Korean J Physiol Pharmacol.  2008 Oct;12(5):225-230. 10.4196/kjpp.2008.12.5.225.

Netrin-1 Specifically Enhances Cell Spreading on Fibronectin in Human Glioblastoma Cells

• Lee HK
• Seo IA
• Shin YK
• Lee SH
• Seo SY
• Suh DJ
• Park HT

• KMID: 1486103
• DOI: http://doi.org/10.4196/kjpp.2008.12.5.225

Abstract

Netrins are secreted molecules and involved in axon guidance, cell migration and tumor development. Recent studies revealed that netrins perform novel functions in such processes as epithelial development and angiogenesis without operating through the classical netrin receptors, DCC (Deleted in Colorectal Cancer) and Unc5h. In the present study, we investigated the roles of netrin-1 and its receptors in cell spreading of human glioblastoma cells, and found that netrin-1 haptotactically enhanced fibronectin-induced cell spreading and focal adhesion formation in U373 glioblastoma cells. Netrin-1 binding to the U373 cell membrane was blocked by an antibody against alpha v integrin subunit, but not by an anti-DCC or anti-Unc5h antibody. In addition, enhancement of the fibronectin response by netrin-1 was abrogated by a function blocking antibody against integrin alpha v beta 3. Since the alpha v subunit of the integrin family plays an important role in the pathophysiological aspects of cell migration, including tumor angiogenesis and metastasis, our data provide important insight into the molecular mechanism of netrin function.

Keyword

Netrin; Fibronectin; Spreading; Integrin alpha v; Glioblastoma

MeSH Terms

Axons
Cell Membrane
Cell Movement
Fibronectins
Focal Adhesions
Glioblastoma
Humans
Integrin alphaV
Integrin alphaVbeta3
Neoplasm Metastasis
Nerve Growth Factors
Receptors, Cell Surface
Tumor Suppressor Proteins
Fibronectins
Integrin alphaV
Integrin alphaVbeta3
Nerve Growth Factors
Receptors, Cell Surface
Tumor Suppressor Proteins

Figure

• 

  Fig. 1. Netrin-1 enhances fibronectin-induced cell spreading. (A) U373 cells were attached to ECM protein-coated dishes for 1 h, and photographed. Netrin-1 (Net, 500 ng/ml) alone had no positive effect on cell spreading, but netrin-1 (50 ng/ml) enhanced fibronectin (FN)-induced cell spreading. (B) The extent of cell spreading was analyzed by measuring areas occupied by randomly selected cells 1 h after plating. C, no coating; N, netrin-1 (500 ng/ml); VN, vitronectin; FN, fibronectin; LA, laminin. Values shown are means±s.d. from three independent experiments. ∗∗p<0.01.

• 

  Fig. 2. Netrin-1 enhances focal adhesion formation without affecting FAK activation. (A) U373 cells were attached to ECM protein-coated dishes for 2 h, fixed and immunostained with an antibody to phosphotyrosine to localize focal adhesion. Arrows indicate focal adhesions, and asterisks indicate the areas magnified in the panels to the right. (B) U373 cells were attached to ECM protein-coated dishes for 1 h, and cell lysates were immunoblotted with an anti-phospho-FAK (pY397) antibody. FN, fibronectin; Net, netrin-1.

• 

  Fig. 3. Netrin enhancement of cell spreading on fibronectin is blocked by a function blocking antibody against α v β3. (A) A live cell binding assay with netrin-1-His (Net) demonstrates specific membrane binding of netrin-1-His in controls. Pretreatment with a function blocking antibody against α 3 or α v significantly reduced netrin-1 binding, whereas anti-α 5 or anti-Unc5h3 did not affect the cell surface binding of netrin-1-His. (B) Cell spreading on fibronectin (FN) or fibronectin/netrin-1 (FN/Net) substrate was inhibited by incubation with an antibody to α v β3. (C) Quantitative result showing the specific effect of an antibody to α v β3 on cell spreading. AB, antibody; mIg, mouse Ig G. Values shown are means±s.d. from three independent experiments. ∗∗p<0.01.

Reference

Barry ST., Critchley DR. The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions. J Cell Sci. 107:2033–2045. 1994.

Cirulli V., Yebra M. Netrins: beyond the brain. Nat Rev Mol Cell Bio. 8:296–306. 2007.

Colamarino SA., Tessier-Lavigne M. The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Cell. 81:621–629. 1995.

Danen EH., Yamada KM. Fibronectin, integrins, and growth control. J Cell Physiol. 189:1–13. 2001.

Eliceiri BP., Cheresh DA. Role of alpha v integrins during angiogenesis. Cancer J. 3:S245–249. 2000.

Hong K., Hinck L., Nishiyama M., Poo MM., Tessier-Lavigne M., Stein E. A ligand gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell. 97:927–941. 1999.

Keino-Masu K., Masu M., Hinck L., Leonardo ED., Chan SS., Culotti JG., Tessier-Lavigne M. Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell. 87:175–185. 1996.

Kennedy TE., Serafini T., dela Torre Jr., Tessier-Lavigne M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell. 78:425–435. 1994.

Kim TH., Lee HK., Seo IA., Bae HR., Suh DJ., Wu J., Rao Y., Hwang KG., Park HT. Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin-proteasome pathway in the embryonic cortical neuron. J Neurochem. 95:1–8. 2005.

Lee HK., Seo IA., Park HK., Park HT. Identification of the basement membrane protein nidogen as a candidate ligand for tumor endothelial marker 7 in vitro and in vivo. FEBS Lett. 580:2253–2257. 2006.

Leonardo ED., Hinck L., Masu M., Keino-Masu K., Ackerman SL., Tessier-Lavigne M. Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature. 386:833–838. 1997.

Liu G., Beggs H., Jürgensen C., Park HT., Tang H., Gorski J., Jones KR., Reichardt LF., Wu J., Rao Y. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction. Nat Neurosci. 7:1222–1232. 2004.

Ly NP., Komatsuzaki K., Fraser IP., Tseng AA., Prodhan P., Moore KJ., Kinane TB. Netrin-1 inhibits leukocyte migration in vitro and in vivo. Proc Natl Acad Sci USA. 102:14729–14734. 2005.

Park HT., Wu J., Rao Y. Molecular control of neuronal migration. Bioessays. 24:821–827. 2002.

Park KW., Crouse D., Lee M., Karnik SK., Sorensen LK., Murphy KJ., Kuo CJ., Li DY. The axonal attractant Netrin-1 is an angiogenic factor. Proc Natl Acad Sci USA. 101:16210–16215. 2004.

Rodrigues S., De Wever O., Bruyneel E., Rooney RJ., Gespach C. Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis. Oncogene. 26:5615–5625. 2007.

Schneiders FI., Maertens B., Böse K., Li Y., Brunken WJ., Paulsson M., Smyth N., Koch M. Binding of netrin-4 to laminin short arms regulates basement membrane assembly. J Biol Chem. 282:23750–23758. 2007.

Serafini T., Kennedy TE., Galko MJ., Mirzayan C., Jessell TM., Tessier-Lavigne M. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell. 78:409–424. 1994.

Serini G., Valdembri D., Bussolino F. Integrins and angiogenesis: a sticky business. Exp Cell Res. 312:651–658. 2006.

Wheeler AP., Ridley AJ. RhoB affects macrophage adhesion, integrin expression and migration. Exp Cell Res. 313:3505–3516. 2007.

Wilson BD., Ii M., Park KW., Suli A., Sorensen LK., Larrieu-Lahargue F., Urness LD., Suh W., Asai J., Kock GA., Thorne T., Silver M., Thomas KR., Chien CB., Losordo DW., Li DY. Netrins promote developmental and therapeutic angiogenesis. Science. 313:640–644. 2006.

Yebra M., Montgomery AM., Diaferia GR., Kaido T., Silletti S., Perez B., Just ML., Hildbrand S., Hurford R., Florkiewicz E., Tessier-Lavigne M., Cirulli V. Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration. Dev Cell. 5:695–707. 2003.