Materials and Methods

Cell cultures

Human aortic smooth muscle cells (hASMC) from Clonetics (San Diego) were obtained from 2 donors. Cells were used between passages 4-8 and were characterized by morphological criteria and smooth muscle a-actin expression. hASMC were grown in culture medium supplemented with FBS (5%), insulin (5 µg/mL), recombinant human basic FGF (2 ng/mL), recombinant human EGF (10 ng/mL), gentamycin (50 µg/mL) and amphotericin B (50 ng/mL). hASMC were cultured into 12-well plates at 3.8 x104 cells/well until subconfluence was obtained after 7 days in culture. The culture medium was changed and hASMC were incubated in complete culture medium with 5% FBS before exposure for a subsequent period of time (from 8 hours to 72 hours) in the presence of the different cytokines and reagents tested.

Mitogenic assay and cell morphology analysis

Proliferation of hASMC was assessed by cell number. Proliferating (about 50% confluence) or subconfluent (80-90% confluence) cells were made quiescent for 24 hours in serum-free culture medium. After replenishing with fresh culture medium with or without 5% FBS, cells were further incubated up to 4 days in the absence or presence of OSM (1, 10 or100 ng/mL), with or without 1 mM NS-398, a specific COX-2 inhibitor (13). At the end of incubation, the medium was removed, and cells were treated with 0.05% trypsin, suspended into 10 mL of culture medium, and counted manually. Experiments were repeated at least three times.

To characterize the effect of OSM on cell morphology, focal adhesions were analyzed by using vinculin staining. Cells grown onto glass slides at 50% confluence were incubated for 4 days in the absence or presence of OSM (10 ng/mL). They were fixed in 4% paraformaldehyde solution and immunostained using anti-mouse vinculin antibodies (Sigma). Bound primary antibody was detected with biotin-conjugated anti-mouse IgG, amplified by the streptavidin-Texas red system (Amersham International PLC). Fluorescence was visualized using a Leitz microscope equipped with an epifluorescence system (Leica).

IL-6 bioassay

IL-6 activity was measured by a specific cell proliferation bioassay using an IL-6 dependent B9 hybridoma cell line (14), as previously described (15). The sensitivity of this bioassay had a range of 0.1-0.2 U/mL (100-200 pg/mL) and the coefficient of variation was <15%. Although the B9 assay is considered as relatively specific for IL-6, some factors may induce B9 proliferation such as soluble IL-6 receptors and IL-11, and we therefore verified the absence of OSM action on B9 proliferation. In a small series of experiments, we used in parallel IL-6 bioassay and IL-6 ELISA (from Genzyme) and obtained similar results with the 2 methods.

RNA analysis

RNA extractions (16) and northern blot analysis (17) of total cellular RNA were performed as described. 350 base pairs of the COX-2 cDNA (18) and 240 base pairs of glyceraldehyde-3 phosphate dehydrogenase were used as probes. cDNAs probes were radiolabeled using 32P-dCTP by random priming (Boehringer Mannheim kit) to specific activity >109 cpm/mg DNA. After prehybridization for 4 hours at 42°C, the cDNA probe was added directly to the hybridization solution to a concentration of 106 cpm/mL and incubated at 42°C for 16 hours.

Western blot analysis

hASMC were washed twice in PBS buffer and lysed in 250 µl of ice-cold buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 30 mM octyl glucoside and 0.5 mM Pefabloc) for 30 min. Cells were scraped and centrifuged at 4°C for 10 min at 10,000 x g. The protein content was determined using microbicinchoninic acid assay (Pierce, Rockford, IL) with BSA as standard. Cell lysate (30 µg of protein) was mixed with Laemmli reagent under reducing conditions. SDS-PAGE, using 7% bis-acrylamide for the separation gel and transfer of proteins was performed as previously described (19). Nitrocellulose membranes (Amersham Corp.) were saturated for 2h at room temperature in Tris buffer saline (50 mM Tris-HCL (pH7.5), 250 mM NaCl and 0.1% Tween saline) containing 5% of fat-free dry milk. For COX-1 and COX-2 detections, we used mAbs specific to either hCOX-1 or hCOX-2 (19). Chemiluminescence substrates (ECL, Western blotting; Amersham Corp.) were used to reveal positive bands according to the manufacturer's instructions, and bands were visualized after a fixed time exposure (usually 1-2 minutes) to Hyperfilm ECL (Amersham Corp.). Protein bands were quantified by densitometry. Immunodetection was linear between 15 and 75 µg for COX-2.

Immunoprecipitation

For analysis of the Janus kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway, hASMC were incubated in the presence of OSM (10 ng/mL) for 0, 10 or 30 min at 37°C. Cells were washed twice in PBS buffer then lysed in 500 ml ice-cold buffer (20 mmol/L Tris-HCl pH-7.5, 5 mmol/L EGTA, 150 mmol/L NaCl, 20mmol/L glycerophosphate, 10 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1% Triton X-100, 0.1% Tween-20, 1 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L N-tosyl-l-phenylalanine chloromethyl ketone, and 0.5 mmol/L N(a)-p-tosyl-L-lysine chloromethyl ketone). Cells were scraped and extracts were incubated on ice for 15 minutes and then centrifuged (14,000 x g, 15 minutes, 4oC). The detergent-soluble supernatant fractions were retained, and protein concentrations in samples were equalized using a Bio-Rad protein assay. Immunoprecipitation was performed overnight in the presence of anti-JAK1 antibodies and protein A/G agarose (both Santa Cruz; 40 ml/mL lysate). Beads were washed four times with the lysis buffer, then resuspended in Laemmli reagent. A SDS-PAGE was performed using 6% bis-acrylamide. Nitrocellulose membranes were incubated 1 hour with 5% BSA and 5% low-fat milk, then 1 hour in the same solution containing phosphotyrosine (PY20) or JAK1 polyclonal antibodies (Santa Cruz). Positive bands were revealed by chemiluminescense as described above.

Electrophoretic mobility shift assays (EMSA)

Nuclear extracts were prepared as previously described (20). Protein concentration was evaluated using the BCA protein assay kit (Pierce). 5-10 µg proteins were incubated for 30 min at 4°C with 10-15 fmol of a 32P-labeled oligonucleotide. Complexes were separated on a 4% non-denaturating polyacrylamide gel as reported (20). The oligonucleotide probes used (m67 SIE, 5'-CATTTCCCGTAAATC-3'; IRF1-GAS, 5'-GATCCATTTCCCCGAAATGA-3') were end-labeled using T4 polynucleotide kinase to a specific activity of 3000 cpm/fmol. As a positive control for STAT5 activation, we used nuclear extracts from human hematopoietic UT7-mpl cells treated with thrombopoietin for 10 min (20)

PGE2 and 6-keto-PGF1a assays

PGE2 and 6-keto-PGF1a were determined in hASMC supernatants using immunoassay with acetylcholinesterase-labeled PGE2/6-keto-PGF1a as tracers (19).

Materials

Recombinant human Oncostatin M, recombinant human IL-6, polyclonal rabbit anti-human oncostatin M antibodies were from Genzyme, recombinant human LIF from RxD. hASMC culture medium, including insulin, recombinant human basic FGF, recombinant human EGF, was from Clonetics, and genistein, daidzein, actinomycin-D and fludarabine were from Sigma.

Statistics

Results are presented as mean±SEM. Effects of OSM and/or IL-1 were tested using ANOVA or t test.