2.1 Reagents, cells and culture conditions
Lipopolysaccharide (LPS) was obtained from Sigma Chemical Co. (St. Louis, MO). The Pyk2 inhibitor (Tyrphostin A9) and the p38MAP kinase inhibitor (SB203580) were obtained from Calbiochem (San Diego, CA). Phospho-Pyk2 and p-FAK antibodies were obtained from Biosource (Carlsbad, CA), while Py99, p-ERK, ERK, p-p38, and p38 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Pyk2 antibodies were obtained from BD Transduction Laboratories (San Jose, CA). Human dermal microvascular endothelial cells (HMVEC) (Clonetics, San Diego, CA) were maintained in EGM-2MV growth medium containing growth factors, antimicrobials, cytokines and 5% FBS at 37°C in a humidified atmosphere containing 5% CO2. To avoid phenotypic drift associated with decreasing expression of surface receptor molecules, HMVEC was not used beyond passage 4. Human umbilical vein endothelial cells (HUVEC) were also purchased from Clonetics (San Diego, CA) and grown in EGM growth medium containing supplements and 2% FBS.
In all experiments, HMVEC were grown to 80% confluence in 6-well assay plates. The cells were stimulated with LPS in the presence of 0.5% FBS. In the case of inhibitor treatments (Tyrphostin A9, SB203580), HMVEC were pretreated with the inhibitor for 1 hour after which they were stimulated with LPS for various time periods. The supernatant was used for the MCP-1 or trans-endothelial migration (TEM) assays and the cell lysates were used for the Western blotting analyses.
2.3 Recombinant adeno-associated virus transduction
High-efficiency gene delivery of the dominant-negative Pyk2 mutant, Pyk2K457A (Pyk2MT) or a control gene (β-galactosidase) was accomplished using a recombinant adeno-associated virus (rAAV)-based method. The AAV vectors were prepared as described previously (Madry et al., 2003
). Before being exposed to the virus, the HMVEC were cultured overnight in complete medium. HMVEC were transduced by application of the AAV in a minimal amount of serum-free medium for 90 min at 37°C in a cell culture incubator. Equal volumes of complete EGM containing 10% serum were added to the cells to achieve a final serum concentration of 5%. The cells were cultured for 36 hours before being used for the experiments described later. After transduction, LPS was added to the medium and the cells were incubated for an additional 24 hours. The culture supernatant was removed and evaluated for MCP-1 content. Alternatively, the cells were lysed and subjected to Western blot analysis by using rabbit anti-human Pyk2 antibody or β-galactosidase staining in the case of the control.
2.4 MCP-1 ELISA
After various stimulations, the culture supernatants were collected, centrifuged, and processed for MCP-1 quantification by commercially available ELISA kits (Endogen), per the manufacturer’s instructions.
2.5 Isolation of monocytes
The CD14+ monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) by using the CD14 negative isolation kit (Dynal, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, human PBMCs were isolated by density-gradient centrifugation with Ficoll/Hypaque (GE Lifesciences, Piscataway, NJ) and then mixed with microbeads. The monocytes were isolated by magnetic bead separation. Flow cytometric analysis revealed a purity of the CD14+ cells to be more than 92%. (data not shown)
2.6 Trans-endothelial migration (TEM) assay of monocytes
Briefly, about 100,000 HMVEC cells were added to fibronectin-coated 24-well Transwell chambers with a pore size of 8 μm (Costar Corp., Corning, NY) and grown for 3 days in 5% CO2 at 37 °C. 0.6 ml of medium from the untreated or LPS/Tyrphostin A9- or AAV-Pyk2MT-treated HMVEC cells were added to the lower compartment. In the upper compartment, 1 × 106 monocytes in 0.1 ml of 0.5% FBS containing EBM medium were added onto the HMVEC monolayer. Supernatants pretreated with 20 μg/ml MCP-1 antibody served as controls. The chambers were incubated for 4 hours at 37°C in 5% CO2. The cells in the lower compartment were counted on a hemocytometer. The results are presented as the means ± S.D. of three separate experiments and are expressed as the number of cells migrating toward the lower compartment.
2.7 Western blotting and immunoprecipitation
Total cellular extracts from the LPS-treated cells were prepared by lysing the cells in radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 10 μg/ml aprotinin, leupeptin, and pepstatin, 10 mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate). Proteins (50 μg) were size-fractionated by 8% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 2–3 hours with 5% nonfat milk and then incubated with the respective primary and secondary Abs for 2–3 h each. The membranes were washed three to four times for 15 min each with TBS and 0.05% Tween 20, and later developed by chemiluminescence (ECL System; GE Healthcare, Piscataway, NJ).
2.8 siRNA-mediated knockdown of Pyk2
RNA interference-mediated knockdown of Pyk2 was performed using SMARTpool Pyk2 duplex RNA oligonucleotides obtained from Dharmacon (Lafayette, CO). A non-targeting siRNA was used as the control. HMVEC were transfected with the siRNA using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Pyk2 siRNA-mediated knockdown was estimated by detection of Pyk2 expression using Western blot analysis, 48 hours after the initial transfection.
2.9 NF-κB assay
At the end of the stimulation, cell and nuclear extracts were obtained by using a Nuclear Extract Kit (Active Motif, Carlsbad, CA). Briefly, cells were collected in the PBS/phosphatase inhibitors solution and lysed in a buffer (Active Motif, Carlsbad, CA) containing 10 mM DTT and a cocktail of protease inhibitors as per the manufacturer’s recommendations. Solubilized proteins were then separated from cell debris by centrifugation (20 min at 14,000×g). The protein concentration in the cytoplasmic and nuclear fraction was measured by Bradford assay and the protein content was adjusted to have the same concentration in all the samples. NF-κB activation was measured by the NF-κB ELISA kit (Active Motif, CA, USA) according to the manufacturer’s recommendations. The levels of NF-κB activation were measured by a Spectrophotometer at OD450.
2.10 Statistical Analysis
Reported data are the means ± S.D. of at least three independent experiments performed in duplicate or triplicate. The statistical significance was determined by the Student’s t test.