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First published online 29 April 2008
doi: 10.1242/jcs.020149

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PAI1 stimulates assembly of the fibronectin matrix in osteosarcoma cells through crosstalk between the vβ5 and 5β1 integrins

Center for Cell Biology and Cancer Research (MC-165), Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA

View larger version (16K): [in this window] [in a new window]   Fig. 1. PAI1 increases fibronectin-matrix assembly and β1-integrin activation in MG-63 cells. (A) MG-63 cells were incubated with [125I]fibronectin for 6 hours in the presence of the indicated concentrations of PAI1, PAI1R or PAI1 (Q123K) in DMEM supplemented with 0.02% BSA and 20 mM HEPES. Cell layers were extracted with 1% DOC, and [125I]fibronectin that was incorporated into the detergent-insoluble matrix was recovered by centrifugation and measured by -scintillation. This experiment is representative of three separate experiments. Data are the mean ± s.e. of two experiments performed in duplicate. *P<0.05 (n=4), significantly different than control cells (t-test). (B) MG-63 cells were pretreated with 20 nM PAI1 for 20 minutes and incubated with 50 µM of uPAR agonist P25 or S25 for 1 hour in DMEM. Activation of β1 integrin was assessed by ELISA using the HUTS-4 antibody. Total levels of β1 integrin were measured using the P5D2 antibody against 5β1 integrin. The graph shows the levels of β1-integrin activation after normalization to total β1 levels. Neither P25 nor PAI1 had any effect on the levels of total β1 integrin. Data represent one of three different experiments performed in triplicate. *P<0.05 (n=3), significantly different than control or P25-treated cells (t-test).

View larger version (20K): [in this window] [in a new window]   Fig. 2. The PAI1-mediated increase in fibronectin matrix assembly does not require uPAR. (A) Cell lysates were prepared from MG-63 cells transfected with control siRNA or uPAR siRNA. uPAR and β5 integrin levels were analyzed by western blotting. The membrane was stripped and reprobed with anti-β-actin antibody as a loading control. (B) Cells layers from MG-63 cells transfected with control siRNA or uPAR siRNA were incubated with [125I]fibronectin for 6 hours in the absence or presence of PAI1 (20 nM). Cell layers were extracted with 1% DOC, and [125I]fibronectin that had been incorporated into the detergent-insoluble matrix was recovered by centrifugation and measured by -scintillation. Data are the mean ± s.e. of two experiments performed in duplicate. *P<0.05 (n=4), significantly different than untreated cells (t-test). (C) Cells layers from MG-63 cells transfected with control siRNA or uPAR siRNA were incubated with [125I]fibronectin for 6 hours in the presence of the uPAR agonist P25 or the control peptide S25. Cell layers were washed and scraped into 1% DOC and cell-layer associated [125I]fibronectin was measured by -scintillation. Data are the mean ± s.e. of two experiments performed in duplicate. *P<0.05 (n=4), significantly different than control cells (t-test).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Inhibition of the interaction between vβ5-integrin and vitronectin increases fibronectin-matrix assembly. (A) MG-63 cells were pre-incubated for 45 minutes with the cyclic peptide RGDfV (20 µM) or its inactive analog RADfV (20 µM) or for 2 hours with 20 µg/ml of normal mouse IgG, β3-integrin-blocking antibody LM609 or β5-integrin-blocking antibody P1F6; cells were then incubated with [125I]fibronectin (Fn) for 6 hours. The data shown here are representative of three separate experiments. Data are the mean ± s.e. of two different experiments performed in duplicate. *Significantly different than control cells, t-test, P<0.05 (n=4), significantly different than control cells (t-test). (B) Same as A only under conditions of uPAR stimulation with P25. Cell layers were extracted in 1% DOC, and [125I]fibronectin that was incorporated into the detergent-insoluble matrix was recovered by centrifugation and measured by -scintillation. This experiment is representative of three separate experiments. Data are the mean ± s.e. of two experiments performed in duplicate. *P<0.05 (n=4), significantly different than P25-treated cells (t-test).

View larger version (21K): [in this window] [in a new window]   Fig. 4. PAI1 stimulates fibronectin-matrix assembly in osteosarcoma and osteoblast cells. (A) Cells (HOS, Saos-2, hFOB) were pre-incubated for 20 minutes with PAI1 (20 nM), or for 45 minutes with the cyclic peptide RGDfV (20 µM) or its inactive analog RADfV (20 µM); cells were then incubated with [125I]fibronectin (Fn) for 6 hours. Cell layers were extracted in 1% DOC, and [125I]fibronectin that had been incorporated into the detergent-insoluble matrix was recovered by centrifugation and measured by -scintillation. This experiment is representative of three separate experiments. Data are the mean ± s.e. of two different experiments performed in duplicate. *P<0.05 (n=4), significantly different than control cells (t-test). (B) Same as A only under conditions of uPAR stimulation with 50 µM P25. This experiment is representative of three separate experiments. Data are the mean ± s.e. of two experiments performed in duplicate. *P<0.05 (n=4), significantly different than P25-treated cells (t-test).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Activation of β1 integrin by β5-integrin-blocking agents. (A) MG-63 cells were pretreated for 45 minutes with 20 µM of the cyclic peptides RGDfV or RADfV and incubated with 50 µM of P25 or S25 in DMEM. Activation of β1 integrin was assessed by ELISA using the HUTS-4 antibody. (B) MG-63 cells were preincubated for 2 hours with 20 µg/ml of normal mouse IgG, or β5 integrin blocking antibody P1F6 before treatment with P25 or S25. Integrin activation was assessed using monoclonal antibody 9EG7. All graphs show the levels of β1-integrin activation after normalization to total β1 levels as described in Fig. 1. Total β1-integrin levels did not change. Data represent one of three different experiments performed in triplicate. *P<0.05 (n=3), significantly different than control or P25-treated cells (t-test).

View larger version (51K): [in this window] [in a new window]   Fig. 6. PAI1 disrupts focal adhesions. Monolayers of MG-63 cells were plated overnight onto coverslips in complete medium and then incubated for 3 hours with 20 nM of PAI1 or PAI1 mutants (PAI-1R and PAI-1 Q123K). Cells were subsequently fixed, permeabilized and stained for 1 hour with the clone 15F11 antibody against β5 integrin (panels a-d). After 1-hour staining using Alexa-Fluor-594-derivatized anti-mouse secondary antibody, cells were washed and labeled for an additional hour with a paxillin-FITC antibody (panels e-h). Panels i-l show merged images that indicate colocalization of β5 integrin with paxillin. Scale bar, 20 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 7. PAI1 disrupts actin stress fibers. MG-63 cells were plated overnight onto coverslips in complete medium and then incubated for 3 hours with 20 nM of PAI1 or PAI1 mutants (PAI-1R and PAI-1 Q123K). Cells were subsequently fixed, permeabilized and stained for 1 hour with the clone 15F11 antibody against β5 integrin. Cells were then washed and labeled for an additional hour using Alexa-Fluor-488-derivatized anti-mouse secondary antibody and Alexa-Fluor-594–phalloidin. Actin and β5 integrin were visualized by indirect immunofluorescence. Scale bar, 20 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 8. PAI1-mediated disruption of focal adhesions does not require uPAR. (A) MG-63 cells were plated overnight in complete medium. Cells were subsequently fixed, permeabilized and stained with uPAR polyclonal antibody (panel a) and 15F11 monoclonal antibody against β5 integrin (panel b). uPAR and β5 integrin were visualized by indirect immunofluorescence. Panel c shows the merged image. Scale bar, 20 µm. (B) Cells stained for uPAR and paxillin. Panels a and b show uPAR and paxillin staining in cells transfected with control siRNA. Panels d and e show uPAR and paxillin staining in uPAR knockdown cells. Panels c and f are the merged images. Yellow indicates colocalization of uPAR and β5 integrin. Scale bar, 20 µm. (C) Cells transfected with uPAR siRNA were plated overnight in complete medium and then incubated for 3 hours in DMEM in the presence or absence of PAI1. Scale bar, 20 µm. Cells were subsequently fixed, permeabilized and stained. uPAR and β5 integrin were visualized by indirect immunofluorescence in untreated cells (panels a and b) and PAI1 treated cells (panels c and d).

View larger version (29K): [in this window] [in a new window]   Fig. 9. PAI1 does not disrupt the association of 5β1 integrin with fibronectin matrix. Cells were incubated for 3 hours in DMEM in the presence or absence of PAI1, subsequently fixed, permeabilized and immunostained. (A) Fibronectin matrix and β5 integrin were visualized by indirect immunofluorescence in untreated cells (panels a and b) and PAI1 treated cells (panels c and d). Scale bar, 20 µm. (B) Fibronectin and activated β1 integrin (detected with 9EG7 antibody) were visualized by indirect immunofluorescence in untreated cells (panels a and b) and PAI1 treated cells (panels d and e). Panels c and f shows merged pictures. Yellow staining indicates areas of colocalization of β1 integrin with fibronectin matrix. Scale bar, 20 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 10. PAI1 does not disrupt matrix adhesions. Cells were incubated overnight on coverslips in complete medium and then incubated for 3 hours in DMEM in the presence or absence of PAI1 (20 nM). Cells were subsequently fixed, and permeabilized. (A) Fibronectin and tensin were visualized by indirect immunofluorescence in untreated cells (panels a and b) and PAI1 treated cells (panels d and e). Panels c and e show the merged pictures. Yellow staining represents area of colocalization of tensin and fibronectin. Scale bar, 20 µm. (B) Paxillin and tensin were visualized by direct and indirect immunofluorescence in untreated cells (panels a and b) and PAI1 treated cells (panels d and e). Panels c and f shows merged pictures. Scale bar, 20 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 11. β5 knockdown inhibits PAI1 stimulation of matrix assembly. (A) Lysates from MG-63 cells transfected with either control siRNA or β5 siRNA were analyzed for β5 integrin and actin using western blotting. (B,C) MG-63 cells treated with either control or β5 siRNA were incubated with [125I]fibronectin for 6 hours in the presence of 20 nM PAI. Cells were extracted with 1% DOC, and [125I]fibronectin that had been incorporated into detergent-insoluble matrix was recovered by centrifugation and measured by -scintillation. Experiments are representative of separate experiments performed in triplicate. Data are the mean ± s.e. of one experiment performed in triplicate. P<0.05 (n=3), t-test. All values were normalized to protein content. No significant difference in protein content was observed between control siRNA and β5 siRNA-treated cells.

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