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Hyperproliferation, induction of c-Myc and 14-3-3, but no cell fragility in keratin-10-null mice

Institute of Physiological Chemistry and Bonner Forum Biomedizin, University of Bonn, Nussallee 11, 53115 Bonn, Germany

View larger version (91K): [in a new window]   Fig. 5. Immunohistochemical analysis showed an increase in cyclin D1 (B) and c-Myc (D) in K10-/- epidermis compared with the wild-type, where both proteins are exclusively found in a restricted number of basal cells (A and C, respectively). The expression of ß-catenin remained restricted to keratinocyte cell membranes (E, wild-type; F, K10-/-). (G) Northern blotting revealed that the expression of Wnt-3 (lane 3, wt; lane 4, K10-/-; loading control: lane 1, wt; lane 2, K10-/-), Wnt-4 (lane 7, wt; lane 8, K10-/-; loading control: lane 5, wt; lane 6, K10-/-1) and Wnt-5a (lane 11, wt; lane 12, K10-/-; loading control: lane 9, wt; lane 10, K10-/-) were not altered in K10-/- skin. Arrowheads point to distinct RNA species. Bar, 40 µm.

View larger version (99K): [in a new window]   Fig. 1. The epidermis of adult K10-/- mice showed hyperkeratosis and acanthosis; cells and nuclei were enlarged (B). Keratohyalin was increased (B, inset, arrow) compared with that in the wild-type (A). A and B show H&E-stained paraffin sections of ear skin. DAPI staining of normal ear epidermis showed epidermal proliferative units (C, epidermal sheet, small arrow; E, higher magnification, arrow; G, section, arrow; large arrow in C and D, hair follicle), which were absent in knockout mice (D, epidermal sheet; F, higher magnification of D; H, section). In normal epidermis Ki-67 was restricted to basal cells (I), whereas it was expressed throughout the basal layer and in two to three suprabasal layers in K10-/- mice (K). sb, stratum basale; sg, stratum granulosum; ss, stratum spinosum. Bars, 40 µm. The respective wild-type and knockout photos are of the same magnification.

View larger version (49K): [in a new window]   Fig. 2. Western blotting showed that the amount of K1 was slightly increased in K10-/- skin (A, lane 4) compared with that in wild-type skin (A, lane 3; lanes1,2, corresponding Coomassie-stained blot). K5 (B, lane 4, K10-/-; lane 3, wild-type; lanes 1,2, corresponding blot) and K14 (C, lane 4, K10-/-; lane 3, wild-type; lanes 1,2, corresponding blot) were also slightly increased, whereas K15 was unchanged (D, lane 4, K10-/-; lane 3, wild-type; lanes 1,2, corresponding blot). Immunofluorescence analysis of ear epidermis showed unaltered suprabasal K1 localization (E, wild-type; F, K10-/-), K5 and K14 were detected in basal and suprabasal layers (K10-/-, H and K, respectively; wild-type, G and I). K15 remained restricted to the basal layer (K10-/-, M; and wild-type, L). Bars, 50 µm.

View larger version (45K): [in a new window]   Fig. 3. K6 and K16, while hardly detectable in wild-type western blots (A, lane 3 and B, lane 3, respectively), were strongly increased in K10-/- skin (A, lane 4 and B, lane 4; A and B, lanes 1,2 are the corresponding coomassie-stained blot; dots indicate marker lanes at 66, 56 and 43 kDa). Immunofluorescence analysis of K6 showed that its expression in the wild-type was restricted to hair follicles (C, arrow) whereas K10-/- mice showed additional strong expression through all epidermal layers (D, arrow on hair follicle). In contrast to K6, K16 was induced only in the suprabasal layers of K10-/- epidermis (E, arrow on hair follicle; broken line indicates basement membrane). The sections show ear epidermis. Bar, 40 µm.

View larger version (94K): [in a new window]   Fig. 4. Two hours after a BrdU pulse, wild-type mice showed 8% labelled basal cells (A), while more than 50% of basal cells were labelled in K10-/- mice (B). 24 hours after the pulse the wild-type showed predominantly labelling of daughter cells in the basal layer (C). In contrast, K10-/- mice showed massive suprabasal labelling (D) indicating increased turnover of keratinocytes. The line depicts the dermo-epidermal junction. Bar, 40 µm.

View larger version (73K): [in a new window]   Fig. 7. Western blot analysis of proliferation-associated proteins showed no alterations in p53 (A, lane 4, wild-type; lane 5, K10-/-; lanes 1,2, a corresponding coomassie-stained blot; lane 3, marker; dot, 56 kDa), a slight decrease in p21 (B, lane 4, wild-type; lane 5, K10-/-; lanes 2,3, loading control; lane 1, marker; dots, 36 and 26 kDa) and p27 (C, lane 4, wild-type; lane 5, K10-/-; lanes 1,2, loading control; lane 3, marker; dots, 36 and 26 kDa), no changes in ß-catenin (D, lane 4, wild-type; lane 5, K10-/-; lanes 2,3, loading control; lane 1, marker; dots from top to bottom, 116, 97 and 66 kDa), Rb (E, lane 4, arrowhead, wild-type; lane 5, K10-/-; lanes 2,3, loading control; lane 1, marker; dots, 116 and 97 kDa) or phosphorylated Rb (Ser780) (F, lane 4, arrowhead, wild-type; lane 5, K10-/-; lanes 1,2, loading control; lane 3, marker; dots, 158 and 116 kDa). (G) The level of unphosphorylated Akt kinase was unaltered (lane 8, wild-type; lane 9, K10-/-, upper band; lanes 2,3, loading control; lane 1, marker; dots from top to bottom, 66 and 56 kDa) while phosphorylated Akt (Thr308-specific detection: lane 10, wild-type; lane 11, K10-/-; Ser473-specific detection; lane 12, wild-type; lane 13, K10-/-; lanes 4-7, loading control) was reduced. A and B show blots of 18%, E and F of 8% and C, D, G and H of 10% polyacrylamide gels.

View larger version (76K): [in a new window]   Fig. 6. 14-3-3 was markedly increased in the cytoplasm of suprabasal K10-/- keratinocytes (B) compared with the wild-type (A). (C) Protein extracts of skin revealed a strong increase in 14-3-3 expression in K10-/- mice (lane 5) compared with the wild-type (lane 4; lane 1, marker; lanes 2,3, Coomassie-stained blot of lanes 4,5; dots from top to bottom: 36 kDa and 26 kDa). (D) Northern analysis showed an equally strong increase in 14-3-3 mRNA (lane 4) over the wild-type (lane 3; lanes 1,2, ethidium-stained agarose gel corresponding to lanes 3,4; dots from top to bottom: 2.8, 1.8 and 1.5 kb). Bar, 40 µm.

View larger version (68K): [in a new window]   Fig. 8. The restriction patterns for NlaIII in exon 1 (A) and BsaAI in exon 2 (B) of the p16Ink4a gene revealed the presence of polymorphisms in K10-/- mice that were on a mixed BALB/cx129/Ola background. Lanes 2,4,6,8 (A,B) show undigested PCR products of the respective exons. p16 exon 1 of BALB/c mice (A, lane 3) showed a different restriction pattern from the 129/Ola p16 gene (A, lane 5). K10-/- mice as well as wild-type control animals of a mixed strain background showed an intermediate pattern (A, lane 7 and lane 9, respectively). Exon 2 of the BALB/c p16 gene (B, lane 3) lacked a restriction site that was present in the 129/Ola p16 gene (B, lane 5). K10-/- mice, as well as the wild-type mice of the same mixed strain background, also showed an intermediate pattern (B, lane 7 and lane 9, respectively). Size markers from top to bottom: (A) 200 bp, 100 bp and 80 bp; (B) 400 bp and 300 bp.

View larger version (20K): [in a new window]   Fig. 9. Model for K10-mediated regulation of cell proliferation.

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