Vitamin D3 receptor ablation sensitizes skin to chemically induced tumorigenesis
Glendon M. Zinser1,
John P. Sundberg2 and
JoEllen Welsh1,3
1 Department of Biology, University of Notre Dame, Notre Dame, IN 46556, USA and
2 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500, USA
 |
Abstract
|
---|
1,25-Dihydroxyvitamin D3 (1,25D3) is the biologically active form of vitamin D3 that interacts with the nuclear vitamin D3 receptor (VDR) to modulate gene expression in a tissue-specific fashion. 1,25D3 is a potent regulator of cell proliferation, differentiation and apoptosis in a variety of cell types, including keratinocytes. In these studies, we assessed the sensitivity of mice homozygous for a null allele of the VDR (VDR-/- mice) and their wild-type counterparts (VDR+/+ mice) to oral administration of the carcinogen 7,12-dimethylbenzanthracene (DMBA). Although the protocol was optimized for the induction of mammary tumors, 85% of VDR-/- mice developed persistent skin tumors within 60 days of carcinogen exposure. In VDR-/- mice exposed to DMBA, papillomas arose on all areas of the body, with an average tumor burden of 5.3 papillomas/mouse. No papillomas or any other skin lesions were observed in age- and sex-matched VDR+/+ mice dosed with DMBA and followed for 6 months. The majority (80%) of skin tumors that developed in VDR-/- mice were classified histologically as sebaceous, squamous or follicular papillomas. Other types of lesions, including basal cell carcinoma, hemangioma and melanotic foci, were occasionally observed in VDR-/- mice (but not in VDR+/+ mice) exposed to DMBA. Quantification of epidermal thickness and BrdU incorporation indicated that skin from VDR-/- mice exhibited hyperproliferation beginning at 7 weeks of age, which was exacerbated by DMBA treatment. Untreated aging VDR-/- mice did not exhibit tumor formation, but did develop a progressive skin phenotype characterized by thickened wrinkled skin, dermoid cysts and long curly nails. Together with previous reports that 1,25D3 inhibits papilloma formation induced by topical DMBA-TPA regimens, our observation of enhanced sensitivity of VDR-/- mice to chemically induced skin carcinogenesis offers compelling evidence that disruption of VDR signaling predisposes to neoplasia.
Abbreviations: 1,25D3, 1,25-dihydroxyvitamin D3; DMBA, 7,12-dimethylbenzanthracene; MPA, medroxyprogesterone acetate; ODC, ornithine decarboxylase; VDR, vitamin D3 receptor
 |
Introduction
|
---|
It has been recognized for some time that skin not only generates vitamin D3, but is also an important target organ for its biologically active form, 1,25-dihydroxyvitamin D3 (1,25D3). Natural mutations in the vitamin D3 receptor (VDR) in humans, and targeted ablation of the VDR in mice, result in alopecia and generalized atrichia (13), indicating a role of the VDR in epidermis and hair follicles. The VDR has been localized to normal mouse and human keratinocytes as well as human basal cell carcinomas, squamous cell carcinomas and melanomas. In vitro, 1,25D3 induces cell-cycle arrest and differentiation in normal keratinocytes and in human cell lines derived from squamous cell carcinomas and malignant melanomas (47). The observation that 1,25D3 exerts anti-proliferative and pro-differentiating effects on keratinocytes, coupled with data showing that 1,25D3 reduces the incidence and multiplicity of papilloma formation in topical two-stage skin carcinogenesis assays (8,9) support the concept that the vitamin D3 endocrine system exerts protective effects against skin transformation. In these studies we have utilized the VDR null (/) mouse (1) to provide evidence of a role for the VDR in protection against cancer development. Our laboratory has a long-standing interest in the role of the VDR in breast cancer, and we initiated studies to compare the incidence of mammary carcinoma in VDR-/- mice and their wild-type (VDR+/+) counterparts following exposure to the chemical carcinogen 7,12-dimethylbenzanthracene (DMBA). In this report, we show that prior to the development of mammary tumors, 85% of VDR-/- mice (but no VDR+/+ control mice) developed persistent skin tumors, which included squamous, sebaceous and follicular papillomas. The data provide the first direct evidence that disruption of the VDR signaling pathway contributes to neoplasia.
 |
Materials and methods
|
---|
Animals and diets
The VDR-/- mice used in these studies were derived from animals originally obtained from Dr Marie Demay (Massachusetts General Hospital, Boston, MA), who generated the VDR knockout on the C57BL/6J background by targeted ablation of the second zinc finger of the DNA binding domain (1). The breeding colony of VDR-/- mice established at Notre Dame is maintained in a barrier facility and fed a purified diet containing 2% calcium, 1.25% phosphorus and 20% lactose supplemented with 2.2 IU vitamin D3/g (TD96348, Teklad, Madison, WI). This diet has been shown to normalize serum mineral homeostasis, bone growth and body weight in VDR-/- mice (10). We have found no differences in litter size or pup survival rates between VDR+/+ and VDR-/- mice maintained on this diet (11). Cancer development in response to DMBA was compared in female VDR+/+ and VDR-/- mice generated from both heterozygote and homozygote crosses.
Carcinogen treatment and tumor monitoring
The experimental design for these studies was chosen to enhance the development of mammary carcinoma in response to DMBA. The protocol involves pre-treatment of mice with medroxyprogesterone acetate (MPA, a synthetic progesterone analog) to enhance proliferation of epithelial cells in the mammary gland prior to carcinogen exposure (12). Briefly, 4-week-old VDR+/+ (n = 59) and VDR-/- (n = 60) mice were implanted subcutaneously with 21-day continuous release MPA pellets (50 mg, Innovative Research, Sarasota, FL) prior to gavage treatment with DMBA (1 mg/10 g body wt) at 5.5 and 7 weeks of age. Although VDR-/- mice experience hair loss with age, both VDR+/+ and VDR-/- mice had full hair coats at the time of DMBA administration. Mice were examined weekly for tumor development by visual inspection and palpation. For the data reported here, mice bearing raised skin lesions (>1 mm in diameter) that persisted for >2 weeks were scored as tumor positive. In this manuscript, we have compiled data on the incidence, multiplicity and histopathology of skin tumors induced by DMBA (data on mammary and other carcinomas are still being evaluated and will be reported independently).
Sample procurement and histological analysis
Six months after the second dose of DMBA, VDR+/+ and VDR-/- mice were killed by CO2 asphyxiation. Two hours prior to death, all DMBA-treated mice were dosed with BrdU (1 mg) to evaluate cell proliferation rate. Skin tumors as well as dorsal skin biopsies were removed for histological analysis from all DMBA-treated mice and from three age-matched, untreated animals of each genotype. Dorsal skin biopsies were also obtained from VDR+/+ and VDR-/- mice that received no treatment or that received MPA pellets but were not challenged with DMBA (six to nine mice of each genotype were biopsied at 7 and 10 weeks of age). Skin lesions and non-tumor bearing skin were fixed in 4% neutral-buffered paraformaldehyde, embedded in paraffin, sectioned at 5 µM and stained with hematoxylin and eosin. Skin, lesions and tumors were classified histologically as hyperplasias (infundibulum hyperplasia, epidermal hyperplasia), papillomas (sebaceous, squamous or follicular), melanotic foci, basal cell carinomas or hemangiomas according to previously described criteria (13,14).
BrdU was localized in sections with a mouse monoclonal biotinylated anti-BrdU (Zymed Laboratories, San Francisco, CA) and the ABC technique followed by diaminobenzidine (Sigma, St Louis, MO). Labeling index (defined as the number of BrdU positive cells per 100 total cells) was quantified in interfollicular epidermal basal cells of non-tumor bearing skin by counting the number of total and BrdU-positive nuclei in a 40x field. A minimum of three slides was evaluated for each genotype/treatment, with over 200 cells counted in at least three separate fields on each slide. Epidermal thickness was measured in triplicate on at least three separate photographs from each animal. For samples taken from young mice (7 and 10 weeks of age), data on epidermal thickness and BrdU incorporation were pooled for statistical evaluation. For all samples, statistical evaluation of labeling index and epidermal thickness was by one-way ANOVA or Students t-test, as appropriate, using the Graph Pad Instat computer program (1998, GraphPad Software, San Diego, CA).
 |
Results
|
---|
Development of skin lesions in VDR-/- mice exposed to DMBA
In studies designed to examine the effect of VDR ablation on sensitivity to DMBA-induced mammary carcinogenesis, female VDR-/- mice (n = 60) and their wild-type (VDR+/+) counterparts (n = 59) were pre-treated with MPA and gavaged with DMBA as described in the Materials and methods. Within 60 days of carcinogen exposure, VDR-/- mice began to develop firm, pigmented, ulcerated skin masses. The appearance and diversity of the skin lesions observed in VDR-/- mice is illustrated in Figure 1
. Lesions included raised projections supported by thin stalks, horn-like cornified structures, globular, wart-like growths (Figure 1A and C
), and both flat (sessile) and raised, patchy, pigmented masses (Figure 1B and D
). At the end of the 6 month trial, we observed 85% frequency of skin tumors in VDR-/- mice (51 of 60 mice developed persistent skin lesions), whereas age- and sex-matched VDR+/+ mice on the same genetic background (C57BL/6J) did not develop persistent skin lesions in response to DMBA, even if followed for up to 6 months. The resistance of VDR+/+ mice on the C57BL/6J background to DMBA-induced tumorigenesis appeared to be selective for skin, since these mice did develop mammary and other tumors in response to the MPA-DMBA regimen (Zinser et al., manuscript in preparation).

View larger version (128K):
[in this window]
[in a new window]
|
Fig. 1. Gross patterns of skin lesions in VDR-/- mice 6 months after oral administration of DMBA. Unpigmented (A) and pigmented (B) raised masses which ranged in size from <0.5 to >2.0 mm in diameter developed in VDR-/- mice within 60 days of carcinogen administration. Utriculi, which were detected even in untreated VDR-/- mice, appeared as small raised nodules of various sizes under the skin (C), while DMBA-induced papillomas were raised above the skin (AC). Large pigmented papillomas developed in some VDR-/- mice in response to DMBA (D).
|
|
In VDR-/- mice, skin tumors developed on all areas of the body, with a tumor burden of up to 21 lesions/mouse. The average tumor burden in the 51 VDR-/- mice that developed tumors was 5.3 tumors/mouse (range 121 tumors/mouse). The latency for skin tumor development in VDR-/- mice was ~60 days. Although we did not specifically assess whether MPA pretreatment was required for skin tumor development, tumors did develop in four VDR-/- mice in which MPA pellets accidentally dislodged after the first DMBA dose but before the second dose of DMBA. Skin tumors did not develop in mice of either genotype treated with MPA alone and followed for up to 6 months.
Histopathologic analysis of skin lesions in VDR-/- mice
A total of 94 skin tumors which developed in VDR-/- mice were categorized as to histological subtype (Figure 2
). The most frequent lesions observed (40% of all tumors) were sebaceous papillomas (Figure 2A
), benign lesions that involve the hair follicles, sebaceous glands and interfollicular dermis. The next most frequent class of tumors (25% of lesions) were the squamous papillomas (Figure 2B
), followed by follicular papillomas (15% of lesions, Figure 2C
). Other types of lesions, that were infrequently observed, included basal cell carcinoma (Figure 2D
) and hemangioma (Figure 2E
). Pigmented lesions classified as melanotic foci, that are not normally observed in mouse skin, were frequently observed in VDR-/- mice (11% of lesions). Melanotic foci (Figure 2F
) consisted of aggregations of heavily pigmented cells with poorly defined cytoplasmic features. Detailed descriptions of the histopathology of these classes of lesions have been published elsewhere (13,14).

View larger version (202K):
[in this window]
[in a new window]
|
Fig. 2. Histopathology of DMBA-induced skin lesions in VDR-/- mice. Hematoxylin and eosin stained sections of skin tumors obtained from VDR-/- mice 6 months after combined MPA-DMBA treatment regimen. Raised lesions, including those on stalks above the level of the skin, contained a predominance of well-differentiated sebaceous glands and were labeled sebaceous papillomas (A). Those consisting predominantly of squamous cells were labeled squamous papillomas (B). Those containing a predominance of abortive hair follicles were folliclular papillomas (C). Masses with basal cells straying into the dermis were classified as basal cell carcinomas (D). Occasional tumors, which consisted of fine capillary networks were termed hemangiomas (E). Multiple dermal accumulations of melanin containing macrophages were termed melanotic foci (F).
|
|
Effects of MPA and DMBA on epidermal histology and proliferation
Since 1,25D3 exerts anti-proliferative and pro-differentiating effects on keratinocytes, we hypothesized that VDR ablation might be associated with deregulation of proliferation in the skin which could have enhanced cellular sensitivity to DMBA-induced carcinogenesis. To address this hypothesis, we examined histology and quantitated epidermal basal cell proliferation in VDR+/+ and VDR-/- mice in response to various treatments. Since the protocol utilized in these studies included pre-treatment of mice with MPA prior to DMBA challenge, we first considered the possibility that MPA may have differentially affected the skin of VDR+/+ and VDR-/- mice, sensitizing VDR-/- mice to DMBA-induced carcinogenesis. To test this possibility, additional groups of VDR+/+ and VDR-/- mice were implanted with MPA pellets at 4 weeks of age, or left untreated, and skin biopsies were taken at both 7 and 10 weeks of age. Biopsies were assessed histologically, and BrdU incorporation and epidermal thickness were quantified. In Figure 3
, histology of skin from young untreated VDR+/+ and VDR-/- mice is compared with that of age-matched mice implanted with MPA pellets but not dosed with DMBA. Skin from VDR+/+ mice exhibited normal morphology (Figure 3A, panel a
), whereas deep dermal cyst formation and degeneration of hair follicles (utriculi) were prevalent in epidermis of untreated VDR-/- mice (Figure 3A, panel b
). These features are similar to those reported previously for both hypocalcemic and normocalcemic VDR-/- mice (15,16), confirming that the rescue diet that maintains serum calcium and bone development does not improve the histologic appearance of skin from VDR-/- mice. Consistent with the observation that MPA treatment was not associated with tumor formation, the histology of skin from MPA pre-treated VDR+/+ (Figure 3A, panel c
) or VDR-/- mice (Figure 3A, panel d
) was similar to that of skin from their age-matched untreated counterparts.

View larger version (90K):
[in this window]
[in a new window]
|
Fig. 3. Histology and proliferation in dorsal skin of young untreated and MPA-treated VDR+/+ and VDR-/- mice. (A) Hematoxylin and eosin stained sections of dorsal skin obtained from untreated VDR+/+ (WT) and VDR-/- (KO) mice at 10 weeks of age are shown in panels (a) and (b), respectively. As compared with normal epidermal morphology in VDR+/+ mice, VDR-/- mice exhibited hyperplasia, utricle formation, and degeneration of hair follicles. The histological features of dorsal skin obtained from VDR+/+ (c) and VDR-/- (d) mice implanted with 21 day release MPA pellets were similar to those of untreated mice of the same genotype. (B) Epidermal thickness was measured on hematoxylin and eosin stained sections of dorsal skin from untreated VDR+/+ (WT) and VDR-/- (KO) mice (left two bars) and mice implanted with 21 day release MPA pellets (right two bars). Triplicate measurements on at least three independent photographs were made for each mouse, with six to nine animals sampled per group. Data was analyzed by one-way ANOVA and Tukeys multiple comparison test. *Significantly different, VDR+/+ versus VDR-/-, P < 0.01. (C) BrdU labeling index in dorsal skin of 10-week-old untreated VDR+/+ and VDR-/- mice. The number of BrdU positive cells per 100 total cells was quantitated in interfollicular epidermal basal cells of dorsal skin. Data means represent a minimum of 200 cells from four independent areas on slides obtained from VDR+/+ (n = 5) and VDR-/- (n = 6) mice. Data were analyzed by unpaired Students t-test. *Significantly different, VDR+/+ versus VDR-/-, P < 0.05.
|
|
To assess whether genotype or MPA treatment altered cell turnover in the epidermis, epidermal thickness and BrdU incorporation were assessed. Since no differences in dermal histology were noted between samples removed at 7 and 10 weeks of age, the data for the two age groups was pooled for statistical analysis. As presented in Figure 3B
, skin from VDR-/- mice exhibited hyperplasia relative to skin from VDR+/+ mice that was evident as early as 7 weeks of age, and MPA treatment did not affect epidermal thickness in either VDR+/+ or VDR-/- mice. To quantify cell proliferation in epidermis of VDR+/+ and VDR-/- mice, mice were injected with BrdU prior to being killed, and sections were analyzed for BrdU incorporation by immunoperoxidase staining. In young untreated mice, BrdU labeling index of the interfollicular epidermal basal cells was significantly higher in VDR-/- mice than VDR+/+ mice (Figure 3C
).
We next compared dorsal skin histology, epidermal thickness and labeling index of mice 6 months after DMBA treatment to that of age-matched (8 month old) untreated control mice of each genotype. Representative photographs of sections processed for H&E staining and BrdU incorporation from DMBA-treated mice and age-matched controls of each genotype are shown in Figure 4
. In H&E stained sections from VDR+/+ mice (Figure 4A and E
), there was no obvious effect of DMBA on epidermal histology, a finding consistent with the observation that DMBA did not induce tumors in VDR+/+ mice. In 8-month-old, untreated VDR-/- mice (Figure 4C
), the epidermal hyperplasia and dermal cyst formation which was evident in younger VDR-/- mice (Figure 3
) persisted but was not aggravated with age. In contrast, DMBA treatment markedly exacerbated the epidermal hyperplasia in VDR-/- mice (Figure 4G
). In 8-month-old VDR+/+ mice, BrdU was detected in epidermal basal cells and in hair follicles, but positive cells were infrequent, and the effect of DMBA was minimal (Figure 4B and F
). In 8-month-old untreated VDR-/- mice (Figures 4D
), BrdU incorporation was more extensive than in age-matched VDR+/+ mice (Figure 4B
), and proliferation was further enhanced by DMBA treatment (Figure 4H
). In DMBA-treated VDR-/- mice, BrdU incorporation was detected not only in the epidermal basal cells but also in the areas of subdermal fibrosis (Figure 4H
). Quantification of epidermal thickness and BrdU labeling (Figure 5
), followed by ANOVA, indicated that hyperplasia and basal cell proliferation were significantly higher in DMBA-treated VDR-/- mice than in any other group. Thus, the skin of VDR-/- mice appears to be hypersensitive to the proliferative effects of DMBA.

View larger version (147K):
[in this window]
[in a new window]
|
Fig. 4. Effect of combined treatment with MPA and DMBA on epidermal histology and BrdU labeling in VDR+/+ and VDR-/- mice. Representative hematoxylin and eosin stained sections (A, C, E, and G) and BrdU labeling (B, D, F and H) of non-tumor bearing skin obtained from VDR+/+ (A, B, E and F) and VDR-/- (C, D, G and H) mice are shown. Sections from dorsal skin of untreated VDR+/+ and VDR-/- mice are shown in the upper panels, and sections from mice implanted with MPA pellets and orally dosed with DMBA are shown in the lower panels. BrdU was injected 2 h prior to death and visualized on sections with a biotinylated monoclonal antibody directed against BrdU and the ABC technique. BrdU labeled cells appear brown against the blue hematoxylin counterstain.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5. Quantification of epidermal thickness and BrdU labeling index in VDR+/+ and VDR-/- mice pre-treated with MPA and dosed with DMBA. (A) Epidermal thickness was measured in non-tumor bearing skin from VDR+/+ (WT) and VDR-/- (KO) mice treated with DMBA (right two bars) and in age-matched untreated control (Con) mice (left two bars). Triplicate measurements on two to seven independent photographs were made for each mouse, with 1216 treated animals and three age-matched controls sampled per group. (B) BrdU labeling index in non-tumor bearing skin from VDR+/+ (WT) and VDR-/- (KO) mice treated with DMBA regimen (right two bars) and in age-matched untreated mice (left two bars). The number of BrdU positive cells per 100 total cells was quantified in interfollicular epidermal basal cells of dorsal skin. A minimum of 200 cells from two to six independent photographs were counted for each mouse, with 1216 treated animals and three age-matched controls sampled per group.
|
|
To examine patterns of cell proliferation in DMBA-induced tumors, sections from tumors which developed in VDR-/- mice were analyzed for BrdU incorporation by immunoperoxidase staining. Shown are sections from a squamous papilloma (Figure 6A
), a sebaceous papilloma (Figure 6B
), a follicular papilloma (Figure 6C
) and a basal cell carcinoma (Figure 6D
). In all tumors BrdU incorporation was consistently higher than that observed in adjacent non-tumor bearing skin (compare with Figure 4
), as expected. In most tumors, cell proliferation was restricted to single layers; however, in others, particularly the squamous papillomas, BrdU positive cells clustered in multiple layers. The data suggest that tumor expansion is associated with further enhancement of epidermal cell proliferation in VDR-/- mice.

View larger version (113K):
[in this window]
[in a new window]
|
Fig. 6. Patterns of cell proliferation in DMBA-induced tumors from VDR-/- mice. BrdU labeling of tumors from VDR-/- mice 6 months after combined MPA-DMBA treatment regimen. Representative sections from a squamous papilloma (A), sebaceous papilloma (B), follicular papilloma (C) and basal cell carcinoma (D), which developed in VDR-/- mice. BrdU was injected 2 h prior to death and visualized on sections with a biotinylated monoclonal antibody directed against BrdU and the ABC technique. BrdU labeled cells appear brown against the blue hematoxylin counterstain.
|
|
Rhino-like phenotype in aging VDR-/- mice
It has been reported previously that young VDR-/- mice develop utriculi, deep dermoid cysts and alopecia, a skin phenotype that mimics that of the hairless (hr/h) mutant mouse (17,18). In the current studies, the gross and histological features of skin from 10-month-old VDR-/- mice were examined. In addition to alopecia, utriculi and deep dermoid cysts, VDR-/- mice developed excessive amounts of thickened, loose, wrinkled skin and ~20% of VDR-/- mice over 10 months of age displayed the severe phenotype depicted in Figure 7A and B
. This phenotype mimics that of a severe form of the hairless mutation known as the rhino mouse (19,20). Histological analysis of skin from VDR-/- mice indicated that these mice developed progressive lesions very similar to those present in rhino mouse skin, including utricle formation, deep dermal cysts, follicular rupture with foreign body granuloma formation due to hair fibers and cornified debris in the dermis, and pseudoepitheliomatous hyperplasia secondary to healing around ulcers. Deep dermal cysts containing cornified debris were visible in H&E sections of VDR-/- mice of all ages, regardless of treatment (see Figures 3B and D and 4C and G
). Utricle formation was grossly visible as uniform white nodules on the subcutaneous surface of reflected skin (Figure 7C
). These lesions developed in both male and female VDR-/- mice and were not dependent on exposure to either MPA or DMBA (data not shown). In addition, nails from older VDR-/- mice were excessively long and unusually curved (Figure 7D
), another characteristic feature of the rhino phenotype (19).

View larger version (150K):
[in this window]
[in a new window]
|
Fig. 7. Rhino-like phenotype in untreated VDR-/- mice. Cutaneous features of the VDR-/- phenotype in older (>10 months old) mice. (A) Whole mouse view demonstrating excessive thick skin forming wrinkles and folds that can completely cover the face and underside of the animal. (B) Closer view of head area showing excessive skin folds covering the face, often forcing closure of the eyes. (C) Gross view of inner skin surface of VDR-/- mouse, showing large utriculi and pigmented lesions. (D) Excessively long, curved nails of VDR-/- mouse.
|
|
 |
Discussion
|
---|
In the present studies, mice with targeted ablation of the VDR have provided evidence of a role for the vitamin D3 signaling pathway in suppression of DMBA-induced tumorigenesis in skin. Although we intentionally used a protocol optimized for induction of mammary carcinomas, here we report that skin of VDR-/- mice are hypersensitive to tumorigenesis in response to oral administration of DMBA. While wild-type (VDR+/+) mice on the C57BL/6J background are completely resistant to skin tumor induction in response to DMBA, 85% of VDR-/- mice develop papillomas in response to DMBA. The frequency (85%), latency (60 days) and multiplicity (5.3 tumors/mouse) of papilloma formation in VDR-/- mice in response to orally administered DMBA is comparable with that observed in strains of mice classified as highly responsive when subjected to topical two-stage (DMBA-TPA) carcinogenesis protocols (13). These results support the concept that the VDR, a nuclear receptor, contributes to negative growth regulation of skin that suppresses tumor formation, and that down regulation of the vitamin D3 endocrine system may be permissive for skin tumorigenesis. This concept is consistent with reports that 1,25D3, the ligand for the VDR, exerts anti-proliferative and pro-differentiating effects on keratinocytes and inhibits papilloma formation in mouse skin subjected to topical two-stage carcinogenesis regimens (8,9). Clinical studies have also supported a role for the vitamin D3 endocrine system in human skin lesions, for example, topical vitamin D3 analog therapy was shown to induce regression of Kaposi sarcoma lesions in skin (21), and polymorphisms of the VDR gene, which may result in reduced VDR function, have been associated with susceptibility and prognosis in malignant melanoma (22). In addition, a vitamin D3 analog (Dovonex) has long been a successful topical treatment for psoriasis, a hyperproliferative skin disorder (23).
The cellular and molecular basis for the enhanced sensitivity of VDR-/- mice to DMBA-induced skin tumorigenesis is currently unclear. All mice in our study were maintained on a high calcium diet that normalizes growth, reproduction and extracellular calcium homeostasis, therefore it is unlikely that VDR ablation affected epidermal sensitivity to tumorigenesis via secondary effects on calcium homeostasis. Since the VDR is expressed in keratinocytes and dermal papilla cells of the hair follicle, where it exerts anti-proliferative and pro-differentiating effects, VDR ablation might be anticipated to lead to deregulation of proliferation or differentiation in the skin. Consistent with this concept, recent studies have demonstrated reduced expression of epidermal differentiation markers, such as involucrin, profilaggrin and loricrin, in VDR-/- mice (16). Furthermore, in the present studies, quantification of epidermal thickness indicated that skin from VDR-/- mice exhibited enhanced basal cell proliferation and epidermal hyperplasia beginning at 7 weeks of age that was exacerbated by DMBA treatment but not by MPA alone. Although previous studies failed to detect differences in basal proliferation rates of keratinocytes from neonatal VDR+/+ and VDR-/- mice (15), further studies are necessary to determine at what time between 3 and 7 weeks of age the effect of VDR ablation on keratinocyte proliferation becomes manifest.
Despite the presence of epidermal hyperproliferation in untreated VDR-/- mice, we have rarely detected spontaneous cutaneous tumors, and only a few isolated skin lesions have been observed in our colony of over 1500 VDR-/- mice to date (with over 20 mice 14 months of age examined). Thus, the epidermal hyperproliferation observed in VDR-/- mice does not appear to be sufficient for tumor formation. Rather, our data suggest that, like other hyperproliferative phenotypes, skin of VDR-/- mice is hypersensitive to carcinogenesis. This suggestion is consistent with our finding that basal cell proliferation and epidermal thickness continue to increase in VDR-/- mice, but not VDR+/+ mice, in response to the MPA-DMBA regimen. The higher rate of proliferation in skin of VDR-/- mice could enhance the efficiency of initiation, and/or could act as an endogenous promoter to facilitate the progression of cells initiated by DMBA treatment; further studies will be necessary to distinguish between these two possible mechanisms. In either case, these studies suggest that the VDR normally functions to suppress one or more signaling networks that drive proliferation during DMBA-induced tumorigenesis.
It is possible that the enhanced sensitivity to tumorigenesis in the VDR-/- mouse is related to the defective hair cycling associated with VDR ablation that leads to deep dermal cyst and utricle formation. The tumor spectrum observed in VDR-/- mice (predominantly benign sebaceous, squamous and follicular papillomas) is similar to that induced by topical DMBA-TPA regimens in other mutant mice with defects in the hair cycle (13). In addition, previous studies have demonstrated that most, but not all, mutant mice with abnormalities in hair follicles display enhanced susceptibility to topical two-stage carcinogenesis (13). Further studies will be necessary to determine if sensitivity to topically applied DMBA differs between VDR ablated mice and their wild-type controls. Mice transgenic for ornithine decarboxylase (ODC), another hairless-like phenotypic mutant, exhibit alopecia and, like VDR-/- mice, display enhanced sensitivity to skin tumorigenesis (24,25). The similarity of ODC transgenic mice and VDR-/- mice is particularly interesting since vitamin D analog inhibition of skin carcinogenesis is associated with inhibition of ODC activation in skin (26).
Previous studies have indicated that hair loss in young VDR-/- mice is associated with generalized atrichia and development of cutaneous deep dermal cysts and utriculi (1,6,15), similar to mice null for the hairless gene (17). In the present studies, we observed progressive deterioration of the skin of VDR-/- mice with age even in the absence of DMBA. Older VDR-/- mice exhibit long coiled nails and excessive amounts of thickened, wrinkled, waxy skin secondary to formation of large utriculi, mimicking the severe form of the hairless mutation known as the rhino mouse (19). There are numerous mouse allelic mutations at the hairless locus that result in functional knockout of hairless and the rhino-like phenotype (17,20). Humans with mutations in the hairless gene present clinically with generalized atrichia, and the same phenotype has been noted in a patient with a normal hairless gene but with mutations in both alleles of the VDR (3). The recent identification of the hairless gene product as a nuclear receptor co-repressor protein that interacts with histone deacetylases (27) raises the possibility of functional interactions between hairless and VDR in the regulation of the hair cycle. Curiously, a similar skin phenotype of progressive alopecia, degeneration of hair follicles, dermal cyst formation and focal melanosis has been described in mice with targeted disruption of RXR
, a nuclear receptor, which dimerizes with VDR (28). While one possible explanation for these observations is that the RXR
:VDR complex transcriptionally regulates the hairless gene, hairless mRNA expression was found to be normal in both VDR-/- and RXR
-/- mice (15,28). Also, despite phenotypic similarities, the underlying defect in the hair cycle of VDR-/- and RXR
-/- mice occurs during initiation of anagen (15,28), whereas mice with mutations at the hairless locus show a defect during the first catagen stage. Furthermore, although alopecia develops in VDR-/- mice, it does not develop in mice depleted of 1,25D3, the ligand for the VDR, suggesting that VDR function in hair cycling may be ligand independent (15,29). Thus, while emerging data support a link between VDR, RXR
and hairless in regulation of the hair cycle, the specific molecular interactions between these nuclear proteins have yet to be characterized, and further studies will be required to determine whether these interactions have any relevance to the enhanced epidermal sensitivity to DMBA-induced transformation observed in the current study of VDR-/- mice.
In summary, our studies provide the first report that VDR ablation is associated with enhanced sensitivity to tumor formation. The development of skin tumors in VDR-/- mice, but not their wild-type counterparts, in response to the chemical carcinogen DMBA suggests that the VDR acts as a tumor suppressor gene in the epidermis. Although the molecular basis for papilloma formation in the absence of the VDR is unclear, phenotypic similarities between VDR-/- mice and mice with mutations in the hairless gene suggest that the enhanced sensitivity to tumorigenesis may be related to generalized atrichia, deep dermal cyst formation, and/or alopecia. However, since not all mutant mice with similar hairless phenotypes display enhanced sensitivity to chemical carcinogens, possibly due to the influence of strain specific modifications (30), further studies are necessary to clarify the connection between hair follicle biology, keratinocyte function and papilloma formation. The high frequency (85%) and relatively rapid onset (within 60 days) of papillomas in VDR-/- mice in response to orally administered DMBA provides a unique and convenient model for studying the cellular and molecular role of the VDR in skin tumorigenesis.
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: jwelsh{at}nd.edu 
 |
Acknowledgments
|
---|
This work was supported by The National Institutes of Health (CA69700 to J.Welsh, RR173 to J.P.Sundberg). The authors are grateful to Mark Suckow, DVM, for assistance with DMBA dosing of mice, to Emily Tribble for specimen processing and staining, and to Lindsay Barnett and Valerie Schroeder of the Freimann Life Science Center at the University of Notre Dame for care of the VDR-/- mouse colony.
 |
References
|
---|
- Li,Y.C., Pirro,A.E., Amling,M., Delling,G., Baron,R., Bronson,R. and Demay,M.B. (1997) Targeted ablation of the vitamin D receptor: an animal model of vitamin D dependent rickets type II with alopecia. Proc. Natl Acad. Sci. USA, 94, 98319835.[Abstract/Free Full Text]
- Yoshizawa,T., Handa,Y, Uematsu,Y. et al. (1997) Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nature Genet., 16, 391396.[ISI][Medline]
- Miller,J., Djabali,K., Chen,T., Liu,Y., Ioffreda,M., Lyle,S., Christiano,A.M., Holick,M. and Cotsarelis,G. (2001) Atrichia caused by mutations in the vitamin D receptor gene is a phenocopy of generalized atrichia caused by mutations in the hairless gene. J. Invest. Dermatol., 117, 612617.[Abstract/Free Full Text]
- Colston,K., Colston,M. and Feldman,D. (1981) 1,25 Dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of growth in culture. Endocrinology, 108, 10831089.[Abstract]
- Riza-Rabin,M., Sineb,R., Shor,B., Michele,G. and Jana,P. (1994) Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: evidence of a paracrine system for 1,25 dihydroxycholecalciferol. J. Cell Physiol., 159, 131141.[ISI][Medline]
- Sakai,Y. and Demay,M. (2000) Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice. Endocrinology, 141, 20432049.[Abstract/Free Full Text]
- Hagar,G., Formanek,M., Gedlicka,C., Thurnher,D., Knerer,B. and Kornfehl,J. (2001) 1,25 (OH)2D3 induces elevated expression of the cell cycle regulating genes p21 and p27 in squamous carcinoma cell lines of the head and neck. Acta Otolaryngol., 121, 103109.[ISI][Medline]
- Chida,K., Hashiba,H., Fukushima,M., Suda,T. and Kuroki,T. (1985) Inhibition of tumor promotion in mouse skin by 1,25 dihydroxyvitamin D. Cancer Res., 45, 54265430.[Abstract]
- Kensler,T.W., Dloan,P.M., Gange,S.J., Lee,J.K., Wang,Q. and Posner,G.H. (2000) Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis. Carcinogenesis, 21, 13411345.[Abstract/Free Full Text]
- Li,Y.C., Amling,M., Pirro,A.E., Priemel,M., Meuse,J., Baron,R., Delling,G. and Demay,M.B. (1998) Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets and osteomalacia, but not alopecia, in vitamin D receptor ablated mice. Endocrinology, 139, 43914396.[Abstract/Free Full Text]
- Zinser,G.M., Packman,K. and Welsh,J.E. (2002) Vitamin D3 receptor ablation alters mammary gland morphogenesis. Development, 129, 30673076.[Abstract/Free Full Text]
- Aldaz,C.M., Liao,Q., LaBate,M. and Johnston,D.A. (1996) Medroxyprogesterone acetate accelerates the development and increases the incidence of mouse mammary tumors induced by dimethylbenzanthracene. Carcinogenesis, 17, 20692072.[Abstract]
- Sundberg,J.P., Sundberg,B.A. and Beamer,W.G. (1997) Comparison of chemical carcinogen skin tumor induction efficiency in inbred, mutant and hybrid strains of mice: morphologic variations of induced tumors and absence of a papillomavirus cocarcinogen. Mol. Carcinogen., 20, 1932.[ISI][Medline]
- Booth,C.J. and Sunberg,J.P. (1996) Hemangiomas and hemangiosarcomas. In Mohr,U., Dungworth,D.L., Capen,C.C., Carlton,W., Sundberg,J. and Ward,J. (eds) Pathobiology of the Aging Mouse. ILSI Press, Washington DC, Vol. I, pp. 393401.
- Sakai,Y., Kishimoto,J. and Demay,M. (2001) Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice. J. Clin. Invest., 107, 961966.[Abstract/Free Full Text]
- Xie,Z., Komuves,L., Yu,Q.C. et al. (2002) Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth. J. Invest. Dermatol., 118, 1116.[Abstract/Free Full Text]
- Sundberg,J.P. (1994) Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools. CRC Press, Boca Raton, CA.
- Ahmad,W., Panteleyev,A. and Christiano,A.M. (1999) Molecular basis of congenital atrichia in humans and mice. Cutis, 64, 269276.[ISI][Medline]
- Sundberg,J.P. and Boggess,D. (1998) Rhino-9J (hrrh9J): a new allele at the hairless locus. Vet. Pathol., 35, 297299.[Abstract]
- Panteleyev,A., Paus,R., Sundberg,J.P. and Christiano,A.M. Molecular basis for the rhino (hrrh8K) phenotype: a nonsense mutation in the hairless gene. Genomics, 53, 383386.
- Masood,R., Nagpal,S.M., Zheng,T., Cai,J., Tulpule,A., Smith,L. and Gill,P. (2000) Kaposi sarcoma is a therapeutic target for vitamin D3 receptor agonist. Blood, 96, 31883194.[Abstract/Free Full Text]
- Hutchinson,P., Osborne,J., Lear,J., Smith,A., Bowers,P., Morris,P., Jones,P., York,C., Strange,R. and Fryer,A. (2000) Vitamin D receptor polymorphisms are associated with altered prognosis in patients with malignant melanoma. Clin. Cancer Res., 6, 498504.[Abstract/Free Full Text]
- Scott,L.J., Dunn,C.J. and Goa,K.L. (2001) Calcipotriol ointment. A review of its use in the management of psoriasis. Am. J. Clin. Dermatol., 2, 95120.[Medline]
- Guo,Y., Zhao,J., Sawicki,J., Soler,A. and OBrien,T.G. (1999) Conversion of C57BL/6 mice from a tumor promotion resistant to a sensitive phenotype by enhanced ornithine decarboxylase expression. Mol. Carcinogen., 26, 3236.[ISI][Medline]
- Panteleyev,A., Christiano,A.M., OBrien,T.G. and Sundberg,J.P. (2000) Ornithine decarboxylase transgenic mice as a model for human atrichia with papular lesions. Exp. Dermatol., 9, 146151.[ISI][Medline]
- Hashiba,H., Fukushima,M., Chida,K. and Kuroki,T. (1987) Systemic inhibition of tumor promoter induced ornithine decarboxylase in 1-alpha-hydroxyvitamin D3 treated animals. Cancer Res., 47, 50315035.[Abstract]
- Potter,G.B., Beaudoin,G. III, DeRenzo,C., Zarach,J., Chen,S. and Thompson,C. (2001) The hairless gene mutated in congenital hair loss disorders encodes a novel nuclear corepressor. Genes Dev., 15, 26872701.[Abstract/Free Full Text]
- Li,M., Chiba,H., Warot,X., Messaddeq,N., Gerard,C., Chambon,P. and Metzger,D. (2001) RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations. Development, 128, 675688.[Abstract/Free Full Text]
- Panda,D., Miao,D., Tremblay,M., Sirois,J., Farookhi,R., Hendy,G. and Goltzman,D. (2001) Targeted ablation of the 25-hydroxyvitamin D 1-hydroxylase enzyme: evidence for skeletal, reproductive and immune dysfunction. Proc. Natl Acad. Sci. USA, 98, 74987503.[Abstract/Free Full Text]
- Pietila,M., Parkkinen,J., Alhonen,L. and Janne,J. (2001) Relation of skin polyamines to the hairless phenotype in transgenic mice overexpressing spermidine/spermine N-acetyltransferase. J. Invest. Dermatol., 116, 801805.[Abstract/Free Full Text]
Received June 25, 2002;
revised August 27, 2002;
accepted August 29, 2002.