HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 18/3/592 03-0957fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by BARBERIS, D. Articles by TAMAGNONE, L. Search for Related Content PubMed PubMed Citation Articles by BARBERIS, D. Articles by TAMAGNONE, L.

Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility ¹

Institute for Cancer Research and Treatment, University of Torino School of Medicine, Candiolo, Italy; and
^* Cancer Research UK Receptor Structure Group, Wellcome Building of Genomic Medicine, Oxford, UK

²Correspondence: Institute for Cancer Research and Treatment, University of Torino, SP. 142, 10060 Candiolo (TO), Italy. E-mail: luca.tamagnone{at}ircc.it

SPECIFIC AIMS

The molecular mechanisms mediating semaphorin are poorly understood. We hypothesized that semaphorins could regulate lamellipodia extension and cell motility by hampering cell adhesion to the extracellular matrix (ECM).

PRINCIPAL FINDINGS

1. The cytoplasmic domain of Plexin-B1 mediates retraction of lamellipodia and cell rounding induced by Semaphorin 4D
Plexins encode receptors for semaphorins, a family of molecular signals implicated in developmental morphogenesis, angiogenesis, and cancer progression. The plexin gene family includes nine members in vertebrates, grouped into four subfamilies (plexins A to D).

We analyzed the functional response mediated by Semaphorin 4D (Sema4D) in cells expressing its specific receptor, Plexin-B1. Plexin-B1 signaling in spread cells induces a rapid and generalized retraction of pseudopodia and lamellipodia, leading to cell rounding ("cellular collapse"). Time-lapsed movies of two representative experiments in mouse fibroblasts are provided as supplemental data (available at http://www.fasebj.org). It has been shown that Sema3A elicits the collapse of cells expressing Neuropilin-1 and Plexin-A1 (forming Sema3A functionally competent receptor complexes). We concluded that Plexin-B1 alone is fully functional and coexpression of neuropilins is not required. By analyzing truncated and chimeric receptors, we demonstrated that the cytoplasmic domain of plexins is required and sufficient to mediate the collapsing response.

2. Plexin-mediated cellular collapse does not require the activity of Rho-dependent kinase or myosin-mediated contraction of stress fibers whereas it is antagonized by stiff focal adhesive structures
The activation of RhoA signaling pathway has been implicated in the axonal collapse mediated by plexins of the B subfamily, but not by other family members. The suggested mechanism is by myosin-mediated pulling on F-actin cables, triggered by the effector molecule Rho-dependent kinase (ROCK). Surprisingly, we found that the collapsing response mediated by Plexin-B1 in fibroblasts is not blocked by a selective ROCK inhibitor (Y-27632) or by an inhibitor of myosin light-chain kinase (ML-7). Actin depolymerization induced by cytochalasin D did not interfere with the cellular collapse whereas stabilization of F-actin cables by treatment with jasplakinolide antagonized the functional response.

Cytoskeletal dynamics are functionally linked to integrin-mediated adhesion. Plexin-mediated cell collapse was inhibited by lysophosphatidic acid (LPA), an activator of Rho signaling known to induce larger and more abundant focal adhesions. We found that the cellular collapse mediated by Plexin-B1 is hampered by inhibition of 12/15-lipoxigenase, whose eicosanoid products are known to release cell-substrate adhesion.

3. Plexin activation by semaphorins rapidly induces disassembly of integrin-based adhesive structures, followed by F-actin depolymerization
We analyzed the early events affecting actin cytoskeleton and focal adhesive complexes of 3T3 mouse fibroblasts on Plexin-B1 activation. Cells growing in the presence of serum displayed a prominent actin meshwork of stress fibers and typical clustering of focal adhesion components, such as paxillin (shown in Fig. 1 ). Upon treatment with purified Sema4D, focal adhesion components were dispersed in 1–2 min and actin stress fibers disappeared after 5 min. Then, pseudopodia and lamellipodia retracted and cells became rounded featuring the overt collapsed phenotype.

View larger version (78K): [in this window] [in a new window]   Figure 1. A) NIH-3T3 fibroblasts expressing Plexin-B1 were treated with 5 nM Sema4D. Focal adhesions and F-actin cables are revealed by immunofluorescence with anti-paxillin antibodies and staining with phalloidin-FITC, respectively. Focal adhesion was dispersed after 1 min; F-actin cables disappeared after 5 min. Cellular collapse ensued after 10 min. Quantification of the effects (shown on the right) was done by scoring the presence of focal adhesions and stress fibers in 2 duplicated wells (n>100). Scale bar: 40 µm. B) NIH-3T3 fibroblasts, mock transfected or expressing the indicated receptors, were treated with Sema4D for 2 or 10 min and analyzed by immunofluorescence staining with anti-paxillin antibodies. Scale bar: 40 µm.

Cells expressing a chimeric Plexin-B1/A1 receptor were studied to compare the functional response elicited by the cytoplasmic domain of two different plexins. Intracellular signaling elicited by Plexin-B1 or Plexin-A1 led to rapid disassembly of adhesive structures (compare Fig. 1A, B ) and cellular collapse. We analyzed the functional response of a receptor mutated, in the conserved arginine 1687 (Plexin-B1-R1mut). Disassembly of focal adhesive structures and stress fibers, as well as the cellular collapse were abrogated, suggesting these events depend on the same signaling pathway (Fig. 1B ). Furthermore, Sema4D stimulation dispersed integrin-based focal adhesive complexes in cells expressing endogenous Plexin-B1, such as mammary carcinoma cells SKBR3 and neuroblastoma cells NB100. Receptor specificity of the functional response was demonstrated by using anti-Plexin-B1 blocking antibodies.

4. Plexin activation inhibits integrin-based adhesion, lamellipodia extension, and cell migration on the extracellular matrix
We assayed whether the disassembly of focal adhesive complexes mediated by plexin activation could hinder substrate adhesion of suspended cells. As shown in Fig. 2 A, plexin activation soon inhibited the adhesion to fibronectin of 3T3 fibroblasts while not interfering with adhesion to the integrin-independent substrate poly-L-lysine. As shown in Fig. 2A , at early time points, plexin activation inhibited the adhesion to fibronectin of 3T3 fibroblasts, while not interfering with adhesion to the integrin-independent substrate poly-L-lysine. At later times, many cells could adhere also in presence of the semaphorin; however they never spread on the substrate. Instead of forming lamellipodia, semaphorin-treated cells displayed several long and branched extensions (Fig. 2B ). Sema4D inhibited substrate adhesion and spreading of cells expressing chimeric receptor Plexin-B1/A1, but not of cells expressing a truncated receptor lacking the cytosolic domain or the inactive receptor Plexin-B1-R1mut (not shown). We observed semaphorin-dependent inhibition of substrate adhesion and cell spreading in epithelial SKBR3 cells expressing endogenous Plexin-B1 (not shown). That semaphorin treatment blocks cell spreading suggests that plexin signaling impinges on the functional coupling between integrin-mediated adhesion and cytoskeletal dynamics.

View larger version (85K): [in this window] [in a new window]   Figure 2. A, B) NIH-3T3 cells expressing the indicated proteins were harvested with 1 mM EDTA and seeded in microtiter wells coated with 10 µg/mL fibronectin or 1 µg/mL poly-L-lysine, PLL. After a period in the presence or absence of 5 nM Sema4D, adherent cells were fixed and stained with crystal violet. Cell adhesion was measured by eluting the dye and reading the absorbance at 595 nm. Before that, the percentage of cell spreading at 1 h was determined by evaluating >100 cells in 2 distinct wells; micrographs at bottom depict representative fields of cells stimulated with Sema4D. Results are representative of at least 3 experiments performed in duplicate. C) Haptotactic migration of engineered NIH-3T3 cells on a gradient of fibronectin in the absence of calf serum and the presence or absence of 5 nM Sema4D. After 4 h, migrated cells were fixed and stained with crystal violet. Cell migration was quantified by A595nm.

We tested whether plexin-dependent inhibition of integrin-based adhesion would lead to reduced cell migration on a gradient of ECM substrate ("haptotaxis"). Whereas NIH-3T3 cells efficiently moved along a gradient of fibronectin, plexin activation inhibited this haptotactic migration (Fig. 2D ). Consistent with the results on adhesion and spreading, Plexin-B1 and Plexin-A1 cytoplasmic domains were able to mediate this response.

CONCLUSIONS

The role of semaphorins as regulators of cell migration has been addressed only recently. Most prominent phenotypes observed in semaphorin-deficient mice have been attributed to defective migration of neural crest cells and endothelial cells.

We postulated that semaphorins could mediate a localized retraction of pseudopodia on the leading edge of migrating cells, affecting their motility. What are the cellular mechanisms mediating this function? A role for Rho GTPase has been proposed, leading to the activation of Rho-dependent kinase and to myosin-dependent contraction of F-actin cables. Our results do not confirm this hypothesis. Instead, we show here that semaphorin stimulation rapidly induces disassembly of focal adhesive structures and of F-actin cables in adherent cells. Plexin activation inhibits cell adhesion, lamellipodia extension, and cell migration. We suggest that this mechanism may function as a molecular clutch, putting on hold adhesion-dependent protrusion of cellular processes and redirecting cell migration or axonal extension. Although further studies are required to establish the functional role in vivo of this mechanism, our findings indicate that it could account for the regulation of cell migration and axon guidance by semaphorins.

View larger version (36K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proposed mechanism mediating plexin function. The conserved cytoplasmic domain of plexins mediates a fast disassembly of adhesive structures and loss of integrin-mediated adhesion. This functional response is hampered by strong adhesion to the ECM by a selective inhibitor of 12/15 lipoxygenase (CDC) and by lysophosphatidic acid (LPA), known to activate Rho signaling pathway and stabilize focal adhesive structures. Although plexins of B subfamily have been shown to activate the Rho signaling pathway, we report that an inhibitor of Rho-dependent kinase (Y-27632) does not block semaphorin-induced cell rounding and collapse.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0957fje;

This article has been cited by other articles:

				J. R. Sierra, S. Corso, L. Caione, V. Cepero, P. Conrotto, A. Cignetti, W. Piacibello, A. Kumanogoh, H. Kikutani, P. M. Comoglio, et al. Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages J. Exp. Med., July 7, 2008; 205(7): 1673 - 1685. [Abstract] [Full Text] [PDF]

				J. M. Swiercz, T. Worzfeld, and S. Offermanns ErbB-2 and Met Reciprocally Regulate Cellular Signaling via Plexin-B1 J. Biol. Chem., January 25, 2008; 283(4): 1893 - 1901. [Abstract] [Full Text] [PDF]

				O. G.-W. Wong, T. Nitkunan, I. Oinuma, C. Zhou, V. Blanc, R. S. D. Brown, S. R. J. Bott, J. Nariculam, G. Box, P. Munson, et al. Plexin-B1 mutations in prostate cancer PNAS, November 27, 2007; 104(48): 19040 - 19045. [Abstract] [Full Text] [PDF]

				L. Chung, T.-L. Yang, H.-R. Huang, S.-M. Hsu, H.-J. Cheng, and P.-H. Huang Semaphorin signaling facilitates cleft formation in the developing salivary gland Development, August 15, 2007; 134(16): 2935 - 2945. [Abstract] [Full Text] [PDF]

				S. Deng, A. Hirschberg, T. Worzfeld, J. Y. Penachioni, A. Korostylev, J. M. Swiercz, P. Vodrazka, O. Mauti, E. T. Stoeckli, L. Tamagnone, et al. Plexin-B2, But Not Plexin-B1, Critically Modulates Neuronal Migration and Patterning of the Developing Nervous System In Vivo J. Neurosci., June 6, 2007; 27(23): 6333 - 6347. [Abstract] [Full Text] [PDF]

				A. Rody, U. Holtrich, R. Gaetje, M. Gehrmann, K. Engels, G. von Minckwitz, S. Loibl, R. Diallo-Danebrock, E. Ruckhaberle, D. Metzler, et al. Poor Outcome in Estrogen Receptor-Positive Breast Cancers Predicted by Loss of Plexin B1 Clin. Cancer Res., February 15, 2007; 13(4): 1115 - 1122. [Abstract] [Full Text] [PDF]

				L. Zhu, W. Bergmeier, J. Wu, H. Jiang, T. J. Stalker, M. Cieslak, R. Fan, L. Boumsell, A. Kumanogoh, H. Kikutani, et al. Regulated surface expression and shedding support a dual role for semaphorin 4D in platelet responses to vascular injury PNAS, January 30, 2007; 104(5): 1621 - 1626. [Abstract] [Full Text] [PDF]

				J. C. Gatlin, A. Estrada-Bernal, S. D. Sanford, and K. H. Pfenninger Myristoylated, Alanine-rich C-Kinase Substrate Phosphorylation Regulates Growth Cone Adhesion and Pathfinding Mol. Biol. Cell, December 1, 2006; 17(12): 5115 - 5130. [Abstract] [Full Text] [PDF]

				J. D. Berndt and M. C. Halloran Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain Development, October 15, 2006; 133(20): 3983 - 3992. [Abstract] [Full Text] [PDF]

				N. Banu, J. Teichman, M. Dunlap-Brown, G. Villegas, and A. Tufro Semaphorin 3C regulates endothelial cell function by increasing integrin activity FASEB J, October 1, 2006; 20(12): 2150 - 2152. [Abstract] [Full Text] [PDF]

				I. Oinuma, H. Katoh, and M. Negishi Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating {beta}1 integrin activity J. Cell Biol., May 22, 2006; 173(4): 601 - 613. [Abstract] [Full Text] [PDF]

				D. Barberis, A. Casazza, R. Sordella, S. Corso, S. Artigiani, J. Settleman, P. M. Comoglio, and L. Tamagnone p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling J. Cell Sci., October 15, 2005; 118(20): 4689 - 4700. [Abstract] [Full Text] [PDF]

				H. Kashiwagi, M. Shiraga, H. Kato, T. Kamae, N. Yamamoto, S. Tadokoro, Y. Kurata, Y. Tomiyama, and Y. Kanakura Negative regulation of platelet function by a secreted cell repulsive protein, semaphorin 3A Blood, August 1, 2005; 106(3): 913 - 921. [Abstract] [Full Text] [PDF]

				C. Christensen, N. Ambartsumian, G. Gilestro, B. Thomsen, P. Comoglio, L. Tamagnone, P. Guldberg, and E. Lukanidin Proteolytic Processing Converts the Repelling Signal Sema3E into an Inducer of Invasive Growth and Lung Metastasis Cancer Res., July 15, 2005; 65(14): 6167 - 6177. [Abstract] [Full Text] [PDF]

				T. Walzer, L. Galibert, and T. De Smedt Dendritic cell function in mice lacking Plexin C1 Int. Immunol., July 1, 2005; 17(7): 943 - 950. [Abstract] [Full Text] [PDF]

				P. Conrotto, D. Valdembri, S. Corso, G. Serini, L. Tamagnone, P. M. Comoglio, F. Bussolino, and S. Giordano Sema4D induces angiogenesis through Met recruitment by Plexin B1 Blood, June 1, 2005; 105(11): 4321 - 4329. [Abstract] [Full Text] [PDF]

				V. Potiron and J. Roche Class 3 Semaphorin Signaling: The End of a Dogma Sci. Signal., May 24, 2005; 2005(285): pe24 - pe24. [Abstract] [Full Text] [PDF]

				M. L. Lemons, S. Barua, M. L. Abanto, W. Halfter, and M. L. Condic Adaptation of Sensory Neurons to Hyalectin and Decorin Proteoglycans J. Neurosci., May 18, 2005; 25(20): 4964 - 4973. [Abstract] [Full Text] [PDF]

				T. Walzer, L. Galibert, M. R. Comeau, and T. De Smedt Plexin C1 Engagement on Mouse Dendritic Cells by Viral Semaphorin A39R Induces Actin Cytoskeleton Rearrangement and Inhibits Integrin-Mediated Adhesion and Chemokine-Induced Migration J. Immunol., January 1, 2005; 174(1): 51 - 59. [Abstract] [Full Text] [PDF]

				I. Oinuma, H. Katoh, and M. Negishi Molecular Dissection of the Semaphorin 4D Receptor Plexin-B1-Stimulated R-Ras GTPase-Activating Protein Activity and Neurite Remodeling in Hippocampal Neurons J. Neurosci., December 15, 2004; 24(50): 11473 - 11480. [Abstract] [Full Text] [PDF]

				G. Serini and F. Bussolino Common Cues in Vascular and Axon Guidance Physiology, December 1, 2004; 19(6): 348 - 354. [Abstract] [Full Text] [PDF]

				I. Oinuma, Y. Ishikawa, H. Katoh, and M. Negishi The Semaphorin 4D Receptor Plexin-B1 Is a GTPase Activating Protein for R-Ras Science, August 6, 2004; 305(5685): 862 - 865. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 18/3/592 03-0957fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by BARBERIS, D. Articles by TAMAGNONE, L. Search for Related Content PubMed PubMed Citation Articles by BARBERIS, D. Articles by TAMAGNONE, L.