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First published online 2 March 2004
doi: 10.1242/jcs.01037

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Membrane-associated HB-EGF modulates HGF-induced cellular responses in MDCK cells

Department of Medicine, Vanderbilt University, Nashville, TN 37232-4794, USA

View larger version (25K): [in a new window]   Fig. 1. Graphical presentation of cDNA constructs for membrane-bound HB-EGF (proHB-EGF), soluble HB-EGF (sHB-EGF)and the deletion mutant constructs. The sequence of the juxtamembrane extracellular domain and the amino acids deleted in the mutant constructs are shown. A C-terminal FLAG epitope (M2) was added to each construct.

View larger version (61K): [in a new window]   Fig. 2. (A) Western blot analysis using a polyclonal rabbit anti-rat HB-EGF antibody and total cellular lysate from the cells used in the studies. (B) Release of soluble HB-EGF in the culture medium. Serum free conditioned medium from the control and HB-EGF-transfected cells was loaded onto pre-equilibrated heparin-sepharose columns, washed with 10 mM Tris-cl, pH 7.4 and 100 mM NaCl and eluted with 2 M NaCl. Equal amount of eluants was resolved on 12% SDS-PAGE and probed with anti-HB-EGF antibody. The observations are representative of data from two independent clones for each transfected molecule and three independent experiments. (C) A Paracrine assay to establish the lack of cleavage of proHB-EGF in cells expressing deletion mutant constructs of HB-EGF. Equal amounts of the eluants from the purified conditioned media of control and HB-EGF-transfected cells were added to the quiescent A431 cells for 5 minutes. Cells were washed with PBS and lysed in RIPA buffer. Equal amounts of protein were resolved on 6% SDS-PAGE and probed with anti-phospho-EGFR antibody. The observations are representative of data from two independent clones for each transfected molecule and at least three independent experiments. The same blot was stripped and probed with the anti-EGFR antibody to verify equal protein loading. (D) Effect of phorbol myristate acetate ester (PMA; 1 µM) on the cleavage of proHB-EGF and deletion mutant constructs of HB-EGF. Quiescent cells were treated with 1 µM of PMA for 30 minutes. Cells were washed with 2 M NaCl to remove any soluble HB-EGF bound to the cell surface. Equal amount of protein was immunoprecipitated with anti-FLAG (M2) antibody. Samples were resolved on 10% Tricine gel and probed with anti-FLAG antibody. The (+) indicates cells treated with PMA and (–) indicates nontreated cells. The full-length HB-EGF and residual transmembrane/cytoplasmic domain remaining after cleavage of proHB-EGF are indicated by arrows and arrowheads, respectively. The observations are representative of data from two independent clones for each transfected molecule and at least three independent experiments. (E) EGF receptor expression in confluent MDCK control and HB-EGF-transfected cells. The basal EGF receptor tyrosine phosphorylation in quiescent MDCK control and HB-EGF-transfected cells. The observations are representative of data from two independent clones for each transfected molecule and five independent experiments.

View larger version (73K): [in a new window]   Fig. 3. (A) Juxtacrine assay with transiently transfected MDCK II cells. Lane 1: vector transfected cells; 2: proHB-EGF; 3: 5 aa deletion; 4: 12 aa deletion; 5: soluble HB-EGF administration. Subconfluent A431 cells were overlain with formalin fixed MDCK cells transiently transfected with different HB-EGF constructs. After 10 minutes, the MDCK cells were washed away, membranes were prepared from A431 cells, EGF receptor was immunoprecipitated with an anti-EGFR antibody and an in vitro kinase assay was performed. Soluble HB-EGF-induced EGF receptor tyrosine phosphorylation was used as a positive control. (B) Immunofluorescent staining of the control, MDCK5aa and MDCKpro cells with the anti-FLAG (M2) antibody. Cells were plated on transwells and stained with a polyclonal anti-FLAG (M2) antibody followed by an anti-rabbit conjugated with Rhodamine-X. Photographs were taken using a Zeiss-410 confocal microscope. The photographs shown are three-dimensional reconstructions of the Z-stack captured by confocal microscope and are representative of observations from two independent clones for each transfected molecule. Original magnification x400. (C) The YZ orthogonal view of the sectioning for the FLAG-immunofluorescent staining for the MDCK5aa and MDCKpro cells through the stacks obtained using confocal microscopy.

View larger version (63K): [in a new window]   Fig. 4. (A) Collagen I-dependent cell adhesion. Equal numbers of cells were plated on collagen I-coated culture wells (U-bottom 96-well plates) for one hour at 37°C/5%CO2. Cells were washed with PBS, fixed with 3.7% formaldehyde and stained with crystal violet (0.1% solution). Thereafter, cells were lysed in 20% acetic acid and values were determined at 570 nm in a plate reader. Values shown represent the mean of the values obtained from at least four independent experiments ± s.e.m. *P<0.001. (B) Cell spreading. Assay was performed as described in Materials and Methods. These photographs are representative of data from two independent clones for each transfected mutant molecule and at least four independent experiments. MDCK5aa cells showed extended cellular extensions, whereas MDCKcon cells were all rounded. Original magnification x100. (C) Cell migration. Experiments were performed as described in Materials and Methods. Values shown represent the mean of the values obtained from at least four independent experiments ± s.e.m. *P<0.05; **P<0.001.

View larger version (20K): [in a new window]   Fig. 5. Transepithelial resistance in polarized cells (cells grown on transwells). Final values were obtained by subtracting the value from blank wells. Values shown represent the mean of the values obtained from at least three independent experiments and are representative of the data obtained from two independent clones for each transfected molecule ± s.e.m. *P<0.01; **P<0.001.

View larger version (125K): [in a new window]   Fig. 6. Cell scattering. Experiments were performed as described in Materials and Methods. Upper panel (–HGF) shows cells without HGF/SF treatment; lower panel (+ HGF) shows cells 16 hours after HGF/SF (20 ng/ml) treatment. These findings are representative of at least five independent experiments from two independent clones for each transfected molecule. Original magnificatiox100.

View larger version (53K): [in a new window]   Fig. 7. (A) Effect of PD153035, a specific and stable inhibitor of EGF receptor tyrosine phosphorylation, on EGF-induced EGFR and ERK 1/2 phosphorylation. Quiescent MDCK II cells were treated with 20 ng/ml of EGF with or without PD153035 (2.5 µM) for 30 minutes before EGF treatment. (B) Effect of PD153035 on HGF/SF-induced cell scattering in MDCKcon and MDCK5aa cells. Experiments were performed as in Fig. 6 except that one group of cells received PD 153035 (2.5 µM) 30 minutes before the addition of HGF (20 ng/ml). The top panel shows MDCKcon cells and the lower panel shows MDCK5aa cells. Original magnificatiox100.

View larger version (108K): [in a new window]   Fig. 8. (A) Effect of Batimastat on HGF/SF-induced cell scattering in MDCKpro cells. Experiments were performed as in Fig. 6 except that one group of cell received Batimastat (20 µM) for 3-5 hours before the addition of HGF (20 ng/ml). (B) Effects of anti-CD9 antibody and heparinase I on HGF/SF-induced cell scattering in MDCK5aa cells. Experiments were performed as above except that cells received anti-CD9 antibody (1 µg/ml) or Heparinase I (1 U/ml) for 3-5 hours before the addition of HGF (20 ng/ml). Original magnification x100.

View larger version (113K): [in a new window]   Fig. 9. Cells grown in collagen. Experiment was performed as described in Materials and Methods. After 9 days of culture, cells were fixed with 3.7% formaldehyde and photographed. Upper panel (–) shows cells without treatment and lower panel (+) shows cells grown in the presence of HGF/SF (20 ng /ml). These findings are representative of at least six independent experiments and are representative of the data obtained from two independent clones for each transfected molecule. Original magnificatiox200.

View larger version (82K): [in a new window]   Fig. 10. Disruption of adherent and tight junctions by HGF/SF was prevented by expression of membrane-anchored HB-EGF mutant. Experiments were performed as for the scattering experiment except that the cells were plated on glass coverslips. Forty-eight hours after the plating, cells were exposed to HGF/SF 20 ng/ml and cells were washed and fixed at 4, 8 and 12 hours after the HGF/SF treatment and subjected to immunofluorescent staining. (a) Adherens junction's integrity was assessed using anti-E-cadherin antibody followed by an anti-mouse FITC-conjugated antibody. 1-4: MDCKcon cells at 0, 4, 8 and 12 hours after HGF/SF treatment. The adherent junctions began dissociating 4 hours after HGF/SF treatment (see arrowheads) and had mostly disappeared after 8 hours, with the majority of E-cadherin translocated into the cytoplasm (see arrows), and after 12 hours post-treatment cells had begun to move apart. However, they were still connected with individual strands of adherent junctions (see double arrows). At this time-point most of the E-cadherin staining was lost possibly because of endocytosis. 5-8: MDCK5aa cells at 0, 4, 8 and 12 hours after HGF/SF treatment. Although there was a small amount of cytoplasmic translocation of E-cadherin in response to HGF/SF treatment, the junctions were intact even after 12 hours after the treatment. Original magnificatiox400. (b) Tight-junction integrity was assessed using anti-ZO-1 antibody followed by anti-rabbit-rhodamine-X conjugated antibody. 1-4: MDCKcon cells at 0, 4, 8 and 12 hours after HGF/SF treatment The cytoplasmic translocation of ZO-1 was observed by 4 hours after HGF/SF treatment (see arrowhead) but the tight junctions were still largely intact. However, by 8 hours of HGF/SF treatment, tight junctions were markedly disrupted (see arrows) and by 12 hours post-treatment, almost all of junctional ZO-1 had translocated into the cytoplasm and cells had begun to move apart. However, they were still connected with strands of tight junctions (see double arrows). 5-8: MDCK5aa cells at 0, 4, 8 and 12 hours after HGF/SF treatment. Although there was little cytoplasmic translocation of ZO-1 in response to HGF/SF treatment, the tight junctions were intact like adherent junctions in the MDCK5aa cells even at 12 hours after the treatment. Original magnificatiox400.

View larger version (61K): [in a new window]   Fig. 11. Effects of HGF/SF on phosphorylation of ERK 1/2 and Akt proteins. Quiescent cells were treated for the indicated times with HGF/SF (20 ng/ml). One group of cells did not receive any HGF and were considered 0 hours. Immunoblot analysis was performed with the use of the (A) anti-phospho-Akt kinase antibody or (B) anti-phospho-ERK 1/2 kinase antibody. The filters used for examination of phospho-ERK 1/2 and Akt were stripped and reprobed with their respective anti-ERK 1/2 and Akt antibodies to determine equal protein loading. The bar diagrams on the right panel represent the densitometric values for the respective western blot analysis.

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