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Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow.

Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O, Kinashi T - J. Cell Biol. (2003)

Bottom Line: However, the key regulatory molecules regulating this process have remained elusive.Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration.Spa1 effectively suppressed this polarization after SLC treatment.

View Article: PubMed Central - PubMed

Affiliation: Bayer-chair, Dept. of Molecular Immunology and Allergy, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.

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Effects of the Ras/Rho family of GTPases on adhesion and migration on immobilized ICAM-1. (A) Effects of the Ras/Rho family on the adhesion of BAF/LFA-1 to ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for adhesion on immobilized human ICAM-1–Fc. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1 and measured as in Fig. 3. The mean and SE of triplicate determinations are shown. (B) Effects of the Ras/Rho family on the migration of BAF/LFA-1 on ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for migration on immobilized human ICAM-1–Fc as described in Materials and methods. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1.
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fig4: Effects of the Ras/Rho family of GTPases on adhesion and migration on immobilized ICAM-1. (A) Effects of the Ras/Rho family on the adhesion of BAF/LFA-1 to ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for adhesion on immobilized human ICAM-1–Fc. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1 and measured as in Fig. 3. The mean and SE of triplicate determinations are shown. (B) Effects of the Ras/Rho family on the migration of BAF/LFA-1 on ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for migration on immobilized human ICAM-1–Fc as described in Materials and methods. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1.

Mentions: Then, we compared the Rap1V12 promigratory effect with other Ras/Rho family GTPases. We used a proB cell line, BAF/3, reconstituted with human LFA-1 (BAF/LFA-1; Katagiri et al., 2000), which endogenously expresses CXCR4 and migrates on immobilized ICAM-1 in response to SDF-1 stimulation (Fig. 4, A and B) . Rap1V12 expression in BAF/LFA-1 stimulated both adhesion and migration (Fig. 4, A and B) at a migratory speed of 15 μm/min, comparable to that induced by SDF-1. Introduction of constitutively activated Rap2A, Rac, Cdc42, Rho, or H-Ras had little or small stimulatory effect on adhesion. Previously, we demonstrated that mild stimulation of adhesion by Rac1V12 and H-RasV12 required PI3K activity (Katagiri et al., 2000). However, none of these GTPases, with the exception of Rap1V12, enhanced cell migration. Thus, Rap1 has unique characteristics facilitating both adhesion and migration.


Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow.

Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O, Kinashi T - J. Cell Biol. (2003)

Effects of the Ras/Rho family of GTPases on adhesion and migration on immobilized ICAM-1. (A) Effects of the Ras/Rho family on the adhesion of BAF/LFA-1 to ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for adhesion on immobilized human ICAM-1–Fc. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1 and measured as in Fig. 3. The mean and SE of triplicate determinations are shown. (B) Effects of the Ras/Rho family on the migration of BAF/LFA-1 on ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for migration on immobilized human ICAM-1–Fc as described in Materials and methods. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172897&req=5

fig4: Effects of the Ras/Rho family of GTPases on adhesion and migration on immobilized ICAM-1. (A) Effects of the Ras/Rho family on the adhesion of BAF/LFA-1 to ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for adhesion on immobilized human ICAM-1–Fc. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1 and measured as in Fig. 3. The mean and SE of triplicate determinations are shown. (B) Effects of the Ras/Rho family on the migration of BAF/LFA-1 on ICAM-1. BAF/LFA-1 cells expressing constitutively activated small GTPases were measured for migration on immobilized human ICAM-1–Fc as described in Materials and methods. BAF/LFA-1 transfected with vector alone (vector) were stimulated with 20 nM SDF-1.
Mentions: Then, we compared the Rap1V12 promigratory effect with other Ras/Rho family GTPases. We used a proB cell line, BAF/3, reconstituted with human LFA-1 (BAF/LFA-1; Katagiri et al., 2000), which endogenously expresses CXCR4 and migrates on immobilized ICAM-1 in response to SDF-1 stimulation (Fig. 4, A and B) . Rap1V12 expression in BAF/LFA-1 stimulated both adhesion and migration (Fig. 4, A and B) at a migratory speed of 15 μm/min, comparable to that induced by SDF-1. Introduction of constitutively activated Rap2A, Rac, Cdc42, Rho, or H-Ras had little or small stimulatory effect on adhesion. Previously, we demonstrated that mild stimulation of adhesion by Rac1V12 and H-RasV12 required PI3K activity (Katagiri et al., 2000). However, none of these GTPases, with the exception of Rap1V12, enhanced cell migration. Thus, Rap1 has unique characteristics facilitating both adhesion and migration.

Bottom Line: However, the key regulatory molecules regulating this process have remained elusive.Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration.Spa1 effectively suppressed this polarization after SLC treatment.

View Article: PubMed Central - PubMed

Affiliation: Bayer-chair, Dept. of Molecular Immunology and Allergy, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.

Show MeSH
Related in: MedlinePlus