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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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Chemokine-induced Rap1 activation in lymphocytes. (A) Rap1 activation by SLC. LN cells were treated with 100 nM SLC for the indicated time (min). GTP-bound Rap1 was detected with pull-down assays using GST–RalGDS–RBD (top). Total Rap1 is shown (bottom). (B) PTX treatment prevented Rap1 activation. LN cells were cultured for 2 h in 50 ng/ml of PTX, then stimulated with 100 nM SLC for 30 s and analyzed for Rap1 activation as above (A). (C) Spa1-inhibited Rap1 activation. Lymphocytes infected with an adenovirus encoding GFP alone (control) or Spa1 (Spa1) were stimulated with either 100 nM SLC or 100 nM SDF-1 for 30 s and analyzed for Rap1 activation as above. Infection efficiencies as indicated by GFP expression were ∼80%. Rap1 activation was reduced to 20% of the control levels, as estimated by an image analyzer (model LAS1000; Fuji).
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fig1: Chemokine-induced Rap1 activation in lymphocytes. (A) Rap1 activation by SLC. LN cells were treated with 100 nM SLC for the indicated time (min). GTP-bound Rap1 was detected with pull-down assays using GST–RalGDS–RBD (top). Total Rap1 is shown (bottom). (B) PTX treatment prevented Rap1 activation. LN cells were cultured for 2 h in 50 ng/ml of PTX, then stimulated with 100 nM SLC for 30 s and analyzed for Rap1 activation as above (A). (C) Spa1-inhibited Rap1 activation. Lymphocytes infected with an adenovirus encoding GFP alone (control) or Spa1 (Spa1) were stimulated with either 100 nM SLC or 100 nM SDF-1 for 30 s and analyzed for Rap1 activation as above. Infection efficiencies as indicated by GFP expression were ∼80%. Rap1 activation was reduced to 20% of the control levels, as estimated by an image analyzer (model LAS1000; Fuji).

Mentions: To examine the activation of Rap1 in primary lymphocytes, mouse lymph node (LN) cells were treated with secondary lymphoid tissue chemokine (SLC). Rap1 activation was subsequently measured with the pull-down assay using a GST–RalGDS–RBD fusion protein (Zwartkruis et al., 1998; Fig. 1 A). Rap1 was maximally activated by SLC at 30 s, the earliest time point measurable, but down-regulated to basal levels over the minutes after stimulation (Fig. 1 A). Rap1 activation by SLC was completely inhibited by PTX treatment, indicating that Rap1 activation is linked to Gi protein signaling (Fig. 1 B). Stromal-derived factor 1 (SDF-1) also induced rapid Rap1 activation in LN cells (Fig. 1 C).


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)

Chemokine-induced Rap1 activation in lymphocytes. (A) Rap1 activation by SLC. LN cells were treated with 100 nM SLC for the indicated time (min). GTP-bound Rap1 was detected with pull-down assays using GST–RalGDS–RBD (top). Total Rap1 is shown (bottom). (B) PTX treatment prevented Rap1 activation. LN cells were cultured for 2 h in 50 ng/ml of PTX, then stimulated with 100 nM SLC for 30 s and analyzed for Rap1 activation as above (A). (C) Spa1-inhibited Rap1 activation. Lymphocytes infected with an adenovirus encoding GFP alone (control) or Spa1 (Spa1) were stimulated with either 100 nM SLC or 100 nM SDF-1 for 30 s and analyzed for Rap1 activation as above. Infection efficiencies as indicated by GFP expression were ∼80%. Rap1 activation was reduced to 20% of the control levels, as estimated by an image analyzer (model LAS1000; Fuji).
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Related In: Results  -  Collection

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fig1: Chemokine-induced Rap1 activation in lymphocytes. (A) Rap1 activation by SLC. LN cells were treated with 100 nM SLC for the indicated time (min). GTP-bound Rap1 was detected with pull-down assays using GST–RalGDS–RBD (top). Total Rap1 is shown (bottom). (B) PTX treatment prevented Rap1 activation. LN cells were cultured for 2 h in 50 ng/ml of PTX, then stimulated with 100 nM SLC for 30 s and analyzed for Rap1 activation as above (A). (C) Spa1-inhibited Rap1 activation. Lymphocytes infected with an adenovirus encoding GFP alone (control) or Spa1 (Spa1) were stimulated with either 100 nM SLC or 100 nM SDF-1 for 30 s and analyzed for Rap1 activation as above. Infection efficiencies as indicated by GFP expression were ∼80%. Rap1 activation was reduced to 20% of the control levels, as estimated by an image analyzer (model LAS1000; Fuji).
Mentions: To examine the activation of Rap1 in primary lymphocytes, mouse lymph node (LN) cells were treated with secondary lymphoid tissue chemokine (SLC). Rap1 activation was subsequently measured with the pull-down assay using a GST–RalGDS–RBD fusion protein (Zwartkruis et al., 1998; Fig. 1 A). Rap1 was maximally activated by SLC at 30 s, the earliest time point measurable, but down-regulated to basal levels over the minutes after stimulation (Fig. 1 A). Rap1 activation by SLC was completely inhibited by PTX treatment, indicating that Rap1 activation is linked to Gi protein signaling (Fig. 1 B). Stromal-derived factor 1 (SDF-1) also induced rapid Rap1 activation in LN cells (Fig. 1 C).

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