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[Cancer Research 65, 8671-8678, October 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Targeted Disruption of Smad4 in Mouse Epidermis Results in Failure of Hair Follicle Cycling and Formation of Skin Tumors

Leilei Yang1, Chunming Mao1, Yan Teng1, Wenlong Li1, Jishuai Zhang1, Xuan Cheng1, Xiaobing Li3, Xinghai Han4, Zhaofan Xia4, Hongkui Deng5 and Xiao Yang1,2

1 Genetic Laboratory of Development and Diseases, Institute of Biotechnology; 2 Model Organism Division, E-institutes of Shanghai Universities, Shanghai Second Medical University; 3 Department of Pathology, 307 Hospital; 4 Department of Burns, Changhai Hospital, Second Military Medical University, Shanghai, P.R. China; and 5 Department of Cell Biology and Molecular Genetics, Life Science College, Peiking University, Beijing, P.R. China

Requests for reprints: Xiao Yang, Institute of Biotechnology, 20 Dongdajie, Fengtai, Beijing 100071, P.R. China. Phone/Fax: 86-10-63895937; E-mail: yangx{at}nic.bmi.ac.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Smad4 is the common mediator of transforming growth factor-ß (TGF-ß) superfamily signaling, which functions in diverse developmental processes in mammals. To study the role of Smad4 in skin development, a keratinocyte-specific null mutant of Smad4 (Smad4co/co;K5-Cre) was generated in mice using the Cre-loxP system. The Smad4-mutant mice exhibited progressive alopecia as a result of the mutant hair follicles failing to undergo programmed regression. Sonic hedgehog (Shh) was only detected in Smad4-mutant hair follicles at the catagen stage. Seventy percent of Smad4co/co; K5-Cre mice developed spontaneous tumors within 12 months of birth. c-Myc and cyclin D1 were up-regulated whereas p21 and p27 expressions were decreased, which correlated with the epidermal hyperplasia in Smad4 mutants. Interestingly, coordinated deletion of the Smad4 and PTEN genes resulted in accelerated hair loss and skin tumor formation, suggesting that Smad4 and PTEN act synergistically to regulate epidermal proliferation and differentiation. All of our data indicate that Smad4 is essential for catagen induction and acts as a critical suppressor in skin tumorigenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The epidermis is a complex and dynamic tissue consisting of a multilayered epithelium, which is composed mainly of keratinocytes and associated appendages, including hair follicles (1). The postnatal hair follicles continually cycle through three stages: organ growth and hair shaft formation (anagen), apoptosis-driven regression (catagen), and relative quiescence (telogen; ref. 1). During the catagen stage, the hair follicle goes through a highly controlled process of involution that largely reflects a burst of programmed cell death in the majority of the follicular keratinocytes (2). This cyclic activity of the hair follicle is governed by the epithelial-mesenchymal interactions between hair follicle keratinocytes and fibroblasts of the dermal papilla (1). The proliferation and differentiation of keratinocytes need to be tightly regulated and coordinated during these processes. In recent years, there has been tremendous progress in identifying many signaling molecules that play critical roles during the normal development of the epidermis and during malignant transformation. Genetic studies have revealed important functions for fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, hedgehog, and Wnt signaling in epidermal development and hair formation (1).

Transforming growth factor-ß (TGF-ß) signaling has been implicated in the maintenance of epidermal homeostasis. In vitro studies have shown that all TGF-ß isoforms inhibit keratinocyte differentiation (3). Overexpression transgenic studies have revealed the paradoxical role of TGF-ß1 in epidermal proliferation (4, 5). Both TGF-ß1 knockout mice and transgenic mice expressing a dominant-negative TGF-ß type II receptor show epidermal hyperproliferation (6, 7). Studies have also shown that TGF-ß1 signaling is involved in regulating the catagen stage through its induction of apoptosis and inhibition of keratinocyte proliferation in hair follicles (8, 9). In vitro studies have shown that TGF-ß inhibits cell growth through transcriptional repression of c-Myc (10) and up-regulation of the cyclin-dependent kinase inhibitors p21 and p27 (11, 12). Additional signaling molecules have been reported to be essential for the TGF-ß antiproliferative response, but their general importance has not been established (13).

Bone morphogenetic protein (BMP) signaling has been suggested to play important roles in hair follicle induction, differentiation, and skin carcinogenesis (14). BMP signaling inhibits embryonic follicle induction and regulates follicle patterning by repressing the placode fate in adjacent cells (15). Targeted disruption of BMPR1A in the epidermis results in abnormal hair follicle differentiation, cycling failure, and skin tumor formation. These findings provide genetic evidence that BMPR1A-mediated signals play important roles in controlling cell proliferation and differentiation during epidermal development and skin tumorigenesis (16–19).

Phosphatase and tensin homologue (PTEN) is a tumor suppressor that negatively regulates cell survival and proliferation mediated by phosphatidylinositol 3-kinase/protein kinase B signaling (20). Previous studies have found that targeted disruption of PTEN in keratinocytes results in hyperproliferation and neoplastic changes in skin, and thus suggested that PTEN is an important regulator of normal skin development and oncogenesis (21, 22). Recent studies have shown that PTEN mediates the convergence of the BMP and Wnt signaling pathways through phosphatidylinositol 3-kinase-Akt in the regulation intestinal stem cell self-renewal (23). However, the interaction between the TGF-ß/BMP and PTEN pathways during epidermal development and tumorigenesis remains poorly understood.

TGF-ß superfamily members signal through type I and II cell surface receptor serine/threonine kinases and through intracellular mediators called Smads (24). The eight identified Smad proteins have been divided into three functional classes: the receptor-regulated Smads (Smad1, 2, 3, 5, and 8), the comediator Smad (Smad4), and the inhibitory Smads (Smad6 and Smad7). Previous studies have shown that the expression of Smads is markedly decreased in basal cell carcinomas (25), chemically induced squamous cell carcinomas (26), and some head and neck squamous cell carcinomas (27), which suggests that Smads play a role in skin carcinogenesis. Additional evidence has implicated Smad3 in the inhibition of reepithelialization, specifically via effects on keratinocyte proliferation (28). Unfortunately, targeted disruption of Smad4, a central cytoplasmic mediator of TGF-ß signaling, results in early embryonic lethality in mice (29, 30), making it difficult to assess the function of Smad4 in epidermal development and skin tumorigenesis.

To comprehensively understand the roles of Smad4-mediated TGF-ß signaling in skin development and oncogenesis, we specifically deleted the Smad4 gene in keratinocytes using the Cre-loxP system. Our data show that Smad4 is necessary for hair follicle cycling and inhibition of tumor formation. Furthermore, we show here that Smad4 and PTEN act in a synergistic manner to negatively regulate epidermal proliferation and tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse strains and genotyping. Mouse genotyping of the Smad4-loxP and PTEN-loxP loci and detection of Cre-mediated recombination by PCR were done as described in refs 21 and 30. The Cre gene is screened by the primer sets 5'-GCCTGCATTACCGGTCGATGC-3' and 5'-CAGGGTGTTATAAGCAATCCC-3'.

Southern blots. Genomic DNAs isolated from multiple tissues of Smad4co/co;K5-Cre and control mice were digested with EcoRV. About 15 µg of DNA from each sample were electrophoresed on the 0.8% agarose gel and transferred to nitrocellulose membrane. Hybridization was done using the probe described previously (31).

RNA isolation and reverse transcription-PCR. Total RNAs were isolated from epidermis using TRIzol reagent (Life Technologies) based on the suggested protocol. Reverse transcription-PCR (RT-PCR) was done by using the mRNA Selective PCR Kit (TaKaRa). Five-fold dilution of each sample was PCR amplified to achieve signals within the linear amplification range. The Smad4 gene is detected by the primer sets 5'-CAGCCTCCCATTTCCAATC-3' and 5'-CCTTAGTTGAAGCCTTATAACTTCG-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers 5'-ACAGCCGCATCTTCTTGTGC-3' and 5'-TTTGATGTTAGTGGGGTCTCGC-3' were used as internal control.

Western blot analysis. Tissue proteins were obtained from the extracts of epidermis of Smad4co/co;K5-Cre and control mice at P2. Antibody reaction was done with antibodies against Smad4, c-Myc, p21, p27 (Santa Cruz Biotechnology), and ß-actin (Sigma).

Histology and X-gal staining. Skin tissue samples were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin. Sectioned slides were stained with H&E. LacZ staining was done on embryos at embryonic day 14.5 (E14.5) and skin tissues at postnatal day 15 (P15). Stained embryos and tissues were postfixed in 4% paraformaldehyde at 4°C overnight and paraffin embedded for histologic analysis.

In situ hybridization. In situ hybridization was done using standard procedures. Probes were labeled with 35S-labeled UTP using the MAXIscript In vitro Transcription Kit (Ambion). Dorsal anterior skin samples were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned at 8 µm. The probe used was murine Shh as described previously (32). Slides were dipped in emulsion (Amersham Pharmacia) and exposed for 10 days before developing.

Immunohistochemistry and BrdUrd labeling. On dissection, tissues were fixed in 4% paraformaldehyde. To block endogenous peroxidase activity of skin, the sections were treated with 0.3% H2O2 and rinsed with PBS. Primary antibodies against the following proteins were used: proliferating cell nuclear antigen (PCNA), keratin 14 (BAbCo), Smad4, c-Myc, p21, and cyclin D1 (Santa Cruz Biotechnology). Bound antibodies were visualized by diaminobenzidine, and sections were counterstained with hematoxylin. For BrdUrd labeling, mice were injected i.p. with 100 µg/g body weight of BrdUrd (Sigma) 2 hours before sacrifice.

Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was done on 8-µm paraffin sections of paraformaldehyde-fixed specimens from P20 mice using the ApopTag Peroxidase In situ Apoptosis Detection Kit following the directions of the manufacturer (Chemicon).

Tissue culture of mouse back skin. Full-thickness back skin tissues (1 x 2 mm) taken from 2-month-old Smad4co/co;K5-Cre mice and wild-type littermates were cultured in defined Keratinocyte SFM medium (Life Technologies) at 37°C in 5% CO2, and were stimulated with 5 ng/mL TGF-ß1 (Sigma) for 0, 24, and 48 hours without or with 1 x 10–5 mol/L TGF-ß1 inhibitor, SB431542 (TOCRIS). For BrdUrd labeling, skin tissues were labeled with 5 µmol/L BrdUrd (Sigma) for 1 hour before paraformaldehyde fixation.

Statistical analysis. All results were presented as mean ± SE. All statistical analyses were done using SPSS software. Statistical differences were determined by Student's t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Targeted disruption of the Smad4 gene in skin epidermis. We generated keratinocyte-specific Smad4-knockout mice using the Cre-loxP system by crossing a mouse strain carrying a Smad4 conditional allele (Smad4Co/+; ref. 31) with transgenic mice expressing Cre under the control of the keratin 5 promoter (K5-Cre; ref. 33). The K5 promoter directs gene expression from E13.5 in the basal layer of epidermal and follicular keratinocytes and has been used to disrupt the floxed genes throughout the epidermis and the outer root sheaths of hair follicles (21). We analyzed the expression pattern of Cre recombinase in the K5-Cre transgenic mouse using the ROSA26 reporter mouse strain (34). LacZ staining done in the K5-Cre and ROSA26 double transgenic mice revealed Cre-mediated recombination in developing hair follicles of E14.5 embryos (Fig. 1A and B). Cre activity was also detected in basal cells of the epidermis and outer root sheaths of hair follicles 15 days after birth (Fig. 1C-E). These data show that the K5-Cre transgenic mice can be used to achieve Cre-mediated recombination in epidermal and hair follicle keratinocytes.



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  		Figure 1. Targeted disruption of Smad4 gene in murine skin. A to E, analysis of Cre recombinase expression in K5-Cre transgenic mice. A and B, whole-mount X-gal staining of E14.5 ROSA26/+ (A) and K5-Cre/ROSA26 mouse embryos (B). C to E, skin tissues from P15 K5-Cre/ ROSA26 mouse were stained with X-gal. LacZ staining was found in the epidermis (C) and hair follicles (D, sagittal section; E, cross section). F, the genotypes were identified by PCR. Primers that amplified a fragment of 481 bp were used to detect the existence of Cre gene. The wild-type Smad4 allele was detected using primers a and b (32) that amplified a fragment of 385 bp from the DNA of wild-type and heterozygous (Smad4Co/+) mice. A fragment of 438 bp was amplified from the floxed allele of heterozygous (Smad4Co/+) and homozygous (Smad4Co/Co) mice. After the Cre-mediated recombination, primers a and c (32) amplified a fragment of 234 bp from the Smad4 allele. G, tissue-specific recombination of the Smad4 gene was detected in eye, tongue, esophagus, tail, and skin by Southern blot analysis. The 4.3 kb fragment was the conditional (floxed) allele; the 7.2 kb fragment was the Smad4 allele after Cre-mediated recombination (32). RT-PCR (H) and Western blot analysis (I) of Smad4 gene in epidermis showed that the expression of Smad4 was remarkably decreased in mutant epidermis. J, percentage of BrdUrd-positive keratinocytes of Smad4-mutant and control mice (n = 6) after treatment with TGF-ß1. +/+, wild-type; +/–, Cre/Co/+ mutant; –/–, Cre/Co/Co mutant. **P < 0.01, significant different from untreated controls. Bar, 20 µm (C); 40 µm (D); 14 µm (E).

 
The K5-Cre transgenic mice were further bred with a strain containing the Smad4 conditional alleles (Smad4Co/Co; ref. 31). The Smad4Co/+;K5-Cre mice were subsequently crossed with Smad4Co/Co mice to generate Smad4Co/Co;K5-Cre mice. In this study, the Smad4Co/+;K5-Cre mice were used as controls because there were no apparent differences observed in skin structure between Smad4Co/+ ;K5-Cre and Smad4Co/Co or Smad4Co/+ or wild-type mice (data not shown). The offspring were genotyped by PCR analysis (Fig. 1F). Cre-mediated excision of exon 8 in different tissues isolated from Smad4Co/Co ;K5-Cre mice was evaluated by PCR (data not shown) and Southern blot analysis (Fig. 1G). Cre-mediated deletion of Smad4 was mainly detected in the skin, but also in the eye, tongue, and esophagus, where keratin 5 is expressed (Fig. 1G). RT-PCR (Fig. 1H) and Western blot analysis (Fig. 1I) showed that the expression of Smad4 in the epidermis of the Smad4Co/Co;K5-Cre mice was markedly decreased compared with that of the wild-type mouse. To evaluate whether knockout of Smad4 blocked the response of keratinocytes to growth inhibition by TGF-ß1, full-thickness skin tissues from Smad4-mutant and control mice were cultured in vitro and treated with TGF-ß1. The numbers of proliferating keratinocytes were significantly decreased in wild-type skin tissues treated with TGF-ß1, whereas the Smad4-mutant skin showed a similar number of BrdUrd-positive keratinocytes after treatment with TGF-ß1 (Fig. 1J). A TGF-ß inhibitor, SB431542, antagonized the TGF-ß1 effect of growth inhibition of wild-type keratinocytes (Fig. 1J). These results indicated that the Smad4 gene was disrupted efficiently in keratinocytes by K5-Cre–mediated recombination.

Smad4-deficient mice exhibit progressive hair loss. The Smad4co/co;K5-Cre mice could not be distinguished from their wild-type and heterozygous littermate controls before P20 (Fig. 2A). Histologic analysis showed that loss of Smad4 did not affect the initiation or development of the primary hair follicles (Fig. 2B). However, the hair loss of Smad4-mutant mice became evident after P20. Obvious hair loss was observed in 3-month-old Smad4co/co;K5-Cre mice (Fig. 2C). H&E-stained histologic sections of the skin revealed disordered and hypertrophied hair follicles (Fig. 2D). The Smad4co/co;K5-Cre mice exhibited generalized alopecia by 7 months of age (Fig. 2E). The skin surface throughout the body exhibited white and black cysts in mutants (Fig. 2H) but not in control mice (Fig. 2G). The cysts could be seen in hypodermis of the mutant back skin but were absent from controls (Fig. 2I and J). The mutant skin was also abnormally thick and stiff. The sections showed that many cysts containing pigments or keratinized debris were located under the epidermis (Fig. 2F). The mutant mice exhibited a shortened life span; none of the Smad4co/co;K5-Cre mice survived more than 15 months (Fig. 2K). Histologic examination of the esophagus in Smad4co/co;K5-Cre mice revealed marked hyperkeratosis (Fig. 2M) compared with littermate controls (Fig. 2L). It is possible that Smad4-mutant mice may have died of malnutrition, which was the result of esophageal dysfunction.



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  		Figure 2. Keratin 5-Cre–induced deletion of the Smad4 gene caused progressive alopecia in mice. G,I, and L, control mice were labeled with Cre/Co/+. The remaining unlabeled figures represent mutants. A,C, and E, gross appearance of Smad4co/co;K5-Cre mice at the stages of 15 days, 3 months, and 7 months after birth, respectively. B,D, and F, H&E-stained histologic sections of back skin tissues from A, C, and E. The skin tissues of Smad4co/co;K5-Cre mice exhibited white and black cysts all over the body (H and J) and were stiffer than the ones of Smad4co/+;K5-Cre mice (G and I). K, lethality of the Smad4co/co;K5-Cre and control mice. No mutant mice survived for more than 15 months. L and M, severe hyperkeratosis (arrow) in the esophagus of Smad4co/co;K5-Cre mice at 7 months after birth. Bar, 200 µm (B, D, and F); 50 µm (L and M).

 
K5-Cre–induced deletion of Smad4 blocks hair follicle regression. We next did a detailed analysis of the morphologic changes in the hair follicles of Smad4co/co;K5-Cre and control mice. Analysis of histologic sections revealed that Smad4-inactivated hair follicles in first anagen were relatively normal compared with controls (Fig. 3A and B). During catagen, all of the hair follicles of heterozygotes began to undergo apoptosis and lifted up from the dermis to epidermis (Fig. 3C and E), whereas the Smad4-mutant hair follicles failed to regress and persisted in an abnormal anagen phase (Fig. 3D and F). A new anagen phase was initiated in the skin of control mice at P30, and new hair follicles grew down into the subcutis (Fig. 3G). In contrast, the Smad4-mutant follicles were found to be in an abnormal, anagen-like state (Fig. 3H), and became further thickened and distorted, and eventually lost their follicular characteristics by the end of the third month (Fig. 2D). TUNEL staining revealed that Smad4 mutants exhibited a lower number of apoptotic cells in the hair bulb of early catagen hair follicles (Fig. 3I and J). As shown in Fig. 3K, there was a lower percentage of TUNEL-positive cells per hair follicle in Smad4 mutants (10.6 ± 5.5) than in controls (35.3 10.7; n = 6, P < 0.001). These data indicate that Smad4 is essential for the regression of hair follicles during catagen.



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  		Figure 3. The hair cycling of Smad4co/co;K5-Cre mice was blocked before catagen. A to H, sagittal sections of Smad4co/+;K5-Cre (A, C, E, and G) and Smad4co/co;K5-Cre mutant (B, D, F, and H) back skin were stained by H&E. A and B, first anagen at P5, catagen (C and D) at P20, telogen at P25 (E and F), and second anagen at P30 (G and H). Note that the mutant hair follicles refused to enter catagen and telogen (D and F). I and J, TUNEL staining showed that there were much less apoptotic cells in hair follicles of Smad4co/co;K5-Cre mutant mice at P20. K, percentage of TUNEL-positive cells per hair follicle. **, P < 0.01, significantly different from littermate controls. Bar, 200 µm (A-H); 100 µm (I and J).

 
Targeted disruption of Smad4 results in increased epidermal and follicular proliferation. Histologic analysis revealed that the epidermis of Smad4-mutant mice, 2 months of age, was thicker than that of controls (Fig. 4A and B). Cross sections of proximal hair follicles in 2-month-old animals showed that the size of mutant hair follicles was significantly increased (Fig. 4E and F). The results of immunohistochemical detection of keratin 14, a marker of the basal layer of epidermis and the outer root sheath of hair follicles, showed that the basal layer of the epidermis (Fig. 4C and D) and the outer root sheath (Fig. 4G and H) of Smad4-mutant hair follicles were abnormally thick. We further investigated whether the increased numbers of keratinocytes in the epidermis and hair follicles were associated with a defect in keratinocyte proliferation. The outer root sheath of hair follicles without Smad4 showed more BrdUrd-positive cells in the first anagen at P15 than the outer root sheaths of controls (Fig. 4I and J). At P25, little proliferation was detected in the skin of littermate controls (Fig. 4K). By contrast, a large number of BrdUrd-positive cells were found in the mutant hair follicle bulbs and outer root sheaths (Fig. 4L). As shown in Fig. 4U, there were greater percentages of BrdUrd-positive cells per hair follicle in the Smad4 mutants (21.5 ± 5.3 and 24.9 ± 8.0) than in the controls (14.5 ± 5.5 and 6.9 ± 4.2) at P15 and P25 (n = 6, P < 0.05 and P < 0.001, respectively).



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  		Figure 4. Increased proliferation of epidermal and follicular keratinocytes in epidermal Smad4 mutants. A to D, H&E staining (A and B) and immunohistochemical analysis of anti–keratin 14 antibody (C and D) showed that the basal cell layer of epidermis in 2-month-old Smad4co/co;K5-Cre mice was much thicker than controls. E to H, H&E staining (E and F) and immunohistochemical analysis of anti–keratin 14 antibody (G and H) showed thickened outer root sheath of hair follicles in 2-month-old Smad4 mutants (F and H). I to L, BrdUrd incorporation in Smad4-mutant mice (J at P15 and L at P25) was greatly increased compared with control mice (I and K). M and N, immunohistochemical staining of c-Myc. O and P, immunohistochemical staining of p21. Q to T,in situ hybridization of Shh showed the expression of Shh was only detected in Smad4-mutant hair matrix (R and T) but not in control hair matrix (Q and S). Q and R, light-field views; S and T, dark-field views. U, percentage of BrdUrd-positive cells per hair follicle at P15 and P25. *, P < 0.05; **, P < 0.01, significant different from littermate controls. V, Western blot showed the up-regulation of c-Myc and the down-regulation of p21 and p27. +/+, wild-type; –/–, Cre/Co/Co mutant. Bar, 40 µm (A-H); 50 µm (I-L); 100 µm (O and P); 200 µm (M, N, and Q-T).

 
In vitro studies have suggested that TGF-ß1 may inhibit cell proliferation by repressing c-Myc expression and up-regulating p21. Therefore, we next examined the expression of c-Myc and p21 in the skin of mutant and control mice by immunohistochemical staining. The expression of c-Myc was significantly increased in mutant epidermal and follicular keratinocytes (Fig. 4M and N), whereas p21 was reduced in the mutants (Fig. 4O and P). These results indicate that Smad4 could mediate TGF-ß–induced inhibition of keratinocyte proliferation by regulating the expression of c-Myc and p21. We also examined the expression of Shh, which has been implicated as a mitogenic factor in keratinocyte proliferation during the initiation of anagen and may be regulated by BMP4 (32, 35). Shh was found to be strongly up-regulated in P20-mutant hair follicles (Fig. 4Q-T). Western blot analysis confirmed the up-regulation of c-Myc and the down-regulation of p21 (Fig. 4V). P27 was also found to be decreased significantly in Smad4-mutant mice (Fig. 4V). These data suggest that the loss of Smad4 caused persistent proliferation of epidermal and follicular keratinocytes, possibly as a result of up-regulation of Shh and c-Myc and down-regulation of p21 and p27.

Spontaneous skin tumor formation in Smad4-deficient mice. Smad4co/co;K5-Cre and control mice were monitored for spontaneous tumorigenesis for 12 months (n = 20 per group). The Smad4co/co;K5-Cre mice started to develop skin tumors around 5 months of age (Fig. 5A and B). By 12 months of age, 70% (14 of 20) of the mutant mice exhibited visible tumors. In contrast, no skin tumors were detected in control mice during the 12-month observation period. Most of these spontaneous tumors were well-differentiated squamous cell carcinomas (8 of 14; Fig. 5C and D) and squamous papillomas (4 of 14; Fig. 5E). One squamous papilloma was found to be accompanied by sebaceous hyperplasia (Fig. 5F). In addition, we observed two basal cell carcinomas characterized by lesions growing downward deep into the dermis as cords of variably basophilic cells (in 14% of tumor-bearing mice; Fig. 5G and H).



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  		Figure 5. Spontaneous tumor formation in Smad4-mutant mice. K and L, control mice were labeled with Cre/Co/+. The remaining unlabeled figures represent mutants. A, a 6-month-old mutant exhibited a big skin tumor. B, incidences of spontaneous tumor formation in 20 Smad4co/co;K5-Cre and control mice. Seventy percent of Smad4co/co;K5-Cre mice developed skin tumors within 12 months of birth. Most of these spontaneous tumors were well-differentiated squamous cell carcinomas (C and D), squamous papillomas (E), sebaceous hyperplasia (F), and basal cell carcinomas (G and H). I, PCNA labeling showed the proliferating cells at the margin of the tumors. J, K14 antibody staining showed that the tumor stemmed from keratinocytes. Cyclin D1 was significantly increased in skin tumor (O) and adjacent noncancerous tissues (M) compared with controls (K). c-Myc was significantly increased in skin tumor (P) and adjacent noncancerous tissues (N) compared with controls (L). Bar, 500 µm (C, E, and G); 100 µm (D, F, and H); 200 µm (I-P).

 
Immunohistochemical staining for PCNA showed that there were more proliferating cells at the margin of the tumors than in the inner region (Fig. 5I). The immunostaining using anti-K14 antibodies showed that most of the tumors stemmed from keratinocytes (Fig. 5J). Cyclin D1 was not expressed in the skin of control mice (Fig. 5K), but was strongly elevated in tumors (Fig. 5O) and noncancerous mutant skin (Fig. 5M). Immunohistochemical staining using an antibody against c-Myc revealed that c-Myc expression was up-regulated in tumors (Fig. 5P) and in noncancerous mutant skin (Fig. 5N) compared with the skin of control mice (Fig. 5L).

Accelerated hair loss and tumor formation in Smad4co/coPTENco/co;K5-Cre mice. Previous studies have shown that targeted deletion of PTEN in keratinocytes results in epidermal hyperplasia and accelerated hair follicle morphogenesis (21, 22). To define the possible synergistic interaction between Smad4 and PTEN, we generated Smad4 and PTEN double knockout mice (Smad4co/coPTENco/co;K5-Cre) and compared the effects on hair follicle morphogenesis and tumor formation to control Smad4co/co;K5-Cre and PTENco/co;K5-Cre mice. The Smad4co/coPTENco/co;K5-Cre mice exhibited obvious hair loss 2 months after birth (Fig. 6A and B), about 1 month earlier than Smad4co/co;K5-Cre mice (Fig. 2C). Histologic analysis of P18 skin revealed that the epidermis and outer root sheaths of the double mutant mice were thicker than that of control mice (Figs. 6C and D and 2D). The hair follicles of the double knockout mutants had significantly more BrdUrd-positive cells at P18 than that of PTENco/co;K5-Cre mice (Fig. 6E and F). Hyperplastic epidermis and outer root sheaths were found in 3-month-old PTENco/co;K5-Cre mice (Fig. 6G), whereas enlarged cysts surrounded by a multilayered epithelium filled with keratinized debris or pigments were found in the double knockout mutants (Fig. 6H). Ten Smad4co/coPTENco/co;K5-Cre double knockout mice were monitored until the age of 3 months for spontaneous tumorigenesis. Three of the mice developed visible squamous papillomas skin tumors on their backs at 2 months of age (Fig. 6B and I). The remaining seven double mutants died of stomach carcinomas by the age of 3 months (data not shown). In contrast, no skin tumors were detected in 23 PTENco/co;K5-Cre and 20 Smad4co/co;K5-Cre at the same age. Large amounts of BrdUrd-positive cells were found in tumor tissues (Fig. 6J). The above data suggest that Smad4 and PTEN are synergistic regulators of normal homeostasis and oncogenesis in the skin.



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  		Figure 6. Accelerated alopecia and tumor formation in Smad4 and PTEN double knockout mice. A and B, gross appearance of PTENCo/Co;K5-Cre (A) and Smad4Co/CoPTENCo/Co;K5-Cre mice (B) at 70 days after birth. The Smad4Co/CoPTENCo/Co;K5-Cre mouse showed dramatic hair loss and skin tumor. C and D, H&E-stained histologic sections of back skin from PTENCo/Co;K5-Cre (C) and Smad4Co/CoPTENCo/Co;K5-Cre mice (D) at P18. Double mutant hair follicles failed to go into catagen stage and exhibited abnormal hair follicle structure. E and F, BrdUrd labeling. Increased BrdUrd-positive cells were detected in the hair follicles of double mutant mice at P18 (F). G and H, H&E-stained histologic sections of PTENCo/Co;K5-Cre (G) and Smad4Co/CoPTENCo/Co;K5-Cre (H) back skin at 3 months. I and J, the tumor from the 3-month-old double mutant mouse was stained by H&E (I) and BrdUrd labeling (J). Bar, 200 µm (C, D, G, and H); 50 µm (E, F, and J); 500 µm (I).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that targeted disruption of Smad4 in the epidermis results in progressive hair loss due to defects in postnatal hair follicle cycling and early onset of neoplasia in the skin. The present results indicate that Smad4 is required for catagen induction and inhibition of epidermal proliferation. These data are consistent with the hypothesis that Smad4 and PTEN act synergistically to negatively modulate the proliferation of epidermal cells and to inhibit tumor formation.

We found defective programmed regression of hair follicles in Smad4 mutants. This finding is consistent with observations that loss of TGF-ß1 or BMPR1A results in failure of catagen induction (8, 17). The Smad4 mutants showed a much lower number of apoptotic cells in the hair bulb during the early catagen stage, which is in line with previous findings suggesting that TGF-ß plays an essential role in catagen induction via activation of an apoptotic pathway (8). These data provide genetic evidence suggesting that Smad4 has an important role in initiating catagen. Previous studies revealed that BMP4 down-regulates Shh, whereas noggin treatment up-regulates the expression of Shh in the hair follicles (35). Consistent with these studies, we found that the Shh transcripts were detected during catagen in Smad4-mutant hair follicles but not in the wild-type hair follicles. These data suggest that Smad4 may mediate BMP4-induced down-regulation of Shh expression during catagen. Our data generally agree with previous results from studies of targeted disruption of BMPR1A in hair follicle (16–19). However, no defect in inner root sheath differentiation was found in the Smad4 mutant. This discrepancy may be due to a compensatory mechanism that allows extracellular BMP and TGF-ß signals to be transduced via Smad4-independent pathways in keratinocytes (36).

One of the most striking phenotypes in Smad4-mutant mice is the spontaneous skin tumor formation. This finding provides genetic evidence that Smad4 acts as tumor suppressor in epidermal development. There are several lines of evidence suggesting that BMP and TGF-ß play important suppressive roles in skin oncogenesis. Transgenic mice overexpressing BMP4 or BMP6 in the epidermis have been shown to suppress skin tumor formation (37, 38), whereas mice with a Cre-mediated mutation of BMPR1A in the epidermis suffer from follicular cysts and various tumors (17, 19). In addition, many studies have shown that TGF-ß acts as a tumor suppressor during the early stages of skin carcinogenesis (6, 7, 39, 40). Furthermore, expression of Smads is found to be down-regulated in chemically induced mouse epidermal tumors and human basal cell carcinomas (25). Although the molecular mechanisms of TGF-ß signaling in the inhibition of epidermal cell proliferation and tumor formation have been uncovered to great molecular details, corroborating in vivo evidence is still lacking (13).

In the current study, we report that inactivation of Smad4 in mouse skin epidermis results in neoplasia due to persistently increased proliferation and reduced apoptosis of follicular keratinocytes. We showed that the expression of c-Myc is dramatically up-regulated and p21 is down-regulated in Smad4-mutant mice. These effects correlated with increased epidermal proliferation, suggesting that Smad4 may mediate TGF-ß signaling in the regulation of the expression of these genes in keratinocytes. This study provides in vivo evidence consistent with the previous finding that TGF-ß inhibits the cell cycle by down-regulation of c-Myc coupled with up-regulation of p21 (41, 42). Targeted expression of c-Myc in the epidermis has also been found to rapidly trigger proliferation and disrupt differentiation of postmitotic keratinocytes and to eventually cause skin tumor formation (43).

TGF-ß1 has been shown to suppress a variety of cell cycle genes, including cyclin D1 (44). Previous studies have revealed that cyclin D1 is overexpressed very early in the development of chemically induced neoplasia in skin (45). Mice overexpressing cyclin D1 in skin exhibit increased papilloma formation in chemical carcinogenesis experiments (46). In agreement with these studies, we showed that cyclin D1 is significantly increased in tumors and in noncancerous epidermis of Smad4 mutants. These data suggest that loss of Smad4 results in an up-regulation of cyclin D1, which induces excessive epidermal proliferation via the altered differentiation state of keratinocytes. On the other hand, persistent proliferation and tumor formation could also result from constitutively expressed Shh via the expansion of follicular stem cells.

Importantly, we provide the first in vivo genetic evidence that Smad4 and PTEN act in a synergistic manner to negatively regulate epidermal and hair follicle morphogenesis. Previous studies have suggested that there is an interaction between PTEN and BMP or TGF-ß pathways during normal development and tumorigenesis. Exposure of breast cancer cells to BMP2 results in decreased PTEN protein degradation and increased PTEN levels (47), whereas inducible PTEN expression in a glioblastoma cell line suppresses expression of the TGF-ß gene (48). In the current study, we found that Smad4co/coPTENco/co;K5-Cre mice exhibited accelerated alopecia and tumor formation. Deletion of both Smad4 and PTEN resulted in tumor formation 2 months earlier than in PTEN mutant mice (21) and 3 months earlier than in Smad4-mutant mice (Fig. 5B), suggesting that Smad4 and PTEN function in a synergistic manner to inhibit skin tumor formation. The significantly thickened epidermis, the early onset of cysts, and the persistently proliferating follicular keratinocytes indicate that the proliferation of epidermal cells is greatly accelerated due to the loss of both Smad4 and PTEN signaling. These results indicate that Smad4 and PTEN function together to inhibit the proliferation of keratinocytes, and are consistent with previous studies demonstrating that both TGF-ß1 and PTEN can inhibit the expression of c-Myc and cyclin D1 (44, 49). The early formation of epithelioid cysts in double knockout mutants also suggests that Smad4 and PTEN act synergistically in influencing stem cell fate and for maintenance during postnatal hair follicle cycling. Recent studies have revealed that PTEN bridges the BMP and Wnt pathways via phosphatidylinositol 3-kinase/Akt and 14-3-3{zeta}, and inhibits intestinal stem cell self-renewal (23). A previous study shows that Akt interacts directly with Smad3 to regulate the sensitivity to TGF-ß induced apoptosis, providing a mechanism underlying the crosstalk between the TGF-ß and phosphatidylinositol 3-kinase signaling pathways (50). Elucidation of the molecular mechanism of the synergistic interaction of Smad4 and PTEN in regulating the proliferation and differentiation of the epidermal stem cells will require further investigation.


   Acknowledgments
 
Grant support: National Science Fund for Distinguished Young Scholars (nos. 30025028 and 30128013), National Natural Science Foundation of China (no. 30400252), Key Technologies R&D Programme (2002BA711A02-5), and National Basic Research Program of China (2001CB510205 and 2005CB522506).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chuxia Deng for Smad4 floxed mouse, and Tak Wah Mak for PTEN floxed mouse. We also thank Xiang Gao (Model animal research center, Nanjing University, China) for mouse strains.


   Footnotes
 
Note: L. Yang, C. Mao, and Y. Teng contributed equally to this work.

Received 3/10/05. Revised 7/ 2/05. Accepted 7/28/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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