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Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins

¹ Asthma and Allergy Research Institute, Nedlands, Western Australia, 6009
² Department of Medicine, University of Western Australia, Nedlands, Western Australia, 6009

View larger version (35K): [in a new window]   Fig. 1. Integrins recognizing VN are present on HFL-1 cells. (A) HFL-1 cells were stained with no antibody (white peaks) or with monoclonal antibodies LM609 (anti-vß3), P1F6 (anti-vß5), P5D2 (anti-ß1) or L230 (anti-v) (black peaks) followed by FITC-labeled rabbit anti-mouse IgG. Immunostaining was then analyzed by flow cytometry. (B) In lane 1, cells were surface biotinylated, lysed and cell extracts immunoprecipitated with L230 (anti v). In lane 2, untreated cells were lysed and extracts were immunoprecipitated with L230. P5D2 (anti ß1) was used in western analysis to establish that the vß1 complex was present in HFl-1 cells.

View larger version (47K): [in a new window]   Fig. 2. Detection of VN on HFL-1 cells. HFL-1 cells were stained with a monoclonal antibody recognizing VN followed by FITC-labeled goat anti-mouse IgG. Sections were counterstained with DAPI to visualize cell nuclei (blue) and analyzed using confocal microscopy. (a) A pseudo coloured z-series projection shows specific VN immunoreactivity (green stain) in the intracellular compartment and on the cell membrane (arrows). DAPI staining of nuclei is shown in blue. (b) A higher magnification single optical section shows intense localization to the cell membrane (arrowhead). Bar, 100 µm.

View larger version (33K): [in a new window]   Fig. 3. Expression of -SMA in HFL-1 cells induced by VN. Equal amounts of protein from HFL-1 cells was exposed to VN (0.01, 0.1, 1 µg/ml), FN (10 µg/ml), CN I (10 µg/ml) or laminin (10 µg/ml), subjected to SDS-PAGE and probed by western blotting using a monoclonal antibody to -SMA. Membranes were stripped and reprobed with a monoclonal antibody for tubulin, as a protein loading control. Blots are representative of at least three separate experiments.

View larger version (21K): [in a new window]   Fig. 4. Expression of -SMA is induced in HFL-1 cells by function-blocking antibodies to VN integrins. (A) Protein from HFL-1 cells exposed to P1F6 (anti-vß5), L230 (anti-v), LM609 (anti-vß3) or P5D2 (anti-ß1) was subjected to SDS-PAGE and probed by western blotting using a monoclonal antibody to -SMA. Membranes were stripped and reprobed with a monoclonal antibody for tubulin, as a protein loading control. Blots are representative of at least three separate experiments. (B) HFL-1 cells were incubated with monoclonal antibodies recognizing specific -subunits: L230 (v), P1E6 (2), P3D10H5 (5); specific ß-subunits: 10D5.8 (ß6), P5D2 (ß1), 25E11 (ß3); integrin heterodimers: LM609 (vß3) and P1F6 (vß5) or RGD peptides GRGDdSP and GPenGRGDSPCA for 24 hours. Cells were permeabilised, fixed in methanol and incubated with a monoclonal antibody to -SMA followed by HRP-conjugated rabbit anti-mouse IgGs. Expression of -SMA was then analyzed by ELISA. Results are calculated from triplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly greater -SMA expression compared to control cells, P<0.05.

View larger version (22K): [in a new window]   Fig. 5. Expression of -SMA in HFL-1 cells is mediated by PI-3 kinase and PKC. HFL-1 cells were incubated with either the MAPK inhibitor PD 98059 (50 µM), the tyrosine kinase inhibitors genestein (10-100 µM) and herbimycin (2 µM), the src inhibitor pp2 (10 µM), the PI-3 kinase inhibitor wortmannin (100 nM) or the PKC inhibitor calphostin c (1 µM) for 60 minutes prior to incubation with P1F6 (A) LM609 (B) or P5D2 (C) for a further 24 hours. HFL-1 cells were incubated with the Rho inhibitor C3-exoenzyme (2.5 µg/ml) for 60 minutes prior to the addition of anti-ß1 (P4C10), LM609 or P1F6 for 24 hours (D). Expression of -SMA was analyzed using ELISA. Results are calculated from triplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly less -SMA expression compared to cells exposed to function-blocking antibody alone, P<0.05.

View larger version (17K): [in a new window]   Fig. 6. Cells were treated with the translation inhibitor cyclohexamide (10 µM) or the transcription inhibitor actinomycin D (1 µM) for 45 minutes prior to incubation with P1F6 (A) LM609 (B) or P5D2 (C) for a further 24 hours. Expression of -SMA was analyzed using ELISA. Results are calculated from triplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly less -SMA expression compared to cells exposed to function-blocking antibody alone, P<0.05.

View larger version (128K): [in a new window]   Fig. 7. Assembly of -SMA and F-actin in HFL-1 cells is induced by function-blocking antibodies to VN integrins. HFL-1 cells were incubated with no antibody (a) or LM609 (anti-vß3, b), P5D2 (anti-ß1, c), P1F6 (anti-vß5, d), 10D5.8 (anti-ß6, e) or with VN itself (f). Dual-label images of HFL-1 cells were obtained by confocal immunofluoresence microscopy of fixed cells following staining with -SMA (green) and TRITC-phalloidin to label filamentous actin (red). Yellow/orange staining represents intense areas of colocalization of green and red fluorescence. Bar, 50 µm.

View larger version (12K): [in a new window]   Fig. 8. Contraction of FPCL is induced following exposure of HFL-1 to function-blocking antibodies against VN integrins. (A) Fibroblast populated collagen gels were exposed to culture medium alone (), P1F6 (), MAPK inhibitor PD98059 () or P1F6 in the presence of PD98059 (), PKC inhibitor calphostin c, PI-3 kinase inhibitor wortmannin, Rho-GTPases inhibitor C3-exoenzyme, F-actin inhibitor cytochalasin D or tyrosine kinase inhibitor genestein (all ). Gel dimensions were determined after 24, 48 and 72 hours. (B) Fibroblast populated collagen gels were exposed to culture medium alone () or VN 1 µg/ml () or 10 µg/ml (). Gel dimensions were determined after 24, 48 and 72 hours. Results are calculated from duplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly less gel area compared to cells exposed to medium alone, P<0.05.

View larger version (118K): [in a new window]   Fig. 9. Assembly of -SMA induced by P1F6 in HFL-1 cells is disrupted by agents that inhibit collagen gel contraction. HFL-1 cells were incubated with or P1F6 in the presence of PKC inhibitor calphostin c (a), tyrosine kinase inhibitor genestein (b), src inhibitor pp2 (c), MAPK inhibitor PD98059 (d), PI-3 kinase inhibitor wortmannin (e) or Rho-GTPases inhibitor C3-exoenzyme (f). Dual-label images of HFL-1 cells were obtained by confocal immunofluoresence microscopy of fixed cells following staining with -SMA (green) and TRITC-phalloidin to label F-actin (red). Bar, 50 µm.