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Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization.

Nunan R, Campbell J, Mori R, Pitulescu ME, Jiang WG, Harding KG, Adams RH, Nobes CD, Martin P - Cell Rep (2015)

Bottom Line: Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back.Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells.If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man.

View Article: PubMed Central - PubMed

Affiliation: Schools of Biochemistry and Physiology & Pharmacology, University of Bristol, Bristol BS8 1TD, UK.

No MeSH data available.


Related in: MedlinePlus

In Vitro KD of Ephrin-B1/B2 Prevents Cell-Cell Loosening and E-cadherin Cleavage(A) Confirmation of ephrin-B1/B2 knockdown (KD) by western blot of human keratinocyte cells (HaCaTs) at 3 days after siRNA transfection is shown.(B) (Top) HaCaT cells transfected with efnB1/B2 siRNA, scratch wounded at confluence, and compared with control wounds at 0 and 15 hr post-scratching (data within each experiment normalized to control). (Bottom) Graph shows relative extent of wound closure at this time point (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 6; four wells per experiment).(C) Graph illustrates the rate of HaCaT wound repair from time-lapse imaging over 3-hr periods from 0–15 hr (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 4).(D) (Left) Colored tracks of follower cells (fourth row cells) superimposed on phase-contrast images of 15-hr scratch wounds. efnB1/B2 KD leads to considerably reduced persistence of migration. (Right) Graphic representation of follower cell persistence is shown (∗∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 4; nine cells per experiment).(E) Snapshot, phase-contrast images show transfected HaCaT cells from 15-hr wounds, taken at 20-min intervals to illustrate how similar are lamellar protrusions of leading-edge cells of KD versus control wounds.(F) (Left) An example of intercellular spaces (red) between cells back from the leading edge (as indicated by wound schematic) at 15 hr post-wounding normalized to control. (Right) Graphic representation of differences in intracellular spaces between control and ephrinB1/B2 KD cells is shown (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 3).(G) Western blot shows E-cadherin from culture media and cell lysates (data within each experiment were normalized to tubulin control (∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 5).(H) WT day 3 wound shows immunostaining for ADAM10 (brown) in the migrating epidermal tongue (yellow arrowheads).(I) In vitro, 50 μM ADAM10 inhibitor TAPI-1 inhibits HaCaT migration 15 hr after scratch wounding (∗∗∗p < 0.001, as determined by one-way ANOVA with Dunnett’s post hoc test; n = 3).(J) (Left) TAPI-1 injected into mouse dorsal skin wounds significantly retards re-epithelialization. Yellow bar indicates length of epidermal tongue. (Right) Graphic representation of the extent of re-epithelialization in control versus TAPI-1-treated wounds is shown (∗p < 0.05, as determined by an unpaired Student’s t test; n = 6 wounds from three mice per condition).Scale bars, 100 μm (B), 50 μm (D), 20 μm (E), 50 μm (F), 200 μm (H), and 100 μm (J).
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fig5: In Vitro KD of Ephrin-B1/B2 Prevents Cell-Cell Loosening and E-cadherin Cleavage(A) Confirmation of ephrin-B1/B2 knockdown (KD) by western blot of human keratinocyte cells (HaCaTs) at 3 days after siRNA transfection is shown.(B) (Top) HaCaT cells transfected with efnB1/B2 siRNA, scratch wounded at confluence, and compared with control wounds at 0 and 15 hr post-scratching (data within each experiment normalized to control). (Bottom) Graph shows relative extent of wound closure at this time point (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 6; four wells per experiment).(C) Graph illustrates the rate of HaCaT wound repair from time-lapse imaging over 3-hr periods from 0–15 hr (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 4).(D) (Left) Colored tracks of follower cells (fourth row cells) superimposed on phase-contrast images of 15-hr scratch wounds. efnB1/B2 KD leads to considerably reduced persistence of migration. (Right) Graphic representation of follower cell persistence is shown (∗∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 4; nine cells per experiment).(E) Snapshot, phase-contrast images show transfected HaCaT cells from 15-hr wounds, taken at 20-min intervals to illustrate how similar are lamellar protrusions of leading-edge cells of KD versus control wounds.(F) (Left) An example of intercellular spaces (red) between cells back from the leading edge (as indicated by wound schematic) at 15 hr post-wounding normalized to control. (Right) Graphic representation of differences in intracellular spaces between control and ephrinB1/B2 KD cells is shown (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 3).(G) Western blot shows E-cadherin from culture media and cell lysates (data within each experiment were normalized to tubulin control (∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 5).(H) WT day 3 wound shows immunostaining for ADAM10 (brown) in the migrating epidermal tongue (yellow arrowheads).(I) In vitro, 50 μM ADAM10 inhibitor TAPI-1 inhibits HaCaT migration 15 hr after scratch wounding (∗∗∗p < 0.001, as determined by one-way ANOVA with Dunnett’s post hoc test; n = 3).(J) (Left) TAPI-1 injected into mouse dorsal skin wounds significantly retards re-epithelialization. Yellow bar indicates length of epidermal tongue. (Right) Graphic representation of the extent of re-epithelialization in control versus TAPI-1-treated wounds is shown (∗p < 0.05, as determined by an unpaired Student’s t test; n = 6 wounds from three mice per condition).Scale bars, 100 μm (B), 50 μm (D), 20 μm (E), 50 μm (F), 200 μm (H), and 100 μm (J).

Mentions: To further analyze the link between ephrin-B signaling and junction dissolution and how this might impact on epidermal sheet migration, we established an in vitro scratch wound assay with HaCaT (human keratinocyte) cells that endogenously express ephrin-B1/B2 localized to cell-cell junctions (Figure S5A). We knocked down ephrin-B1 (91%) and -B2 (74%) using conventional small interfering RNAs (siRNAs) (Figure 5A; Figure S5B) and observed that ephrin-B1/B2 knockdown (KD) scratch wounds had a significantly retarded rate of closure, such that, at 15 hr, repair was 36% less in KD cells compared to control wounds (Figure 5B; Figure S5C). Time course analysis indicated that cell migration stalled from about 3 hr after wounding KD cells, whereas control cells continued advancing forward (Figure 5C; Movie S1). Tracking studies of cells back from the leading edge (follower cells) over 15 hr in the KD wounds showed them to still be moving after 3 hr, but not in a polarized, forward direction (Figure 5D). Stalled wound closure was not a consequence of perturbed lamellipodial assembly in leading-edge cells, because lamellar dynamics appeared identical in control and ephrin-B KD scratch wounds, even after the wound edges had stalled (Figure 5E; Movie S2); neither were there defects in cell polarity (Figure S5D) or proliferation (Figure S5E). Rather, our data suggest that the leading cells might be restrained from moving forward by tension within the epithelial monolayer.


Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization.

Nunan R, Campbell J, Mori R, Pitulescu ME, Jiang WG, Harding KG, Adams RH, Nobes CD, Martin P - Cell Rep (2015)

In Vitro KD of Ephrin-B1/B2 Prevents Cell-Cell Loosening and E-cadherin Cleavage(A) Confirmation of ephrin-B1/B2 knockdown (KD) by western blot of human keratinocyte cells (HaCaTs) at 3 days after siRNA transfection is shown.(B) (Top) HaCaT cells transfected with efnB1/B2 siRNA, scratch wounded at confluence, and compared with control wounds at 0 and 15 hr post-scratching (data within each experiment normalized to control). (Bottom) Graph shows relative extent of wound closure at this time point (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 6; four wells per experiment).(C) Graph illustrates the rate of HaCaT wound repair from time-lapse imaging over 3-hr periods from 0–15 hr (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 4).(D) (Left) Colored tracks of follower cells (fourth row cells) superimposed on phase-contrast images of 15-hr scratch wounds. efnB1/B2 KD leads to considerably reduced persistence of migration. (Right) Graphic representation of follower cell persistence is shown (∗∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 4; nine cells per experiment).(E) Snapshot, phase-contrast images show transfected HaCaT cells from 15-hr wounds, taken at 20-min intervals to illustrate how similar are lamellar protrusions of leading-edge cells of KD versus control wounds.(F) (Left) An example of intercellular spaces (red) between cells back from the leading edge (as indicated by wound schematic) at 15 hr post-wounding normalized to control. (Right) Graphic representation of differences in intracellular spaces between control and ephrinB1/B2 KD cells is shown (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 3).(G) Western blot shows E-cadherin from culture media and cell lysates (data within each experiment were normalized to tubulin control (∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 5).(H) WT day 3 wound shows immunostaining for ADAM10 (brown) in the migrating epidermal tongue (yellow arrowheads).(I) In vitro, 50 μM ADAM10 inhibitor TAPI-1 inhibits HaCaT migration 15 hr after scratch wounding (∗∗∗p < 0.001, as determined by one-way ANOVA with Dunnett’s post hoc test; n = 3).(J) (Left) TAPI-1 injected into mouse dorsal skin wounds significantly retards re-epithelialization. Yellow bar indicates length of epidermal tongue. (Right) Graphic representation of the extent of re-epithelialization in control versus TAPI-1-treated wounds is shown (∗p < 0.05, as determined by an unpaired Student’s t test; n = 6 wounds from three mice per condition).Scale bars, 100 μm (B), 50 μm (D), 20 μm (E), 50 μm (F), 200 μm (H), and 100 μm (J).
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fig5: In Vitro KD of Ephrin-B1/B2 Prevents Cell-Cell Loosening and E-cadherin Cleavage(A) Confirmation of ephrin-B1/B2 knockdown (KD) by western blot of human keratinocyte cells (HaCaTs) at 3 days after siRNA transfection is shown.(B) (Top) HaCaT cells transfected with efnB1/B2 siRNA, scratch wounded at confluence, and compared with control wounds at 0 and 15 hr post-scratching (data within each experiment normalized to control). (Bottom) Graph shows relative extent of wound closure at this time point (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 6; four wells per experiment).(C) Graph illustrates the rate of HaCaT wound repair from time-lapse imaging over 3-hr periods from 0–15 hr (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 4).(D) (Left) Colored tracks of follower cells (fourth row cells) superimposed on phase-contrast images of 15-hr scratch wounds. efnB1/B2 KD leads to considerably reduced persistence of migration. (Right) Graphic representation of follower cell persistence is shown (∗∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 4; nine cells per experiment).(E) Snapshot, phase-contrast images show transfected HaCaT cells from 15-hr wounds, taken at 20-min intervals to illustrate how similar are lamellar protrusions of leading-edge cells of KD versus control wounds.(F) (Left) An example of intercellular spaces (red) between cells back from the leading edge (as indicated by wound schematic) at 15 hr post-wounding normalized to control. (Right) Graphic representation of differences in intracellular spaces between control and ephrinB1/B2 KD cells is shown (∗∗∗p < 0.001, as determined by an unpaired Student’s t test; n = 3).(G) Western blot shows E-cadherin from culture media and cell lysates (data within each experiment were normalized to tubulin control (∗∗p < 0.01, as determined by an unpaired Student’s t test; n = 5).(H) WT day 3 wound shows immunostaining for ADAM10 (brown) in the migrating epidermal tongue (yellow arrowheads).(I) In vitro, 50 μM ADAM10 inhibitor TAPI-1 inhibits HaCaT migration 15 hr after scratch wounding (∗∗∗p < 0.001, as determined by one-way ANOVA with Dunnett’s post hoc test; n = 3).(J) (Left) TAPI-1 injected into mouse dorsal skin wounds significantly retards re-epithelialization. Yellow bar indicates length of epidermal tongue. (Right) Graphic representation of the extent of re-epithelialization in control versus TAPI-1-treated wounds is shown (∗p < 0.05, as determined by an unpaired Student’s t test; n = 6 wounds from three mice per condition).Scale bars, 100 μm (B), 50 μm (D), 20 μm (E), 50 μm (F), 200 μm (H), and 100 μm (J).
Mentions: To further analyze the link between ephrin-B signaling and junction dissolution and how this might impact on epidermal sheet migration, we established an in vitro scratch wound assay with HaCaT (human keratinocyte) cells that endogenously express ephrin-B1/B2 localized to cell-cell junctions (Figure S5A). We knocked down ephrin-B1 (91%) and -B2 (74%) using conventional small interfering RNAs (siRNAs) (Figure 5A; Figure S5B) and observed that ephrin-B1/B2 knockdown (KD) scratch wounds had a significantly retarded rate of closure, such that, at 15 hr, repair was 36% less in KD cells compared to control wounds (Figure 5B; Figure S5C). Time course analysis indicated that cell migration stalled from about 3 hr after wounding KD cells, whereas control cells continued advancing forward (Figure 5C; Movie S1). Tracking studies of cells back from the leading edge (follower cells) over 15 hr in the KD wounds showed them to still be moving after 3 hr, but not in a polarized, forward direction (Figure 5D). Stalled wound closure was not a consequence of perturbed lamellipodial assembly in leading-edge cells, because lamellar dynamics appeared identical in control and ephrin-B KD scratch wounds, even after the wound edges had stalled (Figure 5E; Movie S2); neither were there defects in cell polarity (Figure S5D) or proliferation (Figure S5E). Rather, our data suggest that the leading cells might be restrained from moving forward by tension within the epithelial monolayer.

Bottom Line: Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back.Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells.If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man.

View Article: PubMed Central - PubMed

Affiliation: Schools of Biochemistry and Physiology & Pharmacology, University of Bristol, Bristol BS8 1TD, UK.

No MeSH data available.


Related in: MedlinePlus