Ultraviolet B Inhibits Skin Wound Healing by Affecting Focal Adhesion Dynamics

Abstract

As the most important interface between human body and external environment, skin acts as an essential barrier preventing various environmental damages, among which DNA-damaging UV radiation from the sun remains the major environmental risk factor causing various skin diseases. It has been well documented that wavelengths in the ultraviolet B (UVB) radiation range (290-320 nm) of the solar spectrum can be absorbed by skin and lead to cutaneous injury and various other deleterious effects. During process such as wound healing, the orchestrated movement of cells in a particular direction is essential and highly regulated, integrating signals controlling adhesion, polarity and the cytoskeleton. Cell adhesion and migration are modulated through both of actin and microtubule cytoskeletons. However, little was known about how UVB affects skin wound healing and migration of epidermal keratinocytes. Here, we demonstrate that UVB can delay the wound healing progress in vivo with a murine model of full-thickness skin wound. In addition, UVB significantly inhibited keratinocyte motility by altering focal adhesion turnover and cytoskeletal dynamics. Our results provide new insights into the etiology of UVB exposure-induced skin damages.

Figure 1. Wound healing and histopathological examination in UVB-treated mice

(A) Macroscopic view of wound healing on day 0, 2, 4, 6 and 8. A 6-mm wound was created on the back of mice and wound closure was monitored with or without UVB treatment. (B) Wound healing as monitored by histological staining of skin sections at the wound edges 2, 4 and 6 days after injury with or without UVB treatment (100 mj/cm²). Halves of wound sections are shown. Epi: epidermis; der: dermis; Es: eschar. Dotted lines denote dermal–epidermal boundaries. Scale bar represents 500 µm. (C) Quantification of the length of hyperproliferative epidermis generated at times indicated after wounding. All bars represent mean ± SD from three mice.

Figure 2. Cell migration defects in UVB-treated keratinocytes

(A) Migration of confluent monolayers of mouse keratinocytes cultured from control and UVB treatment was assessed by in vitro scratch-wound assays. (B) The kinetics of in vitro wound healing was quantified. Wounding areas were measured using ImageJ software and expressed as the percentage of the original area size with the bar graph (P < 0.01). A representative experiment is shown. (C) Movements of individual keratinocytes were traced by videomicroscopy. Migration tracks of multiple cells for each group (control or UVB treated) are shown here as scatter plots.

Figure 3. UVB affects focal adhesion size

(A) Immunolabeling of UVB treated and untreated keratinocytes for tubulin (red), and focal adhesion marker vinculin (green). Scale bar represents 10 µm. (B) Box and whisker plot indicating the size distribution of focal adhesions in control and UVB treated cells.

Figure 4. UVB affects focal adhesion dynamics

(A) Representative time-lapse images (montages) of DsRed-Zyxin expressing keratinocytes. Note formation and dissolution of focal adhesions in control cells and very static focal adhesions in UVB treated cells. Scale bar represents 10 µm. (B) Box and whisker plots revealing slow assembly and disassembly rates of focal adhesions in UVB treated cells relative to their control counterparts. (C) Box and whisker plots revealing slow assembly and disassembly rates of focal adhesions in UVB treated skin explants relative to their control counterparts.

Figure 5. UVB effects on FA dynamics by FRAP

(A) Fluorescence recovery after photobleaching (FRAP) was used to visualize reduced dynamics of focal adhesions in UVB treated vs untreated cells expressing DsRed-Zyxin. Fluorescence recovery was recorded for 100 s after photobleaching. Representative time-lapse images (montage) of focal adhesions are shown. Scale bar represents 1 µm. (B) Box-and-whisker diagram quantifying the differences in half-time (T_1/2) of FRAP between control and UVB treated cells.

Figure 6. UVB effects on microtubule (MT) dynamics

Box and whisker plots for EB1 plus end dynamics in control and UVB treated cells. Three parameters of MT plus end dynamics were retrieved from the confocal videos, plus end growth speed (A), growth lifetime (B), and growth displacement (C).

Figure 7. UVB effects on MT targeting to FA

(A) Representative time-lapse images (montages) of keratinocytes expressing GFP-Paxillin and TaqRed-EB1 show MT plus-end growth (red) toward FAs (green). The scale bar represents 1 µm. (B) Behavior of MT targeting to FAs was manually traced, and targeting frequencies to individual FAs were quantified and depicted by bar graph. Note the significant alterations in the ability of MTs targeting to FAs in UVB treatment.