Functional differences between keratins of stratified and simple epithelia

Abstract

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.

Figure 1

Expression of K16-K14 in transgenic mice. (A) A schematic of the replacement vector, showing 2,100 bp of the human K14 promoter (hK14), followed by the 5′ intron and 5′ untranslated sequence from the rabbit β-globin gene (β int), followed by a K16-14 hybrid transgene followed by the K14 3′ untranslated sequence (see Allen et al., 1996 for expression vector). The dotted vertical lines demarcate the relative portions of K16 versus K14 coding sequences used (see Paladini and Coulombe, 1998). (B) IF proteins were isolated from the skins of K16-K14 transgenic (lane 1) and control (lane 2) neonatal mice. Proteins were then subjected to SDS-PAGE. Gels were subjected to immunoblot analysis using an antibody against the COOH terminus of K14, also present in the 49-kD transgene product. Relative amounts of transgenic versus wild-type K14 were determined by densitometry scanning. Sample shown is from the low expressing K16-14 transgenic mouse.

Figure 2

The cytolysis in the basal layer of K14 null epidermis is missing when these cells express the K16-14 transgene. Semithin (0.75 μm) sections of neonatal paw skin biopsies from (A and B) K14 null/K16-14 transgenic and (C) K14 null mice. Skins were embedded in Epon and stained with Toluidine blue. Section in A is from central paw, showing that even in areas rich in rete ridges, the K16-14–expressing, K14 null paw skin appears indistinguishable from wild type. Basal cells from the equivalent region of K14 null skin were fully cytolyzed, leading to complete separation of the upper epidermis (not shown). Areas of paw skin from areas where hair follicles were still sparse, but where rete ridges were no longer present were still normal in the replacement skin (B), but partially blistered in the K14 null skin (C). Bar, 30 μm.

Figure 3

K14 null mice expressing the K16-14 transgene have basal epidermal cells that are rich in keratin filament bundles and similar to wild-type basal cells. Paw skin samples were taken from neonatal age-matched animals that were either K16-14 transgenic/K14 null, K14 null, or wild type. Skins were processed for electron microscopy. (A) Basal cell of K16-14 paw skin in area of rete ridge formation. Basal cells show bundles of keratin filaments (Kf) in cytoplasm. No signs of microblistering or basal cell cytolysis were evident here, or elsewhere throughout the skin. (B) Basal layer of K14 null mouse skin. Note that keratin filament bundles are largely absent. Note also the presence of basal cell cytolysis (asterisks). (C) Basal layer from wild-type mouse skin. Note presence of bundles of keratin filaments (Kf). Nu, nucleus; BL, basal lamina; Mi, mitochondria; De, desmosome; Hd, hemidesmosome. Bar, 0.5 μm.

Figure 4

K18 replacement vector and transgene expression in mouse skin. (Top) Schematic depicting the K18 replacement vector. The backbone of the vector is as described in Fig. 1. The full-length human K18 cDNA (Oshima et al., 1986) was introduced into the BamHI site of the vector. Frozen, methanol-fixed sections of back skins (10 μm) of neonatal K18 transgenic mice (tg) on a wild-type K14 background were subjected to double-immunofluorescence microscopy using antibodies against K18 (FITC-conjugated secondary) and K14 (Texas red–conjugated secondary), respectively (see Materials and Methods). A and B, wild-type skin; C and D, K18 transgenic skin. Bar, 40 μm.

Figure 5

Polyacrylamide gel analysis of IF proteins. IF proteins were extracted from the skins of neonatal transgenic mice expressing the K18 transgene on a wild-type (A and B) or K14 null (C) background. A–C, duplicate gels were either stained with Coomassie blue (A and C) or transferred to nitrocellulose paper for immunoblot analysis with K18 (B) or K14 (not shown) antibodies. Shown are the type I keratins. K10 is far more abundant than K14 (spot below K10 in A). Note that the K14 spot, confirmed by immunoblot analysis, is missing in C. (Note: we do not know the identity of the very acidic spot in C; it does not migrate at a pKi or size known to keratins, and it was not consistently obtained in our 0.6 M KCl–insoluble extracts.) (D and E) Duplicate samples of IF proteins from wild-type (wt) and K18 transgenic (Tg) mouse skins and dilutions of purified recombinant human K18 or K14 were subjected to one-dimensional gel electrophoresis and immunoblot analysis using αK18 or αK14 antibodies. Loadings were: wt, 4 μg extract; Tg, 2 μg and 4 μg extract, respectively. Recombinant protein loadings are as indicated in nanograms.

Figure 6

Immunoblot analyses of skin IF proteins from wild-type, K14 null, K18 transgenic, and K18 rescue mice. Triton X-100– insoluble and –soluble protein extracts were prepared from back skins of 1–2-d-old mice, resolved by electrophoresis through 10% SDS–polyacrylamide gels (Wu et al., 1982) and transferred to nitrocellulose paper. The blot was then sequentially hybridized with antibodies against K18, K14, and K5. After each hybridization, bound antibody was visualized by chemiluminescence (Amersham Corporation), and the blot was then stripped to remove the bound antibody before proceeding with the next antibody. Extracts are as indicated: WT, wild-type; KO, K14 null; K18 tg, K18 transgenic; and K18 res, two different lines of K18 rescue. Note that both transgene protein and endogenous keratins reside in the insoluble fraction. Molecular mass standards at right in kD.

Figure 7

K18 expression is still maintained and does not induce K6 when K18 transgenic mice are bred onto a K14 null background. Neonatal back skins and paw skins of control (not shown) and K18+/K14 null mice (K18 res) were frozen and methanol-fixed, sectioned (10 μm), and then stained with antibodies against the keratins indicated. (Note: depending upon the fixation/processing conditions, antibody staining of K18 sometimes extended into the suprabasal layers. This happened inconsistently in control as well as transgenic skin, and in all cases, was always paralleled by an identical staining pattern with αK5.) Note the wild-type staining pattern of αK6 in the outer root sheath of hair follicles; suprabasal epidermal induction of K6, a typical marker of hyperproliferative disorders did not occur in K18 rescue skin. Bar, 40 μm.

Figure 8

K18 expression does not rescue the blistered paw phenotype of K14 null mice. Neonatal back skins and paw skins from K18+/K14+, K18+/K14−, and wild-type littermates were embedded in Epon, sectioned (0.75 μm), and then stained with Toluidine blue. Sections shown are from K18 transgenic skin on a K14 null background. (A) back skin, showing no obvious abnormalities; (B) paw skin, depicting early signs of basal cell degeneration (arrowheads); (C) paw skin showing clear signs of basal cell cytolysis (arrows); (D) paw skin showing blister resulting from completely degenerated basal epidermal layer. Double arrow, blister; arrowheads in D, fragments of basal cells left on the blister floor, indicative of basal cell rupturing. Bars: (A and B) 40 μm; (C and D) 20 μm.

Figure 9

Ultrastructure of basal cells from the back skins and paw skins of K18 transgenic mice bred on either a wild-type or a K14 null background. Skins of 1–2-d-old K18 transgenic mice on either a wild-type or K14 null background were processed for electron microscopy as described in the Materials and Methods. (A) basal cells from K18 transgenic/K14 wild-type back skin, depicting normal keratin filament bundles (kf) and desmosomes (de). Paw skin showed similar morphology. (B– D) basal cells from K18 transgenic/K14 null back skin (B and C) or paw skin (D). The majority of basal cells in back skin displayed normal morphology and keratin filament bundles, similar to that seen in B. An occasional basal cell from back skin exhibited signs of cytolysis (asterisks and small arrowheads), with some regions of the cytoplasm devoid of keratin filaments (large arrowhead in C) and other regions showing some small aggregates or clumps of keratin (C′, kc). Many cells from paw skin displayed prominent clumping of keratin both in the cytoplasm and associated with the desmosomes (D and inset to D, respectively). D′ and D″ show higher magnification to visualize these clumps of keratin in more detail. Note that spinous cells (SP) contained a largely normal keratin network, reflective of the induction of K1 and K10 in these layers. BL, basal lamina; mi, mitochondria; hd, hemidesmosome; Nu, nucleus; BC, basal cell. Bar in A: (A) 0.4 μm; (B, C′, D′, D″) 0.3 μm; (C) 0.9 μm; (D) 0.8 μm; (inset to D) 0.1 μm.

Figure 10

Keratin clumps in K18 transgenic/K14 null basal cells contain a mixture of K5 and K18. Paw skin from K18 transgenic/ K14 null animals was embedded in Lowicryl and subjected to 30-nm gold labeling with antibodies against either K18 (A) or K5 (B) as described previously (Coulombe et al., 1989). de, desmosome; hd, hemidesmosome; kc, keratin clumps. Bar, 0.2 μm.

Figure 11

In K18 transgenic/K14 null basal cells, the morphological deviations from a wild-type keratin network become more pronounced upon mechanical stress. Two separate litters of neonatal mice were used for these studies. For each litter, two K18 transgenic/K14 null and one wild-type littermates were subjected to lateral rubbing of the skin on one side of the back, and no rubbing on the other side. After the rubbing experiment, skins from each side of the back of each animal were processed for conventional electron microscopy. Skin samples were analyzed for alterations in the morphology of the basal keratin network. Many of the basal cells maintained a proper keratin network (A). Approximately 20% of the basal cells of K18 transgenic/K14 null back skin showed signs of keratin clumping on the rubbed side (B). The majority of the basal cells from unrubbed back skin displayed keratin filaments typical of wild-type skin (not shown). Wild-type skin showed abundant keratin filament bundles and no keratin clumps irrespective of whether the skin was rubbed or not (C). Bar in A: (A and B) 0.4 μm; (C) 0.3 μm.

Figure 12

Assembly of K18 and K5 into 10-nm keratin filaments. (A and B) Pure preparations of K5, K18, and K14 in 6 M urea buffers were obtained by FPLC chromatography, as described by Coulombe and Fuchs (1990). Keratin-rich fractions were pooled, and the protein concentration measured using an assay kit (Bio-Rad Laboratories). Equimolar mixtures of K5/K18 or K5/K14 were subjected to anion-exchange chromatography to purify the heteromeric complexes. Complexes were confirmed by chemical cross-linking with BS3 as described previously (Wawersik et al., 1997). Samples of unlinked (−) and cross-linked (+) proteins were subjected to SDS-PAGE, and the gel was stained with Coomassie blue to visualize the protein. D, dimer; T, tetramer. (B and C) Complexes of either K5/K18 (B) or K5/K14 (C) were subjected to in vitro filament assembly as described in Materials and Methods. Filaments were visualized under a Philips CM10 electron microscope. Bar, 100 nm.