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First published online 27 May 2008
doi: 10.1242/jcs.024620

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Involvement of the nectin-afadin complex in PDGF-induced cell survival

¹ Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Osaka, Japan
² Division of Lipid Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
³ Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

View larger version (78K): [in this window] [in a new window]   Fig. 1. Enhancement of apoptosis in EBs derived from afadin–/– ES cells. EBs formed from wild-type and afadin–/– ES cells were fixed, and apoptotic cells were detected using the TUNEL method and immunostaining with the anti-cleaved-caspase-3 mAb. F-actin (red) and nuclei (blue) were counterstained with rhodamine-phalloidin and DAPI, respectively. (A) EBs at 9 days after the withdrawal of leukemia inhibitory factor (LIF). (B) EBs at 11 days after the withdrawal of LIF. (C) EBs at 15 days after the withdrawal of LIF. Scale bars: 50 µm. The results shown in this figure are representative of three independent experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Involvement of afadin in PDGF-induced Akt activation and cell survival in NIH3T3 cells. (A) Inhibition of the PDGF-induced phosphorylation of Akt by knockdown of afadin in NIH3T3 cells. After 16 hours of serum starvation, control (denoted by C) and afadin-knockdown (KD) NIH3T3 cells were treated with 3 ng/ml PDGF for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. Bars in the graph under each blot represent the relative band intensities of phosphorylated Akt (top), Src (middle) and PDGF receptor (bottom) normalized to the total amount of each protein as compared with the values from control cells treated with PDGF for 5 minutes, which are expressed as 1. Grey bars, control cells; white bars, KD cells. Error bars indicate s.e.m. *P<0.05 vs control NIH3T3 cells. (B) Increase in serum-starvation-induced apoptosis by knockdown of afadin in NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells were serum-starved for 16 hours and then treated with or without 3 ng/ml PDGF or 3 ng/ml EGF for 24 hours. Apoptotic cells were detected using the TUNEL method. Nuclei were counterstained with DAPI and are seen in blue. Bars in the graph to the right represent the mean percentages of TUNEL-positive cells in three independent experiments. In each experiment, the number of TUNEL-positive cells among a total of 100 cells observed in four different fields of view was counted. Error bars indicate s.e.m. *P<0.05 vs paired control cells; #P<0.05 vs control cells without growth-factor treatment; P<0.05 vs afadin-knockdown cells without growth-factor treatment. (C) Increase in Fas-ligand-induced apoptosis by knockdown of afadin in NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand (Jo2 mAb) concomitant with or without 3 ng/ml PDGF or 3 ng/ml EGF for 24 hours. Apoptotic cells were counted as described in B. Error bars indicate s.e.m. and symbols indicate the same as shown in B. (D) Increased production of cleaved caspase-3 by knockdown of afadin in NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells were cultured in the same conditions described in B and C. Cell lysates were subjected to western blotting with the anti-cleaved-caspase-3 pAb. GAPDH was immunoblotted as a loading control. (E) Inhibitory effect of Akt-CA on apoptosis in afadin-knockdown NIH3T3 cells. Confluent afadin-knockdown NIH3T3 cells transfected with GFP or Akt-CA-GFP were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand for 24 hours in the presence of 3 ng/ml PDGF. Cells were immunostained with the anti-cleaved-caspase-3 mAb and nuclei were counterstained with DAPI. The graph represents the mean percentage of cleaved-caspase-3-positive cells assessed as described in B. Error bars indicate s.e.m. *P<0.05 vs control NIH3T3 cells. (F) Reduced production of cleaved caspase-3 by the expression of Akt-CA in afadin-knockdown NIH3T3 cells. Cell lysates from control and afadin-knockdown NIH3T3 cells cultured in the same conditions as described in E were subjected to western blotting with the anti-cleaved-caspase-3 pAb. GAPDH was immunoblotted as a loading control. Scale bars: 50 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Significance of the binding of afadin to nectin for the prevention of apoptosis. (A) Restoration of PDGF-induced phosphorylation of Akt by re-expression of full-length afadin, but not afadinPDZ, in afadin-knockdown NIH3T3 cells. Four types of cells (control NIH3T3 cells, afadin-knockdown NIH3T3 cells, afadin-knockdown NIH3T3 cells re-expressing GFP-tagged full-length afadin and afadin-knockdown NIH3T3 cells re-expressing GFP-tagged afadinPDZ) were treated with PDGF for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. The relative band intensity was calculated as described in Fig. 2A. *P<0.05 vs control NIH3T3 cells. C, control; KD, afadin knockdown. (B) Reduction of apoptosis by re-expression of full-length afadin in afadin-knockdown NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells stably expressing GFP, GFP-afadin or GFP-afadinPDZ were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand for 24 hours with 3 ng/ml PDGF. Cells were immunostained with the anti-cleaved-caspase-3 mAb (red) and nuclei were counterstained with DAPI (blue). The graph represents the mean percentage of cleaved-caspase-3-positive cells in three independent experiments. In each experiment, the number of cleaved-caspase-3-positive cells among a total of 100 cells observed in four different fields of view was counted. Error bars indicate s.e.m. *P<0.05 vs control NIH3T3 cells expressing GFP. Scale bars: 50 µm. (C) Reduced expression of cleaved caspase-3 by re-expression of full-length afadin in afadin-knockdown NIH3T3 cells. Control NIH3T3 cells, afadin-knockdown NIH3T3 cells, afadin-knockdown NIH3T3 cells re-expressing GFP-tagged full-length afadin and afadin-knockdown NIH3T3 cells re-expressing GFP-tagged afadinPDZ were cultured in the same conditions as described in B. Lysates from these cells were subjected to western blotting with the anti-cleaved-caspase-3 pAb. GAPDH was immunoblotted as a loading control.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Involvement of nectin-3 in PDGF-induced phosphorylation of Akt and cell survival in NIH3T3 cells. (A) Inhibition of PDGF-induced phosphorylation of Akt by knockdown of nectin-3 in NIH3T3 cells. Confluent control and nectin-3-knockdown NIH3T3 cells were treated for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. The relative band intensity was calculated as described in Fig. 2A. *P<0.05 vs control NIH3T3 cells. (B) Increased number of apoptotic nectin-3-knockdown NIH3T3 cells. Confluent control and nectin-3-knockdown NIH3T3 cells were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand for 24 hours in the presence of 3 ng/ml PDGF. Apoptotic cells were detected using the TUNEL method. Nuclei were counterstained with DAPI and are seen in blue. The graph represents the mean percentage of TUNEL-positive cells assessed as described in Fig. 2C. *P<0.05 vs control NIH3T3 cells. Scale bars: 50 µm. (C) Requirement for the association of nectin-3 with afadin in order to obtain PDGF-induced phosphorylation of Akt. Four types of cells (control NIH3T3 cells, nectin-3-knockdown NIH3T3 cells, nectin-3-knockdown NIH3T3 cells rescued by FLAG-tagged full-length nectin-3 and nectin-3-knockdown NIH3T3 cells rescued by FLAG-tagged nectin-3 lacking the C-terminal four amino acids) were treated with PDGF for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. The relative band intensity was calculated as described in Fig. 2A. *P<0.05 vs control NIH3T3 cells. C, control; KD, nectin-3 knockdown.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Nectin-3- and afadin-mediated regulation of PI3K activity in NIH3T3 cells. (A) Inhibition of the PDGF-induced increase in PI3K activity by knockdown of nectin-3 and afadin in NIH3T3 cells. After 16 hours of serum starvation, control, nectin-3-knockdown and afadin-knockdown NIH3T3 cells were treated with 3 ng/ml PDGF for 2 minutes. Lysates from cells harvested before and after PDGF treatment were subjected to an assay for PI3K activity. Bars in the graph represent the relative spot intensity compared with the values of control NIH3T3 cells treated with PDGF, which are expressed as 1. KD, knockdown; PI(3)P, phosphatidylinositol 3-phosphate. (B) Association of FLAG-afadin with PI3K. Untransfected HEK293 cells and HEK293 cells transiently transfected with FLAG-afadin were lysed and the cell lysates were immunoprecipitated with the anti-FLAG mAb or the anti-p85 pAb. The immunoprecipitates were subjected to western blotting with the anti-p85 pAb and the anti-FLAG mAb. (C) Association of endogenous afadin and PI3K in NIH3T3 cells. Lysates of NIH3T3 cells were immunoprecipitated with the anti-p85 pAb or non-immunized rabbit IgG as a control, and the immunoprecipitates were subjected to western blotting with the anti-afadin mAb and the anti-p85 pAb. The results shown in this figure are representative of three independent experiments.

View larger version (74K): [in this window] [in a new window]   Fig. 6. Association of the nectin-afadin complex with PDGF receptor. (A) Colocalization of the nectin-afadin complex with PDGF receptor at cell-cell adhesion sites. Confluent NIH3T3 cells (Aa) or nectin-3-knockdown NIH3T3 cells (Ab) were immunostained with the anti-PDGF receptor pAb (Santa Cruz Biotechnology) and the rat anti-nectin-3 or mouse anti-afadin mAb. Arrowheads indicate the colocalization sites of nectin-3 or afadin with PDGF receptor. Scale bars: 10 µm. (B) Recruitment of nectin-3 and PDGF receptor at the contact sites between Nef-1-coated beads and NIH3T3 cells. NIH3T3 cells were incubated with Nef-1-coated microbeads (Ba) or ConA-coated microbeads (Bb) for 1 hour; they were then fixed and immunostained with the anti-PDGF receptor pAb and the anti-nectin-3 mAb. The positions of the beads are marked with asterisks. Insets are higher-magnified images of boxed areas. DIC, differential interference contrast. Scale bars: 10 µm. (C) Co-immunoprecipitation of endogenous PDGF receptor with nectin-3. The cell lysates of NIH3T3 cells treated with a chemical cross-linker DTSSP were immunoprecipitated with the anti-p85 pAb or normal rabbit IgG as a control, and were subjected to western blotting with the anti-nectin-3 pAb and the anti-PDGF receptor pAb. (D) Co-immunoprecipitation of PDGF receptor with nectin-3. Lysates of HEK293 cells transiently expressing PDGF receptor with or without FLAG-nectin molecules were immunoprecipitated with the anti-FLAG mAb. The immunoprecipitates were subjected to western blotting with the anti-PDGF receptor pAb and the anti-FLAG mAb. (E) No co-immunoprecipitation of EGF receptor with nectin-3 occurred. Lysates of HEK293 cells transiently expressing FLAG-nectin-3 with or without EGF receptor were immunoprecipitated with the anti-FLAG mAb, followed by western blotting as described in D. (F) Co-immunoprecipitation of PDGF receptor with nectin-3 through their extracellular regions. The cell lysates of HEK293 cells transiently expressing FLAG–nectin-3CP or FLAG–nectin-3EC with or without PDGF receptor were immunoprecipitated with the anti-FLAG mAb, followed by western blotting, as described in D. (G) Co-immunoprecipitation of PDGF receptor with nectin-3 independent of the phosphorylation of PDGF receptor. HEK293 cells transiently expressing PDGF receptor with or without FLAG–nectin-3 were incubated in the presence or absence of 50 µM AG1296, a PDGF receptor inhibitor, for 1 hour. Cell lysates were immunoprecipitated with the anti-FLAG mAb, followed by western blotting as described in D. The results shown in this figure are representative of three independent experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 7. A schematic model for the crosstalk between the nectin-afadin complex and PDGF-receptor-mediated cell-survival signaling. Details are described in the Discussion.

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