Formation of a normal epidermis supported by increased stability of keratins 5 and 14 in keratin 10 null mice

Abstract

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.

Figure 1

Semithin sections of mouse back skin. Comparison of K10^−/− (B) with wild-type (A) mice revealed a normal differentiation in the K10^−/− animal. In the granular layer of K10^−/− mice, cell size was increased, but the number of cell layers was identical in both genotypes. We noted an increase in the size of keratohyalin granules (B, arrow) in K10^−/− mice. The stratum corneum appeared irregular compared with that of the wild-type mice. In contrast to homozygous neonatal K10T mice (C, asterisk), which carry the dominant-negatively acting K10T, the K10^−/− epidermis did not show any sign of cytolysis (B). In K10T skin the massive suprabasal accumulation of keratin aggregates was clearly visible (C, dark matter), whereas K10^−/− keratinocytes exhibited a clear cytoplasm (B). Bar, 10 μm.

Figure 2

Immunofluorescence analysis of suprabasal keratins. The loss of K10 in the knockout mice (B) was confirmed (A, wild-type) and the suprabasal K1 expression was clearly reduced in in the K10^−/− mice (D) compared with the wild-type mice (C). K6 expression was not induced in K10^−/− (F) but showed the normal expression pattern of wild-type skin in hair follicles (E and F, arrow) as well as in rare single interfollicular cells. K17 expression was patchy in both wild-type (G) and K10^−/− (H) epidermis. The dotted line in A–F marks the basal membrane, ignoring hair follicles. Bar, 64 μm.

Figure 3

Immunofluorescence analysis of basal keratins. We noted a suprabasal increase in the basal cell keratins K5 (A and B), K14 (C and D), and K15 (E and F). In the wild-type mice, these keratins are predominantly found in the basal layer (A, C, and E), whereas the knockout had a significant suprabasal persistence of these keratins (B, D, and F). Bar, 64 μm.

Figure 4

K14 mRNA is increased, but it remains restricted to basal cells. (A) Northern blot analysis revealed that K1 and 5 expression were unaltered in K10 knockout mice (−/−), whereas K14 mRNA was increased by 36%. In addition, the complete loss of K10 in the knockout mice was confirmed. Quantitation was performed by densitometric measurement. Lanes M, marker; lanes 1 and 2, ribosomal RNA; lanes 3 and 4, corresponding autoradiograph; dots from top to bottom, 4.4, 2.9, 1.9, and 1.5 kb. Northern blots for K1, 10, and 14 were derived from the same gel. (B) In situ hybridization showed that in K10^−/− epidermis, both K5 and 14 mRNA remained restricted to basal keratinocytes.

Figure 5

Western blot analysis of epidermal keratins. (A) The loss of K10 expression in K10^−/− epidermis was confirmed by Western blotting, and its reduced expression in heterozygote skin (+/–) was shown. Note a remarkable decrease in K1 in K10^−/− pups and a slight decrease in the skin of heterozygotes. (B) The amount of K5 remained unaltered, whereas its partner K14 was slightly increased. The amount of the third basal keratin, K15, was unaltered. Equal loading was verified by quantitative comparison of Coomassie blue staining. Lanes 1–3, Coomassie blue–stained polyvinylidene difluoride membrane; lanes 4–6, corresponding Western blot; M, marker; dots from top to bottom, 66.4, 55.6, and 42.7 kDa.

Figure 6

Ultrastructure of K10^−/− epidermis. The granular layer of K10^−/− epidermis maintained the typical content and distribution of IF bundles (C, arrow, survey EM). Higher magnification shows the regular shape of these filament bundles (D, arrow). For comparison, see survey micrograph of the wild type in A and a higher magnification in B (arrows on filament bundles) and the completely different setting in K10T mice, where the cytoplasm of granular layer cells was filled with a large amount of keratin aggregates (E, arrows, survey; F, details). The bundling otherwise typical of K1/10 was also noted for K5/14 in granular cells. Interestingly, in a few cells, we noted small keratin aggregates in the knockout epidermis (D, arrowhead), which were absent in wild-type littermates. These small aggregates closely resembled the aggregates observed in K10T mice (E and F, arrowheads). There were no signs of epidermal cytolysis in K10^−/− neonates. Bars: A (also valid for C and E), 1 μm; B (also valid for D and F), 0.25 μm; kh, keratohyalin.

Figure 7

Immunogold EM revealed the formation of K1/K14 filaments in K10^−/− mice. (A–C) IFs in the granular layer of K10^−/− epidermis. (D–F) Basal–suprabasal transition zone of wild-type epidermis, with the basal cell at the bottom. Desmosomes mark the level of the cell membranes, which have been lost upon fixation. (A) Survey of keratin bundles, which were labeled with both K1 and 14 antibodies. Filaments that were composed of K1 and 14 were also found attached to desmosomes (B). (C) Higher magnification of a filament bundle showing that K1 and 14 were in close proximity in the filaments. In single-antibody–labeling experiments in wild-type skin, K14 was detected in the basal epidermal layer (D), whereas K1 was exclusively found in subrabasal cells (E). This was confirmed in a double-labeling experiment with both antibodies (F). K1, 10-nm gold particles; K14, 5-nm gold particles. Bars: A and D–F, 0.5 μm; B, 0.1 μm; C, 0.24 μm.

Figure 8

Two-dimensional gel electrophoresis of keratin complexes and subsequent Western blotting revealed that only a minor portion of the residual K1 formed filaments with K14. Keratin complexes were extracted from neonatal epidermis and resuspended in 5.5 M urea (A and C). To visualize keratins, the blots shown were incubated with a mixture of corresponding antibodies. At 5.5 M urea, the keratins of wild-type extracts still formed complexes with their partners and migrated at the complex-specific isoelectric point in the first dimension (A; IEF). Note that all keratins migrated at the same isoelectric position. Although K5 and 14 behaved in K10^−/− null extracts (C), as in the control (A), and were exclusively migrating as a complex, most of K1 focused at its own basic IP (C, black arrow). Only a small amount of K1 (white arrow) was observed at the complex-specific IP. Dialysis to 6.5 M urea (B and D) resulted in almost complete dissociation of K1/K10 in the wild-type epidermis (exemplified by K10) and partial dissociation of K5/K14 complexes in both wild-type (B) and K10^−/− (D) epidermis.

Figure 9

Normal profilaggrin processing in neonatal K10^−/− epidermis. In contrast to the impairment of profilaggrin processing in K10T neonates (B), neither the amount of profilaggrin (PF) nor that of its processing intermediates (I) or mature filaggrin (F) was altered in neonatal K10^−/− mice (A).

Figure 10

Transient barrier defect of forepaw sole epidermis in K10^−/− neonates. During the dye perfusion assay, the skin of control neonates did not take up the color (A), whereas the forepaw sole skin of K10 null neonates was stained (B). K10T neonates were much more affected than K10 null mice, showing dye penetration at multiple body sites (C).