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Vol. 19, Issue 1, 137-149, January 2008
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*Molecular Oncology Unit, Division of Biomedicine, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, E-28040 Madrid, Spain; Department of Carcinogenesis, Science Park-Research Division, University of Texas M.D. Anderson Cancer Center, Smithville, TX 78957; Department of Veterinary Clinical Sciences, Veterinary Pathology Unit, Veterinary Faculty, University of Santiago de Compostela, E-27002 Lugo, Spain; and ||Flow Cytometry Unit, Division of Hematopoiesis, CIEMAT, E-28040 Madrid, Spain
Submitted August 7, 2007;
Revised September 21, 2007;
Accepted October 17, 2007
Monitoring Editor: M. Bishr Omary
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Three Akt isoforms sharing common structural features (Hanada et al., 2004) have been found in mammals (Akt1, Akt2, Akt3). Although the relevance of Akt signaling in cancer is widely recognized (Bellacosa et al., 2005; Manning and Cantley, 2007), its involvement in development has only recently been highlighted. Akt1 and Akt2 knockout (KO) mice are viable and exhibit mild phenotypes characterized by growth retardation and diabetes (Chen et al., 2001; Cho et al., 2001), suggesting functional redundancy among Akt isoforms. Further proof of these overlapping roles comes from analysis of compound mutant mice. Akt1 and Akt2 double KO mice displayed a much more severe phenotype: dwarfism, impaired skin development, delayed bone development, reduced adipogenesis, and early lethality after birth (Peng et al., 2003). More recently, mice with combined mutant alleles of Akt1 and Akt3 have also been generated. Double KO of Akt1 and Akt3 causes embryonic lethality at around embryonic days 11 and 12, and Akt1–/–;Akt3–/– mice have severe developmental defects in the cardiovascular and nervous systems (Yang et al., 2005). Akt1–/–;Akt3+/– mice display multiple defects in the thymus, heart, and skin and die within several days after birth, whereas Akt1+/–;Akt3–/– mice are viable (Yang et al., 2005). These data demonstrate that the three Akt isoforms have overlapping functions but also may have unique functions in specific organs.
Besides the skin phenotype observed in different compound-deficient mice, several lines of evidence have highlighted the importance of the phophoinositide 3 kinase (PI3K)/Akt pathway in epidermis. Of particular interest is the relevant role of Akt during mouse skin carcinogenesis. We have demonstrated that Akt is a key molecule in insulin growth factor 1 (IGF-1)-mediated mouse skin tumor promotion (Wilker et al., 2005). In addition, Akt exerts essential roles in two-stage carcinogenesis protocols affecting tumor proliferation and apoptosis (Segrelles et al., 2002) and also modulates the tumor-stroma cross-talk leading to an increase in angiogenesis (Segrelles et al., 2004). Recently, using cultured cell systems, we provided evidence indicating that the functions of Akt in epidermal tumors are exerted by transcriptional and posttranscriptional mechanisms and show several parallels with human head and neck squamous cell carcinomas (Segrelles et al., 2006). Of relevance, we observed that increased Akt expression modulates β-catenin and 
Np63 expression, two essential modulators of epidermal development, in this system (Segrelles et al., 2006).
To further explore the role of Akt in skin, we have generated transgenic mice expressing either a wild-type form of Akt1 (Aktwt) or a form of Akt1 that is constitutively activated by means of a myristoylation sequence (myrAkt), directed to the basal layer of the stratified epithelia by the K5 promoter (Segrelles et al., 2007). These approaches, together with the tissue-specific knock out of PTEN tumor suppressor gene, have been widely used to explore the functions of the Akt signaling pathway in vivo (Yang et al., 2004). Notably, there are functional differences among these three approaches. Because PTEN is a negative modulator of PI3K signaling, elimination of PTEN leads to the permanent up-regulation of the PI3K pathway, whereas increased expression of wild-type Akt leads to the amplification of PI3K signaling, and the expression of constitutively active Akt generates a permanent, PI3K-independent signal. Although performing our previous analysis, we observed that many of the founders expressing K5myrAkt, but not those expressing K5Aktwt, displayed developmental defects in multiple ectodermal organs in parallel with the level of transgene expression and Akt activity. Here we have characterized these developmental alterations. We provide evidence that deregulated Akt activity in stratified epithelia mediated by myrAkt expression leads to aberrant bone morphogenetic protein 4 (BMP4) signaling and altered adult epidermal stem cell homeostasis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Orientation and integrity of the inserts were confirmed by restriction digests. Purified fragments were used in microinjection. Mice were genotyped using primers specific for β-globin on genomic DNA isolated from tails, as previously described (Bol et al., 1998). Transgenic and control mice in a C57BL/DBA/FVB mixed background were housed in a 12/12 light/dark cycle at 24°C and given standard mouse chow and water ad libitum. In some cases, where alterations in oral cavity or teeth were observed, food was supplemented with jelly. In addition to the founders, at least 40 mice of each transgenic line or controls were analyzed in search for developmental defects.
Histology and Akt Kinase Analyses
For histological analysis, samples were fixed in Formalin and embedded in paraffin before sectioning. Sections of 5 µm were cut and stained with H&E. At least five different samples were analyzed for each time point. The expression of BMP2 (1/200 diluted mAb; Abcam, Cambridge, UK), BMP4 (1/50 diluted goat polyclonal; AbCam) and Np63 (1/200 diluted mAb 4A4; Santa Cruz Biotechnology, Santa Cruz, CA) was monitored in deparaffined sections using conventional protocols. For expression of active Akt (phospho Ser 473, 1/50 diluted; Cell Signaling, Beverly, MA), phospho Smad1/5/8 (phosphorylated in Ser463 and 465, 1/100 diluted; Cell Signaling), Foxo3a (1/200 diluted; Upstate, Lake Placid, NY), and BMPRIA (1/100; Santa Cruz Biotechnology), the slides were microwaved for 10 min after deparaffinization to enhance the staining. Keratin 15 (mouse mAb LHK15; Neomarkers, Lab Vision, Fremont, CA; diluted 1/50), and CD34 (rat mAb; eBioscience, San Diego, CA; diluted 1/50) were monitored in 70% ethanol-fixed tissues by double immunofluorescence. Before incubation overnight at 4°C with primary antibodies diluted in bovine serum albumin (BSA)/phosphate-buffered saline (PBS), sections were incubated with 5% horse serum for 45 min to block the Fc receptor in tissue and then washed three times with sterile PBS (pH 7.5). Horseradish peroxidase–, Texas red–, or fluorescein isothiocyanate–conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA) and used at dilutions of 1/4000, 1/500, and 1/100, respectively. Peroxidase was visualized using a DAB kit (Vector, Burlingame, CA). Control slides were obtained by replacing primary antibodies with PBS (data not shown). Akt activity in mouse skin was determined after immunoprecipitation with anti-Akt (Santa Cruz Biotechnology, C-20 antibody 1 ml/25 mg protein) essentially as previously described (Segrelles et al., 2002), using histone 2B (H2B; Roche Molecular Biochemicals, Indianapolis, IN) as the substrate for Akt or by Western blot (see below). Autoradiograms were scanned and subsequently quantified using a Phosphorimager (Bio-Rad, Richmond, CA).
Scanning Electron Microscope Studies of Hair Shafts
Hair shafts from the dorsal skin of 30-d-old transgenic and nontransgenic littermates were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.5). After dehydration, samples were dried by critical point drying (Balzers, Hudson, NH; CPD020), coated with gold palladium by a Jeol JFC-1100 Ion Sputter, and examined with a Jeol JSM-T220A scanning electron microscope (SEM; Peabody, MA). At least 10 samples from each genotype were analyzed.
RNA Purification and Affymetrix Mouse Gene Chip 430A Analysis
Mouse skin tissue, obtained 30 d after birth, was preserved in RNAlater (Ambion, Austin, TX) and disrupted and homogenized using Mixer Mill MM301 (Retsch). Total RNA was extracted and purified from 30 mg of skin using RNeasy Fibrous Tissue Mini kit (Qiagen, Chatsworth, CA) following the manufacturer's recommendations. The integrity of the RNA populations was tested in the Bioanalyzer (Agilent, Wilmington, DE). Two (transgenic mouse lines L60, L84 and LA) or three pools (control mice) from RNA whole skin extracts of same genotype were done and analyzed, individually, in mouse microarrays. We exported CEL files from Affymetrix GCOS software (Santa Clara, CA), and using the web-based tool Gene Expression Profile Analysis Suite (GEPAS, http://www.gepas.org; Vaquerizas et al., 2005), we subtracted the background intensity values with RMA (Irizarry et al., 2003), normalized the chips using quantile method (Bolstad et al., 2003), and finally log2-transformed and mean-centered the intensity values. Differentially expressed genes with normal variability were extracted between the four mouse genotypes with CLEAR test (2183 Affymetrix probes, p < 0.05; Valls et al., 2007). Further analyses were performed using the MeV software (Saeed et al., 2003). The selection of genes belonging to clusters 1–4 was made using Template Matching (Pearson R > 0.8, p < 0.01; Pavlidis and Noble, 2001). Genes selected in more than one cluster were included in the cluster in which they displayed the best R and p values. The cluster Gene Ontology (GO) analysis of Biological Processes using DAVID software (Dennis et al., 2003; http://david.abcc.ncifcrf.gov/home.jsp) from the National Institute of Allergy and Infectious Diseases/NIH was used to identify functional categories for each cluster. The search for GO terms was made using all themes, listed by p value based on EASE score (Hosack et al., 2003) and manually curated. EASE score identifies functional categories overrepresented in a gene list relative to the representation within the proteome of a given species.(Hosack et al., 2003). Pathway Architect software (Stratagene, La Jolla, CA) was used to identify and characterize possible pathways affected by specific alterations in gene expression. Gene expression microarray dataset has been submitted in GEO database under the accession number GSE9054.
Western Blot Analysis
Extracts, prepared from skin of 30-d-old mice, were ground with a mortar on liquid nitrogen and homogenized in buffer P (Tris, pH 7.5, NaCl 150 mM, EDTA 1 mM, EGTA 1 mM, β-glycerophosphate 40 mM, sodium orthovanadate 1 mM, PMSF 0.1 mM, aprotinin 2 mg/ml, leupeptin 2 mg/ml, NP-40 1%). Total protein (35 µg) from at least three age- and genotype-matched pooled skin samples was used for NuPAGE 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA), transferred to nitrocellulose membrane (Invitrogen), and probed with primary antibodies against Akt1/2 (Santa Cruz Biotechnology; 1/500), phosphorylated Akt (Ser 473, diluted 1/50 and Thr 308, diluted 1/10; Cell Signaling), BMPR1 (Santa Cruz Biotechnology; 1/100), BMP4 (Abcam; 1/300), p63 (Santa Cruz Biotechnology; 1/500), and phospho-Smad1/5/8 (recognizing Smad1/5/8 phosphorylated in Ser463 and 465; Cell Signaling; 1/500). Actin (Santa Cruz Biotechnology; 1/1000) was used to normalize protein loading. Secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG) were purchased from Jackson ImmunoResearch. Chemiluminescence was performed using manufacturer's recommendations (Pierce, Rockford, IL).
Label-retaining Cell Analysis
Ten-day-old nontransgenic and myrAkt pups (six mice of each class) were injected with BrdU (20 µl of a 12.5 mg/ml dilution in 0.9% NaCl every 12 h for a total of four injections). Skin sections were collected at 30 d after the last injection and bromodeoxyuridine (BrdU) incorporation was measured as percentage of hair follicles (HF) containing positive cells as previously reported (Ruiz et al., 2004). Experiments were performed in triplicate (n 
3 per group) and at least 100 follicles were scored in each section.
Fluorescence-activated Cell Sorting Analysis
For each experiment, dorsal skin of four adult animals of each genotype was shaved and treated with trypsin to separate dermis from epidermis. Cell suspensions were strained (40 -µm nylon, Falcon, Oxnard, CA) and stained in PBS containing 2% fetal bovine serum with anti-CD34 coupled to biotin (BD-PharMingen, San Diego, CA) and anti-
6 integrin (CD49f) coupled to PE (PharMingen). Cells were washed and stained with streptavidin-Tricolor (Caltag Laboratories, Burlingame, CA) and washed again. Cells were resuspended in PBS containing 2 µg/ml propidium iodide to exclude dead cells and analyzed in an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is worth mentioning that, because of the deleterious effects of myrAkt or wtAkt expression, it was impossible to establish lines from many of the founders. Eventually, we were able to establish two lines with different expression levels of the myrAkt transgene (lines 60 and 84, hereafter L60 and L84) and one line expressing the wtAkt transgene (Line A, hereafter LA). Our analysis was then focused on these three lines and six myrAkt founders. MyrAkt founders and L84 transgenic mice showing high levels of Akt kinase activity (Figure 1, A and A') displayed developmental defects that affected multiple ectodermally derived organs such as hair, teeth, nails and several ectodermal glands. Of note, these alterations were not observed in mice expressing wtAkt or low levels of myrAkt, associated with lower Akt activity (Figure 1, A' and B). This suggests that a certain threshold of Akt activity is necessary to promote the deregulated development of ectodermal organs. As expected, the increased kinase activity was restricted to those tissues expressing the transgene, such as epidermis (Figure 1C' and data not shown).
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; Table 2). Interestingly, the concept "development" appears as significantly represented in the clusters including those genes that only showed altered expression in L84 skin samples (clusters 3 and 4). Utilizing the Entrez gene database, we narrowed the dataset to those genes that were involved in skin or ectodermal morphogenesis (Table 3). Overall, these data indicate that deregulated Akt activity results in gene expression changes that can modulate epithelial development.
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) and Twsg1 (Scott et al., 2001) were up-regulated. In addition, several genes found in these clusters are involved in BMP signaling (Figure 6C and highlighted in bold in Table 3) either as integral pathway effectors or as downstream targets (Figure 6C), thus suggesting that this developmental pathway is altered by myrAkt expression.
The forkhead transcription factor family has been widely described as a target of Akt (Burgering and Kops, 2002). Direct phosphorylation of these factors by Akt results in their cytoplasmic retention and inactivation, inhibiting the expression of forkhead transcription factor-regulated genes (Burgering and Kops, 2002). We thus studied whether the expression and localization of Foxo3a, a representative member of this family, is affected in the epidermis of the different transgenic mice by postnatal day (pnd) 28. In control, nontransgenic mice (Figure 6D), we observed a positive staining throughout the epithelium, and a number of positive nuclei were detected in the hair follicle and the basal layer of the interfollicular epidermis (denoted by arrows in Figure 6D). A similar staining pattern was observed in L60 (Figure 6E) and LA (Figure 6F) transgenic mice epidermis, although a minor decrease in the interfollicular epidermal positive nuclei was observed in L60 samples. On the contrary, in L84 skin (Figure 6G), the overall staining was decreased and very few positive nuclei were observed (denoted by arrows in Figure 6G). Therefore, in agreement with our previous data (Segrelles et al., 2006), the expression of Akt, upon a certain threshold, leads to the reduction and cytoplasmic localization of Foxo3a in keratinocytes (Segrelles et al., 2006). This might help to explain, at least in part, some of the changes observed in microarray analyses.
Altered BMP Signaling Mediated by myrAkt in Skin
BMP signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of HFs (Botchkarev, 2003). BMPs function by binding type 1 (BMPR1A and BMPR1B) and type 2 (BMPR2) transmembrane serine/threonine kinase receptors, resulting in phosphorylation of the intracellular proteins Smad 1, 5, and 8. Therefore, to further confirm the possible alterations in BMP signaling, we carried out a detailed histology analysis of several components of this pathway.
As shown in Figure 7, expression of BMP4 (Figure 7A) and BMP2 (Figure 7B) was predominant in the HF matrix and outer and inner root sheath (denoted by ORS and IRS, respectively) in nontransgenic mice with very little expression observed in the interfollicular epidermis (denoted by IE). With respect to myrAkt transgenic mice, we observed altered distribution and localization of both morphogens (Figure 7, C and D, respectively), because scattered expression was observed in only a few cells. Western blot analysis of whole skin extracts (Figure 7E) confirmed the dramatic decrease in the level of BMP4.
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; Andl et al., 2004; Yuhki et al., 2004; Sharov et al., 2005). In contrast, phospho-Smad staining in myrAkt transgenic mice was faint, and only a few cells expressed very low amounts at the tip of the hair (Figure 8C). The apparent decrease in phospho-Smad was confirmed by Western blot of whole skin extracts (Figure 7E). In nontransgenic mice and mice expressing the wtAkt transgene (LA), BMPRIA expression was restricted to a subset of cells in the outer root sheath and hair matrix (Figure 8, D and E), as previously reported (Kobielak et al., 2003; Andl et al., 2004; Yuhki et al., 2004; Sharov et al., 2005), whereas in myrAkt L84 mice (Figure 8F), BMPRIA expression was observed in the outer and inner root sheath (denoted by ORS and IRS, respectively in Figure 8F) and almost absent in hair tips. Moreover, immunohistochemistry also suggested a possible increased expression of BMPRIA that was confirmed by Western blotting (Figure 7E).
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Np63. This protein is an ectoderm-specific direct transcriptional target of BMP signaling (Bakkers et al., 2002) and is essential for skin development (Mills et al., 1999; Yang et al., 1999), and it can be induced by increased Akt activity (Barbieri et al., 2003; Segrelles et al., 2006). Compared with nontransgenic mice (Figure 8G) and LA mice (not shown), L84 mice did not display any obvious difference in localization, although L84 mice also displayed some Np63-positive cells located in the inner root sheath (denoted by IRS in Figure 8H) in addition to the outer root sheath and the interfollicular epidermis (denoted by ORS and IE in Figure 8H), or overall expression level (Figure 7E). Notably, we found that in control mice Np63 expression correlated with increased phospho-Akt in the bulge areas (Figure 8I), but not in the hair bulb (Figure 8I'), whereas in myrAkt L84 mice, Np63 was coexpressed with phospho-Akt throughout the HF (Figure 8J). These data indicated that, despite the alterations in BMP signaling observed in mice with high expression of the myrAkt transgene the overall expression of Np63 was not affected and discounted the possibility that the phenotypic alterations were primarily mediated by altered Np63 expression.
Deregulated Akt Activity Alters Epidermal Stem Cell Homeostasis
There are several lines of evidence indicating that BMP signaling may influence the behavior of epidermal stem cells (Sharov et al., 2006; Zhang et al., 2006b; Kobielak et al., 2007) It has also been shown that other adult stem cell populations, for example, as in hematopoietic tissue (Rossi and Weissman, 2006) may be impacted by PTEN/Akt pathway signaling. Because the K5 promoter is also active in these cells, we have thus studied possible alterations in the stem cell population in the skin of L84 transgenic mice. Initially we determined the expression of two putative epidermal stem cell markers, K15 and CD34 (Liu et al., 2003; Cotsarelis, 2006). The pattern of double immunofluorescence staining suggested an increase in the population of cells in the hair follicle of L84 (Figure 9A') compared with control mice (Figure 9A). We next determined whether the epidermal stem cell compartment was affected by myrAkt expression using a label-retaining (LR) technique (Cotsarelis et al., 1990; Taylor et al., 2000). In L84 skin we found a consistent increase in the number of labeled cells 30 d after BrdU administration (Figure 9, B' and C) compared with control mice (Figure 9, B and C). To further substantiate these observations, we also performed fluorescence-activated cell sorting (FACS) analysis to determine the proportion of cells expressing integrin 6 and CD34, which is considered characteristic of epidermal bulge stem cells (Blanpain et al., 2004; Blanpain and Fuchs, 2006). These analyses demonstrate that the proportion of 6+CD34+ cells is similar between control and L84 skin samples; however, L84 epidermis had a dramatic increase in the population characterized as 6low CD34+ at the expense of a partial reduction in the 6high CD34+ cell population (Figure 9, D and E). This result is in agreement with the presence of BrdU positive cells with clear suprabasal localization observed during the LR experiments (denoted by arrows in Figure 9B'). Finally, we also performed a colony-forming efficiency experiment using adult epidermis as the source of primary keratinocytes. Five days after plating, multiple abortive colonies were observed in cultures from control mice (Figure 9F), and very few rendered productive colonies after long-term culture (Figure 9G). In contrast, cells from L84 epidermis displayed undifferentiated morphology (Figure 9F') and produced multiple colonies (Figure 9G). Collectively, these data indicate that constitutively active Akt expression results in an expansion of the stem cell population.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), does not lead to a similar phenotype points to a finely regulated mechanism of control affecting Akt in the development of these organs. Our results are in agreement with the reported alterations found in different mouse models of Akt deficiency (Di-Poi et al., 2002; Peng et al., 2003; Yang et al., 2005) and the keratinocyte-specific null mutation of epidermal Pten (Suzuki et al., 2003). Of note, data obtained from double Akt KO mice, besides arguing for partially overlapping functions of Akt isoforms in vivo, also revealed that some functions of Akt are only discernible when total Akt levels are below a critical threshold in specific cell types and tissues (Dummler et al., 2006). Further evaluation of ectodermal derived organs in compound Akt KO mice may reveal additional roles.
The Akt signaling pathway has been widely studied in the context of carcinogenesis (Manning and Cantley, 2007) and is associated with increased cell proliferation and survival. Consistently, these functions are altered in L84 mice and also in LA and in L60 (Segrelles et al., 2007); however, the major developmental defects are primarily found in L84 mice and several founders. This would indicate that the observed development defects due to increased Akt activity can be delineated from proliferative or antiapoptotic effects. We have taken this aspect as a starting hypothesis for the analysis of the microarray data. Indeed, the consideration of genes that do not display altered expression in LA epidermis certainly restricts the analyses. The list of genes found is relatively small and includes multiple genes previously associated with ectoderm or skin development. Furthermore, in unconstrained analysis of possible pathways involved, we detected the BMP-dependent pathway, indicating that this may be a major mediator in the skin phenotypic alterations found in myrAkt mice. The possibility that this pathway is also a target mediating other ectodermal alterations seems plausible but undoubtedly will require further investigations.
It is widely recognized that BMP signaling is required for proper development of several ectodermal structures (reviewed in Jernvall and Thesleff, 2000; Botchkarev, 2003). Of note, cre-mediated mutation of the BMPR1A gene causes altered tooth morphogenesis, defective postnatal development of HFs, and abnormal nail growth (Andl et al., 2004) and leads to the formation of epidermal tumors (Kobielak et al., 2003; Andl et al., 2004; Sharov et al., 2006; Zhang et al., 2006b), probably through the altered homeostasis of epidermal stem cells (Sharov et al., 2006; Zhang et al., 2006b; Kobielak et al., 2007). In agreement, we also observed the development of epithelial tumors in Akt transgenic mice that in many cases were associated with hair follicle structures (Segrelles et al., 2007). Although epidermal-specific deletion of the Bmpr1a gene or Noggin overexpression caused severe alterations in the expression of several genes associated with development and cell cycle (Kobielak et al., 2003; Andl et al., 2004; Sharov et al., 2006; Zhang et al., 2006b), our microarray analysis did not produce similar results. This difference may be due to the fact that ablation of BmprIa or Noggin overexpression completely abrogates BMP signaling, whereas our data support a possible deregulation together with decreased signaling rather than complete inhibition. As an alternative explanation, searching for genes displaying a selected pattern of expression may obscure or lead to an incomplete analysis of the data. Indeed, as mentioned above, the genes found through the unrestricted analysis of the microarray data also included most of the genes reportedly altered in BmprIa conditional KO and Noggin transgenic mice. Nevertheless this group of genes was also altered in wtAkt transgenic mice that do not display ectodermal defects.
Several lines of evidence have previously shown an association between BMP and PI3K/Akt signaling pathways (Waite and Eng, 2003; He et al., 2004; Tian et al., 2005). In particular it has been shown that altered BMP signaling can, through the modulation of PTEN expression and activity, control the activity of the PI3K/Akt signaling pathway (Tian et al., 2005; Zhang et al., 2006b; Kobielak et al., 2007). The present data complement these observations and show through microarray and histology analyses that deregulated Akt activity affects BMP signaling and, as an overall consequence, the BMP pathway is at least partially inhibited. Furthermore, the existence of an autoregulatory loop between BMP and PI3K/PTEN/Akt signaling may exist such that each element is subject to the control of the other, and importantly, disruption of this balance may lead to altered ectodermal development and tumor formation. The molecular bases of this alteration in Bmp4 expression are not known at present; however, among the multiple regulators of Bmp4 gene expression there are several putative candidates that can be modulated by Akt activity. In this regard, p65 RelA, which can be activated by Akt (Madrid et al., 2000, 2001), is a transcriptional repressor of Bmp4 gene expression in vivo (Zhu et al., 2007), whereas Bmp4 transcription is activated by forkhead and NKX2 transcription factors (Zhu et al., 2004; Begum et al., 2005), which are inactivated by Akt (Burgering and Kops, 2002; Naito et al., 2003). In this regard, the altered expression and distribution of Foxo3a observed in L84 epidermis might explain the reduced expression of Bmp4. Further studies will help to clarify the functional impact of Akt activity on Bmp4 expression.
Many of the alterations observed in the ectodermal organs of myrAkt mice were similar to the defects present in mice resembling human ectodermal dysplasia syndromes (Thesleff, 2006). The possibility that Akt may be involved in these disorders is very intriguing and would certainly merit further investigations. In some cases this group of diseases is associated with altered Np63 expression (Koster and Roop, 2004). The finding that expression level of this protein was not altered in myrAkt transgenic mice relative to nontransgenic mice indicates that other targets of Akt kinase may be responsible for the observed phenotype. In this regard, the previously reported cross talk between the glucocorticoid receptor and Akt (Leis et al., 2004) and the involvement of the glucocorticoid receptor in certain ectodermal dysplasia syndromes (Perez et al., 2001; Cascallana et al., 2005) reinforces the possibility that Akt may also be involved in some of these disorders.
In the current study we observed that deregulated Akt activity results in altered homeostasis of adult epidermal stem cells. This result is in agreement with the reported involvement of the PTEN/Akt pathway in the maintenance of other adult stem cells (Li et al., 2002, 2003; Cheung and Mak, 2006; Rossi and Weissman, 2006; Yilmaz et al., 2006; Zhang et al., 2006a). On the other hand, the alterations in stem cells observed in the epidermis of myrAkt mice also agree with the reported modulation of these cells by BMP signaling in epidermis and other tissues (Kobielak et al., 2003; Rajan et al., 2003; He et al., 2004; Zhang et al., 2006b; Kobielak et al., 2007). Unexpectedly, we also observed that expression of myrAkt specifically affects the subpopulation of epidermal stem cells characterized by low integrin 6 expression. It has been reported that this suprabasal cell population is derived from that which maintains basal lamina contact and arises only after the start of the first postnatal hair cycle (Blanpain et al., 2004). Our data implicate that Akt may affect the transition between these two cell populations and would suggest that Akt may control the cross talk between stem cells and the niche microenvironment.
Collectively, we present evidence that ectodermal organ development is dependent on accurate Akt signaling and that deregulation of this activity results in altered development of these organs, which in the case of skin proceeds through altered BMP signaling and affects epidermal stem cell population.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Address correspondence to: John DiGiovanni (jdigiovanni{at}mdanderson.org) or Jesús M. Paramio (jesusm.paramio{at}ciemat.es)
Abbreviations used: BMP, bone morphogenetic protein; HF, hair follicle.
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) 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.