Keratinocyte-Specific Pten Deficiency Results in Epidermal Hyperplasia, Accelerated Hair Follicle Morphogenesis and Tumor Formation1

[CANCER RESEARCH 63, 674–681, February 1, 2003] Keratinocyte-specific Pten Deficiency Results in Epidermal Hyperplasia, Accelerated Hair Follicle Morphogenesis and Tumor Formation1 Akira Suzuki, Satoshi Itami,2 Minako Ohishi,2 Koichi Hamada, Tae Inoue, Nobuyasu Komazawa, Haruki Senoo, Takehiko Sasaki, Junji Takeda, Motomu Manabe, Tak Wah Mak,3, 4 and Toru Nakano3, 5 Departments of Biochemistry [A. S., K. H.], Dermatology [T. I., M. M.], and Anatomy [H. S.], Akita University School of Medicine, Akita 010-8543, Japan; Department of Molecular Cell Biology, Research Institute for Microbial Disease [M. O., T. N.], Department of Dermatology [S. I.], and Department of Social and Environmental Medicine [N. K., J. T.], Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan; Department of Pharmacology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan [T. S.]; and Advanced Medical Discovery Institute, University of Toronto, Toronto, Ontario, M5G 2C1 Canada [T. W. M.] ABSTRACT The epidermis of the skin is a keratinized and stratified squamous epithelium composed mainly of keratinocytes, cells whose prolifera- PTEN is a tumor suppressor gene mutated in many human cancers. We tion and differentiation must be tightly regulated and coordinated used the Cre-loxP system to generate a keratinocyte-specific null mutation (13–15). Keratinocytes first attach to the basal membrane of the of Pten in mice (k5Ptenflox/flox mice). k5Ptenflox/flox mice exhibit wrinkled skin because of epidermal hyperplasia and hyperkeratosis and ruffled, epidermis as undifferentiated precursor cells. These precursors mi- shaggy, and curly hair. Histological examination revealed that skin mor- grate toward the surface of the epidermis and subsequently form its phogenesis is accelerated in k5Ptenflox/flox mice. Within 3 weeks of birth, outermost layer (16). Both the normal development of keratinocytes 90% of k5Ptenflox/flox mice die of malnutrition possibly caused by hyper- and the onset of tumors involving these cells require complex prolif- keratosis of the esophagus. All k5Ptenflox/flox mice develop spontaneous erative/apoptotic events. The biochemical pathways underlying many tumors within 8.5 months of birth, and chemical treatment accelerates the of these events have been delineated through the use of transgenic and onset of tumors. k5Ptenflox/flox keratinocytes are hyperproliferative and knockout mice (17). Hair follicle morphogenesis and hair remodeling resistant to apoptosis and show increased activation of the Pten down- also depend on a balance of proliferative and apoptotic events. How- stream signaling mediators Akt/protein kinase B (PKB) and extracellular ever, the molecular mechanisms underlying hair morphogenesis are signal-regulated kinase. Pten is thus an important regulator of normal not well understood. A normal hair follicle cyclically traverses three development and oncogenesis in the skin. phases of organ growth: hair shaft formation (anagen); organ involu- tion (catagen); and relative quiescence (telogen). Hair follicle mor- INTRODUCTION phogenesis continues until the first hair cycle after birth (18), after PTEN (also called MMAC1 or TEP1) is a tumor suppressor gene which periodical hair remodeling occurs throughout the life of the mutated in both human sporadic cancers (1) and in hereditary cancer animal. syndromes such as Cowden disease and Bannayan-Zonana syndrome The importance of EGFR, IGF-1, and Ha-ras signaling in morpho- (2, 3). Among the characteristic symptoms of Cowden disease are genesis and carcinogenesis is clearly evident in skin (19–21). Acti- ␣ those involving the skin such as trichilemmomas in the face papules, vation of the transforming growth factor /EGFR pathway (22–24), papillomatosis in the mucosal and cutaneous tissues, and hyperkera- IGF-1 (20, 25), or v-Ha-ras (26) in the epidermis of transgenic mice tosis in the acral region of the skin (4). The association of cutaneous leads to skin hyperplasia, hyperkeratosis, and tumor formation. Trans- squamous cell carcinomas with Cowden disease has also been re- genic animals overexpressing IGF-1 also show accelerated hair ported (5, 6). growth (27) and mice lacking IGF-1R exhibit hypoplastic skin (28). Ј PTEN is a dual protein and lipid phosphatase (7, 8). PTENЈs major Because each of EGFR, IGF-R, and Ras triggers PI3 K signaling and substrate is PIP36, a second messenger molecule generated by PI3ЈK PTEN regulates this pathway, we felt it likely that the PTEN gene activated in response to numerous growth factors such as EGF (9), would play an important role in skin development and oncogenesis. In previous work, we used gene targeting to create a complete null hepatocyte growth factor (10), fibroblast growth factors (11), and IGF-1 Ϫ/Ϫ (12). PIP3 in turn activates the serine-threonine kinase Akt/PKB, which mutation of Pten in mice (Pten mice; Ref. 29). However, total is involved in antiapoptosis, proliferation and oncogenesis. Thus, by deficiency for Pten proved to be embryonic lethal in the very early dephosphorylating PIP3, PTEN negatively regulates cell survival. stages of development, precluding the functional analysis of Pten in various organs. We subsequently showed in PtenϪ/Ϫ MEFs that Akt/PKB is a key player downstream of Pten and that Akt/PKB is Received 8/5/02; accepted 12/16/02. The costs of publication of this article were defrayed in part by the payment of page hyperactivated in the absence of Pten (30). Mice heterozygous for the ϩ Ϫ charges. This article must therefore be hereby marked advertisement in accordance with Pten null mutation (Pten / mice) were frequently found to develop 18 U.S.C. Section 1734 solely to indicate this fact. lymphoid hyperplasia and endometrial, prostatic, and breast cancers. 1 This work was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan; Japan Medical Association; the Naito Foundation; the ONO Medical With respect to skin anomalies, focal hyperkeratosis was reported in Research Foundation; the Japanese Society for Promotion of Science (JSPS- a proportion of Ptenϩ/Ϫ mice (29, 31, 32). Because Pten is expressed RAT98L01101); the Sumitomo Foundation; and the Public Trust Haraguchi Memorial Cancer Research Foundation. in keratinocytes and decreased Pten activity contributes to the malig- 2 These individuals contributed equally as second authors. nant conversion stage in chemically induced mouse skin cancers (33), 3 These individuals contributed equally as last authors. we decided to investigate the role of Pten in skin development and 4 To whom requests for reprints should be addressed, at Advanced Medical Discovery Institute, 620 University, Suite 706, Toronto, Ontario, M5G 2C1 Canada. Phone: oncogenesis using the Cre-loxP system. (416) 204-2236; Fax: (416) 204-5300; E-mail: [email protected]. 5 To whom requests for reprints should be addressed, at Department of Molecular Cell Biology, Research Institute for Microbial Disease, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8361; Fax: 81-6-6879-8362; E-mail: MATERIALS AND METHODS [email protected]. 6 The abbreviations used are: PIP3, phosphatidylinositol 3,4,5-triphosphate; PI3ЈK, Generation of k5Ptenflox/flox Mice. Ptenflox/flox mice (129 Ola x C57BL6/J phosphoinositide-3-kinase; EGF, epidermal growth factor; EGFR, epidermal growth F2) generated as previously described (29) were mated to Keratin5Cre trans- factor receptor; IGF, insulin-like growth factor; MEF, murine embryonic fibroblast; SE, genic mice (C57BL6/J background; Ref. 34) in which expression of Cre is scanning electron microscopy; DMBA, 7,12-dimethylbenz(a)anthracene; TPA, 12-O- tetradecanoylphorbol-13-acetate; MAPK, mitogen-activated protein kinase; LOH, loss of controlled by the endogenous promoter of the keratinocyte-specific gene heterozygosity; PKB, protein kinase B. Keratin5. Offspring carrying Keratin5Cre and two copies of the floxed Pten 674 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2003 American Association for Cancer Research. ROLE OF PTEN IN SKIN MORPHOGENESIS AND ONCOGENESIS allele (k5CrePtenflox/flox), Keratin5Cre plus one copy of the floxed Pten allele medium without supplements and cultured in the presence of EGF (30 ␮g/ml) (k5CrePtenflox/ϩ), and Keratin5Cre plus two copies of the wild- for 72 h. Cells were pulsed with 1␮Ci [3H]thymidine (Amersham) for another type Pten allele (k5CrePtenϩ/ϩ) were used in the analyses as homozygous 16 h before harvesting by trypsinization. Incorporated radioactivity was meas- ϩ mutant (k5Ptenflox/flox), heterozygous mutant (k5Ptenflox/ ) and wild-type ured using a ␤-scintillation counter. ϩ ϩ (k5Pten / ) mice, respectively. Apoptosis Assay. Keratinocytes cultured in 6-well plates coated with type PCR Analysis of Pten Genotypes. Genomic DNA from mouse tails was I collagen were treated with either UV- or ␥-irradiation at the doses indicated isolated and amplified by PCR following a published protocol (29). Sense in Fig. 4. One day after treatment, cell viability was determined by staining primer [5Ј-GTCACCAGGATGCTTCTGAC-3Ј] and antisense primer [5Ј- with 7-amino-actinomycin D (Sigma) as described previously (35). GAAACGGCCTTAACGACGTAG-3Ј] were used to detect the floxed Pten SE. Hairs plucked from the anterior backs of mice were attached to alu- allele; sense primer [5Ј-GTCACCAGGATGCTTCTGAC-3Ј] and antisense minum mounts and coated with 350 angstrom gold using EIKO IB-5 sputter primer [5Ј-GTGACATCAACATGCAACACTG-3Ј] were used to detect

Load more