Increased levels of keratin 16 alter epithelialization potential of mouse skin keratinocytes in vivo and ex vivo

Abstract

The process of wound repair in adult skin is complex, involving dermal contraction and epithelial migration to repair the lesion and restore the skin's barrier properties. At the wound edge, keratinocytes undergo many changes that engender an epithelialization behavior. The type II keratin 6 and type I keratins 16 and 17 are induced well before cell migration begins, but the role of these proteins is not understood. Forced expression of human K16 in skin epithelia of transgenic mice has been shown to cause dose-dependent skin lesions concomitant with alterations in keratin filament organization and in cell adhesion. Here we show, with the use of a quantitative assay, that these transgenic mice show a delay in the closure of full-thickness skin wounds in situ compared with wild-type and low-expressing K16 transgenic mice. We adapted and validated an ex vivo skin explant culture system to better assess epithelialization in a wound-like environment. Transgenic K16 explants exhibit a significant reduction of keratinocyte outgrowth in this setting. This delay is transgene dose-dependent, and is more severe when K16 is expressed in mitotic compared with post-mitotic keratinocytes. Various lines of evidence suggest that the mechanism(s) involved is complex and not strictly cell autonomous. These findings have important implications for the function of K16 in vivo.

Figure 1

Analysis of full-thickness skin wounds in vivo. (A) In vivo wound closure assay was performed to determine the average rate of linear ingrowth for wild-type (n = 4), 6-17-sbK16 (n = 3), and 5-7-sbK16 (n = 3) mice. Mice from the 5-7-sbK16 line display significantly (p < 0.04) slower ingrowth than wild type. The 6-17-sbK16 mice do not vary significantly from wild type. (B–F) Skin wounds were fixed at various time points for histological analysis. C and E show wounds from wild-type mice, whereas B, D, and F show wounds from 5-7-sbK16 mice. Immunostaining with antibodies that preferentially recognize human K16 shows transgene induction 27 h after wounding (B). C and D are hematoxylin and eosin stainings of sections from 5-d-old wounds. E and F are 5-d-old wounds that have been immunostained with antibodies directed against K17. Black arrows denote the wound edge. The open arrowhead shows nonwounded skin, which does not express the transgene (B). HF, hair follicles. Bars, 100 μm.

Figure 2

Analysis of explants from newborn mice. (A) A 4-mm full-thickness punch biopsy was isolated, grown in medium for 7 d, and immunostained with antibodies directed against K17 to confirm the cells are keratinocytes. The solid line denotes the explant edge. The dotted line denotes the cells that are most distal to the explant. (B) Phase contrast microscopy of an unstained explant. Cells that have migrated onto the culture dish have an epithelioid appearance. Bar, 200 μm. (C) Distance between the explant edge and the most distal cells of the outgrowth was measured over time. The heavy solid line is a linear curve fit to the keratinocyte measurements (●). The dashed line is a linear curve fit to measurements of fibroblast outgrowth (×). A 24-h (⋄) or 36-h (♦) cell cycle time was used to calculate the theoretical curve for distance due to cell proliferation, distance = (A_total/π)^1/2 · (e^t/2τ).

Figure 3

BrdU pulse-chase labeling of skin explants. (A–F) Keratinocyte outgrowth from wild-type explants was pulse-labeled with BrdU after 3, 6, or 8 d of culture, fixed after a 5-, 2-, or 0-d chase period, respectively, then immunostained to assess movement of BrdU-positive cells over time. (A and D) BrdU labeling after 8 d, with no chase, shows that most BrdU-positive keratinocytes localize proximal (solid line) rather than distal (dotted line) to the explant. Labeling after 6 d of culture followed by a 2-d chase (B and E), or after 3 d with a 5-d chase (C and F), reveals that the majority of BrdU-positive keratinocytes have moved away from the proximal explant edge (E and F) and closer to the distal edge (B and C). White arrows indicate the direction of outgrowth movement. Bar, 170 μm. (G–J) Modified Boyden chamber assay. Wild-type explants were cultured on a porous membrane for 8 d, fixed, and immunostained for K17 to assess migration of keratinocytes through the membrane. After immunostaining, keratinocytes were removed from the bottom, top, or both sides of the membrane. Outgrowth of keratinocytes (black arrow) out of the explant (white arrow) appeared normal on top of the membrane (G). Keratinocytes were also observed to have moved through membrane pores and localized on the bottom side of the membrane (H) as well as on the culture dish below the membrane (black arrow) alongside fibroblasts (J). Wiping both sides of the membrane shows cell removal to be efficient (I). Bar, 300 μm. (K) Mitomycin C treatment. Keratinocyte outgrowth after 8 d in culture was quantitated for wild-type explants previously treated with mitomycin C to permanently block cell proliferation. Treatment was performed for 2 h at either 24, 48, or 72 h after explants were placed into culture, and resulted in an ∼48, ∼43, and ∼17% reduction in keratinocyte outgrowth, respectively.

Figure 4

Keratin expression in skin explants. (A–F) Immunofluorescence antibody staining of explant outgrowth. A filamentous pattern is seen for K14 (A), K16 (B), K17 (C), and K6 (D). K6, K17, and K14 staining appears homogeneous throughout the outgrowth, whereas K16 shows a heterogeneous pattern. Only background staining is seen for K10 (E). Bar, 85 μm. Dashed line indicates the distal edge of the outgrowth. Arrows indicate the direction of cell migration. (F) SDS-PAGE and Western analysis show that levels of K10 are decreased relative to K14 in the explant keratinocytes compared with keratinocytes grown in primary culture. (G) X-gal staining of a skin puncture wound in an adult KT1–1p mouse whose genome harbors a wound-inducible promoter fused to lacZ. This piece of skin was fixed and stained 24 h after wounding. The blue color indicates β-galactosidase activity. (H and I) Explants were removed from KT1-1p mouse pups, placed into culture, and incubated with X-gal until blue color developed. At 8 h (H), there is already strong induction of the transgene at the edge of the tissue. By 6 d (I) in culture, the cells that have grown out of the explant, and the explant wound edge itself, are positive for the transgene. Bar, 200 μm.

Figure 5

Effect of keratin transgene expression on keratinocyte migration in the skin explant setting. Outgrowth of keratinocytes from wild-type or transgenic explants was quantitated after 8 d in culture. Homozygous 5-7-sbK16 explants show a 30% reduction compared with wild-type explants, whereas homozygous 10-bK16 explants show a 57% reduction. In comparison, explants isolated from heterozygous 10-bK16 and homozygous or heterozygous B1-bK16-C14 mice reveal only a 15, 20, and 15% reduction in outgrowth relative to wild-type, respectively.

Figure 6

Characterization of K16 expression in transgenic explants. (A) Cell extracts isolated from homozygous 5-7-sbK16 and 10-bK16 explant outgrowth were isolated and subjected to Western analysis. Blots probed with antibodies directed toward K14 and K16 reveal total levels of the hK16 transgene to be similar between keratinocytes from these two types of transgenic explants. (B–D) The distribution and filament reorganizing properties of the hK16 transgene product in homozygous 5-7-sbK16 and 10-bK16 explants were assessed via immunofluorescence. (B) Heterogeneity of hK16 staining at the distal edge of 5-7-sbK16 explants. The asterisk identifies a nonreactive keratinocyte. (C) hK16 staining is less heterogeneous at the proximal edge in these explants, but a subset of keratinocytes shows a punctate staining pattern, indicating alterations in keratin assembly or organization (black arrow). (D) Immunostaining for hK16 in keratinocytes from 10-bK16 explants reveals filament reorganization (black arrows) almost exclusively in keratinocytes distal to the explant as well as a more homogeneous transgene expression pattern throughout the outgrowth. Solid lines indicate the proximal explant edge, dotted lines the distal edge, and white arrows indicate the direction of keratinocyte migration. Bar, 42.5 μm.