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. 2006 Feb 16;25(7):1081-9.
doi: 10.1038/sj.onc.1209130.

Hair cycle and wound healing in mice with a keratinocyte-restricted deletion of FAK

S Essayem  1 , B Kovacic-Milivojevic, C Baumbusch, S McDonagh, G Dolganov, K Howerton, N Larocque, T Mauro, A Ramirez, D M Ramos, S J Fisher, J L Jorcano, H E Beggs, L F Reichardt, D Ilic
Affiliations

Hair cycle and wound healing in mice with a keratinocyte-restricted deletion of FAK

S Essayem et al. Oncogene. .

Abstract

Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.

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Figures

Figure 1
Figure 1
FAK is expressed in keratin 5-positive cells of adult mouse skin. Immunostaining for FAK was carried out with the anti-FAK antibody from LabVision. A similar staining pattern was obtained with the anti-FAK antibodies from Santa Cruz Biotechnology (data not shown).
Figure 2
Figure 2
DNA, RNA, and protein analyses confirmed disruption of full-length FAK in FAK conditional knockout mice. (a) Possible genotypes from crossing of male K5Cre+FAK+/− and female K5Cre−FAKloxP/− mouse. WT, wild type; HE, heterozygote; KO, knockout. (b) Distribution of the wild-type (18.2%), heterozygote (64.7%), and knockout (17.1%) genotype among first 286 pups is according to the Mendelian law (16.7, 66.6, and 16.7%, respectively). (c) RNA quality and quantity were analysed using the Agilent 2100 bioanalyser and RNA 6000 Nano LabChip Kit (Agilent Technologies). Only samples with the highest quality of RNA (i.e. 3, 4, and 5) were used for real-time PCR analyses. RNA was of comparable quality in both whole skin and epidermal prep from the same animal. (d) Real-time PCR analyses of FAK expression. Only samples from FAKK5 KO and FAKloxP/+ littermates that had similar mRNA levels of E-cadherin (E-cad), desmoglein 3 (Dgl3), and K5, genes expressed specifically in epidermis but not in dermis, were compared for FAK mRNA levels. Data are shown as fold increase/decrease of mRNA levels in samples from FAKK5 KO animals using log l0 scale. The differences are less pronounced and error bars are bigger in samples prepared from whole skin because of the presence of dermal fibroblasts, which do not have deleted FAK. Scheme of FAK, outlining in red the deleted exon, which encodes amino acids 445–472, and the location of primers for real-time PCR (arrows) are shown. (e) Northern blot analyses of FAK expression. Northern blots with both FAK N- and C-terminal probes demonstrated that the epidermis of FAKK5 KO mice has greatly reduced intensity of bends corresponding to full-length FAK mRNA. As a positive control for FRNK expression, we used RNA isolated from primary mouse embryonic fibroblasts transduced with FRNK-expressing adenovirus. Although the total amount of loaded RNA (20 µg/lane) was the same in all samples (see ethidium bromide staining of the gel), levels of GAPDH mRNA and FAK were higher in fibroblasts. Therefore, image of the blot with a C-terminal probe is a combination of shorter and longer exposure. FRNK was not detected in mouse epidermis at the age examined (2–4-day-old animals). (f) Western blot analyses of FAK expression. Whole lysates from the epidermis of newborn pups were blotted with antibodies against FAK N-terminal, kinase, and C-terminal domains. Lower molecular weight bands in FAKloxP/+ lysates are likely products of FAK degradation/cleavage by calpain or caspases (Mitra et al., 2005). UBI, N-terminal monoclonal 4.41 antibody from Upstate Biotechnologies; TL, monoclonal antibody from BD Transduction Laboratories; LV, polyclonal antibody from Lab Vision.
Figure 3
Figure 3
Hair phenotype in FAKK5 KO mice. (a) At initial stages, pelage of FAKK5 KO mice seems to have less hair (P9 and P12). By P17, there is no difference in the appearance of FAKloxP/+ and FAKK5 KO littermates. Images of the same area of the same FAKloxP/+ and FAKK5 KO mice were taken over a period of 9 days. (b) Hematoxylin/eosin-stained sections of FAKloxP/+ and FAKK5 KO mice at P12 and P22. Deeper follicles of P12 FAKK5 KO mice are misaligned and their skin is thinner than the skin of their FAKloxP/+ littermates. At P22, however, the skin is somewhat thicker, and this could be explained by the abnormal presence of hair follicles in the hypodermis (red arrowheads). Deposits of keratinous material are more abundant at the surface of the FAKK5 KO epidermis (yellow arrowheads). (c) Diagrammatic reconstruction of the hair follicle distribution on histological sections parallel with the skin surface hair count in FAKloxP/+ and FAKK5 KO littermates at P12 and P22 shows that FAKK5 KO mice, when compared with their littermates, have constantly about 25% less hair even though the appearance is different before and after PI7. (d) Similar mitosis level in hair follicles of P12 FAKloxP/+ and FAKK5 KO littermates. (e) Lack of FAK does not induce epithelial cell apoptosis in vivo in P07 mice. Comparable mitosis and almost nonexisting apoptosis were observed in all samples throughout the period analysed (P07–P22).
Figure 4
Figure 4
Sebaceous gland hypoplasia and thinner epidermis in FAKK5 KO mice. (a) Sebaceous glands are less in number and hypoplastic in FAKK5 KO mice (yellow arrows). Insets: Oil Red staining of sebaceous glands (black arrowheads). (b) Western blot analyses of epidermis from P1 and P4 littermates show less keratin 10 in lysates from FAKK5 KO mice. Ezrin is a loading control. (c) Thinner epidermis in FAKK5 KO mice. There are fewer keratin 10-positive suprabasal layers in the epidermis of E18.5-day-old FAKK5 KO embryos. A similar difference in thickness was observed in all samples throughout the period analysed (E16.5–P14). (d) β4 integrin is confined to the dermal–epidermal junction in both FAKloxP/+ and FAKK5 KO mice. (e) Ki67-positive cycling cells are present at a similar level in the basal layer (SB) of epidermis in both FAKloxP/+ and FAKK5 KO mice. De, dermis; Ep, epidermis; SB, stratum basale; SC, stratum corneum.
Figure 5
Figure 5
Lack of FAK does not affect wound healing in vivo, although it is essential for keratinocyte survival in vitro. (a) Wound closure over 10 days; mm-scale is at the left. (b) Similar rate of wound healing in FAKloxP/+ and FAKK5 KO mice. (c) Hematoxylin and eosin staining of sections from 4-day wounds reveals similar histology of wound edge in FAKloxP/+ and FAKK5 KO mice. Es, eschar; HE, hyperproliferative epithelium. (d) Activated β1 integrin, recognized by immunostaining of fresh frozen unfixed tissue sections by 9EG7 antibody, is localized in the basal layer (SB) of epidermis in both FAKloxP/+ and FAKK5 KO mice. De, dermis; Ep, epidermis. (e) FAKK5 KO mouse keratinocytes could not be maintained in vitro. They neither proliferate nor survive in primary culture.
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