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[Cancer Research 64, 7836-7845, November 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Smad3 Knockout Mice Exhibit a Resistance to Skin Chemical Carcinogenesis

Allen G. Li1,2,3, Shi-Long Lu1,2,3, Ming-Xiang Zhang1,2,3, Chuxia Deng4 and Xiao-Jing Wang1,2,3

Departments of 1 Otolaryngology, 2 Dermatology, and 3 Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon; and 4 Mammalian Genetics Section, Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADDENDUM
 REFERENCES
 
It has been shown that Smad3 exerts both tumor-suppressive and -promoting roles. To evaluate the role of Smad3 in skin carcinogenesis in vivo, we applied a chemical skin carcinogenesis protocol to Smad3 knockout mice (Smad3–/– and Smad3+/–) and wild-type littermates (Smad3+/+). Smad3–/– mice exhibited reduced papilloma formation in comparison with Smad3+/+ mice and did not develop any squamous cell carcinomas. Further analysis revealed that Smad3 knockout mice were resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced epidermal hyperproliferation. Concurrently, increased apoptosis was observed in TPA-treated Smad3–/– skin and papillomas when compared with those of wild-type mice. Expression levels of activator protein-1 family members (c-jun, junB, junD, and c-fos) and transforming growth factor (TGF)-{alpha} were significantly lower in TPA-treated Smad3–/– skin, cultured keratinocytes, and papillomas, as compared with Smad3+/+ controls. Smad3–/– papillomas also exhibited reduced leukocyte infiltration, particularly a reduction of tumor-associated macrophage infiltration, in comparison with Smad3+/+ papillomas. All of these molecular and cellular alterations also occurred to a lesser extent in Smad3+/– mice as compared with Smad3+/+ mice, suggesting a Smad3 gene dosage effect. Given that TGF-ß1 is a well-documented TPA-responsive gene and also has a potent chemotactic effect on macrophages, our study suggests that Smad3 may be required for TPA-mediated tumor promotion through inducing TGF-ß1–responsive genes, which are required for tumor promotion, and through mediating TGF-ß1–induced macrophage infiltration.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADDENDUM
 REFERENCES
 
Transforming growth factor (TGF)-ß is a multifunctional cytokine that regulates cell proliferation, apoptosis, tissue remodeling, and angiogenesis, all of which are associated with cancer development (for a recent review, see ref. 1 ). Smad family members are the major intracellular mediators of TGF-ß signaling (for a recent review, see ref. 1 ). Among the Smad family members, Smad2 and Smad3 are responsible for TGF-ß and activin signaling (2) . On binding of TGF-ß to its receptors, Smad2 and Smad3 are phosphorylated and subsequently form heteromeric complexes with Smad4, which then translocate from the cytoplasm to the nucleus. The Smad complex functions in the nucleus as a transcription factor and regulates expression of TGF-ß downstream targets (2) .

TGF-ß signaling plays a complex role in cancer development. As a potent growth inhibitor for keratinocytes, TGF-ß1 is considered to play a tumor-suppressive role at early stages of skin carcinogenesis. For instance, loss of TGF-ß1 expression results in early progression to malignancy (3) . Similarly, transgenic mice overexpressing the dominant negative TGF-ß type II receptor ({Delta}ßRII) in the epidermis exhibited a higher susceptibility to skin chemical carcinogenesis (4 , 5) . Paradoxically, TGF-ß1 is overexpressed in many cancer types in humans (6) . In experimental carcinogenesis models, expression of TGF-ß1 is also induced after application of tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate [TPA (7) ]. Studies from transgenic mice show that overexpression of TGF-ß1 in the epidermis results in inhibition of tumor formation at early stages but acceleration of tumor progression at later stages (8 , 9) . These studies suggest that TGF-ß1 has a dual role in skin carcinogenesis. It remains to be determined, however, whether TGF-ß1 overexpression at an early stage of skin carcinogenesis exerts any tumor promotion effect.

As the major signaling mediators of the TGF-ß superfamily, Smads may be actively involved in mediating the effects of TGF-ß1 in carcinogenesis. In vitro studies have shown that TGF-ß–induced growth inhibition in epithelial cells is preferentially mediated by Smad3 (10 , 11) and that cyclin-dependent kinase (cdk) 2 and cdk4 can phosphorylate Smad3, thus inactivating its ability to instigate cell cycle arrest (12) . Therefore, Smad3 may mediate TGF-ß1–induced growth inhibition during cancer development. Supporting this notion, Smad3-null keratinocytes transduced by v-rasHa undergo accelerated malignant conversion when they are transplanted onto nude mice (13) . Further analyses revealed that the loss of Smad3 abrogates the functions of TGF-ß1 in regulating genes critical for cell cycle control, such as p15 and c-myc (13) , suggesting that Smad3 is required for TGF-ß–induced growth inhibition in keratinocytes. However, Smad3 knockout mice do not exhibit epithelial hyperproliferation (14) , suggesting that the role of Smad3 in mediating TGF-ß1–induced growth inhibition is dispensable in vivo. In addition, TGF-ß1 overexpression in keratinocytes in vivo induces severe skin inflammation (15 , 16) , whereas Smad3 knockout mice exhibit accelerated wound healing with decreased inflammation in vivo (17) . These studies suggest that Smad3 may also mediate TGF-ß1–induced skin inflammation. Because inflammation plays an important role in tumor promotion (18) , affecting TGF-ß1–induced skin inflammation may significantly affect the role of TGF-ß1 in skin carcinogenesis. Furthermore, a recent study using breast cancer cell lines showed that Smad3 exhibits both tumor-suppressive and -promoting roles (19) . To evaluate the role of Smad3 in TGF-ß signaling during skin carcinogenesis in vivo, we applied a chemical carcinogenesis protocol to Smad3 knockout mice (designated Smad3+/– and Smad3–/– hereafter), in which TGF-ß1 expression is induced by TPA promotion (7) . Here we demonstrate that Smad3 knockout mice exhibited a resistance to skin chemical carcinogenesis, correlating with decreased proliferation and increased apoptosis in Smad3 knockout keratinocytes during TPA promotion and reduced tumor-associated macrophage (TAM) infiltration in Smad3 knockout tumors. Consistently, TGF-{alpha} and activator protein (AP)-1 family members, which are critical factors for TPA-induced tumor promotion, were not up-regulated in Smad3 knockout skin and tumors. Our results suggest that Smad3 is required for TPA-mediated tumor promotion, possibly by regulating TGF-ß–responsive genes that are involved in tumor promotion and by mediating TGF-ß1–induced inflammation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADDENDUM
 REFERENCES
 
Skin Chemical Carcinogenesis Protocol.
The Smad3 knockout mice were produced in a C57BL/6 x 129SVEV background (20) and backcrossed to 129SVEV for >12 generations during a 2-year period before their use in this skin carcinogenesis study. Twelve hemizygous (Smad3+/–) breeding pairs were set up to generate mice with Smad3+/+, Smad3+/–, and Smad3–/– genotypes. In this study, 35 Smad3+/+ (15 males and 20 females), 44 Smad3+/– (23 males and 21 females), and 16 Smad3–/– (7 males and 9 females) mice were used. The chemical carcinogenesis protocol was applied as described previously (21) . Briefly, neonatal pups at day 3 were treated topically with 20 µg of 7,12-dimethylbenz(a)anthracene (DMBA; dissolved in 50 µL of acetone; Sigma, St. Louis, MO). TPA (5 µg; dissolved in 50 µL of acetone; Sigma) was applied to pup skin at day 4 and day 6, and application was continued twice a week for 30 weeks.

Acute 12-O-Tetradecanoylphorbol-13-acetate Treatment on Adult Skin.
The dorsal skins of 6-week–old mice were shaved and treated with 5 µg of TPA (dissolved in 50 µL of acetone). Forty-eight hours after TPA treatment, mice received injection with 0.125 mg/g bromodeoxyuridine (BrdUrd; Sigma). One hour later, the mice were euthanized, and the dorsal skins were harvested for histologic analysis, BrdUrd labeling, immunohistochemical staining, and RNA and protein extractions.

Tissue Histology.
Dissected skin and tumor samples were fixed in 10% neutral-buffered formalin at 4°C overnight, embedded in paraffin, sectioned to 6-µm thickness, and stained with hematoxylin and eosin (H&E). Tumor types were determined by H&E analysis using the criteria described previously (22) and confirmed by Dr. C. Corless, a pathologist and the director of the Cancer Pathology Core facility at Oregon Health and Science University. Generally, papillomas appeared as exophytic, pedunculated proliferations consisting of multiple finger-like hyperkeratotic epidermal projections having an intact basement membrane creating a distinct border from the underlying dermis. Squamous cell carcinomas (SCCs) were characterized by invasive tumor cell proliferation into the dermis in a lobular pattern and numerous mitotic figures within tumor lobules. SCCs were further classified into well-, moderately, and poorly differentiated groups based on the criteria described previously (22) .

Double Stain Immunofluorescence.
Double stain immunofluorescence was performed as we have described previously (23) . The primary antibodies included fluorescein isothiocyanate-conjugated BrdUrd (undiluted; Becton-Dickinson, Franklin Lakes, NJ), keratin 1 (K1; 1:500; ref. 24 ), keratin 13 (K13; 1:500; ref. 24 ), and nuclear factor (NF)-{kappa}B p50 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, paraffin-embedded sections were deparaffinized in fresh xylene and rehydrated. Antigen retrieval was performed by microwaving slides in 10 mmol/L sodium citrate solution for 10 minutes. Each section was incubated overnight at 4°C with a primary antibody diluted in PBS containing 12% bovine serum albumin, together with a guinea pig antiserum against mouse keratin 14 (K14; 1:500), the latter of which highlights the epithelial compartment of the skin (9) . The sections were then washed with PBS and incubated with fluorescence dye-conjugated secondary antibodies, an Alexa Fluo 488-conjugated (green) secondary antibody against the species of the primary antibody [1:100; Molecular Probes, Eugene, OR (except for the sections incubated with fluorescein isothiocyanate-conjugated BrdUrd antibody)] and Alexa Fluo 594-conjugated (red) anti-guinea pig secondary antibody (1:100; Molecular Probes). The fluorescence dye-conjugated secondary antibodies were diluted in 10% bovine serum albumin-PBS and applied to the tissue sections at room temperature for 30 minutes. After several PBS washes, sections were visualized under a Nikon Eclipse E600W fluorescence microscope (Nikon, Melville, NY). The BrdUrd labeling index in the epidermis was expressed as the mean number of BrdUrd-positive cells per millimeter of basement membrane ± SD.

Immunohistochemistry.
To examine inflammatory cell subtypes, immunohistochemistry was performed on frozen sections, as we have described previously (16) , using primary antibodies for different leukocyte markers including CD45 (1:20), CD4 (1:20), and Ly-6G [1:20; ref. 25 (all from BD Biosciences, San Diego, CA)]. The BM8 antibody (1:400; BMA Biomedicals, Augst, Switzerland), which recognizes macrophages (26) , was also used. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and phosphorylated Smad2 was performed on paraffin-embedded sections using a PCNA antibody (1:100; Santa Cruz Biotechnology) and a phosphorylated Smad2 antibody (1:50; Cell Signaling, Beverly, MA). After deparaffinization, the sections were subjected to antigen retrieval as described above, followed by incubation with 5% serum (from the species in which the secondary antibody was developed) for 1 hour at room temperature. Incubation with primary antibodies was carried out at 4°C overnight. The sections were then sequentially incubated with biotinylated secondary antibodies (1:250) and an avidin-peroxidase reagent (Vector Laboratories, Burlingame, CA) at room temperature for 10 and 5 minutes, respectively. The immune complexes in the sections were visualized using diaminobenzidine (DAKO, Carpinteria, CA). Quantitative measurement of positively stained cells was performed using MetaMorph software (Universal Imaging Corp., Burnaby, British Columbia, Canada) and expressed as the mean number of PCNA-positive cells per mm2 tumor area ± SD.

Apoptosis Assays.
Following the manufacturer’s instructions, apoptosis was evaluated using terminal deoxynucleotidyltransferase-mediated uridine nick end labeling (TUNEL) assay with the DeadEnd Fluorometric TUNEL kit (Promega, Madison, WI). Briefly, paraffin-embedded sections were deparaffinized, rehydrated, and washed in 0.85% NaCl. The sections were then sequentially prefixed (in 4% formaldehyde-PBS), permeabilized (in 20 µg/mL proteinase K), and postfixed (in 4% formaldehyde-PBS). After several PBS washes, the sections were incubated in a buffer that contained fluorescein-dUTP and recombinant terminal deoxynucleotidyltransferase at 37°C for 1 hour in a humidified chamber in the dark. The reaction was then terminated in 2x SSC for 15 minutes at room temperature, and the slides were washed in PBS. The slides were then immersed in a 1 µg/mL propidium iodide solution for 15 minutes at room temperature to counterstain the nuclei. The samples were then analyzed under a fluorescence microscope. Quantitative measurement of apoptotic cells was performed using MetaMorph software (Universal Imaging Corp.) and expressed as the mean number of apoptotic cells per mm2 tumor area ± SD.

Keratinocyte Culture and 12-O-Tetradecanoylphorbol-13-acetate Treatment.
Primary keratinocytes were isolated from neonatal Smad3+/+, Smad3+/–, and Smad3–/– skin and cultured in conditioned medium supplemented with 0.05 mmol/L Ca2+, as we have described previously (27) . On reaching subconfluence, the cells were treated with TPA (100 nmol/L) for 12 hours before harvesting (28) .

RNA Extraction, Quantitative Reverse Transcription-Polymerase Chain Reaction, and RNase Protection Assay.
Total RNA was isolated from skin, chemically induced tumors, and cultured keratinocytes using RNAzol B (Tel-Test, Friendswood, TX), as we have described previously (11) , and further purified using a Qiagen RNeasy Mini kit (Qiagen, Valencia, CA). The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was achieved by combining in vitro reverse transcription with quantitative polymerase chain reaction, which was performed in a Stratagene Mx3000P thermal cycler (Stratagene, La Jolla, CA; ref. 29 ). Briefly, 5 µg of RNA from each sample were treated with DNase (Ambion, Austin, TX). The RNA was then subjected to a reverse transcription reaction using avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, MN). The resultant cDNA products were used as templates for quantitative polymerase chain reaction to examine the levels of transcripts of mouse Smad3, TGF-{alpha}, and AP-1 family members including c-jun, junB, junD, and c-fos using corresponding TaqMan Assays-on-Demand probes (Applied Biosystems, Foster City, CA). Tumor RNA samples were also assayed for gene expression levels of interleukin (IL)-1ß and monocyte chemotactic protein (MCP-1). An 18S RNA probe was used as an internal control, and the data (CT values) were analyzed using Stratagene Mx3000P Comparative Quantitation software. The expression level of each gene was normalized with 18S using a comparative CT ({Delta}CT) and expressed as the difference of the CT values from a test gene (e.g., TGF-{alpha}) and 18S [CT(18S) minus CT(TGF-{alpha})]. The relative RNA expression levels were calculated using the {Delta}CT method (30) , and the average results from three to five samples from three to five mice of each genotype are shown, with the exception that only two Smad3–/– mice developed tumors. For Smad3 gene expression assays, the expression levels from all Smad3+/+ samples were set as 1 arbitrary unit, which was used as a baseline to compare expression levels of the same gene in samples with different Smad3 genotypes. In analyzing the relative expression levels of other genes, the expression level from one Smad3–/– sample (unless otherwise specified) of each particular gene being analyzed was set as 1 arbitrary unit. To examine expression levels of various cell cycle control genes, RNase protection assay was performed using RNase protection assay III kits (Ambion), as we have described previously (31) . The probes used included multiprobe gene sets [mCC1c, mCYC-1, and mCYC-2 (BD Biosciences)] and individual probes for c-myc, p15, and p21 (31) .

Statistics.
Significant differences between the values obtained in each assay on samples from various genotypes were determined using Student’s t test throughout this study.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADDENDUM
 REFERENCES
 
Smad3 Knockout Mice Exhibited Reduced Tumor Formation and Malignant Progression during Skin Chemical Carcinogenesis.
To determine the effect of loss of Smad3 on skin carcinogenesis in vivo, we applied a chemical carcinogenesis protocol to the skin of Smad3 knockout mice (Smad3+/– and Smad3–/–). Because adult Smad3–/– mice develop a wasting syndrome (20) , which could potentially affect the outcome of the skin carcinogenesis experiment, we applied DMBA to Smad3 knockout mice 3 days after birth, followed by applications of TPA twice a week for 30 weeks. Before 30 weeks of age, animals of all three genotypes remained apparently healthy, with the exception of 10% to 20% lower body weights in Smad3–/– mice in comparison with Smad3+/– and Smad3+/+ mice. By 30 weeks of age, the Smad3–/– mice exhibited an approximately 30% reduction in body weight (males, 22 ± 3.3 g; females, 20 ± 2.1 g; P < 0.01), as compared with Smad3+/– mice (males, 39 ± 4.1 g; females, 37 ± 2.1 g) or Smad3+/+ mice (males, 36 ± 3.7 g; females, 34 ± 4.2 g). After 30 weeks of age, the wasting syndrome in some of the Smad3–/– mice became dominant, resulting in a rapid decline in overall health of these mice, as described previously (20) . Therefore, tumorigenesis in mice with different Smad3 genotypes was monitored until 30 weeks after DMBA initiation.

Smad3+/+ mice began to develop benign papillomas 6 weeks after DMBA initiation, and by 16 weeks, approximately 60% of Smad3+/+ mice had developed papillomas (Fig. 1A)Citation . The first papillomas on Smad3–/– mice began to develop at 11 weeks after DMBA initiation, and only 2 of 16 (~20%) Smad3–/– mice developed papillomas by 16 weeks (Fig. 1A)Citation . Smad3+/– mice also exhibited reduced tumorigenesis (47% by 20 weeks; Fig. 1ACitation ) as compared with Smad3+/+ mice. Additionally, Smad3–/– mice developed significantly fewer tumors as compared with Smad3+/+ mice. Smad3+/+ mice averaged 2.5 tumors per mouse at the end of the promotion stage (30 weeks; Fig. 1BCitation ). In contrast, Smad3–/– mice only averaged 0.5 tumor per mouse by the end of TPA promotion, a 5-fold reduction in comparison with Smad3+/+ mice (Fig. 1BCitation ; P < 0.01). Smad3+/– mice averaged 1 tumor per mouse by 30 weeks, which was also a significant reduction in tumor number when compared with Smad3+/+ mice (Fig. 1BCitation ; P < 0.01). Furthermore, 40% of the tumors that developed on Smad3+/+ mice progressed to SCCs after termination of TPA treatment (30 weeks; Fig. 1CCitation ), with an average of 0.8 ± 0.04 SCCs per mouse (Fig. 1D)Citation . In contrast, all of the tumors dissected from Smad3–/– mice at the same time point were benign papillomas (Fig. 1C and D)Citation . Smad3+/– mice also exhibited a decreased frequency of SCC development, with only 27% of Smad3+/– tumors progressing to SCC (Fig. 1C)Citation and a reduced average number of SCCs per mouse (0.4 ± 0.03; P < 0.01) in comparison with Smad3+/+ mice. Histologically, Smad3+/+ SCCs appeared more advanced and less differentiated than Smad3+/– SCCs, which displayed features of typical carcinomas in situ or well-differentiated SCCs (Fig. 2)Citation . Consistently, the Smad3+/+ and Smad3+/– tumors classified as SCCs showed a lack of K1 expression, a marker of benign papillomas (24) , whereas all of the Smad3–/– tumors retained uniform or at least focal K1 expression (Fig. 2)Citation . In contrast, strongly positive immunofluorescence staining for K13, a malignancy marker in squamous epithelia (32) , was observed in tumors classified as SCCs from both Smad3+/+ and Smad3+/– mice, but not in tumors from Smad3–/– mice (Fig. 2)Citation , further confirming that the tumors from the Smad3–/– mice were benign papillomas.



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  		Fig. 1. Tumor kinetics. A, percentage of tumor-bearing mice in each group. B, average number of tumors per mouse. C, percentage of mice that developed carcinoma by 30 weeks after TPA treatment. None of the Smad3–/– mice papillomas converted to carcinomas. D, average number of SCCs per mouse.

 


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  		Fig. 2. Histology and immunofluorescence analyses of the most advanced tumor types from each genotype. H&E examination revealed a poorly differentiated SCC, an early-stage SCC, and a papilloma in Smad3+/+, Smad3+/–, and Smad3–/– mice, respectively. A K14 antibody (red) was used as a counterstain for immunofluorescence. The bar in the top left panel represents 200 µm for H&E sections and 60 µm for immunofluorescence sections.

 
Smad3 Deletion in Keratinocytes Resulted in a Resistance to 12-O-Tetradecanoylphorbol-13-acetate–Induced Epidermal Hyperplasia and an Increased Rate of Apoptosis.
To determine whether the resistance to skin chemical carcinogenesis in Smad3 knockout mice is due to the blockade of DMBA-induced initiation of tumor development, we examined tumor samples from different Smad3 genotypes for c-rasHa mutations, which are induced by DMBA in >90% of papillomas (33) . Sequence analysis for a c-rasHa genomic fragment (5) revealed that all tumor samples examined from the different Smad3 genotypes possessed a c-rasHa mutation at codon 12, 13, or 61 (data not shown), suggesting that loss of Smad3 likely does not affect DMBA initiation. Next, we analyzed whether Smad3 deletion contributes to a reduction of susceptibility to TPA-induced tumor promotion by examining adult skin with different Smad3 genotypes 48 hours after a single TPA treatment. As compared with TPA-treated Smad3+/+ skin, which was characterized by obvious epidermal hyperplasia (8.1 ± 1.6 cell layers in epidermis), Smad3+/– (4.7 ± 1.2 layers; P < 0.01; n = 3) and Smad3–/– (2.3 ± 0.4 layers; P < 0.01; n = 3) skin showed a resistance to TPA-induced epidermal hyperplasia (Fig. 3A)Citation . Accordingly, the number of BrdUrd-labeled keratinocytes was significantly reduced in TPA-treated Smad3 knockout skin. The number of BrdUrd-labeled keratinocytes was 189 ± 23.4 per mm of epidermis in TPA-treated Smad3+/+skin, whereas it was reduced to 122 ± 14.2 (P < 0.01; n = 3) and 35.6 ± 9.0 per mm of epidermis (P < 0.01; n = 3) in Smad3+/ and Smad3–/– skins, respectively (Fig. 3A)Citation . In contrast to the change in epidermal proliferation, CD45 immunostaining showed no difference in the degree of inflammatory cell infiltration in TPA-treated skin from three groups (Fig. 3A)Citation . The degree of infiltration by different leukocyte subsets, including CD4+ cells, granulocytes, and macrophages, in TPA-treated skin of three groups was also indistinguishable (data not shown). These results suggest that loss of Smad3 has a direct effect on keratinocyte proliferation during the promotion stage of skin chemical carcinogenesis. Analysis of cell proliferation in Smad3 knockout papillomas revealed that the number of PCNA-positive cells was reduced by >50% in Smad3+/– papillomas (163 ± 12.2/mm2 tumor area; P < 0.01; n = 3) in comparison with Smad3+/+ papillomas (389 ± 28.9/mm2) at the same stage and further reduced by 90% in Smad3–/– papillomas (37.2 ± 4.5/mm2) as compared with Smad3+/+ papillomas (Fig. 3B)Citation .



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  		Fig. 3. Proliferation and apoptosis in TPA-treated skin and papillomas from each group. Dotted lines denote the epidermal–dermal junction. A, histology, BrdUrd labeling (green), and CD45 staining on TPA-treated skin. Arrows point to typical BrdUrd-labeled cells. A K14 antibody (red) was used as a counterstain for immunofluorescence. Sections stained by immunohistochemistry for CD45 were counterstained with hematoxylin. The bar in the top left panel represents 100 µm for H&E- and BrdUrd-stained sections and 40 µm for CD45-stained sections. B, PCNA staining of representative papillomas from each genotype. The sections are counterstained with hematoxylin. Red arrows point to examples of positively stained nuclei. The bar in the top left panel represents 100 µm. C, TUNEL assay on TPA-treated skin (top panels) and papillomas (bottom panels) from each group. Arrows point to representative apoptotic nuclei (green or yellowish). The bar in the top left panel represents 40 µm for all sections.

 
To determine whether Smad3 deletion alters the apoptotic process, TUNEL assay was performed on TPA-treated adult skin and papillomas with different Smad3 genotypes. The numbers of apoptotic cells in the epidermis of Smad3–/– (24.8 ± 3.53/mm2 epidermis; n = 3) and Smad3+/– skin (19.8 ± 2.65/mm2 epidermis; n = 3) were significantly increased as compared with the number of apoptotic cells in Smad3+/+ skin (5.25 ± 1.28/mm2 epidermis; n = 3; P < 0.01) 48 hours after TPA treatment (Fig. 3C)Citation . The apoptotic cells in these samples were primarily located in the suprabasal layers of the epidermis (Fig. 3C)Citation . Similarly, apoptotic cells, located mainly in suprabasal layers, were prominent in Smad3+/– (31.3 ± 2.83/mm2 tumor epithelia) and Smad3–/– papillomas (38.7 ± 3.69/mm2) but sparse in Smad3+/+ papillomas (11.6 ± 1.47/mm2; n = 3; P < 0.01; Fig. 3CCitation ).

To further confirm the correlation between the above-mentioned effects and the inactivation of the Smad3 gene, we examined the relative expression levels of the Smad3 transcript by qRT-PCR. As shown in Fig. 4ACitation , Smad3 expression levels in nontreated skin, TPA-treated skin, and papillomas, all from Smad3+/– mice, were reduced by about 50% as compared with the levels detected in the same tissues from Smad3+/+ mice. Expression of Smad3 was undetectable in all samples from Smad3–/– mice (Fig. 4A)Citation . To elucidate whether the effect of Smad3 deletion on skin carcinogenesis involves altered activation of its signaling partner, Smad2, the presence of phosphorylated Smad2 (pSmad2) was examined by immunohistochemistry using a pSmad2 antibody. Nontreated skins from different Smad3 genotypes exhibited comparable pSmad2 nuclear staining (Fig. 4B)Citation . Noticeably, TPA-treated skin from all three Smad3 genotypes exhibited increased pSmad2 in both the epidermis and dermis (Fig. 4B)Citation , suggesting that TPA-induced endogenous TGF-ß1 activates Smad2. However, both TPA-treated skins and papillomas exhibited comparable pSmad2 nuclear staining among different Smad3 genotypes (Fig. 4B)Citation , suggesting that the effect of Smad3 deletion on skin chemical carcinogenesis is independent of Smad2 activation.



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  		Fig. 4. Smad3 expression and Smad2 phosphorylation. A, qRT-PCR results of Smad3 expression in 6-week–old mouse skin (skin), TPA-treated skin, and papillomas from the three different genotypes. The Smad3 expression level of all samples from Smad3+/+ mice was set as 1 arbitrary unit. B, immunohistostaining of pSmad2. The bar in the top left panel represents 40 µm for all sections. Hematoxylin was used as a counterstain.

 
Reduced Expression of Activator Protein-1 Family Members and Transforming Growth Factor {alpha} in 12-O-Tetradecanoylphorbol-13-acetate–Treated Skin and Tumors of Smad3 Knockout Mice.
To determine the molecular mechanisms of Smad3 knockout skin resistance to TPA-induced epidermal hyperplasia and increased apoptotic rate, we examined expression levels of proliferative/survival factors that normally respond to TPA. In contrast to a previous report showing decreased expression of the cdk inhibitor p15 and increased expression of c-myc in vrasHa-transduced Smad3-null keratinocytes (13) , we did not find differences among the different Smad3 genotypes in expression of TGF-ß1 target genes involved in cell cycle control, including the cdk inhibitors p15 and p21 and the c-myc oncogene, in TPA-treated skin of each genotype (data not shown). These results suggest that either TPA itself or an endogenous mechanism(s) is able to regulate expression of these molecules in the absence of Smad3. We next examined the expression of major AP-1 family members, which are TGF-ß–responsive genes (34) that are rapidly up-regulated in response to TPA and mediate TPA-induced tumor promotion (35) . We found that expression levels of c-jun, junB, junD, and c-fos in TPA-treated skin and papillomas were highest in Smad3+/+ mice and lowest in Smad3–/– mice (Fig. 5)Citation .



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  		Fig. 5. Relative expression levels of AP-1 family members and TGF-{alpha} as determined by real-time RT-PCR. Results from two representative samples in each group are shown. The expression level of each molecule in one Smad3–/– sample was set as 1 arbitrary unit. An exception was made for TGF-{alpha} expression levels in TPA-treated skins, in which one Smad3+/– skin sample was set as 1 arbitrary unit, because TGF-{alpha} expression was not detectable in TPA-treated Smad3–/– skin.

 
Because TGF-{alpha} overexpression induced by TPA is thought to be critical for TPA-induced tumor promotion (36) and for protection of tumor cells from apoptosis (37, 38, 39) , we examined expression of TGF-{alpha} in TPA-treated skin and tumors. Expression of TGF-{alpha} was almost undetectable in untreated skin (data not shown) but was induced in Smad3+/+ skin after TPA treatment (Fig. 5)Citation . However, expression of TGF-{alpha} was 8- to 10-fold lower in TPA-treated Smad3+/– skin and almost undetectable in TPA-treated Smad3–/– skin. A similar pattern of TGF-{alpha} expression was observed in tumors from the three genotypes (Fig. 5)Citation .

To further determine whether changes in expression levels of these genes represent a direct effect of Smad3 deletion on keratinocytes, we cultured primary keratinocytes isolated from mice with different Smad3 genotypes and treated these cells with TPA for 12 hours. Expression of the above-mentioned five genes was significantly lower in TPA-treated Smad3–/– keratinocytes than in Smad3+/+ cells (Fig. 5)Citation . TPA-treated Smad3+/– keratinocytes exhibited a reduction in the expression of these genes by about 45% to 66% relative to Smad3+/+ samples (Fig. 5)Citation , suggesting a dose-dependent effect of Smad3 inactivation on the transcription of these genes in keratinocytes in response to TPA.

Reduced Tumor-Associated Macrophages and Inflammation in Smad3 Knockout Papillomas.
Because Smad3 can affect infiltration of inflammatory cells (20) , we examined leukocyte infiltration in TPA-treated skin and papillomas from different Smad3 genotypes. Immunohistochemistry was performed using antibodies that recognize different surface markers of leukocytes. At the acute phase of TPA-induced inflammation (48 hours after a single TPA treatment), we did not observe any differences in the number or type of infiltrating inflammatory cells among different Smad3 genotypes (Fig. 3A)Citation . However, CD45 immunostaining revealed a dramatic reduction in the number of inflammatory cells present in Smad3+/– (216 ± 19.6/mm2 tumor area; P < 0.01) and Smad3–/– (103.3 ± 9.31/mm2 tumor area; P < 0.01) tumors as compared with Smad3+/+ tumors (723 ± 67.5/mm2 tumor area). Numerous BM8+ TAMs were present within Smad3+/+ tumor epithelia, whereas only sporadic TAMs were present in tumor epithelia and stroma of Smad3–/– papillomas (Fig. 6)Citation . Smad3+/– papillomas also exhibited a reduction in the number of TAMs in comparison with Smad3+/+ tumors. In particular, the number of TAMs within tumor epithelia of Smad3+/– (62.3 ± 8.91/mm2 tumor epithelia; P < 0.01; n = 3) or Smad3–/– papillomas (30.4 ± 2.92; P < 0.01; n = 3) was significantly reduced as compared with that of Smad3+/+ papillomas (119 ± 10.7). In contrast, the number of CD4+ T cells (Smad3+/+, 76 ± 4.8/mm2 tumor area; Smad3+/–, 83 ± 9.1/mm2 tumor area; Smad3–/–, 79 ± 8.5/mm2 tumor area; P > 0.05; n = 3) or Ly6-G+ granulocytes (Smad3+/+, 67 ± 5.3/mm2 tumor area; Smad3+/–, 73 ± 8.0/mm2 tumor area; Smad3–/–, 75 ± 7.7/mm2 tumor area; P > 0.05; n = 3) was statistically similar for each genotype (Fig. 6)Citation .



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  		Fig. 6. Inflammatory response in papillomas from different Smad3 genotypes. Immunohistochemistry was carried out to determine the presence of different leukocyte subsets. Note the reduced number of CD45+ cells and macrophages (BM8) in Smad3+/– and Smad3–/– papillomas as compared with Smad3+/+ papillomas. Immunofluorescence was performed to detect p50 expression (green) in papillomas from each group. A K14 antibody (red) was used as a counterstain. The bar in the top left panel represents 40 µm for immunohistochemistry sections and 100 µm for p50-stained sections.

 
Furthermore, we examined nuclear translocation of the NF-{kappa}B subunit p50, an end point of the inflammatory cascade (40) . Immunofluorescence staining showed that >90% of the Smad3+/+ papilloma cells stained positive for p50 in the nucleus (Fig. 6)Citation . In contrast, nuclear staining of p50 was only observed in a few Smad3–/– papilloma cells and in only approximately 50% of Smad3+/– papilloma cells (Fig. 6)Citation .

Consistent with decreased inflammation in the Smad3–/– papillomas, IL-1ß, a proinflammatory cytokine that is mainly expressed in keratinocytes on inflammation (41) , was expressed at a very low level in Smad3–/– papillomas (Fig. 7)Citation . In contrast, IL-1ß expression was >500-fold higher in Smad3+/+ papillomas as compared with Smad3–/– papillomas (P < 0.01). IL-1ß expression levels in Smad3+/– papillomas were approximately 400-fold less than IL-1ß expression levels in Smad3+/+ papillomas (P < 0.01; Fig. 7Citation ). In addition, the expression level of MCP-1, a major monocyte/macrophage-attracting chemokine (42) , was expressed at approximately 8-fold higher levels in Smad3+/+ papillomas than in Smad3–/– papillomas (P < 0.01; Fig. 7Citation ), and expression of MCP-1 in Smad3+/– papillomas was only 2-fold higher than that in Smad3–/– papillomas (P < 0.01; Fig. 7Citation ).



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  		Fig. 7. Relative expression levels of IL-1ß and MCP-1 in papillomas as determined by qRT-PCR. The expression level in one Smad3–/– sample was set as 1 arbitrary unit.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADDENDUM
 REFERENCES
 
In this report, we have shown that mice lacking Smad3 exhibited resistance to skin chemical carcinogenesis compared with wild-type littermates. This result contradicts a previous report that loss of Smad3 in keratinocytes accelerates malignant conversion in a tumor transplant model (13) . Because nutritional status can greatly affect skin carcinogenesis (43) , we were concerned that results from a skin chemical carcinogenesis study on adult Smad3 knockout mice, which develop a wasting syndrome (20) , may not accurately reflect the effect of the loss Smad3 on susceptibility to skin chemical carcinogenesis. To avoid this problem, we used neonatal mice and applied a previously described chemical carcinogenesis protocol (44) that is initiated 3 days after birth, when Smad3–/– pups are morphologically normal (20) . By the time Smad3+/+ mice began to develop papillomas (6 to 15 weeks), Smad3–/– mice remained free of obvious wasting syndrome symptoms yet demonstrated a resistance to tumorigenesis. In addition, Smad3+/– mice, which did not develop wasting syndrome and maintained body weight similar to wild-type littermates, showed a reduction in tumor formation and malignant conversion. Furthermore, Smad3–/– and Smad3+/– skin exhibited a gene dosage-dependent resistance to epidermal proliferation after acute TPA treatment. In fact, Smad3 gene dosage effects during skin chemical carcinogenesis were seen in almost every variable we analyzed on littermates with different Smad3 genotypes. Because activation of a Smad3 partner, Smad2, remained unaffected by Smad3 deletion (Fig. 4)Citation , our results suggest that the resistance to skin chemical carcinogenesis in Smad3 knockout mice is likely a direct effect of the loss of Smad3 gene expression. Supporting our observations, a recent report shows that Smad3+/– mice were resistant to skin chemical carcinogenesis, whereas Smad2+/– mice, with the same genetic background, exhibited accelerated skin carcinogenesis (45) .

Lack of AP-1 Activation and TGF-{alpha} Overexpression in Smad3 Knockout Keratinocytes Contributes to Resistance to TPA-Induced Tumor Promotion.
Unlike v-rasHa-transduced keratinocytes, which can progress to benign papillomas on grafting onto the athymic mouse skin (13) , normal keratinocytes harboring a point mutation in the c-rasHa gene induced in vivo by a subcarcinogenic dose of DMBA do not typically form papillomas in normal mice (5) , and further tumor promotion is required for these cells to undergo tumorigenesis. The tumor promotion effect of TPA depends mainly on the ability of TPA to induce keratinocyte hyperproliferation, which expands the population of initiated stem cells [e.g., stem cells carrying a c-rasHa mutation (46 , 47) ]. On TPA application, TGF-ß1 expression is elevated (7) . Although TGF-ß1 directly inhibits keratinocyte proliferation, it also activates expression of genes closely related to TPA-induced tumor promotion, e.g., the AP-1 family members (34) . Smad3 has been found to mediate the effect of TGF-ß1 on transactivation of c-fos, c-jun, junB, and junD by binding to their promoters (48, 49, 50) . In our current study, we show that induction of these AP-1 family members did not occur in TPA-treated Smad3 knockout skin and tumors or TPA-treated Smad3 knockout keratinocytes in vitro. Thus, Smad3 appears to be required for transcriptional activation of these genes during TPA promotion. In addition to direct transcriptional regulation by Smad3, the AP-1 family members are also transcriptionally regulated on activation of epidermal growth factor receptor (51) . Therefore, the lack of overexpression of TGF-{alpha}, the epidermal growth factor receptor ligand, in Smad3 knockout skin and tumors during TPA promotion may also contribute to the low levels of AP-1 family members in Smad3 knockout skin and tumors. Each AP-1 family member has been suggested to play an essential role in skin carcinogenesis through regulating cellular proliferation and apoptosis (52) . For instance, c-fos, c-jun, and junD proteins positively regulate cell proliferation, whereas junB may negatively regulate cell proliferation in the presence of c-jun (52) . Thus, it could be predicted that decreased expression of these molecules in TPA-treated skin and tumors of Smad3 knockout mice can cause a decrease in keratinocyte proliferation. With respect to apoptosis, the effects of AP-1 family members are somewhat contradictory and highly tissue specific. c-jun knockout embryos exhibit massive apoptosis in hepatocytes, indicating an antiapoptotic activity of c-jun protein. A previous study has shown an antiapoptotic effect of jun/fos proteins on skin carcinogenesis (53) . Overall, the role of AP-1 family members in skin carcinogenesis has been at least partially attributed to their ability to promote keratinocyte proliferation and inhibit apoptosis (52 , 54) . In vivo experiments showed that c-jun is required for the development of papillomas (55) , whereas c-fos is required for malignant transformation (56) . Thus, lack of AP-1 expression/activation during TPA promotion in Smad3 knockout skin appears to greatly contribute to resistance to papilloma formation as well as malignant progression during skin chemical carcinogenesis. In the case of constant overexpression of v-rasHa in transplanted keratinocytes (13) , the tumor promotion event can bypass TPA-induced AP-1 activation; thus, the effect of Smad3 on AP-1 gene expression is dispensable. Under this circumstance, the effect of the loss of Smad3 appears to predominantly abrogate TGF-ß1–induced growth inhibition, resulting in accelerated malignant conversion.

Another growth factor that is typically rapidly induced by TPA treatment of normal keratinocytes is TGF-{alpha}. TPA up-regulates TGF-{alpha} expression through signaling via protein kinase C (PKC)-dependent and -independent pathways (57) . In the latter case, it is well-documented that TGF-{alpha} is an autoinductive cytokine and that the TGF-{alpha} gene promoter contains a TGF-{alpha}/epidermal growth factor-responsive element (58) . It has also been shown that TGF-ß1 transcriptionally regulates TGF-{alpha} expression (59) . Because PKC is rapidly down-regulated after TPA application (60) , it is reasonable to believe that the PKC-independent pathways are primarily responsible for the persistent TGF-{alpha} overexpression. Although the exact mechanism by which Smad3 deletion blocks TGF-{alpha} overexpression remains to be determined, it is likely that loss of Smad3 abrogates TGF-ß1–induced TGF-{alpha} overexpression, consequently perturbing the autoinduction of TGF-{alpha} expression. TGF-{alpha} is overexpressed in many cancer types and functions as a potent mitogen for cancer cell proliferation (61 , 62) . In addition, TGF-{alpha} is a critical survival factor against apoptosis for cancer cells (37 , 38) . Thus, persistent TGF-{alpha} overexpression is thought to be critical for the TPA-induced tumor promotion effect (36) . Supporting this, TGF-{alpha}–deficient mice (Wa-1 mutant) exhibit a resistance to TPA promotion (63) . Consistent with the above-mentioned studies, lack of TPA- induced TGF-{alpha} overexpression in Smad3 knockout skin and papillomas correlated with decreased proliferation and increased apoptosis in keratinocytes, which apparently contributed to the resistance of Smad3 knockout mice to skin chemical carcinogenesis. Because TGF-{alpha} overexpression is critical for activation of several signal transduction pathways, such as the ras-mitogen-activated protein kinase pathway (64) , the lack of TGF-{alpha} overexpression in Smad3 knockout skin/tumors may also explain the contradictory data between our present study and a previous report showing accelerated malignant conversion in skin tumors that resulted from transplantation of Smad3-null keratinocytes transduced with v-rasHa (13) . In that study, consistently high levels of v-rasHa expression in keratinocytes should be able to bypass the effect of TGF-{alpha} overexpression as seen during TPA-induced tumor promotion.

Reduced Inflammation in Smad3 Knockout Skin May Also Contribute to Resistance to Skin Carcinogenesis In vivo.
Given the importance of inflammation in cancer development (18) , TPA-induced skin inflammation is expected to contribute to the tumor promotion effect of TPA. In keratinocytes, TPA application induces expression of inflammatory cytokines, including IL-1 (65) , which not only induces inflammation but also stimulates keratinocyte proliferation (66) . In TPA-treated preneoplastic skin, we did not find differences in inflammation among the different Smad3 genotypes. This suggests that TPA-induced inflammation may not be completely mediated by TGF-ß1 overexpression, and therefore the loss of Smad3 cannot attenuate TPA-induced inflammation, at least at the acute phase. At this stage, the resistance to skin carcinogenesis in Smad3 knockout mice may be mainly a result of altered keratinocyte properties (i.e., resistance to proliferation and increased apoptosis). However, because TGF-ß1 overexpression has been shown to induce skin inflammation (15 , 16) , persistent TGF-ß1 overexpression at later stages of skin carcinogenesis may maintain a certain level of inflammation that is required for tumor development (18) . In particular, TAMs play an important role in tumor promotion and progression (18) . Thus, a reduction in TAM infiltration in Smad3 knockout tumors may also explain, at least in part, the resistance of these tumors to malignant conversion. The impaired chemotactic effect of TGF-ß1 on Smad3–/– monocytes (20) is possibly responsible for reduced TAM infiltration in Smad3 knockout tumors. Supporting this notion, reduced local inflammation associated with decreased monocyte infiltration has also been reported in Smad3 knockout wounds as compared with Smad3+/+ wounds (17) . TGF-ß1 has a potent chemotactic effect on monocytes (67) and may induce expression of multiple chemokines in the skin (16 , 68) . MCP-1, which was down-regulated in Smad3 knockout tumors, is a chemokine that attracts monocyte-macrophage infiltration to sites of inflammation and can be up-regulated on TGF-ß1 overexpression in the skin (16) . It is thus likely that Smad3–/– monocytes do not respond to the chemotactic effect of TGF-ß1. Alternatively, Smad3–/– stromal cells and endothelial cells may affect monocyte infiltration. In either case, reduced TAM infiltration would not occur in tumors generated in mice in which Smad3 is deleted only in keratinocytes, as indicated in a previous study that reported accelerated skin carcinogenesis of transplanted Smad3-null keratinocytes (13) . However, because inflammation was reduced in Smad3 knockout papillomas, as evidenced by reduced IL-1ß and the lack of NF-{kappa}B activation, reduced TAM infiltration in Smad3 knockout papillomas may also be a consequence of reduced expression levels of inflammatory cytokines and chemokines in Smad3–/– tumor epithelia. All of the above-mentioned possibilities can be tested in the future by generating mice with epidermal-specific deletion of Smad3 and performing carcinogenesis experiments on these mice.

In summary, the present study shows that loss of Smad3 in vivo significantly reduced tumor formation and malignant conversion during skin chemical carcinogenesis. Our study suggests that, at early stages of skin carcinogenesis, TGF-ß1 overexpression induced by TPA may also have a tumor promotion effect via activation of AP-1 family members in keratinocytes and inflammation in the stroma, both of which may require wild-type Smad3. Our study instigates future studies to investigate the complex nature of the role of Smad3 in cancer development and its underlying molecular mechanisms under different pathological conditions.


   ADDENDUM
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADDENDUM
REFERENCES
 
While this article was under review, a report was published (45) showing that Smad3+/– mice were resistant to skin chemical carcinogenesis.


   FOOTNOTES
 
Grant support: National Institutes of Health grants CA87849 and CA79998 (X-J. Wang).

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.

Note: M-X. Zhang is presently in the Department of Physiology, Baylor College of Medicine, Houston, Texas.

Requests for reprints: Xiao-Jing Wang, 3710 SW US Veterans Hospital Road, Mail code R&D46, Portland, OR 97239. Phone: 503-220-8262, ext. 54273; Fax: 503-402-2817; E-mail: wangxiao{at}ohsu.edu

Received 4/14/04. Revised 8/16/04. Accepted 9/ 2/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADDENDUM
 REFERENCES
 

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