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. 2005 Aug;6(8):755-61.
doi: 10.1038/sj.embor.7400458.

Targeted expression of RALT in mouse skin inhibits epidermal growth factor receptor signalling and generates a Waved-like phenotype

Costanza Ballarò  1 , Sara Ceccarelli, Cecilia Tiveron, Laura Tatangelo, Anna Maria Salvatore, Oreste Segatto, Stefano Alemà
Affiliations

Targeted expression of RALT in mouse skin inhibits epidermal growth factor receptor signalling and generates a Waved-like phenotype

Costanza Ballarò et al. EMBO Rep. 2005 Aug.

Abstract

Although it has been clearly established that negative feedback loops have a fundamental role in the regulation of epidermal growth factor receptor (EGFR) signalling in flies, their role in the regulation of mammalian EGFR has been inferred only recently from in vitro studies. Here, we report on the forced expression of RALT/MIG-6, a negative feedback regulator of ErbB receptors, in mouse skin. A RALT transgene driven by the K14 promoter generated a dose-dependent phenotype resembling that caused by hypomorphic and antimorphic Egfr alleles-that is, wavy coat, curly whiskers and open eyes at birth. Ex vivo keratinocytes from K14-RALT mice showed reduced biochemical and biological responses when stimulated by ErbB ligands. Conversely, knockdown of RALT by RNA interference enhanced ErbB mitogenic signalling. Thus, RALT behaves as a suppressor of EGFR signalling in mouse skin.

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Figures

Figure 1
Figure 1
Generation of K14-RALT mice. (A) Southern blot analysis of genomic DNA isolated from founder mice. The sizes of the hybridized fragments from the endogenous RALT gene (9 kb) and transgenic K14-RALT (4.6 kb) are indicated. (B) Expression of RALT protein in the dorsal skin from wild-type (wt) mice and the heterozygous progeny of selected founders. Expression levels are compared with those in control and RALT-overexpressing NIH 3T3 cells. Immunoblotting was performed on total protein extracts using a polyclonal antiserum that recognizes rat and mouse RALT. (C) Photograph of 4-week-old A9 and littermate control mice showing that A9 mice have a typical Waved phenotype. (D) Newborn pups of the A9 line show defective closure of eyelids. (E) Adult mice from the A9 line show fuzzy and wavy hair with areas of alopecia and inflammation of the skin. (F) A 3-week-old mouse of the A24 line showing a wavy first hair coat. (G) Segments of hairs from wt (upper) and heterozygous (lower) adults of the A9 line. Note septulation and irregular pattern of pigmentation in the medulla.
Figure 2
Figure 2
Hair-follicle cycle and morphogenesis are severely impaired in K14-RALT mice. (A) Histological sections through the skin of control and A24 and A9 lines at different ages. The skin of newborn K14-RALT mice and that of age-matched controls appear similar. In 18-day-old K14-RALT mice, hair follicles fail to progress to catagen and accumulate in the subdermal tissue, whereas follicles from control mice are in telogen. As a result, overall skin thickness is markedly different in the two groups. Scale bar, 100 μm. (B) Sections of the dorsal skin from newborn and 18-day-old mice (control and A9 littermates) were immunostained with the indicated antibodies. In some cases, H33258 was used to stain nuclei. Note normal proliferation and differentiation patterns in the newborn skin; however, Ki67 staining is increased in the basal layer of epidermis in 18-day-old A9 mice. The dotted line is superimposed on the basement membrane. Scale bars, 100 μm. (C) Representative micrographs of TdT-mediated dUTP nick end-labelling (TUNEL)-positive cells in cryosections from the skin of 18-day-old A9 mice. The blue H33258 image is merged with the green FITC image (TUNEL). Scale bar, 50 μm. (D) Expression levels of RALT and ErbB family members detected by immunoblotting in skin extracts from 3-day-old and 14-day-old pups of the indicated genotypes.
Figure 3
Figure 3
RALT regulates ErbB-driven keratinocyte proliferation. (A) Western blot analysis of RALT expression in normal keratinocytes and NIH 3T3 cells after treatment with the indicated agents. (B) Expression levels of RALT protein in keratinocytes explanted from individual littermates (b–i) from crossing of heterozygous A24 mice; the two rows of numbers below each lane refer to the degree of inhibition of basal and EGF-induced (0.5 ng/ml) S-phase entry measured by bromodeoxyuridine (BrdU) incorporation in sister cultures. (C) Effect of ectopic RALT expression on BrdU incorporation of primary keratinocytes (wild type (wt), A9 and adenovirus-infected) and adenovirus-infected HaCaT cells stimulated with the indicated agents (see Methods). Note that the degree of inhibition is comparable with that shown by 0.2 μM PD168393. The data are mean±s.d. from three independent experiments. (D) Stable ablation of endogenous RALT in HaCaT cells by RNA interference (short hairpin RALT, shRALT; left panel) leads to threefold enhancement in S-phase entry after treatment with EGF (0.3 ng/ml; centre panel). In the same cells, EGF-induced (5 ng/ml) tyrosine phosphorylation of epidermal growth factor receptor (EGFR), evaluated by immunoblotting with anti-PY1068, is enhanced (right panel). The data are representative of three independent experiments. bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; FBS, fetal bovine serum; NRG1, neuregulin 1; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Figure 4
Figure 4
Expression levels of transgenic K14-RALT correlate with autophosphorylation of EGFR and downstream pathway activation. Primary keratinocytes explanted from A9 (A) and A24 (B) mice were mitogen-starved for 1 day and exposed to epidermal growth factor (EGF) for various lengths of time. Lysates were immunoblotted with anti-PY1068 for detection of phosphorylated EGFR and with the other antibodies indicated. Levels of activation of extracellular-signal-regulated kinase (ERK) were determined by immunoblotting with anti-P-ERK. Note that concentrations of EGF were 10 ng/ml (A) and 1.0 ng/ml (B). wt, wild type. (C) Primary keratinocytes from wt littermates were infected with Ad-RALT, mitogen-starved and subjected to the same treatment and analysis as in (A,B). GFP, green fluorescent protein.
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