Plakoglobin suppresses keratinocyte motility through both cell-cell adhesion-dependent and -independent mechanisms

Abstract

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins and has been shown to play a critical role in the organization of desmosomes and tissue integrity. Because dissolution of intercellular junctions is frequently an initial step in the onset of epithelial cell migration, we examined whether loss of PG promotes cell motility by compromising adhesive strength. Keratinocyte cultures established from PG-/-mice exhibited weakened adhesion and increased motility in transwell migration assays; both were restored by reintroducing PG through adenoviral infection. Interestingly, single PG-/- cells also exhibited increased motility, which was suppressed by reintroducing PG, but not the closely related beta-catenin. Whereas both N- and C-terminally truncated PG deletion mutants restored adhesion, only N-terminally deleted PG, but not C-terminally deleted PG, suppressed single-cell migration. Furthermore, both the chemical inhibitor PP2 and dominant-negative Src tyrosine kinase inhibited single-cell motility in PG-/- cells, whereas constitutively active Src overcame the inhibitory effect of PG. These data demonstrate that PG strengthens adhesion and suppresses motility in mouse keratinocytes, through both intercellular adhesion-dependent and -independent mechanisms, the latter of which may involve suppression of Src signaling through a mechanism requiring the PG C terminus.

Fig. 1.

PG strengthens intercellular adhesion in mouse keratinocytes in a calcium-dependent manner. (A) Integrity of PG+/– and PG–/– cell monolayers at the indicated calcium concentration was assessed by using a dispasebased mechanical dissociation assay. Because the total fragment number at 0.05 mM calcium exceeded 500 for both PG+/– and PG–/– cells, precise values were not determined. (B) Adhesive strength of PG+/–, PG–/–, and PG–/– cells 24 h after infection with adenoviruses encoding GFP, β-catenin, or PG at 0.65 mM calcium concentration was examined by using the dispase-based dissociation assay. The y axis represents the total number of fragments in A and the percentage of fragments generated in response to mechanical stress compared with PG+/– cells in B.

Fig. 2.

PG suppresses keratinocyte motility in a transwell migration assay in a calcium-dependent manner. (A) Motility of PG+/– and PG–/– cells was compared in a transwell migration assay at 0.2 and 1.2 mM calcium. The y axis represents the percentage of migrated cells compared with PG+/– cells at 0.2 mM calcium. (B) Motility of PG–/– cells transduced with adenoviruses encoding GFP, β-catenin, or PG was examined by using a transwell migration assay at 0.2 and 1.2 mM calcium. The y axis represents the percentage of migrated cells compared with vGFP-transduced PG–/– cells at 0.2 mM calcium. Asterisks represent statistically significant difference when compared with vGFP-transduced cells at the same calcium concentration by using Student's t test (t <0.05).

Fig. 3.

PG suppresses single-keratinocyte motility in a calcium-independent manner. (A) Single-cell motility of PG+/– and PG–/– cells at 0.07 and 0.65 mM calcium concentration, respectively. (B) Single-cell motility of PG+/–, PG–/–, and PG–/– cells transduced with adenoviruses encoding GFP, β-catenin, or PG at a 0.07-mM calcium concentration. In A and B, the y axis represents the percentage of the average area of individual tracks compared with PG+/– cells at 0.07 mM calcium. Asterisks represent a statistically significant difference by using Student's t test (t <0.05).

Fig. 4.

Restoration of adhesive strength in cells expressing PG deletion mutants. (A) A diagram representing the N- or C-terminal deletion mutants of PG. PG+/–, PG–/–, and PG–/– cells transduced with vGFP, vβ-catenin, vPG, vΔNPG, or vΔCPG were subjected to Western blot and immunofluorescence analyses to examine the expression of exogenous protein in B, and adhesive strength at the 0.65-mM calcium level was examined by using dispase-based dissociation assay in C. (D) Adhesive strength at 0.65 mM calcium in PG–/– cells transduced with vPG, vΔNPG, or vΔCPG was examined by using a dispase-based dissociation assay. The number of rotations was increased to generate more sheer stress in this experiment.

Fig. 5.

The PG C terminus is required to suppress motility in the colloidal gold assay, but not the transwell assay. Motility of PG–/– cells transduced with vGFP, vPG, vΔNPG, or vΔCPG was examined by using the colloidal gold assay at 0.07 mM calcium in A and the transwell migration assay at 0.65 mM in B. The y axis in A represents the percentage of average area of individual tracks compared with vGFP-infected cells. The y axis in B represents the percentage of the average number of migrated cells compared with vGFP-infected cells. The asterisks represent the statistically significant difference from vGFP by using Student's t test (t <0.05). The number of migrating cells expressing vΔNPG and vΔCPG were also compared with vPG by using Student's t test. +, statistically significant difference (t <0.05).

Fig. 6.

Role for Src in PG-dependent regulation of single-keratinocyte motility. (A) Motility of PG–/– cells in the presence of 10 μM of the indicated kinase inhibitors at 0.07 mM calcium. Kinase inhibitors were added 2 h after plating cells onto coverslips. (B) Motility of PG–/– cells infected with indicated adenoviruses at 0.7 mM calcium. Expression level of PG and Src were examined by using Western blot. In both A and B, the y axis represents the percentage of average area of individual tracks compared with control PG–/– cells. The asterisks represent the statistically significant difference by using Student's t test (t <0.01).