Constitutively active Akt induces ectodermal defects and impaired bone morphogenetic protein signaling

Abstract

Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Akt(wt)), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity.

Figure 1.

Akt Kinase activity in transgenic mice. (A) Top panel, representative kinase assay using skin extracts and histone H2B as substrate; middle and bottom panels, Western blot of the same extracts blotted against Akt and Actin. (A′) Western blot of pooled skin samples from Control, L84, and LA transgenic mice obtained by pnd 28 probed against total and phosphorylated Akt (Ser 473 and Thr 308). The increased phosphorylation of Akt in L84 indicates the increased activation of Akt. (B) Summary of three independent kinase assays using skin extracts from the indicated mice. Transgenic mice displaying ectodermal organ development are denoted. Representative example of immunohistochemical detection of phosphorylated Akt (ser473) in skin section of control nontransgenic (C) and myrAkt founder 82 transgenic mouse. Note that only epithelial cells are positive for phospho-Akt in transgenic sample (C′). Bar, 250 μm.

Figure 2.

Hair phenotype of myrAkt transgenic mice. (A) Example of alopecia in L60 mice affecting the snout and some patches of dorsal skin. (B) Histology of vibrissae from control (B), L60 (B′), and L84 (B″) mice. Note the progressive degeneration. Bar, 250 μm. Overall macroscopic (C and C′) and scanning electron microscopic (D and D′) appearance of awl back skin hairs from control (C and D) and L84 (C′ and D′) transgenic mice. Bars, 50 μm. (E–G) Representative histology of skin by postnatal day (pnd) 28 from Control (E), LA (F), and L84 (G) mice. Note the absence of true anagen in L84 transgenic mice. (H) Representative histology of transgenic L84 skin by pnd 45. Not the enlarged anagen HF structures (denoted by arrows). Bars, 250 μm.

Figure 3.

Nail phenotype of myrAkt transgenic mice. (A) Example of nail overgrowth in founder 23. Also the fragility of nails is depicted (inset). Similar observations were obtained in L84 mice. Histology analysis of nails from L84 transgenic (B) and nontransgenic (B′) mice. Note the normal eponychium (e) and hyponychium (h), but an enlarged nail root (r) and hyperplasia of the nail bed epithelium (nb, insets B and B′) in the transgenic mice.

Figure 4.

Tooth phenotype of myrAkt transgenic mice. Representative examples of transgenic mice showing overgrowth and fragility of the incisors (A), supernumerary teeth located in the palate (B), and lack of the second molar in the lower jaw (C).

Figure 5.

Ectodermal gland phenotype of myrAkt transgenic mice. Representative histology sections from meibomian (denoted by mb in A and A′), salivary (denoted by arrows in B and B′), and sweat (denoted by sg in C and C′) glands from control (A–C) and L84 transgenic mice (A′, B′, and C′). Note the hyperplasia of the meibomian, dysplasia of the salivary and hypoplasia of the sweat glands in transgenic mice. Bars, (A′ and C′) 250 μm; (B′) 100 μm.

Figure 6.

Summary of microarray data and Foxo3a expression. (A) Heatmap showing the relative expression of different genes selected for the analysis. (B) Clusters of genes that were regulated in transgenic mice according to the pattern denoted by red (Up) or green (Down) lines, extracted using Template Matching (R > 0.8, p < 0.01). (C) Summary of pathway integration of microarray analyses using Pathway Architect software (Stratagene). (D–G) Representative examples of the expression and distribution of Foxo3a analyzed by immunohistochemistry in Control (D), L60 (E), LA (F), and L84 (G) mouse epidermis by pnd 28. Note the decreased staining and the reduced number of positive nuclei (denoted by arrows) in L84. Bar, 150 μm.

Figure 7.

Altered BMP expression and localization in hair follicles of myrAkt transgenic mice. Representative examples of the expression of BMP4 (A and C) and BMP2 (B and D) in control (A and B) and L84 (C and D) transgenic mice. Bars, 250 μm. IE, interfollicular epidermis, IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. (E) Western blot of whole skin extracts probed for the expression of the indicated proteins.

Figure 8.

Altered BMP signaling in hair follicles of myrAkt transgenic mice. Representative examples of the expression of phospho Smad1/5/8 (A–C) and BMPRIA (D–F) in control (A and D), LA (B and E), and L84 (C and F) transgenic mice. (G and H) Expression and localization of ΔNp63 in control (G) and L84 (H) transgenic mice. (I and J) Double immunofluorescence of ΔNp63 (green) and phospho-Akt (Ser473; red) in bulge (I) and bulb (I′) regions of control HF and in L84 (J) HF by pnd 28. Bars, 250 μm. IE, interfollicular epidermis, IRS, inner root sheath; ORS, outer root sheath.

Figure 9.

Alterations in L84 epidermal stem cell population. (A and A′) Skin sections from control (A) and L84 (A′) mice by pnd 28 stained with color-coded antibodies showing the expression and localization of keratin K15 (red) and CD34 (green). Note the apparent increase in the double positive cells in L84 hair follicle. (B and B′) Skin sections from control (B) and L84 (B′) after label retaining cell (LRC) experiments. Bars, 250 μm. Brackets denote bulge region (b). Sebaceous glands are denoted by sg. Arrowheads denote BrdU-positive follicle cells. C) Summary of three independent LRC experiments showing the percentage of hair follicles containing positive cells as previously reported (Ruiz et al., 2004). (D, D′, and E) FACS analyses of control (D) and L84 (D′) sorted by α6-integrin and CD34 expression. D and D′ are representative examples showing the CD34-positive α6-integrin low and CD34-positive α6-integrin high expressing cells (boxes). (E) Summary of three independent FACS experiments showing the percentage of CD34+α6^high and CD34+α6^low from control (□) and L84 (■) mice (* p ≤ 0.005). (F and F′) Representative examples showing the appearance of the colonies derived from control (F) and L84 (F′) 5 d after plating. Bar, 150 μm. (G) Representative example of the colony-forming efficiency of primary keratinocytes derived from control and L84 adult (8 wk) mice 3 wk after plating. Data in C and E are shown as mean ± SEM.