neural crest and endothelial cells (22) . Ablation of the sema 3C gene resulted in troncus arteriosus and interruption of the aortic arch and demonstrated that sema 3C chemoattractive cues are required for normal outflow tract development (25) . Disruption of plexin D1 revealed a similar phenotype, indicating that plexin D1 functions as an endothelial cell specific sema 3C receptor in vivo (5 , 7) . During nervous system development, sema 3C provides chemoattractive guidance cues to GABAergic migrating axons toward hippocampal interneurons and induces growth cone collapse of sympathetic neurons (8 , 10) . Sema 3C induces survival and neurite outgrowth of rat cerebellar granule neurons in culture (9) . Sema 3C was found to be up-regulated in tumor cell lines with invasive and metastatic behavior (30 31 32) and in sinovial fluid from rheumatoid arthritis patients (33) . Taken together, these studies suggest that sema 3C function promotes cell survival and provides chemoattractive cues in multiple cell types and chemorepulsive cues to some neurons. To our knowledge, the role of sema 3C signaling in endothelial cell function has not been examined previously.

The studies reported here revealed that sema 3C and its signaling receptors, plexins A1, A2, and D1 mRNAs, are expressed in mouse glomerular endothelial cells, suggesting a possible autocrine system. We had previously reported expression of the binding receptors, neuropilin 1 and 2, in these endothelial cells (29) . Previous reports did not detect sema 3C mRNA in human vein and rat aortic endothelial cells (20 , 34) . The discrepancy may be due to a species or vessel size difference in the origin of the endothelial cells (35) . We determined that mouse glomerular endothelial cells express and secrete sema 3C protein. Sema 3C is readily detected by immunoblotting in endothelial cell lysates. Conversely, sema 3C secretion in serum free, control conditions is low, requiring immunoprecipitation to be detected. The regulation of sema 3C expression and secretion remains unknown. Our data clearly indicate that sema 3C signals and has several functions in mouse glomerular endothelial cells: 1) stimulates proliferation and decreases apoptosis, and 2) enhances endothelial cell adhesion, migration, and capillary-like tube formation. We showed that sema 3C-induced increase in endothelial cell adhesion is prevented by ß1 and ß3 integrin function blocking antibodies and that there is a concomitant increase in ß1 integrin phosphorylation on Ser-785 within the time frame of the cell adhesion assay, suggesting that sema 3C function in vitro is mediated by integrins and, at least in part, by ß1 integrin serine785 phosphorylation. These findings are consistent with previous studies indicating that changes in the Ser-785 phosphorylation state modulate ß1 integrin function promoting cell adhesion (36 , 37) . Sema 3C effects on ß1 integrin appear to be independent of VEGF-A and mediated by NP/plexin signaling, as indicated by VEGFR2 and NP1 or NP2 neutralizing Ab experiments, respectively (see Fig. 2G ).

Sema 3C functions in endothelial cells are remarkably similar to well-established VEGF-A functions, e.g., positive regulator of cell proliferation and survival, migration, and tube assembly (38) . Thus, we explored a possible crosstalk between sema 3C and VEGF-A pathways. Data revealed that sema 3C strongly induces VEGF120 secretion by endothelial cells, documenting for the first time a mechanism linking sema 3C and VEGF-A signaling pathways. Time-course experiments showed a maximal stimulation within 30 min of sema 3C exposure and a lack of further VEGF120 accumulation, suggesting that protein synthesis is not required for VEGF120 secretion. VEGF120 secretion is not mediated by ß1 or ß3 integrins, as described in cancer cells (39) and appears to be mediated by a sema 3C-NP/plexin signaling mechanism because it is blocked by anti-NP-1 and anti-NP2 but not by anti-VEGFR2 neutralizing antibodies. VEGF120 does not compete with sema 3C for NP-1 receptor binding (40) . VEGF120 is freely diffusible and does not bind heparin or heparan sulfate proteoglycans; therefore, it does not generate concentration gradients (41) . Accordingly, it is unlikely that VEGF120 secretion may be responsible for sema 3C-induced cell migration. It is unclear whether sema 3C-induced secreted VEGF120 contributes to enhanced endothelial cell adhesion, but it likely promotes endothelial cell proliferation and survival, as previously reported (41 42 43) , and thereby may contribute to enhance sema 3C-induced capillary-like tube formation. Sema 3C does not alter the expression of VEGF164, the major VEGF-A isoform, nor does it induce its secretion. The tissue-specific regulation of VEGF-A isoforms is poorly understood, and VEGF-A secretion has been mostly assessed using ELISA assays that do not discriminate between VEGF120 and VEGF164 (42 , 43) . Thrombin was shown to induce VEGF120 secretion by megakaryocytes and platelets within the same time frame observed in our studies in endothelial cells (44) . Although the physiological role of sema 3C in modulating VEGF120 isoform needs to be further explored, the findings reported here suggest autocrine cooperation between sema 3C and VEGF-A that promotes endothelial cell homeostasis.

Sema 3C functions in endothelial cells described herein are opposite to those reported for sema 3A (6 , 20) . In addition, our data indicate that sema 3C signals differently from sema 3A. First, sema 3C enhanced ß1 integrin function by inducing its phosphorylation, whereas sema 3A inhibits integrin activity (20) . Second, sema 3C did not alter focal adhesion kinase activity (FAK), whereas sema 3A decreased FAK phosphorylation and up-regulated FRNK (Villegas, G., J. Rovin, J. Teichman, J. T. Parsons, and A. Tufro, unpublished observations). Third, sema 3C reduced endothelial cell apoptosis, whereas sema 3A impairs survival pathways and induces apoptosis (this study, refs. 6 , 27 ). Fourth, sema 3C and VEGF165 have similar functions (this study), whereas sema 3A and VEGF165 compete for NP-1 binding and have opposite effects on integrin activity (6 , 20 , 40 , 45) . Lastly, sema 3C induced VEGF120 secretion, whereas sema 3A did not (this study and data not shown).

In summary, we conclude that sema 3C is a positive regulator of endothelial cell function that mediates endothelial cell survival, proliferation, adhesion, migration, and tube formation in vitro by stimulating integrins and VEGF120 secretion. Sema 3C functions are similar to VEGF-A and opposite to sema 3A, suggesting that the combinatorial sum of these proteins’ gradients may be important for endothelial cell migration, morphogenesis, and remodeling in specific vascular beds.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grant RO1-DK-59333 and RO1-DK-64187 and O’Brien Center Grant P50-DK-064236 (A. Tufro).

Received for publication January 3, 2006. Accepted for publication May 15, 2006.

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