Hairless Suppresses Vitamin D Receptor Transactivation in Human Keratinocytes
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《内分泌学杂志》
Endocrine Unit, Veterans Affairs Medical Center, Northern California Institute for Research and Education and University of California, San Francisco, California 94121
Abstract
The vitamin D receptor (VDR) and its ligand 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are required for normal keratinocyte differentiation. Both the epidermis and the hair follicle are disrupted in VDR-null mice. Hairless (Hr), a presumptive transcription factor with no known ligand, when mutated, disrupts hair follicle cycling similar to the effects of VDR mutations. Hr, like VDR, is found in the nuclei of keratinocytes in both epidermis and hair follicle. To investigate the potential interaction between Hr and VDR on keratinocyte differentiation, we examined the effect of Hr expression on vitamin D-responsive genes in normal human keratinocytes. Inhibition of Hr expression in keratinocytes potentiated the induction of vitamin D-responsive genes, including involucrin, transglutaminase, phospholipase C-1, and 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) by 1,25(OH)2D3. Overexpression of Hr in human keratinocytes suppressed the induction of these vitamin D-responsive genes by 1,25(OH)2D3. Coimmunoprecipitation, DNA mobility shift assays, and chromatin immunoprecipitation revealed that Hr binds to VDR in human keratinocytes. Hr binding to the VDR was eliminated by 1,25(OH)2D3, which recruited the coactivator vitamin D receptor-interacting protein 205 (DRIP205) to the VDR/vitamin D response element complex. These data indicate that Hr functions as a corepressor of VDR to block 1,25(OH)2D3 action on keratinocytes.
Introduction
KERATINOCYTES, THE MOST abundant cells of the epidermis, are the source of 7-dehydrocholesterol, which is necessary for the photochemical production of vitamin D3, but they also possess 25- and 1-hydroxylase enzymes and thus can produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] from vitamin D3 (1, 2). 1,25(OH)2D3 is the ligand for the nuclear hormone receptor vitamin D receptor (VDR) that is also found in keratinocytes (3, 4, 5). A number of genes, such as involucrin, transglutaminase, phospholipase C-1 (PLC-1), and 24-hydroxylase in human keratinocytes are transcriptionally regulated by 1,25(OH)2D3 (6, 7, 8). In most of these genes, vitamin D response elements (VDREs) have been identified (6, 8, 9). VDR functions as a transcriptional factor and binds to VDREs to activate transcription of these target genes. VDR is not only expressed in keratinocytes of the basal and suprabasal layers of the epidermis (3, 4, 5, 10), but also in keratinocytes of the outer root sheath of hair follicles (11). Mutations in the VDR gene are associated not only with rickets (12), but with altered epidermal differentiation (13) and failure to initiate anagen after the initial hair follicle cycle, leading to alopecia (13, 14, 15, 16). Unlike rickets, alopecia cannot be prevented by increasing dietary calcium (17). The absence of VDR expression in keratinocytes, but not in dermal papilla cells, is responsible for the loss of hair follicle cycling (18). Targeted expression of the VDR to the keratinocytes of VDR-null mice can restore normal hair follicle cycling (19, 20). These observations indicate that VDR in follicular keratinocytes is required for normal hair follicle cycling, but the mechanism by which VDR is involved in hair follicle cycling is not clear.
The Hairless (Hr) gene was first isolated from Hairless mice that carry a spontaneous proviral integration in the Hr gene (21). The Hr cDNA in humans and rats were subsequently cloned (22, 23). Mutations of the Hr gene were associated with a recessively inherited complete loss of body hair with dermal papules in humans called papular atrichia (22, 24, 25, 26, 27, 28, 29, 30) as well as in several hairless mouse strains (22, 31, 32, 33). The Hr gene is mainly detected in the hair follicles, the suprabasal layers of the interfollicular epidermis, and the brain (21, 26, 34, 35). However, little is known about the precise role of the protein encoded by the Hr gene, although there is evidence that reduced Hr gene function causes an increase in intrafollicular apoptosis in skin (36). The phenotype of mice with Hr gene mutations is quite similar to that of the VDR-null mouse, in that the developmental hair follicle cycle is normal, but the postnatal hair follicle cycle is blocked at the end of catagen (15). Both Hr and VDR are expressed in hair follicle keratinocytes (11, 35). Recent studies demonstrated that VDR directly binds to Hr, and the VDR-mediated transactivation is inhibited by overexpressed rat Hr in an African green monkey kidney cell line (COS) (37, 38). However, whether endogenous Hr in keratinocytes interacts with VDR was not known at the onset of our studies. In this manuscript we demonstrate that Hr can bind to VDR and repress its 1,25(OH)2D3-stimulated transcriptional activity.
Materials and Methods
Polyclonal antibody against Hr
The customized rabbit polyclonal antibody against Hr was made by ImmunoVision Technologies (Brisbane, CA). The antibody was generated using a peptide that contains the region at the C terminus of the human Hr protein. This epitope is highly homologous to rat and mouse Hr. The antibody was characterized by immunocytochemistry and Western analysis using human and mouse skin, and lysates from human keratinocytes and mouse Hr-overexpressing HEK293 cells.
Immunocytochemistry of Hr and VDR
The monoclonal antibody against VDR and the affinity-purified polyclonal antibody against Hr were used. The VDR antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and was used at a concentration of 0.4 μg/ml. The customized Hr antibody was used at a concentration of 2.4 μg/ml. Immunoreagents for VDR were diluted with blocking buffer provided in the Tyramide Signal Amplification Biotin System (PerkinElmer Life Sciences, Boston, MA). Immunoreagents for Hr were diluted in 10 mM Tris buffer (pH 7.6) containing 4% BSA, 1% teleostean skin gelatin, 0.1% Tween 20, and 500 mM NaCl. The human tissue samples were obtained from the chest skin of adult male individuals. Sections used for the VDR antibody were treated with Antigen Unmasking Solution (Vector Laboratories, Inc., Burlingame, CA) at boiling temperature. The binding of VDR was detected by affinity-purified, biotinylated, goat antimouse IgG (Vector Laboratories, Inc.), followed by streptavidin-horseradish peroxidase (PerkinElmer Life Sciences). The sections were subsequently incubated with biotinyl tyramide (PerkinElmer Life Sciences) followed by streptavidin horseradish peroxidase. Enzyme activity was revealed with diaminobenzidine substrate (Vector Laboratories, Inc.). Omitting the first antibodies resulted in no signal, indicating the specificity of the immunodetection. For Hr staining, the primary antibody was detected by affinity-purified, biotinylated, goat antirabbit IgG (Vector Laboratories, Inc.), followed by the Vectastain Elite ABC kit (Vector Laboratories, Inc.). Peroxidase activity was revealed with diaminobenzidine substrate. For a negative control, the antibody was preabsorbed with a 50-fold molar excess of the peptide against which the Hr antibody was raised.
Construction of plasmids
The mouse Hr cDNA fragment spanning from 381-3933 bp in the mouse Hr cDNA with a flag tag (provided by Dr. A. Christiano, Columbia University, New York, NY) was subcloned between EcoRI and NotI sites of the expression plasmid pcDNA 3.1 (–; Invitrogen Life Technologies, Inc.) in a sense orientation. This construct was used to overexpress mouse Hr in HEK293 cells. The human Hr cDNA fragment spanning from 1470–5101 bp in the human Hr cDNA sequence was made by RT-PCR using human keratinocyte RNA and was cloned into the expression vector pcDNA 3.1/NT-green fluorescence protein-TOPO (Invitrogen Life Technologies, Inc.) in an antisense or a sense orientation. The antisense human Hr construct was used to reduce the expression of Hr in human keratinocytes. The sense human Hr construct was used to overexpress Hr in human keratinocytes. Construction of the full-length promoter constructs for vitamin D-responsive genes, including human involucrin, transglutaminase, PLC-1, and 24-hydroxylase, was described previously (6, 7). The location of the VDRE is shown in Fig. 2C, except for the transglutaminase promoter in which VDREs have not yet been identified.
Cell culture
HEK293 cells obtained from American Type Culture Collection (Manassas, VA) were maintained in DMEM (Invitrogen Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.). Normal human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; BD Clonetics, San Diego, CA) as described previously (39). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4 C, overnight), and primary cultures were established in KGM containing 0.07 mM calcium. The second passage keratinocytes were plated with KGM containing 0.03 mM calcium and used for all subsequent experiments.
Transfection, selection, and luciferase assays
For transfection with the mouse Hr construct, HEK293 cells were plated in six-well plates and transfected with the sense mouse Hr cDNA construct using Lipofectamine 2000 (Invitrogen Life Technologies, Inc.). The total protein was isolated, and the protein levels of mouse Hr were analyzed by Western blotting. For transfection with the human Hr constructs, second passage human keratinocytes were transfected with human antisense or human sense Hr constructs using TransIt-Keratinocyte Transfection Reagent (Mirus, PanVera Corp., Madison, WI). The transiently transfected cells were enriched by a 2-d selection with G418 at a concentration of 200 μg/ml starting 2 d after transfection as described previously (40). The total cellular protein was isolated, and the protein levels for involucrin, transglutaminase, PLC-1, and Hr were analyzed by Western blotting. In other experiments, second passage keratinocytes plated in six-well plates were cotransfected with human antisense Hr, sense Hr, or vector only constructs, and luciferase constructs were cotransfected with the promoters of involucrin, transglutaminase, PLC-1, or 24-hydroxylase (7, 41) plus the control construct pRSV-gal (42) using TransIt-Keratinocyte Transfection Reagent (Mirus, PanVera Corp., Madison, WI). The pRSV-gal is a -galactosidase (-gal) expression vector containing a -galactosidase gene driven by a Rous sarcoma virus (RSV) promoter and enhancer, and is used as an internal control to normalize for transfection efficiency. Cells were lysed, and the cell extracts were assayed for luciferase activity using the Luciferase Assay System (Promega Corp., Madison, WI) and were assayed for -galactosidase activity using the Galacto-Light Kit (Tropix, Bedford, MA).
Western analysis and immunoprecipitation
Keratinocytes in cell culture plates were washed twice with PBS and then incubated in PBS containing either 1% Nonidet P-40 (NP-40) and Complete protease inhibitors (Roche, Indianapolis, IN) or RIPA lysis buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and Complete protease inhibitors for 5 min. Cells were scraped into microfuge tubes, incubated at 4 C for 30 min, and pelleted by centrifugation. The supernatant was collected. The protein concentration of the lysate was measured by a bicinchoninic acid protein assay kit (Pierce Chemical Co., Inc., Rockford, IL). Equal amounts of protein were electrophoresed through 7.5% reducing SDS-PAGE at 200 V for 30 min and electroblotted onto polyvinylidene fluoride microporous membranes (0.45 μM pore size; Immobilon-P, Millipore Corp.) in an electroblotting buffer (25 mM Tris, 192 mM glycine, and 5% methanol) at 100 V for 75 min. After incubation in blocking buffer (100 mM Tris base, 150 mM NaCl, 5% nonfat milk or 2% BSA, and 0.5% Tween 20), the blot was incubated overnight at 4 C with the appropriate primary antibodies: monoclonal antibodies against human involucrin (Sigma-Aldrich Corp.) at a dilution of 1:2000, monoclonal antibodies against human transglutaminase (Biomedical Technologies, Inc., Stoughton, MA) at a dilution of 1:200, polyclonal antibodies against PLC-1 (Santa Cruz Biotechnology, Inc.) at a dilution of 1:200, polyclonal antibodies against Hr at a dilution of 1:3000, and polyclonal antibodies against actin (Sigma) at a dilution of 1:5000. After washes in blocking buffer, the membranes were incubated for 1 h with the appropriate anti-IgG secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) in the blocking buffer. After a second series of washes, bound antibody complexes were visualized using the SuperSignal ULTRA Chemiluminescent Kit (Pierce Chemical Co.) and subsequent exposure to x-ray film. In some experiments, equal amounts of protein were incubated with a specific antibody at 4 C for 1 h and then with UltraLink immobilized protein G (Pierce Chemical Co.) at 4 C overnight. Primary antibodies included polyclonal antibodies against VDR (Santa Cruz Biotechnology, Inc.). The lysate-antibody-agarose bead mixture was washed four times with PBS and then analyzed by Western analysis as described above. In a reverse approach, detection of VDR on the gel from the Hr immunoprecipitates was accomplished using the ProFound Coimmunoprecipitation Kit (Pierce Chemical Co., Inc.). This method eliminates the interference from antibody fragments commonly encountered with the methods based on immobilized protein G.
DNA mobility shift assay
The nuclear extracts were made from normal human keratinocytes according to the method described by Abmayr and Workman (43). Synthetic oligonucleotides used for the DNA mobility shift assay were end-labeled with T4 polynucleotide kinase. In a total of 17 μl, nuclear extracts (12 μg protein) were incubated with 2 μg poly-deoxy-inosinic-deoxy-cytidylic-acid (Amersham Biosciences) and 1 μl (0.5 μg) polyclonal anti-Hr antibody in 10 μl binding buffer [20 mM HEPES (pH 7.9), 20% glycerol, 50 mM KCl, and 0.5 mM dithiothreitol] at 30 C for 20 min, and then with 10,000 cpm of 32P-labeled probe for an additional 20 min. Protein-DNA complexes were electrophoresed in a 6% nondenaturing polyacrylamide gel in 1x gel shift running buffer [50 mM Tris, 380 mM glycerin, and 2 mM EDTA (pH 8.5)].
Chromatin immunoprecipitation (ChIP) assay
Keratinocytes in culture plates were cross-linked with 1% formaldehyde at 37 C for 10 min. The cells were then washed and collected with PBS containing Complete protease inhibitors EDTA free (Roche). The cell pellet was resuspended in sodium dodecyl sulfate lysis buffer [1% sodium dodecyl sulfate, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.1)] with Complete protease inhibitors and incubated on ice for 10 min. Samples were sonicated to shear DNA to lengths between 200 and 1000 bp, then centrifuged. The lysates were diluted into ChIP dilution buffer (Upstate USA, Charlottesville, VA) and preabsorbed with protein A agarose/salmon sperm DNA beads (Upstate USA) for 30 min at 4 C. The precleared lysates were immunoprecipitated with antibodies against VDR (Affinity Bioreagents, Golden, CO), vitamin D receptor-interacting protein 205 (DRIP205) (44), acetyl-histone H3 (Upstate, Charlottesville, VA), or Hairless by overnight incubation. The immunoprecipitated complex was collected by adding protein A agarose beads. The beads were thoroughly washed with low-salt, high-salt, LiCl wash buffer and TE buffer according to the manufacturer’s protocol (Upstate USA). The complexes were eluted from the beads by elution buffer (0.1% SDS and 0.1 M NaHCO3). The samples were reverse cross-linked by heating at 65 C for 4 h. The samples were then neutralized by 3 M NaOAc, and the DNA fragments were purified using a QIAquick Spin Kit (Qiagen, Valencia, CA). The specific DNA sequence incorporated into the complex was analyzed by PCR. The primers for the 24-hydroxylase promoter (9) (forward, 5'-GAAGCACACCCGGTGAACTCC-3'; reverse, 5'-GCCAATGAGCACGCAGAGG-3') were designed to span its VDRE sequence. The primers for the involucrin promoter (8) (forward, 5'-GGAGCTGCAGGTCAGACCAC-3'; reverse, 5'-GCCCGGCCAAAC TCAGTTAC-3'), and PLC-1 promoter (6) (forward, 5'-GCTCTTATTATGCCGTGA GT-3'; reverse, 5'-TCTGCCCTGAAGAACTAATC-3') were designed to span their VDRE sequences.
Results
A polyclonal antibody was generated in rabbits using a peptide based on the sequence of the C terminus of human Hr. This epitope has 95% homology with rat Hr and 87% with mouse Hr. A blast search of GenBank revealed little homology with other mammalian proteins. To determine the specificity of the Hr antibody, a full-length sense mouse Hr cDNA or a full-length sense human Hr cDNA construct were transfected into HEK293 cells or normal human keratinocytes. The cell lysate was analyzed by Western blotting using the Hr antibody. The results showed that the Hr antibody recognizes the constitutively expressed Hr in both HEK293 cells and human keratinocytes. An expected major band at 130 kDa was seen on the Western blot with the cell lysates from vector-transfected human keratinocytes and Hr-transfected HEK293 and human keratinocytes. Other bands at 110, 100, and 60 kDa were also seen on the Western blot (Fig. 1A). Only the smaller bands were seen in HEK293 cells transfected with empty vector. Preabsorption with the peptide against which the antibody was raised blocked or markedly reduced the immunoreaction to all bands (Fig. 1A). We also stained human skin sections using our Hr antibody along with the VDR antibody. We found both Hr and VDR in the nuclei of keratinocytes in the stratum basale of the epidermis and outer root sheath of the hair follicle (Fig. 1B). However, we also found Hr in cells of the inner root sheath and hair bulb as well as in cells surrounding the connective tissue sheath, where little or no VDR could be demonstrated. These results are consistent with the pattern of Hr mRNA expression in mice previously reported by Panteleyev et al. (35), including its expression in dermal fibroblasts (22). Preabsorption of the Hr antibody with the peptide against which it was raised reduced the immunoreaction to background (Fig. 1B).
To determine whether Hr regulates 1,25-(OH)2D3 transactivation, normal human keratinocytes were transfected with either the antisense or sense construct of human Hr or with the empty vector and then treated with 1,25-(OH)2D3. The protein levels of Hr and vitamin D-responsive genes in human keratinocytes, such as involucrin, transglutaminase, and PLC-1, were evaluated by Western analysis. Analysis of the protein levels of other vitamin D-responsive genes, such as 24-hydroxylase, was not included in this study due to the unavailability of a specific antibody to 24-hydroxylase. The antisense Hr construct reduced endogenous Hr to undetectable levels, whereas the sense Hr construct markedly increased Hr levels in these cells (Fig. 2A). However, 1,25(OH)2D3 treatment did not alter the expression levels of Hr (Fig. 2A). In contrast, 1,25(OH)2D3 treatment increased the protein levels of involucrin, transglutaminase, and PLC-1 in the empty vector-transfected cells (Fig. 2A). These effects were nearly doubled by cotransfection with the antisense Hr construct, but were abolished by cotransfection with the sense Hr construct (Fig. 2A). Actin was used as a loading control in the experiment (Fig. 2A). To determine whether Hr suppresses the transcription of vitamin D response genes in human keratinocytes, the Hr antisense or sense construct was cotransfected along with the involucrin, transglutaminase, PLC-1, or 24-hydroxylase promoter construct or pGL-3-control vector (Promega Corp.) into normal human keratinocytes, and promoter activities were determined. The pGL-3-control vector containing simian virus 40 promoter and simian virus 40 enhancer was used as a control; it is known not to be induced by 1,25(OH)2D3. The results showed that the activities for these promoters were all stimulated by 1,25(OH)2D3 in the empty vector-transfected cells (Fig. 2B). Stimulation of the promoter activities by 1,25(OH)2D3 was potentiated by the antisense Hr construct approximately 2-fold and was abolished by the sense construct (Fig. 2B). These data indicate that Hr functions as an inhibitor to suppress the transactivation of vitamin D-responsive genes by 1,25(OH)2D3 in human keratinocytes.
To determine whether Hr is able to bind VDR in human keratinocytes, the total cell lysate extracted from normal human keratinocytes using NP-40 buffer was immunoprecipitated by the VDR antibody. The immunoprecipitates were analyzed by Western blotting with Hr antibody. Figure 3A shows that the VDR antibody precipitated the 130- and 110/100-kDa forms of Hr, indicating binding between Hr and VDR in human keratinocytes. Treatment with 1,25-(OH)2D3 reduced this binding. Preabsorption with Hr peptide against which the Hr antibody was raised blocked the signal on the Western blot. These results were confirmed by a reverse approach, in which the total cell lysate was immunoprecipitated by the Hr antibody, followed by Western blotting with the VDR antibody (Fig. 3B). In the control experiments, normal IgG or protein G beads did not pull down Hr or VDR, indicating that the bands detected on Western blotting are caused by specific interactions (Fig. 3).
To determine whether Hr is able to affect the binding of VDR to the VDREs, nuclear extracts isolated from normal human keratinocytes were incubated with Hr antibody or a comparable amount of IgG before incubation with radiolabeled oligonucleotides containing the VDRE from the promoter of 24-hydroxylase, PLC-1, and involucrin as described previously (6, 8, 41). VDREs in the transglutaminase promoter have not yet been identified, so this promoter was not included in the assay. Figure 4 shows that a single main complex formed after the incubation of each oligonucleotide with the nuclear extracts from human keratinocytes. In these complexes, VDR was identified as reported previously (6, 8, 9). The Hr antibody appeared to cause a weak supershift, but the main effect was to increase the intensity of the existing band. This suggests that the Hr antibody relieves inhibition by Hr of VDR binding to these VDREs. To confirm these results, we examined the binding of Hr to these VDREs using the ChIP assay. In this experiment, human keratinocytes were treated with 1,25(OH)2D3 or vehicle. After cross-linking of the complex to DNA and sonication, the protein-DNA complexes were immunoprecipitated with the antibody against VDR, DRIP205, acetyl-histone H3, or Hr using lack of antibody as the negative control. DRIP205 is a transcription coactivator known to be a part of the binding complex for VDR and VDREs in keratinocytes (44). The incorporation of Hr, DRIP205, VDR, and acetyl-histone H3 into the complex binding to the VDRE was analyzed by PCR. The assay detected incorporation of Hr into the 24-hydroxylase VDRE in the absence of 1,25(OH)2D3, but the incorporation of Hr was remarkably diminished in the presence of 1,25(OH)2D3 (Fig. 5A). In contrast, the incorporation of VDR and DRIP205 into the 24-hydroxylase VDRE was stimulated by 1,25(OH)2D3, as expected (Fig. 5A). As a control, the incorporation of acetyl-histone H3 was not changed by 1,25(OH)2D3 exposure (Fig. 5A). The incorporation of Hr into the VDREs for involucrin and PLC-1, as affected by 1,25(OH)2D3, was also determined by the ChIP assay (Fig. 5B). Like the 24-hydroxylase VDRE, Hr incorporation into these VDREs was blocked by 1,25(OH)2D3.
Discussion
The Hr gene encodes a putative zinc finger transcription factor with a predicted molecular mass of 127 kDa. Our Hr antibody recognizes the full-length and smaller forms of Hr protein in both human keratinocytes and Hr-overexpressed HEK293 cells. These smaller forms might represent either alternative splicing or proteolytic degradation. Like other transcription factors, Hr protein has a novel and conserved six-cysteine motif located at the carboxyl-terminal region (21, 22, 23, 45). In the brain, the abundance of Hr mRNA is induced by thyroid hormone (23), and the Hr protein binds to the thyroid hormone receptor (TR). Hr is mainly located in the cell nucleus (46, 47), contains three amino acid LXXLL motifs (46), and functions as a transcriptional corepressor via interaction with the TR (45, 48). The LXXLL motif is characteristic of coactivators for nuclear hormone receptors such as VDR and TR and is the domain by which coactivators bind to these nuclear hormone receptors (49, 50, 51, 52). Recently, Thompson and colleagues (37) used constitutive expression systems to demonstrate that Hr could bind to VDR in a kidney cell line. They found that Hr bound to VDR in the same region predicted for corepressor binding and different from the C-terminal region to which coactivators bind. Binding of Hr to VDR correlated with inhibition of 1,25(OH)2D3 stimulation of a 24-hydroxylase promoter containing the VDRE of this vitamin D target gene (37). These observations indicate that Hr functions as a corepressor of VDR transactivation. We investigated this interaction for VDR and Hr in keratinocytes, the cells in epidermis and hair follicles where VDR and Hr colocalize naturally. Our data demonstrate that VDR and Hr interact. The interaction between Hr and VDR not only involves physical contact, but also a functional outcome. Overexpression of Hr blocks the ability of 1,25(OH)2D3 to stimulate vitamin D target genes in keratinocytes, whereas inhibition of Hr expression enhances the stimulation by 1,25(OH)2D3. ChIP assays demonstrate that 1,25(OH)2D3 inhibits the binding of Hr and enhances the binding of the coactivator DRIP205 to the VDRE-VDR complex. These data indicate that Hr acts as a repressor of 1,25(OH)2D3-dependent VDR function. In the absence of 1,25(OH)2D3, Hr sits on the VDRE, presumably bound to VDR, and inhibits transcription, whereas in the presence of 1,25(OH)2D3, DRIP205 is recruited to the VDRE complex, displacing Hr and activating transcription (Fig. 6).
Alopecia can be found in both humans and mice with VDR deficiency (13, 14, 15, 16, 53), but not in humans and mice deficient in 1,25(OH)2D or in the ability to make 1,25(OH)2D (54, 55, 56, 57, 58, 59, 60). 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 (38). This observation also suggests that hair cycling is independent of 1,25(OH)2D. However, mice null for the 25-hydroxyvitamin D 1-hydroxylase gene and thus unable to make 1,25(OH)2D show abnormalities in epidermal differentiation similar to those in the VDR-null animals (60). Thus, the role of Hr in the epidermis may differ from its role in hair follicles with respect to controlling VDR transcriptional activity and/or its regulation by 1,25(OH)2D. However, 1,25(OH)2D3 has a positive effect on hair cycling. Chemotherapy-induced alopecia and intrafollicular apoptosis can be prevented by topical 1,25(OH)2D3 administration (61, 62). Vitamin D3 analogs stimulate hair growth in nude mice (63). Patients with VDR mutations not only in the DNA binding domains, but also in the ligand binding domain, in some cases are associated with alopecia (64). These observations suggest that hair cycling can be modulated by 1,25(OH)2D to some extent.
VDR dimerizes with the retinoid X receptor or retinoid acid receptor to activate the transcription of target genes. In keratinocytes, retinoid X receptor dimerizes with VDR and binds to DR3-type VDREs of vitamin D target genes such as involucrin and 24-hydroxylase (8, 9). Retinoid acid receptor dimerizes with VDR and binds to the DR6-type VDRE of vitamin D target genes such as PLC-1 (6, 41). Our data demonstrate that Hr represses transcription at both DR3- and DR6-type VDREs. At this point it is unclear what genes required for hair follicle cycling are regulated by VDR and Hr, but it seems likely that VDR and Hr will participate together in that regulation. In the epidermis, however, the role of Hr in blocking 1,25(OH)2D-dependent VDR transcriptional activity and thus blocking 1,25(OH)2D-regulated epidermal differentiation seems clear.
Acknowledgments
We gratefully acknowledge the technical assistance of Mieko H. Ishikawa.
Footnotes
Disclosures: The authors have nothing to declare.
First Published Online November 3, 2005
Abbreviations: ChIP, Chromatin immunoprecipitation; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; DRIP205, vitamin D receptor-interacting protein 205; Hr, hairless; KGM, keratinocyte growth medium; NP-40, Nonidet P-40; PLC-1, phospholipase C-1; TR, thyroid receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.
Accepted for publication October 20, 2005.
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Abstract
The vitamin D receptor (VDR) and its ligand 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are required for normal keratinocyte differentiation. Both the epidermis and the hair follicle are disrupted in VDR-null mice. Hairless (Hr), a presumptive transcription factor with no known ligand, when mutated, disrupts hair follicle cycling similar to the effects of VDR mutations. Hr, like VDR, is found in the nuclei of keratinocytes in both epidermis and hair follicle. To investigate the potential interaction between Hr and VDR on keratinocyte differentiation, we examined the effect of Hr expression on vitamin D-responsive genes in normal human keratinocytes. Inhibition of Hr expression in keratinocytes potentiated the induction of vitamin D-responsive genes, including involucrin, transglutaminase, phospholipase C-1, and 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) by 1,25(OH)2D3. Overexpression of Hr in human keratinocytes suppressed the induction of these vitamin D-responsive genes by 1,25(OH)2D3. Coimmunoprecipitation, DNA mobility shift assays, and chromatin immunoprecipitation revealed that Hr binds to VDR in human keratinocytes. Hr binding to the VDR was eliminated by 1,25(OH)2D3, which recruited the coactivator vitamin D receptor-interacting protein 205 (DRIP205) to the VDR/vitamin D response element complex. These data indicate that Hr functions as a corepressor of VDR to block 1,25(OH)2D3 action on keratinocytes.
Introduction
KERATINOCYTES, THE MOST abundant cells of the epidermis, are the source of 7-dehydrocholesterol, which is necessary for the photochemical production of vitamin D3, but they also possess 25- and 1-hydroxylase enzymes and thus can produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] from vitamin D3 (1, 2). 1,25(OH)2D3 is the ligand for the nuclear hormone receptor vitamin D receptor (VDR) that is also found in keratinocytes (3, 4, 5). A number of genes, such as involucrin, transglutaminase, phospholipase C-1 (PLC-1), and 24-hydroxylase in human keratinocytes are transcriptionally regulated by 1,25(OH)2D3 (6, 7, 8). In most of these genes, vitamin D response elements (VDREs) have been identified (6, 8, 9). VDR functions as a transcriptional factor and binds to VDREs to activate transcription of these target genes. VDR is not only expressed in keratinocytes of the basal and suprabasal layers of the epidermis (3, 4, 5, 10), but also in keratinocytes of the outer root sheath of hair follicles (11). Mutations in the VDR gene are associated not only with rickets (12), but with altered epidermal differentiation (13) and failure to initiate anagen after the initial hair follicle cycle, leading to alopecia (13, 14, 15, 16). Unlike rickets, alopecia cannot be prevented by increasing dietary calcium (17). The absence of VDR expression in keratinocytes, but not in dermal papilla cells, is responsible for the loss of hair follicle cycling (18). Targeted expression of the VDR to the keratinocytes of VDR-null mice can restore normal hair follicle cycling (19, 20). These observations indicate that VDR in follicular keratinocytes is required for normal hair follicle cycling, but the mechanism by which VDR is involved in hair follicle cycling is not clear.
The Hairless (Hr) gene was first isolated from Hairless mice that carry a spontaneous proviral integration in the Hr gene (21). The Hr cDNA in humans and rats were subsequently cloned (22, 23). Mutations of the Hr gene were associated with a recessively inherited complete loss of body hair with dermal papules in humans called papular atrichia (22, 24, 25, 26, 27, 28, 29, 30) as well as in several hairless mouse strains (22, 31, 32, 33). The Hr gene is mainly detected in the hair follicles, the suprabasal layers of the interfollicular epidermis, and the brain (21, 26, 34, 35). However, little is known about the precise role of the protein encoded by the Hr gene, although there is evidence that reduced Hr gene function causes an increase in intrafollicular apoptosis in skin (36). The phenotype of mice with Hr gene mutations is quite similar to that of the VDR-null mouse, in that the developmental hair follicle cycle is normal, but the postnatal hair follicle cycle is blocked at the end of catagen (15). Both Hr and VDR are expressed in hair follicle keratinocytes (11, 35). Recent studies demonstrated that VDR directly binds to Hr, and the VDR-mediated transactivation is inhibited by overexpressed rat Hr in an African green monkey kidney cell line (COS) (37, 38). However, whether endogenous Hr in keratinocytes interacts with VDR was not known at the onset of our studies. In this manuscript we demonstrate that Hr can bind to VDR and repress its 1,25(OH)2D3-stimulated transcriptional activity.
Materials and Methods
Polyclonal antibody against Hr
The customized rabbit polyclonal antibody against Hr was made by ImmunoVision Technologies (Brisbane, CA). The antibody was generated using a peptide that contains the region at the C terminus of the human Hr protein. This epitope is highly homologous to rat and mouse Hr. The antibody was characterized by immunocytochemistry and Western analysis using human and mouse skin, and lysates from human keratinocytes and mouse Hr-overexpressing HEK293 cells.
Immunocytochemistry of Hr and VDR
The monoclonal antibody against VDR and the affinity-purified polyclonal antibody against Hr were used. The VDR antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and was used at a concentration of 0.4 μg/ml. The customized Hr antibody was used at a concentration of 2.4 μg/ml. Immunoreagents for VDR were diluted with blocking buffer provided in the Tyramide Signal Amplification Biotin System (PerkinElmer Life Sciences, Boston, MA). Immunoreagents for Hr were diluted in 10 mM Tris buffer (pH 7.6) containing 4% BSA, 1% teleostean skin gelatin, 0.1% Tween 20, and 500 mM NaCl. The human tissue samples were obtained from the chest skin of adult male individuals. Sections used for the VDR antibody were treated with Antigen Unmasking Solution (Vector Laboratories, Inc., Burlingame, CA) at boiling temperature. The binding of VDR was detected by affinity-purified, biotinylated, goat antimouse IgG (Vector Laboratories, Inc.), followed by streptavidin-horseradish peroxidase (PerkinElmer Life Sciences). The sections were subsequently incubated with biotinyl tyramide (PerkinElmer Life Sciences) followed by streptavidin horseradish peroxidase. Enzyme activity was revealed with diaminobenzidine substrate (Vector Laboratories, Inc.). Omitting the first antibodies resulted in no signal, indicating the specificity of the immunodetection. For Hr staining, the primary antibody was detected by affinity-purified, biotinylated, goat antirabbit IgG (Vector Laboratories, Inc.), followed by the Vectastain Elite ABC kit (Vector Laboratories, Inc.). Peroxidase activity was revealed with diaminobenzidine substrate. For a negative control, the antibody was preabsorbed with a 50-fold molar excess of the peptide against which the Hr antibody was raised.
Construction of plasmids
The mouse Hr cDNA fragment spanning from 381-3933 bp in the mouse Hr cDNA with a flag tag (provided by Dr. A. Christiano, Columbia University, New York, NY) was subcloned between EcoRI and NotI sites of the expression plasmid pcDNA 3.1 (–; Invitrogen Life Technologies, Inc.) in a sense orientation. This construct was used to overexpress mouse Hr in HEK293 cells. The human Hr cDNA fragment spanning from 1470–5101 bp in the human Hr cDNA sequence was made by RT-PCR using human keratinocyte RNA and was cloned into the expression vector pcDNA 3.1/NT-green fluorescence protein-TOPO (Invitrogen Life Technologies, Inc.) in an antisense or a sense orientation. The antisense human Hr construct was used to reduce the expression of Hr in human keratinocytes. The sense human Hr construct was used to overexpress Hr in human keratinocytes. Construction of the full-length promoter constructs for vitamin D-responsive genes, including human involucrin, transglutaminase, PLC-1, and 24-hydroxylase, was described previously (6, 7). The location of the VDRE is shown in Fig. 2C, except for the transglutaminase promoter in which VDREs have not yet been identified.
Cell culture
HEK293 cells obtained from American Type Culture Collection (Manassas, VA) were maintained in DMEM (Invitrogen Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.). Normal human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; BD Clonetics, San Diego, CA) as described previously (39). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4 C, overnight), and primary cultures were established in KGM containing 0.07 mM calcium. The second passage keratinocytes were plated with KGM containing 0.03 mM calcium and used for all subsequent experiments.
Transfection, selection, and luciferase assays
For transfection with the mouse Hr construct, HEK293 cells were plated in six-well plates and transfected with the sense mouse Hr cDNA construct using Lipofectamine 2000 (Invitrogen Life Technologies, Inc.). The total protein was isolated, and the protein levels of mouse Hr were analyzed by Western blotting. For transfection with the human Hr constructs, second passage human keratinocytes were transfected with human antisense or human sense Hr constructs using TransIt-Keratinocyte Transfection Reagent (Mirus, PanVera Corp., Madison, WI). The transiently transfected cells were enriched by a 2-d selection with G418 at a concentration of 200 μg/ml starting 2 d after transfection as described previously (40). The total cellular protein was isolated, and the protein levels for involucrin, transglutaminase, PLC-1, and Hr were analyzed by Western blotting. In other experiments, second passage keratinocytes plated in six-well plates were cotransfected with human antisense Hr, sense Hr, or vector only constructs, and luciferase constructs were cotransfected with the promoters of involucrin, transglutaminase, PLC-1, or 24-hydroxylase (7, 41) plus the control construct pRSV-gal (42) using TransIt-Keratinocyte Transfection Reagent (Mirus, PanVera Corp., Madison, WI). The pRSV-gal is a -galactosidase (-gal) expression vector containing a -galactosidase gene driven by a Rous sarcoma virus (RSV) promoter and enhancer, and is used as an internal control to normalize for transfection efficiency. Cells were lysed, and the cell extracts were assayed for luciferase activity using the Luciferase Assay System (Promega Corp., Madison, WI) and were assayed for -galactosidase activity using the Galacto-Light Kit (Tropix, Bedford, MA).
Western analysis and immunoprecipitation
Keratinocytes in cell culture plates were washed twice with PBS and then incubated in PBS containing either 1% Nonidet P-40 (NP-40) and Complete protease inhibitors (Roche, Indianapolis, IN) or RIPA lysis buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and Complete protease inhibitors for 5 min. Cells were scraped into microfuge tubes, incubated at 4 C for 30 min, and pelleted by centrifugation. The supernatant was collected. The protein concentration of the lysate was measured by a bicinchoninic acid protein assay kit (Pierce Chemical Co., Inc., Rockford, IL). Equal amounts of protein were electrophoresed through 7.5% reducing SDS-PAGE at 200 V for 30 min and electroblotted onto polyvinylidene fluoride microporous membranes (0.45 μM pore size; Immobilon-P, Millipore Corp.) in an electroblotting buffer (25 mM Tris, 192 mM glycine, and 5% methanol) at 100 V for 75 min. After incubation in blocking buffer (100 mM Tris base, 150 mM NaCl, 5% nonfat milk or 2% BSA, and 0.5% Tween 20), the blot was incubated overnight at 4 C with the appropriate primary antibodies: monoclonal antibodies against human involucrin (Sigma-Aldrich Corp.) at a dilution of 1:2000, monoclonal antibodies against human transglutaminase (Biomedical Technologies, Inc., Stoughton, MA) at a dilution of 1:200, polyclonal antibodies against PLC-1 (Santa Cruz Biotechnology, Inc.) at a dilution of 1:200, polyclonal antibodies against Hr at a dilution of 1:3000, and polyclonal antibodies against actin (Sigma) at a dilution of 1:5000. After washes in blocking buffer, the membranes were incubated for 1 h with the appropriate anti-IgG secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) in the blocking buffer. After a second series of washes, bound antibody complexes were visualized using the SuperSignal ULTRA Chemiluminescent Kit (Pierce Chemical Co.) and subsequent exposure to x-ray film. In some experiments, equal amounts of protein were incubated with a specific antibody at 4 C for 1 h and then with UltraLink immobilized protein G (Pierce Chemical Co.) at 4 C overnight. Primary antibodies included polyclonal antibodies against VDR (Santa Cruz Biotechnology, Inc.). The lysate-antibody-agarose bead mixture was washed four times with PBS and then analyzed by Western analysis as described above. In a reverse approach, detection of VDR on the gel from the Hr immunoprecipitates was accomplished using the ProFound Coimmunoprecipitation Kit (Pierce Chemical Co., Inc.). This method eliminates the interference from antibody fragments commonly encountered with the methods based on immobilized protein G.
DNA mobility shift assay
The nuclear extracts were made from normal human keratinocytes according to the method described by Abmayr and Workman (43). Synthetic oligonucleotides used for the DNA mobility shift assay were end-labeled with T4 polynucleotide kinase. In a total of 17 μl, nuclear extracts (12 μg protein) were incubated with 2 μg poly-deoxy-inosinic-deoxy-cytidylic-acid (Amersham Biosciences) and 1 μl (0.5 μg) polyclonal anti-Hr antibody in 10 μl binding buffer [20 mM HEPES (pH 7.9), 20% glycerol, 50 mM KCl, and 0.5 mM dithiothreitol] at 30 C for 20 min, and then with 10,000 cpm of 32P-labeled probe for an additional 20 min. Protein-DNA complexes were electrophoresed in a 6% nondenaturing polyacrylamide gel in 1x gel shift running buffer [50 mM Tris, 380 mM glycerin, and 2 mM EDTA (pH 8.5)].
Chromatin immunoprecipitation (ChIP) assay
Keratinocytes in culture plates were cross-linked with 1% formaldehyde at 37 C for 10 min. The cells were then washed and collected with PBS containing Complete protease inhibitors EDTA free (Roche). The cell pellet was resuspended in sodium dodecyl sulfate lysis buffer [1% sodium dodecyl sulfate, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.1)] with Complete protease inhibitors and incubated on ice for 10 min. Samples were sonicated to shear DNA to lengths between 200 and 1000 bp, then centrifuged. The lysates were diluted into ChIP dilution buffer (Upstate USA, Charlottesville, VA) and preabsorbed with protein A agarose/salmon sperm DNA beads (Upstate USA) for 30 min at 4 C. The precleared lysates were immunoprecipitated with antibodies against VDR (Affinity Bioreagents, Golden, CO), vitamin D receptor-interacting protein 205 (DRIP205) (44), acetyl-histone H3 (Upstate, Charlottesville, VA), or Hairless by overnight incubation. The immunoprecipitated complex was collected by adding protein A agarose beads. The beads were thoroughly washed with low-salt, high-salt, LiCl wash buffer and TE buffer according to the manufacturer’s protocol (Upstate USA). The complexes were eluted from the beads by elution buffer (0.1% SDS and 0.1 M NaHCO3). The samples were reverse cross-linked by heating at 65 C for 4 h. The samples were then neutralized by 3 M NaOAc, and the DNA fragments were purified using a QIAquick Spin Kit (Qiagen, Valencia, CA). The specific DNA sequence incorporated into the complex was analyzed by PCR. The primers for the 24-hydroxylase promoter (9) (forward, 5'-GAAGCACACCCGGTGAACTCC-3'; reverse, 5'-GCCAATGAGCACGCAGAGG-3') were designed to span its VDRE sequence. The primers for the involucrin promoter (8) (forward, 5'-GGAGCTGCAGGTCAGACCAC-3'; reverse, 5'-GCCCGGCCAAAC TCAGTTAC-3'), and PLC-1 promoter (6) (forward, 5'-GCTCTTATTATGCCGTGA GT-3'; reverse, 5'-TCTGCCCTGAAGAACTAATC-3') were designed to span their VDRE sequences.
Results
A polyclonal antibody was generated in rabbits using a peptide based on the sequence of the C terminus of human Hr. This epitope has 95% homology with rat Hr and 87% with mouse Hr. A blast search of GenBank revealed little homology with other mammalian proteins. To determine the specificity of the Hr antibody, a full-length sense mouse Hr cDNA or a full-length sense human Hr cDNA construct were transfected into HEK293 cells or normal human keratinocytes. The cell lysate was analyzed by Western blotting using the Hr antibody. The results showed that the Hr antibody recognizes the constitutively expressed Hr in both HEK293 cells and human keratinocytes. An expected major band at 130 kDa was seen on the Western blot with the cell lysates from vector-transfected human keratinocytes and Hr-transfected HEK293 and human keratinocytes. Other bands at 110, 100, and 60 kDa were also seen on the Western blot (Fig. 1A). Only the smaller bands were seen in HEK293 cells transfected with empty vector. Preabsorption with the peptide against which the antibody was raised blocked or markedly reduced the immunoreaction to all bands (Fig. 1A). We also stained human skin sections using our Hr antibody along with the VDR antibody. We found both Hr and VDR in the nuclei of keratinocytes in the stratum basale of the epidermis and outer root sheath of the hair follicle (Fig. 1B). However, we also found Hr in cells of the inner root sheath and hair bulb as well as in cells surrounding the connective tissue sheath, where little or no VDR could be demonstrated. These results are consistent with the pattern of Hr mRNA expression in mice previously reported by Panteleyev et al. (35), including its expression in dermal fibroblasts (22). Preabsorption of the Hr antibody with the peptide against which it was raised reduced the immunoreaction to background (Fig. 1B).
To determine whether Hr regulates 1,25-(OH)2D3 transactivation, normal human keratinocytes were transfected with either the antisense or sense construct of human Hr or with the empty vector and then treated with 1,25-(OH)2D3. The protein levels of Hr and vitamin D-responsive genes in human keratinocytes, such as involucrin, transglutaminase, and PLC-1, were evaluated by Western analysis. Analysis of the protein levels of other vitamin D-responsive genes, such as 24-hydroxylase, was not included in this study due to the unavailability of a specific antibody to 24-hydroxylase. The antisense Hr construct reduced endogenous Hr to undetectable levels, whereas the sense Hr construct markedly increased Hr levels in these cells (Fig. 2A). However, 1,25(OH)2D3 treatment did not alter the expression levels of Hr (Fig. 2A). In contrast, 1,25(OH)2D3 treatment increased the protein levels of involucrin, transglutaminase, and PLC-1 in the empty vector-transfected cells (Fig. 2A). These effects were nearly doubled by cotransfection with the antisense Hr construct, but were abolished by cotransfection with the sense Hr construct (Fig. 2A). Actin was used as a loading control in the experiment (Fig. 2A). To determine whether Hr suppresses the transcription of vitamin D response genes in human keratinocytes, the Hr antisense or sense construct was cotransfected along with the involucrin, transglutaminase, PLC-1, or 24-hydroxylase promoter construct or pGL-3-control vector (Promega Corp.) into normal human keratinocytes, and promoter activities were determined. The pGL-3-control vector containing simian virus 40 promoter and simian virus 40 enhancer was used as a control; it is known not to be induced by 1,25(OH)2D3. The results showed that the activities for these promoters were all stimulated by 1,25(OH)2D3 in the empty vector-transfected cells (Fig. 2B). Stimulation of the promoter activities by 1,25(OH)2D3 was potentiated by the antisense Hr construct approximately 2-fold and was abolished by the sense construct (Fig. 2B). These data indicate that Hr functions as an inhibitor to suppress the transactivation of vitamin D-responsive genes by 1,25(OH)2D3 in human keratinocytes.
To determine whether Hr is able to bind VDR in human keratinocytes, the total cell lysate extracted from normal human keratinocytes using NP-40 buffer was immunoprecipitated by the VDR antibody. The immunoprecipitates were analyzed by Western blotting with Hr antibody. Figure 3A shows that the VDR antibody precipitated the 130- and 110/100-kDa forms of Hr, indicating binding between Hr and VDR in human keratinocytes. Treatment with 1,25-(OH)2D3 reduced this binding. Preabsorption with Hr peptide against which the Hr antibody was raised blocked the signal on the Western blot. These results were confirmed by a reverse approach, in which the total cell lysate was immunoprecipitated by the Hr antibody, followed by Western blotting with the VDR antibody (Fig. 3B). In the control experiments, normal IgG or protein G beads did not pull down Hr or VDR, indicating that the bands detected on Western blotting are caused by specific interactions (Fig. 3).
To determine whether Hr is able to affect the binding of VDR to the VDREs, nuclear extracts isolated from normal human keratinocytes were incubated with Hr antibody or a comparable amount of IgG before incubation with radiolabeled oligonucleotides containing the VDRE from the promoter of 24-hydroxylase, PLC-1, and involucrin as described previously (6, 8, 41). VDREs in the transglutaminase promoter have not yet been identified, so this promoter was not included in the assay. Figure 4 shows that a single main complex formed after the incubation of each oligonucleotide with the nuclear extracts from human keratinocytes. In these complexes, VDR was identified as reported previously (6, 8, 9). The Hr antibody appeared to cause a weak supershift, but the main effect was to increase the intensity of the existing band. This suggests that the Hr antibody relieves inhibition by Hr of VDR binding to these VDREs. To confirm these results, we examined the binding of Hr to these VDREs using the ChIP assay. In this experiment, human keratinocytes were treated with 1,25(OH)2D3 or vehicle. After cross-linking of the complex to DNA and sonication, the protein-DNA complexes were immunoprecipitated with the antibody against VDR, DRIP205, acetyl-histone H3, or Hr using lack of antibody as the negative control. DRIP205 is a transcription coactivator known to be a part of the binding complex for VDR and VDREs in keratinocytes (44). The incorporation of Hr, DRIP205, VDR, and acetyl-histone H3 into the complex binding to the VDRE was analyzed by PCR. The assay detected incorporation of Hr into the 24-hydroxylase VDRE in the absence of 1,25(OH)2D3, but the incorporation of Hr was remarkably diminished in the presence of 1,25(OH)2D3 (Fig. 5A). In contrast, the incorporation of VDR and DRIP205 into the 24-hydroxylase VDRE was stimulated by 1,25(OH)2D3, as expected (Fig. 5A). As a control, the incorporation of acetyl-histone H3 was not changed by 1,25(OH)2D3 exposure (Fig. 5A). The incorporation of Hr into the VDREs for involucrin and PLC-1, as affected by 1,25(OH)2D3, was also determined by the ChIP assay (Fig. 5B). Like the 24-hydroxylase VDRE, Hr incorporation into these VDREs was blocked by 1,25(OH)2D3.
Discussion
The Hr gene encodes a putative zinc finger transcription factor with a predicted molecular mass of 127 kDa. Our Hr antibody recognizes the full-length and smaller forms of Hr protein in both human keratinocytes and Hr-overexpressed HEK293 cells. These smaller forms might represent either alternative splicing or proteolytic degradation. Like other transcription factors, Hr protein has a novel and conserved six-cysteine motif located at the carboxyl-terminal region (21, 22, 23, 45). In the brain, the abundance of Hr mRNA is induced by thyroid hormone (23), and the Hr protein binds to the thyroid hormone receptor (TR). Hr is mainly located in the cell nucleus (46, 47), contains three amino acid LXXLL motifs (46), and functions as a transcriptional corepressor via interaction with the TR (45, 48). The LXXLL motif is characteristic of coactivators for nuclear hormone receptors such as VDR and TR and is the domain by which coactivators bind to these nuclear hormone receptors (49, 50, 51, 52). Recently, Thompson and colleagues (37) used constitutive expression systems to demonstrate that Hr could bind to VDR in a kidney cell line. They found that Hr bound to VDR in the same region predicted for corepressor binding and different from the C-terminal region to which coactivators bind. Binding of Hr to VDR correlated with inhibition of 1,25(OH)2D3 stimulation of a 24-hydroxylase promoter containing the VDRE of this vitamin D target gene (37). These observations indicate that Hr functions as a corepressor of VDR transactivation. We investigated this interaction for VDR and Hr in keratinocytes, the cells in epidermis and hair follicles where VDR and Hr colocalize naturally. Our data demonstrate that VDR and Hr interact. The interaction between Hr and VDR not only involves physical contact, but also a functional outcome. Overexpression of Hr blocks the ability of 1,25(OH)2D3 to stimulate vitamin D target genes in keratinocytes, whereas inhibition of Hr expression enhances the stimulation by 1,25(OH)2D3. ChIP assays demonstrate that 1,25(OH)2D3 inhibits the binding of Hr and enhances the binding of the coactivator DRIP205 to the VDRE-VDR complex. These data indicate that Hr acts as a repressor of 1,25(OH)2D3-dependent VDR function. In the absence of 1,25(OH)2D3, Hr sits on the VDRE, presumably bound to VDR, and inhibits transcription, whereas in the presence of 1,25(OH)2D3, DRIP205 is recruited to the VDRE complex, displacing Hr and activating transcription (Fig. 6).
Alopecia can be found in both humans and mice with VDR deficiency (13, 14, 15, 16, 53), but not in humans and mice deficient in 1,25(OH)2D or in the ability to make 1,25(OH)2D (54, 55, 56, 57, 58, 59, 60). 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 (38). This observation also suggests that hair cycling is independent of 1,25(OH)2D. However, mice null for the 25-hydroxyvitamin D 1-hydroxylase gene and thus unable to make 1,25(OH)2D show abnormalities in epidermal differentiation similar to those in the VDR-null animals (60). Thus, the role of Hr in the epidermis may differ from its role in hair follicles with respect to controlling VDR transcriptional activity and/or its regulation by 1,25(OH)2D. However, 1,25(OH)2D3 has a positive effect on hair cycling. Chemotherapy-induced alopecia and intrafollicular apoptosis can be prevented by topical 1,25(OH)2D3 administration (61, 62). Vitamin D3 analogs stimulate hair growth in nude mice (63). Patients with VDR mutations not only in the DNA binding domains, but also in the ligand binding domain, in some cases are associated with alopecia (64). These observations suggest that hair cycling can be modulated by 1,25(OH)2D to some extent.
VDR dimerizes with the retinoid X receptor or retinoid acid receptor to activate the transcription of target genes. In keratinocytes, retinoid X receptor dimerizes with VDR and binds to DR3-type VDREs of vitamin D target genes such as involucrin and 24-hydroxylase (8, 9). Retinoid acid receptor dimerizes with VDR and binds to the DR6-type VDRE of vitamin D target genes such as PLC-1 (6, 41). Our data demonstrate that Hr represses transcription at both DR3- and DR6-type VDREs. At this point it is unclear what genes required for hair follicle cycling are regulated by VDR and Hr, but it seems likely that VDR and Hr will participate together in that regulation. In the epidermis, however, the role of Hr in blocking 1,25(OH)2D-dependent VDR transcriptional activity and thus blocking 1,25(OH)2D-regulated epidermal differentiation seems clear.
Acknowledgments
We gratefully acknowledge the technical assistance of Mieko H. Ishikawa.
Footnotes
Disclosures: The authors have nothing to declare.
First Published Online November 3, 2005
Abbreviations: ChIP, Chromatin immunoprecipitation; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; DRIP205, vitamin D receptor-interacting protein 205; Hr, hairless; KGM, keratinocyte growth medium; NP-40, Nonidet P-40; PLC-1, phospholipase C-1; TR, thyroid receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.
Accepted for publication October 20, 2005.
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