Ligand-Independent Actions of the Vitamin D Receptor Maintain Hair Follicle Homeostasis
Kristi Skorija,
Megan Cox,
Jeanne M. Sisk,
Diane R. Dowd,
Paul N. MacDonald,
Catherine C. Thompson and
Marie B. Demay
Endocrine Unit (K.S., M.C., M.B.D.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Kennedy Krieger Institute and Department of Neuroscience (J.M.S., C.C.T.), Johns Hopkins University School of Medicine, Baltimore 21205, Maryland; and Department of Pharmacology (D.R.D., P.N.M.), Case Western Reserve University, Cleveland, Ohio 44106
Address all correspondence and requests for reprints to: Marie B. Demay, Wellman 501, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail:demay{at}helix.mgh.harvard.edu.
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ABSTRACT
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Alopecia is a feature of vitamin D receptor (VDR) mutations in humans and in VDR null mice. This alopecia results from an inability to initiate the anagen phase of the hair cycle after follicle morphogenesis is complete. Thus, once the initial hair is shed it does not regrow. VDR expression in the epidermal component of the hair follicle, the keratinocyte, is critical for maintenance of the hair cycle. To determine which functional domains of the VDR are required for hair cycling, mutant VDR transgenes were targeted to the keratinocytes of VDR null mice. Keratinocyte-specific expression of a VDR transgene with a mutation in the hormone-binding domain that abolishes ligand binding restores normal hair cycling in VDR null mice, whereas a VDR transgene with a mutation in the activation function 2 domain that impairs nuclear receptor coactivator recruitment results in a partial rescue. Mutations in the nuclear receptor corepressor Hairless are also associated with alopecia in humans and mice. Hairless binds the VDR, resulting in transcriptional repression. Neither VDR mutation affects Hairless interactions or its ability to repress transcription. These studies demonstrate that the effects of the VDR on the hair follicle are ligand independent and point to novel molecular and cellular actions of this nuclear receptor.
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INTRODUCTION
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THE ACTIVE METABOLITE of vitamin D, 1,25-dihydroxyvitamin D, exerts its biological actions by binding to a nuclear receptor, the vitamin D receptor (VDR) (1). Although 1,25-dihydroxyvitamin D is the major steroid hormone that controls mineral ion homeostasis, studies in mice lacking the VDR demonstrate that, in the setting of normal mineral ion homeostasis, the nuclear actions of 1,25-dihydroxyvtiamin D are not required for skeletal development or maturation. However, mice lacking the VDR develop alopecia, regardless of circulating mineral ion levels (2).
The VDR is expressed in hair follicle keratinocytes. The level of VDR expression in these cells increases in the late anagen and catagen stages of the hair cycle, correlating with an increase in differentiation and a decrease in proliferation of the follicle keratinocytes (3). We have previously demonstrated that the keratinocytes of neonatal VDR null mice exhibit the same proliferative and differentiation potential as those isolated from their wild-type littermates. However, the VDR null mice are unable to initiate a new hair cycle after the stage of hair follicle morphogenesis which, in mice, extends to the second week of postnatal life (4).
Hair reconstitution assays demonstrate that the absence of VDR expression in the keratinocyte, and not the dermal papilla component of the hair follicle, leads to abnormal postmorphogenic anagen initiation, establishing that expression of the VDR in the epithelial component of the hair follicle is essential for skin homeostasis (5). Furthermore, targeted expression of the VDR to the keratinocytes of VDR null mice rescues the defect in anagen initiation, demonstrating that expression of the VDR in cells of this lineage is both necessary and sufficient for maintenance of the normal hair cycle (6, 7).
Alopecia is a feature of the human disease, hereditary vitamin D-resistant rickets (HVDRR), the molecular basis of which is homozygous mutation of the VDR (8). Because alopecia is not seen uniformly in HVDRR, it has been postulated that the presence of alopecia may reflect a more severe degree of receptor dysfunction. How the VDR maintains normal hair cycling is not understood, and the molecular pathways that regulate postmorphogenic hair cycling have not been identified (9). Studies in humans and animals have failed to demonstrate the presence of alopecia in vitamin D deficiency. Furthermore, neither humans nor animals with mutations in the enzyme required for hormone activation, the 25-hydroxyvitamin D-1
hydroxylase, manifest alopecia (10, 11, 12, 13, 14, 15). These observations suggest that alopecia is a feature of impaired ligand-independent receptor actions.
The VDR is a member of a subfamily of nuclear receptors that regulates gene expression by heterodimerizing with retinoic X receptor (RXR) and binding to consensus DNA sequences on target genes (1). Ligand binding by the VDR promotes its heterodimerization with RXR and binding to target DNA sequences via the DNA-binding domain. Recruitment of nuclear receptor coactivators, via the activation function 2 (AF2) domain, leads to chromatin remodeling and interactions with the basal transcriptional apparatus, resulting in the induction of gene transcription. The mechanism of transcriptional repression by the VDR is less well understood. Unlike the closely related thyroid hormone receptor superfamily, the VDR interacts only weakly with corepressors such as NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor (16, 17). However, recent investigations have demonstrated that the VDR interacts with Hairless, a protein that functions as a corepressor for thyroid hormone receptors (18, 19, 20). Notably, mutations in the Hairless gene lead to alopecia in humans and mice (21, 22, 23).
The generation of mice lacking the first zinc finger of the VDR DNA binding domain has definitively demonstrated that receptor-DNA interactions are essential for the prevention of alopecia. These mice express a truncated VDR (24), which is unable to bind to vitamin D response elements, but which contains intact hormone binding and nuclear receptor coactivator binding (AF2) domains. Mice homozygous for this truncated transcript are a phenocopy of mice lacking the full-length VDR protein (25), demonstrating that the DNA binding domain of the receptor is essential for the prevention of alopecia. To address whether hormone-receptor interactions and/or recruitment of nuclear receptor coactivators are also required to prevent alopecia, VDR transgenes with mutations that abolish hormone binding or recruitment of nuclear receptor coactivators were targeted to the keratinocytes of VDR null mice.
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RESULTS
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To evaluate whether hormone binding or nuclear receptor coactivator recruitment is required for maintenance of hair follicle homeostasis by the VDR, receptors with mutations in the hormone binding domain (L233S) and AF2 domain (L417S) (26), which abolish hormone binding and nuclear receptor coactivator recruitment, respectively, were employed. Functional characterization of the L417S mutation has previously been reported (26). This mutant retains normal ligand binding, heterodimerization with RXR, and DNA binding. However, in transient gene expression assays, this L417S VDR is unable to mediate ligand-dependent transactivation, because this point mutation abolishes interactions with nuclear receptor coactivators, including SUG1 (suppressor of gal1), steroid receptor coactivator 1, and receptor-interacting protein 140.
To characterize the ligand-binding characteristics of the L233S mutation, COS-7 cells were transiently transfected with the wild-type human (h)VDR, L233S-VDR, and vector alone. As shown in Fig. 1A
, the L233S mutation abolished ligand binding. To evaluate whether this mutation also abolished ligand-dependent transactivation, transient gene expression assays were performed, cotransfecting wild-type or L233S VDR expression vectors with a 1,25-dihydroxyvitamin D-responsive reporter gene (27). As anticipated, based on the inability of this mutant receptor to bind ligand, the L233S VDR was unable to mediate ligand-dependent transactivation in response to 108 M 1,25-dihydroxyvitamin D (Fig. 1B
).

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Fig. 1. Characterization of L233S VDR and Expression of Mutant VDR Transgenes in VDR Null Mice
A, The L233S VDR mutation results in loss of hormone binding. Whole-cell extracts of COS-7 cells, transfected with the wild-type (WT) VDR ( ) L233S mutant VDR ( ), or vector alone ( ), were incubated overnight at 0 C with increasing concentrations of 1,25-(OH)2[3H]D3 in the presence or absence of 400-fold molar excess of unlabeled competitor. Unbound hormone was separated using dextran-coated charcoal. Each point represents duplicate measurements of specific binding at each hormone concentration. B, The L233S VDR mutation results in loss of ligand-dependent transactivation. COS-7 cells were transfected with 4x VDRE-GH and either WT or L233S VDR. Cells were treated, or not, with 108 M 1,25-dihydroxyvitamin D for 24 h before quantitation of reporter gene expression. C, Northern analyses of VDR expression. mRNA prepared from dorsal skin of a WT mouse, a transgene-negative VDR null mouse (KO), and VDR null mice carrying the L233S (L233S) or L417S (L417S) transgenes was subject to Northern analysis and hybridized with a probe containing the full-length coding region of the hVDR. Control hybridization with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe is shown below. D, Immunohistochemical detection of the VDR in the skin. Immunohistochemical detection of the VDR was performed on dorsal skin isolated from a WT mouse, a transgene negative VDR null mouse (KO), and VDR null mice carrying the L233S or L417S mutant transgenes. VDR immunoreactivity (red) is detected in the hair follicle outer root sheath keratinocytes (arrow) and is absent from the dermis. No immunoreactivity is detected in the VDR KO mouse lacking the transgene. Data are representative of experiments performed on two mice of each genotype. KO, Knockout.
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To address whether hormone binding or nuclear receptor coactivator recruitment is critical for maintenance of hair follicle homeostasis by the VDR, L233S and L417S mutant VDRs were expressed in mice under the control of the K14 promoter. This promoter has been shown to target several transgenes, including the wild-type hVDR, specifically to the keratinocytes of mice (6, 7, 28). To evaluate the level of transgene expression, dorsal skin was isolated from wild-type mice and VDR knockout mice expressing mutant hVDR transgenes. As shown in Fig. 1C
, the mRNAs encoding the mutant hVDR transgenes were well expressed in the skin of the transgene-positive VDR null mice. To confirm that the cutaneous expression of the transgene was restricted to the keratinocytes, immunohistochemical analyses were performed using an antibody directed against an N-terminal epitope of the hVDR. As shown in Fig. 1D
, immunoreactivity was specifically detected in the hair follicle keratinocytes (arrows) and was undetectable in the transgene-negative knockout mice. RT-PCR analyses confirmed the lack of expression of the transgene in kidney, liver, intestine, spleen, and bone (data not shown).
Although transient gene expression assays have demonstrated that the L233S and L417S mutations are unable to mediate ligand-dependent transactivation, these studies were performed in immortalized nonkeratinocyte cell lines (26). To evaluate the function of these mutant receptors in the relevant cell, primary keratinocytes were isolated from VDR null mice expressing the mutant transgenes. Second-passage keratinocytes were transfected with a 1,25-dihydroxyvitamin D-responsive reporter gene to evaluate whether expression of the mutant VDRs in keratinocytes altered basal expression or hormone-mediated transactivation of this reporter. Basal expression of the 1,25-dihydroxyvitamin D-responsive reporter was not influenced by the presence of the wild-type or mutant VDRs (data not shown). However, the mutant VDR transgenes failed to mediate hormone-dependent transactivation even at doses of 1,25-dihydroxyvitamin D as high as 106 M (Fig. 2
).

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Fig. 2. Evaluation of Ligand-Dependent Transactivation
Second-passage primary keratinocytes were transfected with a 4x VDRE-Luc reporter gene and treated with 1,25-dihydroxyvitamin D for 18 h at the doses indicated. Firefly luciferase was normalized for cotransfected Renilla-luciferase activity. Data represent the mean ± SEM of the relative ratios of normalized firefly luciferase activity of treated and untreated samples from three independent animals of each genotype. Presence of endogenous VDR or mutant transgene did not affect basal expression of the 4x VDRE-Luc reporter gene. Open bars, wild type (WT); black bars, knockout (KO); lightly shaded bars, KO with L233S transgene; darkly shaded bars, KO with L417S transgene.
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To determine whether expression of the mutant VDR transgenes was able to rescue the defect in postnatal hair cycling, transgene-expressing VDR knockout mice were subjected to depilation, an anagen-initiating stimulus. Depilation leads to induction of anagen, characterized by a rapid proliferation of hair follicle keratinocytes, leading to the presence of mature anagen follicles within 10 d, followed by the appearance of a hair shaft. To address whether expression of the mutant transgenes was able to restore this proliferative response in the VDR null mice, bromodeoxyuridine (BrdU) incorporation into hair follicle keratinocytes was evaluated 6 d after anagen induction (24 d of age). These studies revealed a large number of BrdU-positive nuclei in the follicle keratinocytes of the wild-type control littermates (41 ± 5 BrdU-positive cells per follicle) correlating with marked proliferation that characterizes anagen (Fig. 3
). A similar increase in BrdU incorporation was observed in the hair follicle keratinocytes of the mice expressing the L233S (40 ± 6 BrdU-positive cells per follicle) and L417S (33 ± 7 BrdU-positive cells per follicle) mutant transgenes. These data suggest that neither hormone binding nor recruitment of nuclear receptor coactivators by the VDR is essential for the initial proliferative response to an anagen-initiating stimulus. As previously reported (4, 6), rare BrdU-positive cells were seen among the hair follicle keratinocytes of the transgene-negative VDR null mice (4 ± 2 BrdU positive cells per follicle). Quantitation of the proliferative response to anagen initiation in the VDR null mice carrying the mutant transgenes revealed that the number of BrdU-positive cells per hair follicle did not differ significantly from that observed in their control littermates. The increase in proliferative response resulted in the formation of a functional hair shaft, with regrowth of hair in the mice expressing the L233S and L417S VDR transgenes (Fig. 4A
). However, the mice expressing the L417S VDR transgene demonstrated significant hair loss over a period of 8 months (Fig. 4B
) accompanied by histological appearance of dermal cysts and dilated piliary canals (data not shown), demonstrating that expression of this mutant VDR transgene delays, but does not prevent, the cutaneous consequences of VDR ablation. The skin of the mice expressing the L233S transgene remained clinically (Fig. 4B
) and histologically indistinguishable from that of their wild-type transgene negative littermates. Studies performed in wild-type mice and VDR heterozygous mice expressing the mutant transgenes failed to reveal any alteration in the cutaneous phenotype, demonstrating that these mutant receptors do not exert dominant-negative effects in the follicle keratinocytes (data not shown).

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Fig. 3. BrdU Incorporation in Response to Anagen Initiation
Mice were injected ip, 6 d after anagen initiation, with BrdU and killed 2 h later. Immunohistochemical detection of BrdU-positive cells (brown nuclei indicated by arrows) was performed on dorsal skin isolated from a wild type (WT) mouse, a transgene-negative VDR null mouse (KO), and VDR null mice carrying the L233S or L417S transgenes. The number of BrdU-positive cells per hair follicle (±SEM) is shown below the genotypes of the mice. Data are representative of experiments performed twice on two mice of each genotype. KO, Knockout.
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Fig. 4. Phenotype of VDR Null Mice Expressing the L233S and L417S Transgenes
A, Hair regrowth 2 wk after anagen intiation. VDR status of the littermates is indicated above the photographs. Regions of the dorsal fur were subject to depilation, an anagen-initiating stimulus, as seen in the transgene-negative VDR null littermate (KO), where hair regrowth is not observed. Significant regrowth is observed 2 wk after anagen initiation in the wild-type (WT) mice as well as in their VDR null L233S (left panel) and L417S (right panel) transgene-positive littermates. B, Long-term effects of transgenic rescue. VDR status of the 8-month-old mice is indicated above the photographs. Alopecia is observed in the transgene-negative VDR null mouse (KO), but not in the VDR null mouse expressing the L233S mutant VDR transgene. The VDR null mouse expressing the L417S mutant VDR transgene demonstrates an intermediate phenotype. The smaller size of the three KO mice is due to growth retardation secondary to the noncutaneous consequences of VDR ablation. KO, Knockout.
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Rescue of the cutaneous phenotype by the L233S VDR, which completely lacks ligand-dependent transactivation, raises the question as to whether ligand-independent transcriptional repression by the VDR is required for cutaneous homeostasis. Unlike other nuclear receptors, the VDR does not associate strongly with the corepressors NCoR and SMRT (16, 17); however, recent investigations have revealed that the nuclear receptor corepressor, Hairless, interacts with the VDR and that these interactions are unaffected by the presence of ligand (20). Mutations of the Hairless gene in mice and humans result in a cutaneous phenotype similar to that observed with VDR ablation (21, 22), suggesting that Hairless-VDR interactions may be required for cutaneous integrity. Studies were undertaken, therefore, to determine whether the L233S and L417S mutant VDRs retained the ability to interact with Hairless. After transfection of Hairless and VDR expression vectors into COS cells, immunoprecipitation was performed using an anti-VDR monoclonal antibody, and coimmunoprecipitated Hairless was detected by Western analyses. As shown in Fig. 5
, neither mutation impaired Hairless-VDR interactions. Transient gene expression assays were performed to determine whether coexpression of Hairless repressed VDR-mediated transactivation in the absence of ligand. As was observed in the primary keratinocytes isolated from the transgenic mice, neither the L233S nor the L417S mutation altered basal expression of a 1,25-dihydroxyvitamin D-responsive reporter gene. However, analogous to what was observed in cotransfections with the wild-type VDR, coexpression of Hairless with either of the two mutant VDRs resulted in a 50% inhibition of basal expression of the 1,25-dihydroxyvitamin D-responsive reporter gene (Fig. 6
).

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Fig. 5. Coimmunoprecipitation of VDR Mutants with Hairless (HR)
Protein extracts prepared from COS cellstransfected with the indicated expression vectors were used for immunoprecipitation with VDR-specific ( -VDR) or nonspecific (IgG) antibodies. HR was detected by Western analysis with HR-specific antiserum. In, 3% of the input extract used for immunoprecipitation. Molecular mass markers (kDa) are indicated. Hr, Hairless expression vector.
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Fig. 6. Evaluation of Transcriptional Repression by Hairless (Hr)
COS cells, cultured in the absence of 1,25-dihydroxyvitamin D, were transfected with a VDRE-Luc reporter gene, wild-type (WT) or mutant VDR, and a ß-galactosidase control plasmid. After 36 h, cells were harvested, and luciferase activity was assessed and corrected for the ß-galactosidase activity of the extracts. Data represent the mean ± SEM of the relative ratios of normalized luciferase activity of samples from four independent experiments performed in duplicate.
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DISCUSSION
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The presence of alopecia in humans and mice with mutant VDRs suggests that the VDR has a major role in hair follicle homeostasis. Although targeted expression of the wild-type VDR to the keratinocytes of VDR null mice rescues the alopecia (6, 7), the mechanism by which this nuclear receptor preserves hair cycling remains uncertain. Our current studies demonstrate that the maintenance of hair follicle homeostasis by the VDR does not require 1,25-dihydroxyvitamin D-dependent transactivation, but rather, is dependent upon a ligand-independent function of the VDR.
Ligand-independent effects of the VDR on the hair follicle were suggested by previous investigations in profoundly vitamin D-deficient mice. These mice had no detectable circulating levels of 25-hydroxyvitamin D or of 1,25-dihydroxyvitamin D, yet did not develop alopecia (5). A limitation of these studies was that they could not exclude the possibility that locally produced metabolites could activate the VDR in a ligand-dependent fashion. However, our investigations in the VDR null mice carrying the L233S transgene definitively demonstrate that ligand-dependent transactivation by the VDR is not required for hair follicle homeostasis. The L233S VDR completely lacks transcriptional activation, even at high doses of 1,25-dihydroxyvitamin D, yet when expressed in the keratinocytes of VDR null mice, completely rescues the hair loss phenotype.
These findings focus renewed attention on actions of the unliganded VDR. Evidence that the actions of the VDR on the hair follicle do not require ligand-dependent transactivation suggest that transcriptional repression by the VDR may be the mechanism by which this nuclear receptor maintains hair follicle homeostasis. In contrast to the closely related thyroid hormone receptors, ligand-independent actions of the VDR have been less extensively characterized. A number of studies examining the effects of cotransfected receptors on response elements fused to reporter genes, or using the yeast two-hybrid system, have suggested that the unliganded receptor can mediate transcriptional activation (29, 30) or repression (17, 31). However, most of these effects are modest and in vivo correlates of these observations have not been established. The VDR does not associate strongly with the nuclear receptor corepressors, NCoR and SMRT, but has recently been shown to interact with a novel corepressor, Hairless (20). Interestingly, mutation of the Hairless gene in mice and humans results in alopecia similar to that seen in the VDR null mice, raising the question as to whether the same molecular pathway is affected when the function of either of these genes is disrupted (21, 22). Recent studies have demonstrated that the Hairless protein interacts with VDR and dramatically represses both basal and ligand-dependent VDR-mediated transactivation in transient gene expression assays (20). Furthermore, Hairless is expressed in the same hair follicle cells as the VDR suggesting that the physical and functional interaction of Hairless and VDR occurs in vivo. Our data demonstrate that both the L233S and L417S VDR mutants retain the ability to interact with Hairless. Furthermore, Hairless can suppress basal transcription by both mutant receptors, suggesting that repression of basal transcription by a Hairless-VDR complex may play a critical role in the maintenance of the hair cycle.
Although data from the L233S mutant suggest that ligand-independent Hairless-VDR interactions are important for the maintenance of hair follicle homeostasis, data from the L417S mutant indicate that other cofactors are involved as well. Interestingly, the L417S VDR transgene results in a partial rescue, which delays the onset of the alopecia. The L417S mutant used for these studies retains ligand binding affinities that are similar to that of the wild-type receptor and preserves heterodimerization with RXR, as well as binding to transactivation factor IIB (26). However, this mutation renders the VDR incapable of ligand-dependent transactivation due to its inability to recruit nuclear receptor coactivators including steroid receptor coactivator 1. This suggests that VDR interactions with nuclear receptor comodulators are required for long-term hair follicle homeostasis and that although Hairless-VDR interactions may be required, they are not sufficient. Thus, additional factors are likely recruited to the VDR complex, modulating the effects of Hairless/VDR on the hair follicle keratinocyte.
A number of elegant genetic studies in HVDRR kindreds have examined genotype-phenotype correlations in this disorder. Initial clinical observations suggested that the alopecia, which is not uniformly observed in the human disorder, was associated with a more severe vitamin D resistance (32). This proved to be largely true, in that kindreds with premature termination codons, resulting in an unstable mRNA transcript and absence of detectable receptor protein, were uniformly affected by alopecia (33). However, correlation between receptor domains affected by missense mutations and the presence of alopecia has proven to be more complex, perhaps because of the wide distribution of missense mutations among the functional domains of the VDR in the various affected kindreds (33). Analogous to the observation that expression of a truncated VDR, lacking a DNA-binding domain, in mice is associated with alopecia (24), missense mutants of the VDR DNA-binding domains result in alopecia in humans. The presence of alopecia in patients with VDR mutations in the ligand binding domain is less predictable, with several point mutations being associated with alopecia, whereas others are not (33). The single affected individual reported to have a missense mutation in the AF2 domain, which prevents coactivator binding, was not affected by alopecia (34). This is in contrast to our findings in mice, demonstrating that the mutant VDR transgene with the L417S mutation in the AF2 domain only partially rescues the skin phenotype. Further investigations will be required to determine whether L417 is a critical contact site for recruitment of specific nuclear receptor comodulators in the keratinocyte, the interactions of which remain unaffected by the E420K hVDR AF2 mutation, as well as to identify additional mutations in the AF2 domain in humans to permit more extensive genotype-phenotype correlations.
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MATERIALS AND METHODS
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Experimental Animals
All studies were conducted in accordance with accepted standards of humane animal care and were approved by the institutional animal care committee.
Ligand Binding
COS-7 cells were transfected with SG5 (vector), SG5-VDR, or SG5 L233S-VDR. Cells were harvested and washed in ice-cold PBS, and cell pellets were snap frozen. Whole-cell extracts were prepared as previously described (35). Increasing concentrations of 1,25-(OH)2[3H]D3 were incubated overnight at 0 C with 50 µg of protein, in the presence or absence of a 400-fold molar excess of unlabeled competitor. Bound and free hormone were separated with 3% dextran-coated charcoal.
Animal Maintenance
All studies were approved by the institutional animal care committee. Mice were exposed to a 12-h light/12-h dark cycle. Anagen initiation by depilation was performed as previously described (4, 6). Studies were performed in two independent lines for each transgene, using sex-matched littermates.
Generation of Transgenic Mice
The coding region of the hVDR was subjected to site-directed mutagenesis to engineer the L233S and L417S mutations in the hormone binding domain and AF2 domain, respectively. The mutated VDRs were ligated into a blunt-ended BamHI site, downstream of the keratin 14 (K14) promoter in a transgene cassette also containing the human GH polyadenylation signal (a kind gift of Dr. E. Fuchs) (36). Restriction and DNA sequence analyses confirmed the anticipated mutations and the correct orientation of the hVDR. The K14/hVDR/hGH cassette was isolated from vector sequences and injected into the pronuclei of fertilized mouse oocytes. Potential founders were identified by PCR of tail DNA using hVDR-specific primers that do not recognize the murine (m)VDR (5' 613635; 3' 1020999). Southern analyses were performed to confirm a single site of transgene integration. Potential founders were mated to VDR null mice to obtain offspring heterozygous for transgene integration (hVDR+) and for ablation of the endogenous receptor (mVDR+/). These mice were then bred to obtain hVDR+/mVDR/ mice. One hVDR+/mVDR/ mouse from each litter was killed to confirm transgene expression.
Assessment of Transgene Expression
Transgene expression was confirmed by Northern analyses of mRNA isolated from the dorsal skin using a Fast Track mRNA purification system (Invitrogen, Carlsbad, CA). Blots were probed with hVDR coding region and glyceraldehyde-3-phosphate dehydrogenase cDNA probes.
Cell Culture and Transient Reporter Gene Assays
COS cells were cotransfected with 1,25-dihydroxyvitamin D-responsive reporter genes [vitamin D response element (VDRE)-GH or VDRE-Luc], VDR expression vectors (SG5-VDR, SG5-VDRL233S, or SG5-L417S), and/or Hairless expression vectors as previously described (20, 27). Cells were treated or not with 108 M 1,25-dihydroxyvitamin D for 24 h before evaluation of reporter gene expression. Primary keratinocytes were isolated from 2- to 3-d-old receptor-ablated transgene-positive and -negative mice, as well as from wild-type transgene-negative littermates as previously described (4). The skin was isolated and floated on 0.25% trypsin (Life Technologies, Inc., Gaithersburg, MD) at 4 C overnight. The epidermis was then separated from the dermis, minced, and stirred in MEM with 4% Chelex-treated fetal calf serum (Hyclone Laboratories, Inc., Logan UT), 10 ng/ml epidermal growth factor (Collaborative Research, Inc., Cambridge MA), and 0.05 mM CaCl2 for 1 h at 4 C on ice. The cell suspension was filtered through a 70-µm cell strainer, plated onto collagen-coated (Vitrogen 100, Cohesion Technologies, Palo Alto, CA) 100-mm dishes, and incubated at 34 C, 8% CO2 until 80% confluent. Cells were then trypsinized and plated at a density of 2.5 x 105 cells per well of a six-well plate and grown to 60% confluence before transfection. Transfections were performed using Lipofectamine Reagent (Invitrogen Carlsbad, CA) in Opti-MEM medium, with 10 µg of 4x VDRE-Luc and 50 ng Renilla-Luc per well for 6 h at 34 C 8% CO2. Cells were then treated with 1,25-dihydroxyvitamin D or not, for 18 h before harvest and evaluation of firefly and Renilla luciferase activity using Stop and Glo (Promega Corp., Madison WI). Transfection efficiency was normalized by correcting firefly luciferase activity of the 4x VDRE-Luc fusion gene, for Renilla luciferase activity.
Coimmunoprecipitations
COS cells were transfected with Hairless and VDR expression vectors. Coimmunoprecipitations were performed using a VDR-specific monoclonal antibody or IgG control as previously described (20). Western analyses were performed with Hairless-specific antiserum (19) and a VDR monoclonal antibody (37).
Histology
Skin specimens were obtained from the middorsum of female littermates, fixed, processed, and sectioned. Evaluation of proliferation by BrdU incorporation was performed as previously described (4, 6). Immunohistochemical detection of the VDR was performed using a primary antibody directed against the N terminus of the hVDR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), a secondary biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) and a biotinyl-tyramide amplification system (Perkin Elmer LifeSciences, Inc., Boston, MA).
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FOOTNOTES
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This work was supported by National Institutes of Health Grants DK 46974 (to M.B.D.), DK53980 (to P.N.M.), and NS41313 (to C.C.T.) and a grant from the National Alopecia Areata Foundation (to M.B.D.).
First Published Online December 9, 2004
Abbreviations: AF, Activation factor; BrdU, bromodeoxyuridine; HVDRR, hereditary vitamin D-resistant rickets; NCoR, nuclear receptor corepressor; RXR, retinoic X receptor; SMRT, silencing mediator of retinoid and thyroid hormone receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.
Received for publication October 5, 2004.
Accepted for publication December 2, 2004.
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