The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes

Abstract

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

Keywords: Directional migration; ENaC; Keratinocyte; Lamellipodia.

Fig. 1.

Depletion of ENaC inhibits galvanotaxis of keratinocytes, but overexpression of EGFP-ENaC promotes galvanotaxis. (A) Representative images from the wild-type MEK and the ENaC-depleted MEK in EF (also supplementary material Movies 1–4). (B) The migration speeds of keratinocytes are between 1 and 2 µm/minute. Left: The directionality decreases by 80% in human NHK treated with αENaC siRNA (120 cells tracked). Right: αENaC-KO-MEK also lose 94% of their directionality in the EF (+/+ wild type, 104 cells tracked; −/− αENaC knockout, 93 cells tracked). Although αENaC-KO-MEK migrate faster than wild-type MEK, the migration speed of the ENaC siRNA-treated NHK is similar to that of scrambled siRNA-treated NHK (1.2±0.46 µm/minute versus 1.1±0.47 µm/minute; not significantly different; migration speed not shown). (C) Galvanotaxis of parental H441 cell lines H441 (80 cells tracked), GFP7 (EGFP expressing H441, 165 cells tracked) and αC3-3-GFP (EGFP-αENaC expressing H441, 165 cells tracked) were examined. The H441 and GFP7 lines that express very low levels of endogenous αENaC do not respond with directional migration to the EF (cosine is close to zero). However, introduction of a functional αENaC channel (in the αC3-3-GFP) significantly increases the cathodal directionality (cosine) of the cells. *P<0.05.

Fig. 2.

Blocking the open state of ENaC prevents keratinocyte galvanotaxis, whereas increasing the open state of the ENaC channel by S3969 increases galvanotaxis. (A) In the cells pre-treated with 20 µM phenamil, the directionality of NHK in galvanotaxis is inhibited by 30% (110 cells tracked in each group). (B) NHK treated with a control peptide or 20 µM ENaC inhibitory peptide migrate at similar rates (migration speed not shown), but the cathodal directionality is decreased by 30% in the cells treated with the αENaC derived peptide (235 cells tracked in control and 275 cells tracked in the treatment with the inhibitory peptide). Therefore, the open state of αENaC channel is required for NHK galvanotaxis. (C) S3969 was added to NHK 30 minutes prior to the EF exposure. The migration speed was not affected (migration speed not shown), but the directionality (net cosine) of cells increased by 17%. (D) S3969 treatment (open triangles) also increased the slope of the track cosine compared to the DMSO control group (solid circles) in the initial 20 minutes of EF exposure, which suggests that opening the ENaC channel promotes a more persistent, directional migration of NHK in the EF (167 cells tracked). *P<0.05.

Fig. 3.

NHE1 is not required for keratinocyte galvanotaxis. (A) NHK galvanotaxis was examined in the presence of DMSO or 50 µM cariporide in DMSO. The NHK migration speed (left) and directionality (right) were not inhibited in DMSO or in 50 µM cariporide (180 cells tracked in each group). (B) Fibroblasts expressing wild-type NHE1 (PSN) or the E266I-NHE1 were exposed to the EF to examine galvanotaxis. Both fibroblast lines demonstrated statistically equal migration speeds and directionality (110 cells tracked in each group), further supporting the conclusion that NHE1 is not required for keratinocyte galvanotaxis. Note the difference in scale on the y-axis in A and B.

Fig. 4.

αENaC polarizes to the cathodal side of keratinocytes after 60 minutes of galvanotaxis. (A) Immunostaining shows that αENaC is distributed randomly at cell periphery of NHK and MEK before exposure to the EF (0 minutes, cathode on right of the image). After 60 minutes in the EF, ENaC is concentrated at the cathodal side of cells. (B) Both uncleaved (∼80–85 kDa) and cleaved αENaC (60–65 kDa) bands were detected with western blotting on three strains of NHKs. (C) At 0 minutes, the fluorescence intensity between the cathodal side and anodal side of keratinocytes is similar (left), but at 60 minutes, the fluorescence is increased at the cathodal side (right, *P<0.05, 20 human keratinocytes analyzed in each group).

Fig. 5.

ENaC is required to establish stable lamellipodia at the cathodal side of galvanotactic keratinocytes. (A) Mouse keratinocytes were exposed to the EF and filmed for 10 minutes. Fan-shaped cells were selected and a 1-pixel wide line at cell periphery was drawn every 10% of the length (lines were aligned from anode to cathode, numbered 0–10) across the MEK to generated kymographs. (B) The protrusion and retraction of the lamellipodia were tracked and plotted from either wild-type MEK or αENaC-KO-MEK. (C,D) Wild-type MEK lamellipodia protrude faster and further at the distal cathodal side compared to the anodal side (C, n = 13). The asymmetric protrusion is ENaC-dependent and the lamellipodia extended at a similar rate and to a similar distance at the distal sides in the αENaC-KO-MEK (D, n = 7). Quantitative data are presented in Table 1. *P<0.05 for the comparisons indicated.