| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,1
Division of Nephrology, Departments of
* Internal Medicine and
Pediatrics, and
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
SPECIFIC AIMS
Class 3 semaphorins are secreted proteins that generate chemorepellent signals by inducing growth cone collapse of migrating axons, inhibiting endothelial cell migration, and establishing zones of exclusion for cell migration or induce chemoattraction by stimulating migration and survival of axons, neural crest, or epithelial or tumoral cells. Semaphorin 3C (sema 3C) provides chemorepulsive guidance to sympathetic neurons and chemoattractive guidance to GABAergic neurons, is a chemoattractant for neural crest cells, and positively regulates branching of the lung epithelium during development. 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. Although genetic studies clearly indicate that sema 3C plays a distinct role in endothelial cell guidance and vascular morphogenesis, the mechanisms mediating these effects are unknown.
We hypothesized that sema 3C may regulate endothelial cell function. The aims of this study were to 1) identify endothelial cell expression of sema 3C and its receptors and characterize their signaling, and 2) define the function(s) of sema 3C in endothelial cells.
PRINCIPAL FINDINGS
1. Sema 3C and its receptors are expressed in endothelial cells
We determined that previously characterized mouse glomerular endothelial cells (MGEC) express sema 3C mRNA and its signaling receptors, plexins A1, A2, and D1 mRNAs, suggesting that sema 3C may have autocrine functions. We previously showed that MGEC also express the binding coreceptors neuropilin 1 (NP-1) and neuropilin 2 (NP-2). In this study, we observed that mRNA expression of the sema 3C receptors is not regulated by ligand availability and we detected sema 3C protein 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. We documented the specificity of the sema 3C antibody by Western blotting detecting recombinant sema 3C as a single band of the expected Mr (
83 kDa) and absence of cross-reactivity with recombinant sema 3A and sema 3F.
2. Sema 3C is a positive regulator of endothelial cell function that stimulates integrins
We examined the effect of sema 3C on endothelial cell proliferation and survival. Exogenous recombinant sema 3C induced glomerular endothelial cell proliferation in a dose-response manner (180–480 ng/ml; n=4, P<0.05), as assessed by bromodeoxyuridine (BrdU) incorporation. Sema 3C (360 ng/ml) induced MGEC proliferation 18 ± 2% above control (n=3 independent experiments performed in quadruplicate, P<0.05). Sema 3C-induced increase in endothelial cell proliferation was similar to that induced by vascular endothelial growth factor (VEGF)-A (30 ng/ml; n=3, pNS). Using an in situ marker of activated caspase 3, we determined that sema 3C (360 ng/ml) reduced starvation-induced apoptosis by 46 ± 3% (n=3 independent experiments performed in quadruplicate, P<0.05).
It has been reported that sema 3A inhibits endothelial cell adhesion, migration, and vascular morphogenesis. To determine the role of sema 3C on endothelial cells, we first performed cell adhesion assays (Fig. 1
). 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 may mediate this function (Fig. 1A-C
). These changes were similar to those induced by VEGF165 (Fig. 1A, B
). A sema 3C dose response was observed with maximal cell adhesion at 360 ng/ml (Fig. 1D
). To test whether integrins play a role in sema 3C-induced increased adhesion, cell adhesion experiments were performed in the presence or absence of integrin function blocking antibodies (BMA5 and LM609, Chemicon). As shown in Fig. 1E
, anti-
5ßbeta;1 and anti-Vßbeta;3 integrin blocking antibodies prevented sema 3C-induced adhesion, indicating that integrins mediate this effect. Sema 3C induced a significant increase in ßbeta;1 integrin serine phosphorylation (Fig. 1F
), as indicated by Western analysis with a phospho-serine specific pS785 ßbeta;1 integrin antibody. Sema 3C-induced ßbeta;1 integrin serine phosphorylation was not altered by prior exposure to anti-VEGFR2 neutralizing Ab but was abolished by anti-NP1 and anti-NP2 neutralizing antibodies (Fig. 1G
), suggesting that sema 3C effects on ßbeta;1 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 tyrosine397 phosphorylation was altered by sema 3C (Fig. 1H
).
|
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. 2
A). Sema 3C significantly stimulated endothelial cell network and tube formation at 48 h (Fig. 2B, C
).
|
3. Sema 3C induces VEGF-A secretion by endothelial cells
Sema 3C functions in endothelial cells reported here are remarkably similar to well-established VEGF-A functions, e.g., positive regulator of cell proliferation and survival, migration, and tube assembly. Thus, 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. 2D
). The time course of VEGF120 secretion suggests it does not require protein synthesis (Fig. 2E
). Sema 3C-induced VEGF120 secretion was not prevented by ßbeta;1 or ßbeta;3 integrin function blocking antibodies (Fig. 2F
), suggesting that integrins do not mediate VEGF120 secretion. Sema 3C-induced VEGF120 secretion was not prevented by blockade of VEGFR2 signaling but was decreased by NP1 and NP2 neutralizing antibodies (Fig. 2G
), suggesting that sema 3C regulates VEGF120 secretion via NP/ plexin signaling. These data document for the first time a crosstalk between sema 3C and VEGF-A signaling pathways (see Fig. 3
).
|
CONCLUSIONS AND SIGNIFICANCE
To our knowledge, the specific functions of sema 3C in endothelial cells have not been examined previously. Here we report that 1) sema 3C is naturally expressed and secreted by endothelial cells, and 2) sema 3C stimulates glomerular endothelial cell survival and proliferation and promotes their adhesion, migration, and tube formation in vitro by inducing ßbeta;1 integrin phosphorylation and VEGF120 secretion. Mechanistically, we determined that sema 3C signaling stimulates integrin function via NP/plexin and induces VEGF-A secretion in a NP/plexin-dependent, integrin-independent manner, potentially generating a positive feedback loop to activate integrins and promote endothelial cell survival, migration, and tube formation (Fig. 3)
. Our findings imply a new paradigm involving sema 3C and VEGF-A similar functions and antagonistic functions between sema 3C and sema 3A in endothelial cells. The physiological consequences of the crosstalk between VEGF-A and sema 3C signaling are being actively examined.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5698fje
This article has been cited by other articles:
|
|
 |
|
  G. Serini, L. Napione, M. Arese, and F. Bussolino Besides adhesion: new perspectives of integrin functions in angiogenesis Cardiovasc Res, May 1, 2008; 78(2): 213 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Potiron, G. Sharma, P. Nasarre, J. A. Clarhaut, H. G. Augustin, R. M. Gemmill, J. Roche, and H. A. Drabkin Semaphorin SEMA3F Affects Multiple Signaling Pathways in Lung Cancer Cells Cancer Res., September 15, 2007; 67(18): 8708 - 8715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Liu, J. Wang, B. Kinzel, M. Mueller, X. Mao, R. Valdez, Y. Liu, and E. Li Dosage-dependent requirement of BMP type II receptor for maintenance of vascular integrity Blood, September 1, 2007; 110(5): 1502 - 1510. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
3 separate experiments.
