DMEM and glutamine Pen-Strep were obtained from Life Technologies, Inc. FBS was purchased from HyClone Laboratories, Inc. Plasma fibronectin was purified from human plasma by gelatin-agarose affinity chromatography . 4–20% gradient SDS-PAGE gels were from Novex. Anti-phospho-p44/42 MAPK polyclonal antibody and anti-p44/42 ERK polyclonal antibody were purchased from New England Biolabs. Anti-β1 blocking antibody (Ha2/5) was from PharMingen. Peptides GRGDSP and GRADSP were purchased from Life Technologies, Inc. Texas red and FITC labeled secondary antibodies and 35SO4 were from ICN. Heparitinase, chondroitinase ABC and mAb 3G10 were from Seikagaku. Anti-FN antibodies were a gift of Dr. Erkki Ruoslahti (Burnham Institute). ECL plus reagent and Hyperfilm were obtained from Amersham. Complete™ protease inhibitor cocktail tablets were purchased from Boehringer Mannheim Biochemicals. The expression vector pQE-12 was obtained from Qiagen. Heparin and all other chemicals were obtained from Sigma.
The purified glycosaminoglycans used in this study were a generous gift of Dr. Nancy Schwartz (Univ. of Chicago). The glycosaminoglycans were produced under a contract from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH) and serve as reference standards. The tissue sources of the glycosaminoglycans were as follows: hyaluronic acid, human umbilical cord; heparan sulfate, beef lung; dermatan sulfate, hog mucosa; chondroitin-4-sulfate, notochord from river sturgeon (S. platorhynchus), and chondroitin-6-sulfate, cranial cartilage of river sturgeon.
Primary rat aortic smooth muscle cells (RASMCs) were isolated from 12–16 week old Sprague-Dawley rats as described previously . RASMCs were cultured in DMEM supplemented with 10% FBS and glutamine Pen-Strep. RASMCs were typically between passages 2 and 7 for experiments.
Recombinant protein production
Proteins III1-C and III 11-C (previously called III11) were produced in bacteria and purified as described previously . III1-C encompasses fibronectin amino acids 600–674 (Asn-Ala-Pro-Gln-...Thr-Ser-Thr-Pro), and III 11-C amino acids 1,532–1,599 (Leu-Pro-Ser-Ser-...Thr-Ala-Val-Thr), according to the previously published numbering method . The recombinant proteins were expressed by the pQE-12 vector, which places a 6-His coding sequence at the C-terminus of each protein. The proteins were purified on Ni-agarose columns and were >98% pure as judged by SDS-PAGE and coomassie staining. Stock protein solutions were typically 700 μM for III1-C and 520 μM for III 11-C in PBS.
Anti-III1-C antibodies were produced by immunizing rabbits with III1-C. After one month the rabbits were boosted and serum samples were collected every two weeks. The rabbit serum was applied to an affinity column to purify the anti-III 1-C antibodies. In order to avoid purifying antibodies to the 6-His tag in III1-C, the affinity column was made with a GST fusion protein. The 1st type III repeat of fibronectin was expressed as a fusion protein coupled to GST, and this GST-3FN1 fusion protein was coupled to Sepharose and used in affinity purification. Bound antibodies were eluted with 100 mM triethylamine, pH 11.5 and the pH was neutralized with 1 M Tris, pH 8; (the antibody concentration was 0.4 mg/ml). BSA (0.5 mg/ml) was added to stabilize the antibody for subsequent handling. The antibody prep was dialyzed extensively against PBS before use.
Cell attachment and spreading assays
For the cell spreading assay shown in Fig. 1, fibronectin (20 μg/ml) was coated onto 24-well dishes for 1 hr at RT. The wells were washed 3 times with PBS before adding cells. Growing RASMCs were harvested by trypsinization and collected into DMEM + 0.5% BSA + soybean trypsin inhibitor. Cells were washed 3 times with DMEM + 0.5% BSA, then resuspended at 1 × 105 cells/ml in DMEM + 0.5% BSA, in the absence or presence of 25 μM III1-C or 25 μM III 11-C in the medium. Cells were seeded onto the wells (0.5 ml/well, ~30% confluence), placed in a 37°C incubator and allowed to adhere for either 7, 15, 30, 60, 120 or 240 min. Cells were then fixed with 3.7% paraformaldehyde, 60 mM sucrose in TBS, followed by staining with Coomassie Blue dye (1 mg/ml Coomassie Brilliant Blue, 45% MeOH, 10% acetic acid). Cells were destained with water, air-dried, then photographed with a Leica DM IRB phase microscope connected to a Polaroid CCD camera and an Apple Macintosh computer for capturing images. To quantitate the amount of spreading, each image was analyzed with the NIH Image 1.61 application to determine the total amount of area covered by cells and this number was divided by the total number of cells in the image to give an average area per cell (in pixels2/cell).
Cell attachment assays shown in Fig. 2 and 3 were done essentially as described previously . Briefly, fibronectin, III1-C or III 11-C at various concentrations were coated onto 96-well dishes for 1 hr at RT. The wells were then washed 3 times with PBS before adding cells. Growing RASMCs were harvested by trypsinization and collected into DMEM + 0.5% BSA + soybean trypsin inhibitor. Cells were washed 3 times with DMEM + 0.5% BSA, then resuspended at 4 × 105 cells/ml in DMEM + 0.5% BSA with or without inhibitors, as indicated in the figures. Cells were seeded onto the coated wells and allowed to adhere for 30 min at 37°C. The cells were then fixed with 3.7% paraformaldehyde, 60 mM sucrose in TBS, followed by 20% methanol, and staining in 0.5% crystal violet in 20% methanol. Stained cells were washed with water and the dye was solubilized with 0.1 M sodium citrate, pH 4.2, 50% ethanol. Attachment was quantitated by measuring the absorbance at 600 nm.
125I cell surface labeling and affinity chromatography
The procedure was a modification of the procedure used to isolate the fibronectin receptor and other integrins [27, 28]. The affinity resins were prepared by coupling fibronectin, III1-C or III 11-C to CNBr-activated Sepharose CL-4B according to the manufacturer's recommendations. The concentration of protein was typically 4–5 mg fibronectin/ml of resin, or 8–10 mg protein/ml of resin for III1-C and III 11-C. Ten 15-cm dishes of RASMCs were washed twice with PBS, then harvested by detachment of cells in PBS + 10 mM EDTA. The cells were washed twice more with PBS, then resuspended at 4 × 107 cells/ml in PBS. Cells were labeled with 1 mCi Na125I and lactoperoxidase-H2O2, as previously described . After labeling the cell pellet was washed with ice-cold PBS + NaN3, then lysed in 2 volumes of OG Lysis Buffer (200 mM octylglucoside, 1 mM CaCl2, 1 mM MgCl2, TBS, PMSF) at 4°C. The lysate was spun at 14,000 × g for 15 min at 4°C, and the supernatant was used for affinity chromatography. Lysate was applied to the affinity columns (0.2 ml lysate was applied to 0.1 ml affinity matrix bed volume), flow through fractions were collected and passed over the columns twice more. Columns were washed with 10 column volumes of Wash Buffer (40 mM octylglucoside, 1 mM CaCl2, 1 mM MgCl2, TBS), before elution of bound protein with 10 mM EDTA in Wash Buffer. Samples from the final wash and eluted samples were separated on 4–20% gradient Novex gels, the gels were dried and exposed to phosphorimager cassette and the radioactive signal was measured with a Storm phosphorimager instrument.
MAPK assay and immunoblotting
Wells of a 6-well plate were coated with either 20 μg/ml fibronectin, 25 μM III1-C or 25 μM III 11-C for 1 hr at RT. Wells were washed 3 times with PBS before adding cells. Growing RASMCs were harvested by trypsinization and collected into DMEM + 0.5% BSA + soybean trypsin inhibitor. Cells were washed 3 times with DMEM + 0.5% BSA, then resuspended at 3 × 105 cells/ml in DMEM + 0.5% BSA with or without integrin blocking agents or other additions, as described in the figure legends. Cells were either left in suspension (for suspended and PDGF stimulated samples) or were seeded onto the coated wells (1 ml per well) and allowed to adhere for 30 min at 37°C, or for the times indicated in the figure legend (Fig. 5). The cells stimulated with PDGF (2 ng/ml PDGF-BB) received the growth factor 10 min before collecting the cells for lysis, while the other samples received no growth factor or serum. After the adhesion period plates were placed on ice, unattached cells were collected and spun down, while cell monolayers were washed once with ice cold PBS then lysed with SDS-PAGE sample buffer (100 μl/well). Centrifuged pellets of unattached cells were combined with the appropriate monolayer lysate sample and the samples were heated to 100°C for 5 min. Samples were separated on 4–20% gradient Novex gels, transferred to Immobilon P membranes, blocked with 5% nonfat dry milk in TBS-Tween, then probed with anti-phospho-p44/42 MAPK antibody according to the manufacturer's recommendations. Blots were then stripped in Stripping Buffer (2% SDS, 100 mM β-mercaptoethanol, 62.5 mM Tris, pH 6.7) at 50°C for 30 min, washed 4 times with TBS-Tween, blocked with 5% nonfat dry milk in TBS-Tween, then probed with anti-p44/42 ERK antibody according to the manufacturer's recommendations. All blots were developed with the ECL plus reagent and exposed to Hyperfilm. For both antibodies the typical exposure times were under 5 min.
Heparitinase treatment of cells and lysates
6 well plates were coated with either fibronectin, III1-C or III 11-C as described above. Growing RASMCs were harvested by trypsinization and washed in DMEM + BSA as described above. For GAGase treatment of intact cells, the cells remained in suspension and received either no enzyme or 0.1 u/ml heparitinase or 0.1 u/ml chondroitinase ABC for 1 hr at 37°C with constant gentle rotation. Cells were then plated onto precoated dishes and processed for immunoblotting with anti-phospho-p44/42 MAPK antibody as described above. Alternatively, after heparitinase digestion cells were lysed directly in SDS sample buffer and analyzed by immunoblotting with the 3G10 mAb (essentially as described above for anti-phospho-p44/42 MAPK immunoblotting).
For GAGase treatment of cell lysates, cells were lysed on ice in NP40 Lysis buffer (1% Nonidet P 40, 150 mM NaCl, 50 mM Tris, pH 8.0) plus protease inhibitors (Complete™ protease inhibitor cocktail) at 2 × 106 cells/ml. Insoluble material was removed by centrifugation at 14,000 × g for 15 min at 4°C. Lysate was prepared for heparitinase treatment by adding CaCl2 to 10 μM final concentration. Heparitinase was added to 0.1 u/ml final concentration and the lysate was incubated at 37°C for 1 hr. The lysate was then applied to affinity chromatography columns as described below.
35SO4 labeling and affinity chromatography
Growing RASMCs labeled for 20 hr at 37°C with 200 μCi/ml 35S04 in DMEM + 10% dialyzed FCS + Pen-Strep. Cells were placed on ice, washed once with PBS, then lysed in NP40 Lysis buffer as described above. III1-C and III 11-C columns were produced as described above. Cell lysates were applied to III1-C or III 11-C columns (typically 500 μl lysate was applied to 250 μl affinity matrix bed volume), flow through fractions were collected and passed over the columns twice more. The final flow through fractions were then collected. Columns were washed with 10 column volumes of NP40 Lysis buffer, then bound material was removed by boiling the Sepharose beads in 2 column volumes of SDS sample buffer. Alternatively, the bound material was first eluted with 2 column volumes of 8 M urea buffer (8 M urea, 0.1 M NaH2PO4, 10 mM Tris, pH 8.0), followed by washing with 10 column volumes of NP40 Lysis buffer, and then removing any remaining bound material by boiling the Sepharose beads in SDS sample buffer. Samples were separated on 4–20% Novex SDS-PAGE gels, the gels were fixed and dried and the radioactive material was detected by using a phosphorimager.