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First published online 28 October 2008
doi: 10.1242/jcs.028423

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A dual role for caveolin-1 in the regulation of fibronectin matrix assembly by uPAR

¹ Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA
² Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 98557, USA

View larger version (20K): [in this window] [in a new window]   Fig. 1. Cholesterol depletion and caveolin knockdown inhibit the effects of uPAR stimulation on matrix assembly. (A) Monolayers of A1-F cells were treated with increasing doses of MβCD for 30 minutes to deplete cholesterol. After washing, cholesterol was reintroduced into some of the cells (+) by incubating cholesterol-depleted cells with cholesterol (1 mM):MβCD (10 mM). Cells were then incubated with either 50 µM of the uPAR ligand, P25, or the control peptide, S25. Cell layers receiving no peptide served as additional controls (labeled as `C'). 125I-fibronectin (125I-FN) was added to the medium for 6 hours. Cell layers were extracted in 1% deoxycholate, and soluble and insoluble material was separated by centrifugation. The amount of 125I-FN incorporated into the detergent-insoluble matrix was determined by scintillation. Representative data is shown; n=3. The error bars represent the s.e.m. of triplicates. All data were normalized against the control value, which was set at 1. (B) Cells were transfected using oligofectamine with siRNA directed against caveolin-1 or a control non-targeting siRNA for 72 hours. Cells were lysed in gel sample buffer under reducing conditions and lysates were electrophoresed, immunoblotted and analyzed for the expression of caveolin. Blots were then stripped and re-probed for total Erk2 as a loading control. Representative data is shown; n=4. Caveolin knockdown was evident at 72 hours and persisted through to 96 hours. (C) The blots shown in B were scanned and the data was normalized to Erk2. Values from control siRNA cells were set at 1. Error bars represent s.e.m.; n=4. (D) Cells shown in B were treated with 50 µM of P25 or the control peptide, S25, and the 125I–70-kDa-fibronectin-fragment was added to the medium and incubated with the cells for 1 hour. Cells were then rinsed in PBS and solubilized in 1 N NaOH. The amount of 125I–70-kDa that was associated with the cell layers was determined by scintillation. (E) Cells shown in B were incubated with P25 or S25 peptides in the presence of 125I-FN for 6 hours. Cells were then rinsed in PBS and solubilized in 1 N NaOH. The amount of 125I-FN that was associated with the cell layers was determined by scintillation. Data in D and E are presented as fold increase over control, in which cells incubated in the absence of any peptide (labeled as `C') serve as control. Bars represent the standard error of triplicate samples. Asterisk (*) indicates that P25 stimulation of matrix assembly in siRNA-knockdown cells is statistically different from control cells receiving non-targeting siRNA (t-test, P<0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Caveolin knockdown inhibits the effects of uPAR stimulation on β1-integrin activation. Cells were transfected with caveolin siRNA or control non-targeting siRNA for 72 hours. Cells were treated with 50 µM of the uPAR ligand, P25, or the control peptide, S25, for 1 hour. (A) Cells were fixed and incubated with HUTS-4, a monoclonal antibody against the activated β1 integrin, for an additional 1 hour. Bound antibody was detected by ELISA. (B) Cells were fixed and incubated with P5D2, a monoclonal antibody against total β1, for an additional 1 hour. Bound antibody was detected by ELISA. Bars represent the standard error of triplicate samples. Asterisk (*) indicates siRNA knockdown is statistically different from control cells receiving non-targeting siRNA (t-test, P<0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Cholesterol depletion and caveolin knockdown inhibit uPAR-mediated EGFR phosphorylation. (A,B) Confluent fibroblast monolayers were incubated with MβCD (10 mM) for 30 minutes. Cells were washed and incubated with 50 µM of the uPAR ligand, P25, or the control peptide, S25. (A) Cells were fixed and incubated with mAb74, a monoclonal antibody directed against an activated conformation of EGFR, for 1 hour. Bound antibody was detected by ELISA. n=3; error bars indicate s.e.m. Asterisk (*) indicates data that is significantly different from P25 treatment alone (t-test, P<0.05). (B) Cell layers were lysed in gel sample buffer under reducing conditions. Lysates were electrophoresed and immunoblotted using an antibody against the EGFR phosphorylated at Tyr845 (EGFR PY845). The blot was then stripped and re-probed with a total EGFR antibody to assure equal loading. (C,D) Cells were transfected with caveolin siRNA or a non-targeting siRNA for 72 hours. Cells were then incubated with 50 µM S25 or P25 for 1 hour. Cell layers were lysed in sample buffer. (C) Lysates were electrophoresed and immunoblotted using an antibody against EGFR PY845. The blot was then stripped and re-probed with a total EGFR antibody to assure equal loading. (D) Lysates were electrophoresed and immunoblotted in parallel using an antibody against Src family kinases phosphorylated at Tyr418 (Src PY418) or a pan Src antibody to detect total Src. The arrowhead indicates Src.

View larger version (31K): [in this window] [in a new window]   Fig. 4. uPAR stimulation results in Src-dependent phosphorylation of caveolin at Tyr14. (A) Confluent fibroblast monolayers were treated with either 50 µM of the uPAR ligand, P25, or the control peptide, S25, for 1 hour. Cell layers were lysed in sample buffer. Lysates were electrophoresed, immunoblotted and analyzed using an antibody that recognizes caveolin phosphorylated at Tyr14 (caveolin PY14). Blots were then stripped and re-probed with a total caveolin antibody to verify equal loading. (B) Cells were transfected with uPAR siRNA or a non-targeting siRNA for 72 hours. Cell layers were lysed in unreduced sample buffer. Lysates were electrophoresed and immunoblotted using an antibody against uPAR or Erk2. (C) Gels were scanned and uPAR values were normalized to Erk2. Values from control siRNA cells were set at 1. Bars reflect the range of knockdown (n=2). (D) Cells treated with either non-targeting siRNA or uPAR siRNA were incubated with 50 µM P25 or S25 for 1 hour. Cell layers were lysed in sample buffer and lysates were analyzed by western blot for caveolin PY14. Blots were stripped and re-probed for total caveolin to verify equal loading. (E) Fibroblast monolayers were pre-treated with 10 µM PP2 or 5 µM AG1478 (AG) for 1 hour before treatment with 50 µM P25 or S25. Cell lysates were electrophoresed and immunoblotted using an antibody against caveolin PY14. Blots were then stripped and re-probed for total caveolin. (F) Cells were treated with P25 or S25 as described in A and then extracted in RIPA buffer. uPAR was immunoprecipitated from lysates using anti-uPAR antibody 3937. Immune complexes were precipitated with protein A/G agarose beads, solubilized in unreduced sample buffer and electrophoresed into gels. Components of the complexes were analyzed by western blotting.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Treatment of cells with uPAR ligand causes enhanced localization of caveolin PY14 to areas of cell-matrix contact. (A) Fibroblasts were seeded onto glass coverslips coated with 10 µg/ml fibronectin for 2 hours. Cells were then treated with 50 µM P25 or the control peptide, S25, for 1 hour. Cells were fixed, permeabilized and immunostained for caveolin PY14 and the β1 integrin. (B) Confluent monolayers of A1-F cells were incubated with 50 µM S25 or P25 for 1 hour. SAM was isolated by removing cells from the substrate with EGTA and was solubilized in sample buffer under reducing conditions. SAMs were electrophoresed and western blotted. Blots were cut and stained for caveolin PY14, EGFR and paxillin.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Caveolin is required for uPAR-dependent EGFR translocation to the focal adhesion. Cells were transfected with EGFR siRNA (A-C) or caveolin siRNA (D, and see Fig. 2) as described in the Materials and Methods. (A) Cells treated with EGFR siRNA or non-targeting siRNA were lysed in sample buffer under reducing conditions, electrophoresed and analyzed by western blot for EGFR. Blots were stripped and re-probed for Erk2 as a loading control. (B) Blots shown in A were scanned and data was normalized against Erk2. Values from cells receiving control siRNA were set at 1. Bars reflect the range of knockdown (n=2). (C) Cells treated with control siRNA or EGFR siRNA were incubated with 50 µM P25 or S25 for 1 hour. SAMs were isolated by removing cells from the substrate with EGTA treatment and solubilized in sample buffer under reducing conditions. SAMs were electrophoresed, immunoblotted and analyzed for caveolin phosphorylated at Tyr14. Blots were stripped and re-probed for paxillin. (D) SAMs were prepared from cells treated with control siRNA or caveolin siRNA and electrophoresed, immunoblotted and analyzed for EGFR. Blots were stripped and re-probed for paxillin.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Mutation of Tyr14 on caveolin inhibits the effects of P25 on fibronectin matrix assembly and β1-integrin activation. (A) MEF caveolin+/+ (cav+/+) and MEF cav–/– cells infected with adenovirus containing wild-type (WT) or mutant (Y14F) FLAG-tagged caveolins were lysed and analyzed for caveolin expression by western blot. Blots were stripped and re-probed for FLAG to visualize ectopic expression of caveolins. (B) Monolayers of normal MEFs (cav+/+), caveolin-null cells (cav–/–) and caveolin-null cells infected with wild-type caveolin virus (WT) or caveolin Y14F mutant virus (Y14F) for 24 hours were incubated with 50 µM of either S25 or P25 in the presence of 125I-fibronectin (125I-FN) for 6 hours. Cell layers were rinsed in PBS and solubilized in 1 N NaOH. Radioactivity was measured by scintillation. (C) Cells were fixed and incubated with 9EG7, a monoclonal antibody against the activated β1 integrin, for an additional 1 hour. Bound antibody was detected by ELISA. The data are presented as fold increase over control, where the S25 value serves as the control for each experiment. Error bars indicate s.e.m. of triplicate samples. Asterisk (*) indicates statistical difference in effects of P25 on cells expressing wild-type caveolin versus cells expressing either no caveolin or mutant caveolin (t-test, P<0.05).

View larger version (34K): [in this window] [in a new window]   Fig. 8. Phosphorylation of caveolin is required for EGFR translocation but is dispensable for EGFR phosphorylation. Monolayers of caveolin-null cells were infected for 24 hours with virus containing wild-type caveolin or the caveolin Y14F mutant. Confluent fibroblast monolayers were treated with 50 µM of either S25 or P25 for 1 hour. (A) Cell layers were lysed in sample buffer. Lysates were electrophoresed, immunoblotted and analyzed for phosphorylated EGFR at Tyr845 (EGFR PY845). Blots were then stripped and re-probed with a total EGFR antibody to verify equal loading. (B) Western blots shown in A were scanned and the S25 value of EGFR PY845 seen in Cav+/+ cells was set at 1. The bars show the range over two experiments. (C) Cells were then removed with EGTA treatment and SAM was isolated. Lysates were electrophoresed and immunoblotted for EGFR. Blots were then stripped and re-probed for paxillin. (D) Western blots shown in C were scanned and the EGFR value obtained from Cav+/+ cells was set at 1. The bars show the range over two experiments.

View larger version (48K): [in this window] [in a new window]   Fig. 9. uPAR ligation results in dissociation of EGFR from caveolin and increased association of EGFR with phospho-caveolin. Fibroblast monolayers were incubated with 50 µM of either P25 or S25 for 1 hour. Cells were lysed in RIPA buffer. EGFR was immunoprecipitated from cell lysates using a mouse anti-EGFR antibody (3 µg) and the resulting immunoprecipitates were solubilized in gel buffer and analyzed by western blot for EGFR and (A) caveolin or (B) phospho-caveolin.

View larger version (18K): [in this window] [in a new window]   Fig. 10. Model of caveolin function in the regulation of fibronectin matrix assembly by uPAR. This figure shows that, (A) in resting cells, EGFR, uPAR and Src are found in association with caveolin. Both uPAR (via its GPI anchor) and Src (via myristylation) are assumed to partially insert into the lipid bilayer. The binding of an integrin subunit on a neighboring cell (mimicked by P25; B) to uPAR results in the activation of Src, and to the Src-dependent phosphorylation of EGFR (Y845) and caveolin. Phosphorylation of Src and EGFR are dependent on caveolin, suggesting that caveolin functions as a scaffold to facilitate interactions between uPAR, Src and EGFR. (C) Phospho-caveolin and phospho-EGFR are then trafficked to focal adhesions. The redistribution of phospho-EGFR to focal adhesions is dependent on phospho-caveolin, suggesting that phospho-caveolin functions as an accessory molecule to facilitate proper trafficking of EGFR. Activated EGFR is found in complexes with 5β1 integrin, resulting in an increase in activation state. Increased integrin activation results in an increase in assembly of the fibronectin matrix.

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