Rap1A-deficient T and B cells show impaired integrin-mediated cell adhesion

Abstract

Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1-/- cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.

FIG. 1.

Generation of Rap1A knockout mice. (A) Schematic map of the Rap1A gene locus (exons 2 to 4 are shown), targeting construct, and recombinant allele. The black rectangles represent coding exons. The 5-kb targeting cassette contains an IRES sequence, β-galactosidase gene (LacZ), and the neomycin gene (NEO). The 3′ external probe used for probing the genomic blot is shown in panel B as a solid black box under the targeted allele. Restriction enzyme sites: B, BamHI; H, HindIII; P, PstI. (B) Southern blot analysis of the Rap1A gene. DNA was prepared from mouse tails, digested by the ApaI restriction enzyme, and hybridized with 3′ external probe. The wild-type allele generated a 22-kb band, and the targeted allele generated a 14-kb band. (C) PCR-based genotyping of Rap1A knockout. ForPrimer1 and RevPrimer1 generated a 1.2-kb band from the wild-type allele. ForPrimer2 and RevPrimer2 produced 0.6-kb band from targeted allele. (D) β-Galactosidase staining in whole-mount embryonic day 12.5 −/− embryo. (E) Western blot for Rap1A protein. Proteins extracts from thymus and spleen were analyzed by Western blotting with Rap1A-specific antibodies. Rap1A band is absent in protein extracts isolated from −/− mice. α-Tubulin was used as a loading control.

FIG. 1.

Generation of Rap1A knockout mice. (A) Schematic map of the Rap1A gene locus (exons 2 to 4 are shown), targeting construct, and recombinant allele. The black rectangles represent coding exons. The 5-kb targeting cassette contains an IRES sequence, β-galactosidase gene (LacZ), and the neomycin gene (NEO). The 3′ external probe used for probing the genomic blot is shown in panel B as a solid black box under the targeted allele. Restriction enzyme sites: B, BamHI; H, HindIII; P, PstI. (B) Southern blot analysis of the Rap1A gene. DNA was prepared from mouse tails, digested by the ApaI restriction enzyme, and hybridized with 3′ external probe. The wild-type allele generated a 22-kb band, and the targeted allele generated a 14-kb band. (C) PCR-based genotyping of Rap1A knockout. ForPrimer1 and RevPrimer1 generated a 1.2-kb band from the wild-type allele. ForPrimer2 and RevPrimer2 produced 0.6-kb band from targeted allele. (D) β-Galactosidase staining in whole-mount embryonic day 12.5 −/− embryo. (E) Western blot for Rap1A protein. Proteins extracts from thymus and spleen were analyzed by Western blotting with Rap1A-specific antibodies. Rap1A band is absent in protein extracts isolated from −/− mice. α-Tubulin was used as a loading control.

FIG. 1.

Generation of Rap1A knockout mice. (A) Schematic map of the Rap1A gene locus (exons 2 to 4 are shown), targeting construct, and recombinant allele. The black rectangles represent coding exons. The 5-kb targeting cassette contains an IRES sequence, β-galactosidase gene (LacZ), and the neomycin gene (NEO). The 3′ external probe used for probing the genomic blot is shown in panel B as a solid black box under the targeted allele. Restriction enzyme sites: B, BamHI; H, HindIII; P, PstI. (B) Southern blot analysis of the Rap1A gene. DNA was prepared from mouse tails, digested by the ApaI restriction enzyme, and hybridized with 3′ external probe. The wild-type allele generated a 22-kb band, and the targeted allele generated a 14-kb band. (C) PCR-based genotyping of Rap1A knockout. ForPrimer1 and RevPrimer1 generated a 1.2-kb band from the wild-type allele. ForPrimer2 and RevPrimer2 produced 0.6-kb band from targeted allele. (D) β-Galactosidase staining in whole-mount embryonic day 12.5 −/− embryo. (E) Western blot for Rap1A protein. Proteins extracts from thymus and spleen were analyzed by Western blotting with Rap1A-specific antibodies. Rap1A band is absent in protein extracts isolated from −/− mice. α-Tubulin was used as a loading control.

FIG. 1.

Generation of Rap1A knockout mice. (A) Schematic map of the Rap1A gene locus (exons 2 to 4 are shown), targeting construct, and recombinant allele. The black rectangles represent coding exons. The 5-kb targeting cassette contains an IRES sequence, β-galactosidase gene (LacZ), and the neomycin gene (NEO). The 3′ external probe used for probing the genomic blot is shown in panel B as a solid black box under the targeted allele. Restriction enzyme sites: B, BamHI; H, HindIII; P, PstI. (B) Southern blot analysis of the Rap1A gene. DNA was prepared from mouse tails, digested by the ApaI restriction enzyme, and hybridized with 3′ external probe. The wild-type allele generated a 22-kb band, and the targeted allele generated a 14-kb band. (C) PCR-based genotyping of Rap1A knockout. ForPrimer1 and RevPrimer1 generated a 1.2-kb band from the wild-type allele. ForPrimer2 and RevPrimer2 produced 0.6-kb band from targeted allele. (D) β-Galactosidase staining in whole-mount embryonic day 12.5 −/− embryo. (E) Western blot for Rap1A protein. Proteins extracts from thymus and spleen were analyzed by Western blotting with Rap1A-specific antibodies. Rap1A band is absent in protein extracts isolated from −/− mice. α-Tubulin was used as a loading control.

FIG. 1.

Generation of Rap1A knockout mice. (A) Schematic map of the Rap1A gene locus (exons 2 to 4 are shown), targeting construct, and recombinant allele. The black rectangles represent coding exons. The 5-kb targeting cassette contains an IRES sequence, β-galactosidase gene (LacZ), and the neomycin gene (NEO). The 3′ external probe used for probing the genomic blot is shown in panel B as a solid black box under the targeted allele. Restriction enzyme sites: B, BamHI; H, HindIII; P, PstI. (B) Southern blot analysis of the Rap1A gene. DNA was prepared from mouse tails, digested by the ApaI restriction enzyme, and hybridized with 3′ external probe. The wild-type allele generated a 22-kb band, and the targeted allele generated a 14-kb band. (C) PCR-based genotyping of Rap1A knockout. ForPrimer1 and RevPrimer1 generated a 1.2-kb band from the wild-type allele. ForPrimer2 and RevPrimer2 produced 0.6-kb band from targeted allele. (D) β-Galactosidase staining in whole-mount embryonic day 12.5 −/− embryo. (E) Western blot for Rap1A protein. Proteins extracts from thymus and spleen were analyzed by Western blotting with Rap1A-specific antibodies. Rap1A band is absent in protein extracts isolated from −/− mice. α-Tubulin was used as a loading control.

FIG. 2.

Normal differentiation in thymuses of wild-type and Rap1A^−/− mice. (A) Expression pattern for CD4 and CD8. (B) CD44 and CD25 expression in CD4⁻ CD8⁻population. Thymocytes were four-color stained for CD4, CD8, CD44, and CD25 markers. The results are representative for one of 10 sets of wild-type and Rap1A mutant mice. (C) Positive selection in the thymus is intact in Rap1A^−/− mice. The thymus was three-color stained for CD4 PE, CD8 APC, and FITC-labeled αβTCR, CD69, or CD5. Expression profiles for each FITC marker were measured in each CD4/CD8 population.

FIG. 2.

Normal differentiation in thymuses of wild-type and Rap1A^−/− mice. (A) Expression pattern for CD4 and CD8. (B) CD44 and CD25 expression in CD4⁻ CD8⁻population. Thymocytes were four-color stained for CD4, CD8, CD44, and CD25 markers. The results are representative for one of 10 sets of wild-type and Rap1A mutant mice. (C) Positive selection in the thymus is intact in Rap1A^−/− mice. The thymus was three-color stained for CD4 PE, CD8 APC, and FITC-labeled αβTCR, CD69, or CD5. Expression profiles for each FITC marker were measured in each CD4/CD8 population.

FIG. 2.

Normal differentiation in thymuses of wild-type and Rap1A^−/− mice. (A) Expression pattern for CD4 and CD8. (B) CD44 and CD25 expression in CD4⁻ CD8⁻population. Thymocytes were four-color stained for CD4, CD8, CD44, and CD25 markers. The results are representative for one of 10 sets of wild-type and Rap1A mutant mice. (C) Positive selection in the thymus is intact in Rap1A^−/− mice. The thymus was three-color stained for CD4 PE, CD8 APC, and FITC-labeled αβTCR, CD69, or CD5. Expression profiles for each FITC marker were measured in each CD4/CD8 population.

FIG. 3.

Homing of T and B cells into peripheral lymph nodes is unperturbed in Rap1A^−/− mice. Overlay histograms show CD4, CD8 and B220 expressions in peripheral lymph nodes of wild-type mice and −/− mice.

FIG. 4.

LFA-1- and VLA-4-dependent adhesion is impaired in Rap1A^−/−cells from the thymus and spleen. □, adhesion of wild-type mice; ▪, adhesion of Rap1A^−/− mice. Each bar represents an average value of four independently performed experiments. (A) Adhesion of spleen cells on fibronectin and ICAM. (B) Adhesion of separated T and B cells from spleens. The adhesion of T on ICAM and of B cells on ICAM and fibronectin is shown. (C) Adhesion of thymus cells on ICAM and fibronectin. (D) Equal expression of LFA-1 and VLA-4 in wild-type and Rap1A^−/− mouse. The isotype control is shown as a vertical-line histogram. Diagonal-line histograms represent cells stained with anti-LFA-1 or anti-VLA-4.

FIG. 5.

Activity of Rap1 (Rap1GTP) in cell extracts derived from the spleen and thymus is decreased in Rap1-deficient mice. (A) GTP-bound Rap1 was isolated by using RalGDS-RBD from spleen cells before and after stimulation with TPA and detected by Western blotting with a Rap1 antibody (upper panel). Total Rap1 and Rap1A were detected in total lysates (middle two panels) by using an antibody that detects both Rap1 isoforms and one that is specific for Rap1A. Equal loading was confirmed by probing for GAPDH. (B) Detection of GTP-bound Rap1A in thymus cells as described for panel A for spleen cells. (C) Detection of GTP-bound Rap1A in T cells isolated from spleen as described for panel A for spleen cells. (D) T cells were induced with anti-CD3 antibodies for various periods of time. The cell extracts were subjected to Rap1 GST pull-down assay, and then the samples were analyzed by Western blotting. Rap1 activity (Rap1GTP) and total Rap1 (Rap1) was detected by using anti-Rap1 antibodies. In addition, the cell extracts were tested for activation of ERK1 and ERK2 (p-ERK1/2) by using anti-phospho ERK antibodies.

FIG. 6.

Analysis of Rap1a, knockout/wild-type ratios (KO/WT) of proliferation responses and IL-2 production to CD3, CD3/CD28, and TPA/ionomycin stimuli in T cells. The asterisks above the error bars indicate the significance of the results: ✽✽✽, P < 0.001; ✽✽, P < 0.01). (A) Relative proliferation changes in T cells. The estimated KO/WT proliferation ratios lie at 0.78 for CD3 and at 0.9 for CD3/CD28. (B) Relative IL-2 production in Rap1A^−/− T cells measured 48 h after stimulation. The estimated KO/WT IL-2 production ratios lie at 0.28 for CD3 and at 0.81 for CD3/CD28.

FIG. 7.

CD3 induced LFA-1 polarization on the surface of T cells. (A) Examples of polarized and nonpolarized T cells. (B) LFA-1 polarization was assessed by determining the percentage of polarized cells in the fields of about 50 cells. Ten fields were counted for each treatment. Similar results were obtained in three independent experiments. Results for wild-type (□) and Rap1A^−/− (▪) mice are shown.

FIG. 7.

CD3 induced LFA-1 polarization on the surface of T cells. (A) Examples of polarized and nonpolarized T cells. (B) LFA-1 polarization was assessed by determining the percentage of polarized cells in the fields of about 50 cells. Ten fields were counted for each treatment. Similar results were obtained in three independent experiments. Results for wild-type (□) and Rap1A^−/− (▪) mice are shown.