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

This Article Full Text (PDF) All Versions of this Article: 19/6/588 04-2430fjev1    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 Philippova, M. Articles by Resink, T. Search for Related Content PubMed PubMed Citation Articles by Philippova, M. Articles by Resink, T.

RhoA and Rac mediate endothelial cell polarization and detachment induced by T-cadherin

Maria Philippova^*, Danila Ivanov^*, Roy Allenspach^*, Yoh Takuwa, Paul Erne and Thérèse Resink^*,1

^* Department of Research, Cardiovascular Laboratories, Basel University Hospital, Basel, Switzerland;
Division of Physiology, Kanazawa University School of Medicine, Kanazawa, Japan; and
Division of Cardiology, Luzern Kantonsspital, Lucerne, Switzerland

¹Correspondence: Department of Research, Cardiovascular Laboratories, Basel University Hospital, Hebelstrasse 20, CH 4031 Basel, Switzerland. E-mail: therese-j.resink{at}unibas.ch

SPECIFIC AIMS

T-cadherin (T-cad) is an atypical GPI-anchored member of the cadherin superfamily, which does not mediate intercellular adhesion and has been proposed to function as a signaling receptor regulating directed cell migration. During embryogenesis T-cad acts as a negative guidance cue for extending neurites. In vascular tissue T-cad is up-regulated under conditions (atherosclerosis, restenosis, and tumor angiogenesis) associated with abnormal cell migration, growth, and phenotypic modulation. In cultured human umbilical vein endothelial cells (HUVEC), homophilic T-cad ligation induces cell polarization to a migratory phenotype, decreases cell adhesion to the substratum, and facilitates cell motility. Signaling mechanisms mediating T-cad effects have never been investigated. The present study aimed to elucidate a potential role for RhoA/Rho-kinase (ROCK) and Rac pathways in T-cad-induced endothelial cell polarization.

PRINCIPAL FINDINGS

1. RhoA activation is necessary but not sufficient for T-cad-induced cell detachment
HUVEC infected with empty adenoviral vector or with vectors expressing dominant-negative RhoA (N19RhoA) or constitutively active RhoA (RhoA63) were examined with respect to their ability to detach and polarize when plated on substratum containing soluble recombinant T-cad protein without GPI anchor used as a ligand mimicking homophilic T-cad interactions. Adhesion assay demonstrated that adherence of empty vector-infected HUVEC onto gelatine + T-cad substratum was decreased as compared to control gelatine + BSA substratum by 30% at 6 h (Fig. 1 A) and by 50% at 18 h after seeding (Fig. 1B ). Compared with cells adherent on control substratum, cells remaining attached on T-cad substratum were less spread, polarized, and frequently arranged in chains and tubular-like structures. Infection with N19RhoA impaired the ability of T-cad to induce cell detachment and elongation. Six hours after seeding, adhesion of N19RhoA-HUVEC on T-cad and control substrata was equivalent (Fig. 1A ). After 18 h, however, T-cad-induced detachment and elongation of N19RhoA-HUVEC was almost identical to that seen in empty vector cells (Fig. 1B ). RhoA inactivation thus delays the effects of T-cad engagement on adhesion and phenotype but does not abrogate it. Infection of HUVEC with constitutively active RhoA mutant RhoA63 decreases cell attachment and spreading on control substratum, "imitating" T-cad effects (Fig. 1A, B ). However, T-cad substratum is still able to further decrease the adhesion of RhoA63-HUVEC. Involvement of pathway(s) additional to RhoA is implicated in the transmission of T-cad detachment signals.

View larger version (35K): [in this window] [in a new window]   Figure 1. Inhibition of either RhoA or Rac partially prevents T-cad-induced EC detachment. Simultaneous repression of RhoA and Rac pathways completely eliminates EC deadhesion.HUVEC infected with empty adenoviral vector, dominant negative N19RhoA, dominant-active RhoA63, dominant negative N17Rac, and simultaneously with N19RhoA and dN17Rac were seeded on BSA (open bars) or T-cad (striped bars) substrata. Adhesion was measured after 6 h (A) and 18 h (B). *Significant difference in adhesion onto control vs. T-cad-containing substratum (P<0.01). #Significant difference in adhesion of empty- vs. RhoA63-vector infected HUVEC onto control substratum. Significant difference in adhesion of empty- vs. RhoA63- vector infected HUVEC onto T-cad substratum (P<0.01).

2. Both RhoA and Rac pathways are involved in T-cad-induced effects on cell adhesion and shape
Introduction of dominant-negative Rac1 (N17Rac) into HUVEC did not influence cell adhesion or phenotype on control substratum, but impaired T-cad-induced cell detachment and change in phenotype, these responses being completely absent 6 h after seeding (Fig. 1A ) but present after 18 h (Fig. 1B ). Thus similarly to inactivation of RhoA, Rac inactivation delays the ability of T-cad to induce deadhesion and phenotype alterations but does not eliminate it. Coinfection of HUVEC with N19RhoA and N17Rac completely abolished cell responses to T-cad engagement at both 6 and 18 h (Fig. 1A, B ). These data suggest that these two pathways are complementary in mediating changes in endothelial cell adhesion and phenotype after T-cad engagement.

3. T-cad-induced polarization and cytoskeletal changes in cells infected with RhoA and Rac mutants
Empty vector-, and RhoA-, or Rac-mutant-infected HUVEC were plated onto either control or T-cad-containing substratum and stained for actin with TRITC-labeled phalloidin. On control substratum, empty vector-infected cells displayed the spread morphology typical for endothelial cells (Fig. 2 A), whereas on T-cad substratum the cells acquired the migratory phenotype with thick longitudinal stress fibers at the lateral edges and prominent extensions with actin-rich lamellipodia at the leading edges (Fig. 2B, C ). Introduction of dominant-negative N19RhoA led to complete disappearance of stress fibers and improved spreading of cells seeded onto control substratum (Fig. 2D ). In contrast, constitutively active RhoA63 induced formation of very prominent stress fibers and a marked change in the shape of cells, which were contracted and unable to spread even on control substratum (Fig. 2G ). On T-cad substratum, RhoA63-HUVEC were also highly contracted with strong stress fibers, albeit more elongated than on control substratum, and tended to polarize and organize themselves in chains (Fig. 2H, I ). N19RhoA-HUVEC lacked stress fibers and were unable to elongate and contract, although T-cad engagement was still able to induce the formation of actin-rich lamellipodia at the leading edges of membrane protrusions (Fig. 2E, F ). These data suggest that RhoA activation mediates T-cad-promoted contraction and decrease in cell spreading but is not required for T-cad-induced polarization and lamellipodia assembly.

View larger version (69K): [in this window] [in a new window]   Figure 2. T-cad-induced shape and cytoskeletal changes in cells infected with RhoA and Rac mutants. Cells infected with empty vector (A–C), dominant-negative N19RhoA (D–F), constitutively active RhoA63 (G–I), dominant-negative N17Rac (J–L), dominant-negative N19RhoA and N17Rac simultaneously (M–O) were seeded onto BSA or T-cad substratum. Actin is stained with TRITC-labeled phalloidin, nuclei are counterstained with Hoechst. Bar: 20 µm.

HUVEC expressing dominant-negative N17Rac on control substratum displayed normal stress fiber formation but a significantly decreased level of background ruffling (Fig. 2J ). For N17Rac-HUVEC on a T-cad substratum, the stimulatory effects of T-cad on cell contraction and elongation were evident, but there was a complete absence of the actin-rich lamellipodia, membrane protrusions and well defined leading edges (Fig. 2K, L ). Coinfection of HUVEC with N19RhoA and N17Rac completely abolished T-cad effects on cell shape and actin rearrangement (Fig. 2N, O ). Thus, both RhoA and Rac are necessary for the T-cad-induced change in cell shape, with RhoA causing cell contraction and inhibition of spreading and Rac inducing polarization and lamellipodia formation.

4. Rho-kinase is necessary for T-cad-induced cell contraction and elongation
The participation of ROCK, a downstream effector of RhoA, was examined using adenoviral vectors expressing dominant-negative ROCK mutant RB and constitutively active ROCK mutant CAT, as well as specific ROCK inhibitor Y-27632. Inhibition of ROCK by RB mutant or by 10 µM Y-27632 prevented cell deadhesion in response to T-cad engagement and the cells were also markedly more spread than empty-vector cells on a T-cad substratum. TRITC-phalloidin staining in RB-expressing HUVEC revealed a critically reduced amount of stress fibers with prevention of T-cad-induced cell contraction and elongation, but not formation of protrusions and actin-rich lamellipodia at leading cell edges. Irreversible activation of ROCK by CAT resulted in a greater deadhesion response to T-cad engagement than that of empty-vector infected cells. CAT-HUVEC on a T-cad substratum displayed the general stellate appearance typical for ROCK hyperactivation on control substratum, but the cells were more polarized and frequently had prominent leading edges with membrane ruffles. These results are similar to that obtained for RhoA mutants and suggest that ROCK has a limited function in mediating T-cad effects, being necessary for cell contraction but not for polarization and membrane ruffling.

CONCLUSIONS AND SIGNIFICANCE

This study is the first revelation of signaling pathways mediating effects of atypical T-cad on cell adhesion and phenotype. T-cad-induced cell polarization includes two complementary components: RhoA/ROCK pathway is necessary for cell contraction, elongation, stress fiber assembly, and inhibition of spreading, while Rac is required for formation of membrane protrusions and actin-rich lamellipodia at the leading edges of polarized cells (Fig. 3 ). Individual repression of either pathway only partially prevented cell polarization and detachment, while simultaneous repression of RhoA and Rac pathways fully eliminated responses to homophilic T-cad ligation. The data may clarify the mechanisms whereby T-cad regulates vascular and neuronal cell migration, phenotype and guidance.

View larger version (16K): [in this window] [in a new window]   Figure 3. Both RhoA/ROCK and Rac pathways are necessary for T-cad ligation-induced effects on endothelial cell phenotype and adhesion.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2430fje;

This article has been cited by other articles:

				S. A. Dames, E. Bang, D. Haussinger, T. Ahrens, J. Engel, and S. Grzesiek Insights into the Low Adhesive Capacity of Human T-cadherin from the NMR Structure of Its N-terminal Extracellular Domain J. Biol. Chem., August 22, 2008; 283(34): 23485 - 23495. [Abstract] [Full Text] [PDF]

				M. Philippova, D. Ivanov, M. B. Joshi, E. Kyriakakis, K. Rupp, T. Afonyushkin, V. Bochkov, P. Erne, and T. J. Resink Identification of Proteins Associating with Glycosylphosphatidylinositol- Anchored T-Cadherin on the Surface of Vascular Endothelial Cells: Role for Grp78/BiP in T-Cadherin-Dependent Cell Survival Mol. Cell. Biol., June 15, 2008; 28(12): 4004 - 4017. [Abstract] [Full Text] [PDF]

				G. R. Uhl, Q.-R. Liu, T. Drgon, C. Johnson, D. Walther, J. E. Rose, S. P. David, R. Niaura, and C. Lerman Molecular Genetics of Successful Smoking Cessation: Convergent Genome-Wide Association Study Results Arch Gen Psychiatry, June 1, 2008; 65(6): 683 - 693. [Abstract] [Full Text] [PDF]

				L. W. Hebbard, M. Garlatti, L. J.T. Young, R. D. Cardiff, R. G. Oshima, and B. Ranscht T-cadherin Supports Angiogenesis and Adiponectin Association with the Vasculature in a Mouse Mammary Tumor Model Cancer Res., March 1, 2008; 68(5): 1407 - 1416. [Abstract] [Full Text] [PDF]

				G. R. Uhl, T. Drgon, Q.-R. Liu, C. Johnson, D. Walther, T. Komiyama, M. Harano, Y. Sekine, T. Inada, N. Ozaki, et al. Genome-Wide Association for Methamphetamine Dependence: Convergent Results From 2 Samples Arch Gen Psychiatry, March 1, 2008; 65(3): 345 - 355. [Abstract] [Full Text] [PDF]

				M. B. Joshi, D. Ivanov, M. Philippova, P. Erne, and T. J. Resink Integrin-linked kinase is an essential mediator for T-cadherin-dependent signaling via Akt and GSK3{beta} in endothelial cells FASEB J, October 1, 2007; 21(12): 3083 - 3095. [Abstract] [Full Text] [PDF]

				P. Riou, R. Saffroy, C. Chenailler, B. Franc, C. Gentile, E. Rubinstein, T. Resink, B. Debuire, D. Piatier-Tonneau, and A. Lemoine Expression of T-cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma FASEB J, November 1, 2006; 20(13): 2291 - 2301. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/6/588 04-2430fjev1    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 Philippova, M. Articles by Resink, T. Search for Related Content PubMed PubMed Citation Articles by Philippova, M. Articles by Resink, T.