Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells

Abstract

The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane-cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.

Figure 1

Protein levels of endogenous and transfected ezrin. Serial dilutions of total cell extracts (0.1–2 μg) were run on a 7% SDS-PAGE under reducing conditions, and transferred to nitrocellulose membranes. The membranes were probed with polyclonal anti-ezrin antibody (lanes A–C) and monoclonal P5D4 antibody (lane D), followed by peroxidase-conjugated secondary antibody, and developed with chemiluminescence. (Lanes A–D) 1 μg of total cell extract. (Lane A) LLC-PK1 cells; (lane B) E7 cells; (lane C) FF1 cells (see p. 428); (lane D) N2 cells.

Figure 2

Cellular distribution of ezrin and its mutants in transfected LLC-PK1 cells by immunofluorescence analysis. (A and A′) Cells transfected with the cDNA encoding the epitope-tagged, full-length ezrin–E7 clone. (B and B′) Cells transfected with the cDNA encoding the epitope-tagged NH₂-terminal domain of ezrin–N2 clone. (C and C′) Cells transfected with the cDNA encoding the epitope-tagged ezrin with mutations of Y145 and Y353 into phenylalanine–FF1 clone. Paraformaldehyde-fixed and detergent-permeabilized cells were double labeled with the anti-tag mAb P5D4 followed by incubation with FITC-conjugated secondary antibody (A–C), and rhodamine-coupled phalloidin (A′–C′). Bar, 7 μm.

Figure 3

Dominant-negative effects of NH₂-terminal domain of ezrin in LLC-PK1 cells. (A) E7 cells, (top) were labeled with the anti-tag mAb P5D4 (E); N2 cells (bottom) were double labeled with mAb P5D4 (N) and rabbit polyclonal anti–ezrin antibody (E), which does not recognize the NH₂-terminal domain of ezrin. FITC-conjugated anti–mouse IgG and TRITC-conjugated anti–rabbit IgG secondary antibodies were used. Horizontal optical sections (insets) and three-dimensional analyses were obtained by CLSM. For each X–Y section, the intensity of fluorescence was determined with arbitrary units 1–255. All readings were normalized to this scale. All X–Y sections were summed, and the intensities were averaged and represented with the color scale on two dimensions. (B) LLC-PK1 cells (top) and N2 cells (bottom) were analyzed by scanning EM. Bar, 1 μm.

Figure 3

Dominant-negative effects of NH₂-terminal domain of ezrin in LLC-PK1 cells. (A) E7 cells, (top) were labeled with the anti-tag mAb P5D4 (E); N2 cells (bottom) were double labeled with mAb P5D4 (N) and rabbit polyclonal anti–ezrin antibody (E), which does not recognize the NH₂-terminal domain of ezrin. FITC-conjugated anti–mouse IgG and TRITC-conjugated anti–rabbit IgG secondary antibodies were used. Horizontal optical sections (insets) and three-dimensional analyses were obtained by CLSM. For each X–Y section, the intensity of fluorescence was determined with arbitrary units 1–255. All readings were normalized to this scale. All X–Y sections were summed, and the intensities were averaged and represented with the color scale on two dimensions. (B) LLC-PK1 cells (top) and N2 cells (bottom) were analyzed by scanning EM. Bar, 1 μm.

Figure 4

Wild-type ezrin is a substrate of the HGF-tyrosine kinase receptor. (A, left) In vitro kinase assay. GST-fused kinase domain of HGF-receptor (apparent molecular mass, 45 kD) was immobilized on glutathione–sepharose and incubated in the presence of 0.1 mM ATP alone (lane 1), with ezrin NH₂-terminal domain (lane 2; apparent molecular mass, 38 kD), and with wild-type ezrin (lane 3; apparent molecular mass, 80 kD). Beads were washed, eluted in Laemmli buffer, and electrophoresed on 10% SDS-PAGE. Western blots were probed with rabbit anti–phosphotyrosine polyclonal antibodies. (Right) Western blot with anti–glutathione-S-transferase (GST) antibodies. 2 μg of GST proteins were loaded and detected with anti-GST polyclonal antibodies. (B) Tyrosine phosphorylation of ezrin and HGF-receptor and association of p62^c-yes with ezrin and HGF receptor. E7 cells were unstimulated (−) and stimulated (+) in vivo with 120 ng/ml HGF, lysed, and immunocomplexes containing epitope-tagged ezrin and HGF-R were probed in Western blot with anti-phosphotyrosine antibodies (anti-PY; left two panels), and anti-p62^c-yes polyclonal antibodies (right two panels). (C) Tyrosine phosphorylation of the NH₂-terminal domain of ezrin, and association of p62^c-yes with the ezrin NH₂-terminal domain and HGF-R. N2 cells were unstimulated (−) and stimulated (+) in vivo with 120 ng/ml HGF, lysed, and immunocomplexes containing epitope-tagged NH₂-terminal domain of ezrin were probed in Western blot with anti-phosphotyrosine, anti-tag, and anti-p62^c-yes antibodies (left three panels). Immunocomplexes obtained with anti-HGF-R antibody were probed in Western blot with anti-p62^c-yes antibodies (right).

Figure 4

Wild-type ezrin is a substrate of the HGF-tyrosine kinase receptor. (A, left) In vitro kinase assay. GST-fused kinase domain of HGF-receptor (apparent molecular mass, 45 kD) was immobilized on glutathione–sepharose and incubated in the presence of 0.1 mM ATP alone (lane 1), with ezrin NH₂-terminal domain (lane 2; apparent molecular mass, 38 kD), and with wild-type ezrin (lane 3; apparent molecular mass, 80 kD). Beads were washed, eluted in Laemmli buffer, and electrophoresed on 10% SDS-PAGE. Western blots were probed with rabbit anti–phosphotyrosine polyclonal antibodies. (Right) Western blot with anti–glutathione-S-transferase (GST) antibodies. 2 μg of GST proteins were loaded and detected with anti-GST polyclonal antibodies. (B) Tyrosine phosphorylation of ezrin and HGF-receptor and association of p62^c-yes with ezrin and HGF receptor. E7 cells were unstimulated (−) and stimulated (+) in vivo with 120 ng/ml HGF, lysed, and immunocomplexes containing epitope-tagged ezrin and HGF-R were probed in Western blot with anti-phosphotyrosine antibodies (anti-PY; left two panels), and anti-p62^c-yes polyclonal antibodies (right two panels). (C) Tyrosine phosphorylation of the NH₂-terminal domain of ezrin, and association of p62^c-yes with the ezrin NH₂-terminal domain and HGF-R. N2 cells were unstimulated (−) and stimulated (+) in vivo with 120 ng/ml HGF, lysed, and immunocomplexes containing epitope-tagged NH₂-terminal domain of ezrin were probed in Western blot with anti-phosphotyrosine, anti-tag, and anti-p62^c-yes antibodies (left three panels). Immunocomplexes obtained with anti-HGF-R antibody were probed in Western blot with anti-p62^c-yes antibodies (right).

Figure 4

Wild-type ezrin is a substrate of the HGF-tyrosine kinase receptor. (A, left) In vitro kinase assay. GST-fused kinase domain of HGF-receptor (apparent molecular mass, 45 kD) was immobilized on glutathione–sepharose and incubated in the presence of 0.1 mM ATP alone (lane 1), with ezrin NH₂-terminal domain (lane 2; apparent molecular mass, 38 kD), and with wild-type ezrin (lane 3; apparent molecular mass, 80 kD). Beads were washed, eluted in Laemmli buffer, and electrophoresed on 10% SDS-PAGE. Western blots were probed with rabbit anti–phosphotyrosine polyclonal antibodies. (Right) Western blot with anti–glutathione-S-transferase (GST) antibodies. 2 μg of GST proteins were loaded and detected with anti-GST polyclonal antibodies. (B) Tyrosine phosphorylation of ezrin and HGF-receptor and association of p62^c-yes with ezrin and HGF receptor. E7 cells were unstimulated (−) and stimulated (+) in vivo with 120 ng/ml HGF, lysed, and immunocomplexes containing epitope-tagged ezrin and HGF-R were probed in Western blot with anti-phosphotyrosine antibodies (anti-PY; left two panels), and anti-p62^c-yes polyclonal antibodies (right two panels). (C) Tyrosine phosphorylation of the NH₂-terminal domain of ezrin, and association of p62^c-yes with the ezrin NH₂-terminal domain and HGF-R. N2 cells were unstimulated (−) and stimulated (+) in vivo with 120 ng/ml HGF, lysed, and immunocomplexes containing epitope-tagged NH₂-terminal domain of ezrin were probed in Western blot with anti-phosphotyrosine, anti-tag, and anti-p62^c-yes antibodies (left three panels). Immunocomplexes obtained with anti-HGF-R antibody were probed in Western blot with anti-p62^c-yes antibodies (right).

Figure 5

Wound healing abilities by LLC-PK1 cells transfectants. A wound was scored in a confluent monolayer. The average distance traveled by the cell margin during HGF-stimulated wound closure was measured with a micrometer for control cells (LLC-PK1), E7 cells, N2 cells, and FF1 cells. SEM bars are shown.

Figure 6

Tubulogenesis by LLC-PK1 cell transfectants within collagen gels. Spherical cysts were formed by control LLC-PK1 cells (A) and LLC-PK1 cells overproducing wild-type ezrin, grown under control conditions for 3 d. Small colonies were formed by LLC-PK1 cells overproducing the double phosphotyrosine-mutated ezrin-FF1 cells (B), and by cells overproducing the NH₂-terminal domain of ezrin under the same growth conditions. In the presence of 30 ng/ml of HGF, some cysts developed into tubules in LLC-PK1 cells (C); the cysts developed in elongated tubules (three- to fivefold the control LLC-PK1 cells) in E7 cells (D). Inset represents a section of tubules formed and demonstrates lumen formation. N2 cells (E) or the tyrosine mutants (F) did not form tubules. Bars: (A–F) 100 μm; (inset) 25 μm.