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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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Cell adhesion and migration on VCAM-1. (A) SLC-stimulated adhesion to immobilized VCAM-1–Fc. Lymphocytes infected with the control, Spa1-, and Rap1V12-encoding adenovirus were incubated in the absence or presence of 100 nM SLC at 37°C for 10 min on VCAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-VLA-4 or anti-VCAM-1 antibody is also indicated. The numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized VCAM-1–Fc. Migration velocity of T cells infected with control or Spa-1 adenovirus stimulated with or without 100 nM SLC and Rap1V12-expressing T cells are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.
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fig5: Cell adhesion and migration on VCAM-1. (A) SLC-stimulated adhesion to immobilized VCAM-1–Fc. Lymphocytes infected with the control, Spa1-, and Rap1V12-encoding adenovirus were incubated in the absence or presence of 100 nM SLC at 37°C for 10 min on VCAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-VLA-4 or anti-VCAM-1 antibody is also indicated. The numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized VCAM-1–Fc. Migration velocity of T cells infected with control or Spa-1 adenovirus stimulated with or without 100 nM SLC and Rap1V12-expressing T cells are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.

Mentions: We also examined the involvement of Rap1 activation in chemokine-induced VLA-4–dependent adhesion to vascular cell adhesion molecule 1 (VCAM-1). Soluble SLC induced shear-resistant adhesion of lymphocytes to immobilized VCAM-1, which was blocked by treatment with either anti-VLA-4 or anti-VCAM-1 antibodies (Fig. 5 A). Spa1 expression greatly reduced SLC-stimulated adhesion to VCAM-1 (Fig.5 A). Conversely, Rap1V12 expression in T cells increased adhesion to VCAM-1 (Fig. 5 A), as reported in Jurkat cells (Reedquist et al., 2000). As is the case of LFA-1/ICAM-1, Rap1V12 stimulated lymphocyte motility on VCAM-1, the velocity of which was comparable to that stimulated with SLC on VCAM-1 (Fig. 5 B; Videos 3 and 4). The migration velocity of Rap1V12-expressing lymphocytes on VCAM-1 was similar to that on ICAM-1 (Fig. 3 B). The cell motility on VCAM-1 stimulated by SLC was abrogated by Spa-1 expression (Fig. 5 B). Together, these results indicate that Rap1 mediates proadhesive and promigratory effects of SLC through VLA-4 /VCAM-1.


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)

Cell adhesion and migration on VCAM-1. (A) SLC-stimulated adhesion to immobilized VCAM-1–Fc. Lymphocytes infected with the control, Spa1-, and Rap1V12-encoding adenovirus were incubated in the absence or presence of 100 nM SLC at 37°C for 10 min on VCAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-VLA-4 or anti-VCAM-1 antibody is also indicated. The numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized VCAM-1–Fc. Migration velocity of T cells infected with control or Spa-1 adenovirus stimulated with or without 100 nM SLC and Rap1V12-expressing T cells are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.
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Related In: Results  -  Collection

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fig5: Cell adhesion and migration on VCAM-1. (A) SLC-stimulated adhesion to immobilized VCAM-1–Fc. Lymphocytes infected with the control, Spa1-, and Rap1V12-encoding adenovirus were incubated in the absence or presence of 100 nM SLC at 37°C for 10 min on VCAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-VLA-4 or anti-VCAM-1 antibody is also indicated. The numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized VCAM-1–Fc. Migration velocity of T cells infected with control or Spa-1 adenovirus stimulated with or without 100 nM SLC and Rap1V12-expressing T cells are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.
Mentions: We also examined the involvement of Rap1 activation in chemokine-induced VLA-4–dependent adhesion to vascular cell adhesion molecule 1 (VCAM-1). Soluble SLC induced shear-resistant adhesion of lymphocytes to immobilized VCAM-1, which was blocked by treatment with either anti-VLA-4 or anti-VCAM-1 antibodies (Fig. 5 A). Spa1 expression greatly reduced SLC-stimulated adhesion to VCAM-1 (Fig.5 A). Conversely, Rap1V12 expression in T cells increased adhesion to VCAM-1 (Fig. 5 A), as reported in Jurkat cells (Reedquist et al., 2000). As is the case of LFA-1/ICAM-1, Rap1V12 stimulated lymphocyte motility on VCAM-1, the velocity of which was comparable to that stimulated with SLC on VCAM-1 (Fig. 5 B; Videos 3 and 4). The migration velocity of Rap1V12-expressing lymphocytes on VCAM-1 was similar to that on ICAM-1 (Fig. 3 B). The cell motility on VCAM-1 stimulated by SLC was abrogated by Spa-1 expression (Fig. 5 B). Together, these results indicate that Rap1 mediates proadhesive and promigratory effects of SLC through VLA-4 /VCAM-1.

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