Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair

Abstract

The ability to heal wounds is vital to all organisms. In mammalian tissues, alterations in intermediate filament (IF) gene expression represent an early reaction of cells surviving injury. We investigated the role of keratin IFs during the epithelialization of skin wounds using a keratin 6alpha and 6beta (K6alpha/K6beta)-null mouse model. In skin explant culture, null keratinocytes exhibit an enhanced epithelialization potential due to increased migration. The extent of the phenotype is strain dependent, and is accompanied by alterations in keratin IF and F-actin organization. However, in wounded skin in vivo, null keratinocytes rupture as they attempt to migrate under the blood clot. Fragility of the K6alpha/K6beta-null epidermis is confirmed when applying trauma to chemically treated skin. We propose that the alterations in IF gene expression after tissue injury foster a compromise between the need to display the cellular pliability necessary for timely migration and the requirement for resilience sufficient to withstand the rigors of a wound site.

Figure 1.

K6α/K6β-null keratinocytes exhibit enhanced epithelialization potential in skin explant culture. (A) Quantitation of keratinocyte outgrowth from explants. Skin punches were cultured for 8 d, and keratinocytes were identified by K17 immunostaining. Eight skin punches per mouse with a minimum of 13 mice per genotype were analyzed. Mean ± SEM values are shown. Asterisks depict statistically significant difference from wild type (P < 0.0001). (B) Examples of wild-type and K6α/K6β-null skin explants processed for K17 immunostaining after 8 d in culture. Arrow indicates leading edge of keratinocyte outgrowth and double arrowheads denote edge of explant biopsy. (C) The performance of K6α/K6β- and K14-null skin explants was compared by assessing the distance between keratinocytes located at the distal edge of the outgrowth and the explant edge. The error bars represent SD. (D and E) Cellular proteins were prepared from pooled explants in culture for 6 d (n > 60 explants per genotype; see Materials and methods), resolved using SDS-PAGE, and stained with Coomassie blue or Western blotting to determine keratin content. In D, asterisk denotes a band that is present in variable amounts between preparations and which likely represents a degradation product from a larger protein. (F) Semi-quantitative RT-PCR was performed on RNA collected from 6-d-old outgrowths. K16 specific primers were used quantify the amount of K16 mRNA transcripts. β-Tubulin primers were used as an internal control. Samples were collected after 22, 27, 32, and 37 PCR cycles.

Figure 2.

Enhanced K6α/K6β-null epithelialization likely results from increased keratinocyte migration. (A) Immunofluorescence staining for BrdU in cellular outgrowths from 6-d-old skin explants. The region shown is located next to the explant edge (Ex). Arrows depict the direction of outgrowth. Bar, 50 μm. (B) Density of BrdU-positive keratinocytes in wild-type and K6α/K6β-null samples (expressed per millimeter of explant tissue perimeter). The error bars represent SD. (C) Western blot analysis for myc and proliferating cell nuclear antigen in protein extracts prepared from 6-d-old explants. Actin is the loading control. (D and E) Keratinocyte migration in skin explants after mitomycin C treatment. (D) Total area of keratinocyte outgrowth after 8 d, after treatment or not at 24 h, is shown for hemizygous and homozygous K6α/K6β-null explants relative to wild type (n = 62 wt; 132 hemizygous; 35 null explants). (E) Keratinocyte outgrowth in wild-type, hemizygous and homozygous K6α/K6β-null explants as a function of time in live skin explant cultures after treatment at 48 h after seeding. Distance measurements were made at days 2, 4, 6, and 8 d in culture (n = 6 wt; 13 hemizygous; 12 null explants). The error bars represent SEM. (F) Pooled cellular outgrowths from skin explants cultured for 6 d (n > 60 per genotype) were solubilized with lysis buffer, and insoluble proteins were pelleted, solubilized, and electrophoresed (2 μg protein) before Western blot analysis using α-p120^ctn, α-phosphotyrosine, and α-actin antibodies. Phosphotyrosine epitopes that are increased in K6α/K6β-null samples compared with controls are identified with an asterisk, whereas those present in lower amounts are identified with a star. The two bands in the p120^ctn blot likely represents distinct isoforms of p120^ctn (Anastasiadis and Reynolds, 2001).

Figure 3.

Immunofluorescence staining of keratinocyte outgrowths reveals alterations in the keratin and actin cytoskeletons. Wild-type and K6α/K6β-null explants were immunostained for (A) K16, K17, and (B) actin. (A) Single arrows identify pan-cytoplasmic keratin filaments in wild-type samples, whereas arrowheads point to partially collapsed K16- and K17-containing filament networks in K6α/K6β-null keratinocytes at the explant edge. (B) Double arrowheads identify the increased intensity and number of actin filaments in the K6α/K6β-null keratinocytes. Bar, 50 μm.

Figure 4.

Enhanced epithelialization potential in skin explant culture is affected by genetic strain background. Mixed K6α/K6β-null mice (129/Sv × C57Bl/6 × DBA2) were backcrossed into two different inbred strains, 129/SvJ and C57Bl/6, for a minimum of seven generations. The extent of keratinocyte outgrowth after 8 d of skin explant culture was quantitated and compared with the mixed genetic background. Mean ± SEM values are displayed. Genotypes that are statistically distinct from wild-type explants are identified with an asterisk (P ≤ 0.0002).

Figure 5.

Grafted K6α/K6β-null skin tissue exhibit epithelial fragility after incisional wounding. (A and B) Dorsal view of wild-type and K6α/K6β-null backskins grafted onto immunocompromised mice. (C and E) Micrographs of H&E stained sections of unwounded grafted backskin from wild-type and K6α/K6β-null animals. (D and F) Micrographs of similar sections at 3 d after full skin thickness incisional wounding of the grafted tissue. Arrowheads depict migrating keratinocytes at the wound edge. (G and H) Higher magnification of activated epithelium in (G) wild-type and (H) K6α/K6β-null skin grafts at 3 d after injury. Arrows point to the occurrence of degenerative changes in the activated epithelium at the wound edge and to intracellular lysis in migrating keratinocytes. Open arrowheads point to floating nuclei within the suprabasal layers. (I) Immunostaining for K17 in wound edge tissue from a K6α/K6β-null skin graft, confirming that cleavage, as indicated by arrows, occurs within the cytoplasm. epi, epidermis; HF, hair follicle; AK, activated keratinocytes at the wound edge; and Sc, scab. Bars: (C–F) 100 μm; (G–I) 50 μm.

Figure 5.

Grafted K6α/K6β-null skin tissue exhibit epithelial fragility after incisional wounding. (A and B) Dorsal view of wild-type and K6α/K6β-null backskins grafted onto immunocompromised mice. (C and E) Micrographs of H&E stained sections of unwounded grafted backskin from wild-type and K6α/K6β-null animals. (D and F) Micrographs of similar sections at 3 d after full skin thickness incisional wounding of the grafted tissue. Arrowheads depict migrating keratinocytes at the wound edge. (G and H) Higher magnification of activated epithelium in (G) wild-type and (H) K6α/K6β-null skin grafts at 3 d after injury. Arrows point to the occurrence of degenerative changes in the activated epithelium at the wound edge and to intracellular lysis in migrating keratinocytes. Open arrowheads point to floating nuclei within the suprabasal layers. (I) Immunostaining for K17 in wound edge tissue from a K6α/K6β-null skin graft, confirming that cleavage, as indicated by arrows, occurs within the cytoplasm. epi, epidermis; HF, hair follicle; AK, activated keratinocytes at the wound edge; and Sc, scab. Bars: (C–F) 100 μm; (G–I) 50 μm.

Figure 6.

Trauma-induced lysis of K6α/K6β-null keratinocytes in grafted backskin subjected to chemical induction. PMA was applied on days 1, 3, and 5, and the grafted backskins harvested on day 7. (A and D) Micrographs of H&E stained sections of wild-type and K6α/K6β-null PMA treated grafts without mechanical trauma. (B and E) Gentle friction was applied to treated grafts immediately before harvest. Arrows depict intracellular lysis in the suprabasal layers of K6α/K6β-null epidermis. (C and F) Wild-type and K6α/K6β-null grafted tissue were processed for plastic embedding, sectioned (0.5-μm thick), and stained with toluidine blue. epi, epidermis; derm, dermis; exud, exudate; hr, hair; and hf, hair follicle. Bars, 50 μm.