Materials and methods

Generation of Tg mice

The K14 promoter was used to drive hVDR expression in Tg mice. To this end, the full-length coding region of hVDR cDNA (ATTC, Manassas, VA) was inserted into the BamHI site of the K14 -globin cassette (^{Saitou et al, 1995}) (kindly provided by Dr Elaine Fuchs, the University of Chicago) in the sense orientation. This cassette contains 2100 bp human K14 promoter sequence that has been shown to direct transgene expression specifically in the basal layer of the epidermis and the ORS of hair follicles (^{Vassar et al, 1989;Wang et al, 1997}). The K14–hVDR construct (Figure 1a) was released from the plasmid vector by EcoRI and HindIII digestion, purified, and microinjected into FVB mouse embryos at the single cell stage according to the standard procedure (^{Hogan et al, 1986}). Founder Tg mice were identified by polymerase chain reaction analysis of tail genomic DNA with the hVDR primers 5'-CGTGTGAATGATGGTGGAGGGAGCC-3', and 5'-GTCTTGGTTGCCACAGGTCCAGGAC-3', and by western blot analyses of tail skin homogenates using an anti-VDR antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Two Tg lines derived from founders 11 and 14 (line 11 and line 14) were bred into homozygosity Tg^+/+, which was verified by southern and western blot analyses as well as by breeding the Tg^+/+ mice with Wt mice. All offspring from this breeding were heterozygotes (Tg^+/–).

Figure 1.

Generation of Tg mice expressing hVDR.(A) Schematic map of the DNA construct used for microinjection. The 2 kb full-length coding sequence of hVDR cDNA was driven by the human K14 promoter. (B) Southern blot analysis of tail genomic DNA from Wt mice and different Tg founders. The DNA (10 g per lane) was digested with BglII and hybridized with ³²P-labeled hVDR cDNA probe. The transgene copy number in each founder was shown below the blot. (C) Western blot analysis of protein extract (20 g protein per lane) isolated from the tail skin of Wt mice and different Tg founder mice. The blot was analyzed with anti-VDR antibody. Note hVDR and mVDR display different electrophoretic mobility and are well separated. std, hVDR standard.

Full figure and legend (50K)

Generation of Ko mice expressing hVDR

The generation and characterization of Ko^–/– mice have been described elsewhere (^{Li et al, 1997}). Two steps of breeding were used to generate VDR^–/– mice that bear the hVDR transgene: first, Tg^+/+ mice from both line 11 and line 14 were bred with VDR^+/– mice to produce Tg^+/–VDR^+/– mice. Then crossing of the Tg^+/–VDR^+/– mice was used to generate the following littermates that were evaluated in parallel: Tg^+/+VDR^+/– (12.5%), Tg^+/–VDR^+/– (25%), Tg^+/+VDR^–/– (6.25%), Tg^+/–VDR^–/– (12.5%), Tg^+/+ (6.25%), Tg^+/– (12.5%), VDR^+/– (12.5%), VDR^–/– (6.25%), and VDR^+/+ (6.25%). Genotypes obtained in the offspring of both lines approximately followed the expected ratio. All mice were fed an autoclaved rodent chow containing 1% calcium, 0.85% phosphorus, and 4 IU per g vitamin D₃.

Southern blot

Mouse tail genomic DNA was prepared as described (^{Hogan et al, 1986}). Genomic DNA was digested with BglII at 37°C overnight, separated on a 0.7% agarose gel, and transferred on to a nylon membrane. Hybridization was performed using a ³²P-labeled hVDR cDNA probe as described previously (^{YC Li et al, 2001}).

Protein extraction and western blot

Freshly dissected tissues were placed in ice-cold Laemmli sample buffer (^{Laemmli, 1970}) and processed as described previously (^{YC Li et al, 2001}). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis according to Laemmli (^{Laemmli, 1970}), and western blot analyses were performed using an antibody against VDR (Santa Cruz Biotechnology) as described before (^{YC Li et al, 2001}).

Blood parameters

Blood ionized calcium levels were determined using a Ciba/Corning 634 calcium-pH analyzer (Chiron Diagnostics, East Walpole, MA). Serum intact parathyroid hormone levels were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (Immutopics, San Clemente, CA).

Bone analysis

The X-ray radiographic analysis of the long bone was conducted using a MX20 Specimen Radiograph System (Faxitron X-ray Corporation, Wheeling, IL) at 18 kVp. For histologic evaluation, the femur and tibia were fixed in 4% formaldehyde in phosphate-buffered saline (pH 7.2) overnight. Specimens were dehydrated in graded concentrations of alcohol, and embedded in methyl methacrylate. Ten microgram consecutive sagittal longitudinal sections were cut from the mid-plane of each bone with a Polycut Microtome (Leica Inc., Deerfield, IL), and stained with either Goldner's trichrome or Von Kossa/toluidine blue for histologic evaluations.

Anagen induction

At day 20 after birth, mouse littermates were anesthetized and subject to depilation of the dorsal hair using wax strips according to the method described previously (^{Paus et al, 1990}). Skin biopsies were taken from the mid-dorsum at days 4, 7, and 18 after depilation for histologic evaluations.

Histology and immunohistochemistry

Skin specimen were fixed in 4% formaldehyde in phosphate-buffered saline (pH 7.2) overnight, processed, embedded in paraffin wax, and cut into 6 m sections with a Leica Microtome 2030. Hematoxylin and eosin staining was performed according to standard procedures. Skin sections were immunostained with the anti-VDR antibody and visualized with a peroxidase substrate DAB kit (Vector Laboratories, Burlingame, CA) according to manufacturer's instructions.

Bromodeoxyuridine (BrdU) labeling

Mice were injected i.p. with 50 mg per kg body weight of BrdU 2 h before skin samples were taken. The skin samples were fixed, embedded in paraffin, and cut into 5 m sections. The sections were stained with a peroxidase-conjugated anti-BrdU monoclonal antibody (Roche Molecular Biochemicals, Indianapolis, IN) and visualized with the Vector DAB substrate kit.

Scanning electron microscopy

Hair fibers were collected from 2 mo old mice, and placed directly on to aluminum stubs using double-stick tape. They were then sputter-coated with gold (approximately 15–20 nm thick) and viewed under a JEOL 840a scanning electron microscope (Peabody, MA) operated at 10 kV. Images were collected with a digital scan generator, DSG-1 (JEOL), at a working distance of 15 mm.

Results

Generation of Tg and Ko/hVDR mice

We chose the human K14 promoter to drive hVDR expression in Tg mice because this promoter has been shown to target transgene expression specifically in the basal keratinocytes and the ORS of the hair follicles (^{Vassar et al, 1989;Wang et al, 1997}), where a high level of VDR expression was seen previously (^{Stumpf et al, 1984;Reichrath et al, 1994}). Embryonic injection of the K14–hVDR construct (Figure 1a) resulted in eight pups positive for the transgene as identified by genomic polymerase chain reaction and confirmed by southern blot analyses (Figure 1b), among which four (founders 5, 11, 14, and 15) expressed the hVDR protein as identified by western blot analyses of tail skin homogenates with an anti-VDR antibody (Figure 1c). Although hVDR and the endogenous mouse VDR (mVDR) are highly homologous, they can be clearly separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Figure 1c). The transgene copy number for founders 5, 11, 14, and 15 was estimated to be 2, 1, 20, and 4, respectively (Figure 1b), and the ratio of hVDR to mVDR content in these Tg^+/– mice was about 1, 0.5, 2, and 1, respectively (Figure 1c). Therefore, we chose founder 11 and 14 (their offspring were hereby designated as line 11 and line 14), which expressed hVDR at half and twice the amount of endogenous mVDR, for further investigations.

Line 11 and line 14 Tg^+/+ mice were used to breed with the VDR^+/– mice ultimately to generate Tg^+/–VDR^–/– and Tg^+/+VDR^–/– mice, which were the Ko mice that expressed the hVDR transgene (hereby collectively designated as Ko/hVDR mice). As shown in Figure 2(a), in both lines, as expected, Tg^+/–VDR^+/+, Tg^+/+VDR^+/+, Tg^+/–VDR^+/–, and Tg^+/+VDR^+/– mice expressed both the hVDR and mVDR in the skin, Tg^+/–VDR^–/– and Tg^+/+VDR^–/– mice expressed only the hVDR, VDR^+/+ (Wt) mice expressed only the mVDR, and VDR^–/– (Ko) mice did not express VDR at all. The tissue distribution of hVDR expression was examined using Tg^+/–VDR^–/– mice to avoid the interference of the endogenous mVDR. As shown in Figure 2b, no hVDR was detected in the duodenum and kidney, two organs crucial for calcium metabolism, as well as in the spleen, muscle, and heart; however, as reported previously (^{Wang et al, 1997}), low expression of the transgene was seen in the tongue, brain, and esophagus, and in line 14, in the lung and thymus as well (Figure 2b and data not shown). In addition, no hVDR expression was seen in the testis, ovary, and uterus in both Tg lines (not shown). Thus, a high level of hVDR expression was specifically targeted to the skin in Tg and Ko/hVDR mice.

Figure 2.

Generation of Ko mice expressing the hVDR transgene.(A) Western blot analysis of tail skin protein extract (20 g protein per lane) isolated from line 11 and line 14 littermates generated by the breeding strategy described in Materials and Methods. The genotype of each mouse is shown on the top of each lane. (B) Tissue-specific expression of the hVDR transgene. Protein extracts were isolated from different tissues of Tg^+/–VDR^–/– mice in both Tg lines and the expression of hVDR was determined by western blot with anti-VDR antibody (20 g protein per lane). Ko/hVDR, Tg^+/–VDR^–/–; SK, skin; DU, duodenum; K, kidney; SP, spleen; L, liver; B, brain; M, skeletal muscle; H, heart; TH, thymus; TO, tongue.

Full figure and legend (82K)

Impaired calcium homeostasis in Ko/hVDR mice

The Ko/hVDR mice from both line 11 and line 14 were evaluated in parallel with Wt, Ko, and Tg littermates. Similar to the Ko mice (^{Li et al, 1997}), the Ko/hVDR mice displayed growth retardation, and developed hypocalcemia after weaning (Figure 3a), with the blood ionized calcium level decreasing by about 30% at 35 d of age compared with the Wt littermates. Secondary hyperparathyroidism was evident, as the serum parathyroid hormone level was continually elevated with age, reaching a concentration 200 times higher than the Wt level by 90 d (Figure 3a). Contact X-ray radiographic analyses of the long bone revealed a clear decrease in bone calcification in Ko and Ko/hVDR mice, with an expansion in the epiphyseal growth plate and a decrease in cortical width (Figure 3b). Histologic evaluation confirmed a dramatic increase in the width of the hypertropic zone of the growth plate, poor mineralization of the cartilage and cortical bone, and an increase in osteoid in trabecular and cortical bone (Figure 3c), which are typical features of advanced rickets. The calcemic and skeletal phenotypes of the Tg mice in both lines were indistinguishable from those of the Wt mice (Figure 3). These results demonstrated that targeted expression of hVDR in Ko mice had no effect on their calcium metabolism.

Figure 3.

Impaired calcium metabolism in Ko/hVDR mice.(A) The Ko/hVDR mice are growth retarded and develop hypocalcemia and secondary hyperparathyroidism. (a) Growth curves of Wt, Ko, Tg, and Ko/hVDR mice. The data were generated from male mice. n = 7 in each group. (b) Blood ionized calcium levels of Wt, Ko, Tg, and Ko/hVDR mice determined at different times after birth. n = 7 in each group. (c) Serum intact parathyroid hormone (iPTH) concentrations of Wt, Ko, Tg, and Ko/hVDR mice determined at different ages. n = 5 in each group. Curve legend: Wt, ; Ko, Tg, ; Ko/hVDR, . (B) The Ko/hVDR mice develop rickets. Contact X-ray radiograph of the long bones from 75 d old Wt, Ko, Tg, and Ko/hVDR mice in both lines. Arrowheads indicate the cortical bone, and arrows indicate the epiphyseal growth plates. (C) Von Kossa/toluidine blue staining of their undecalcified bones. Panel a, Wt; panel b, Ko; panel c, Tg line 14; panel d, Ko/hVDR line 14; panel e, Tg line 11; and panel f, Ko/hVDR line 11. GP, growth plate. Scale bar: 100 m.

Full figure and legend (81K)

Rescue of alopecia and skin abnormalities by the hVDR transgene

One dramatic difference between the Ko and Ko/hVDR littermates was that, in both Tg lines, the Ko/hVDR mice displayed a normal hair coat like the Wt mice. No alopecia was seen in the Ko/hVDR mice 8 mo after birth, whereas alopecia was already prominent in the Ko mice at 4 mo of age (Figure 4a). The hair loss in the Ko mice was evident on both dorsal and ventral skins, with dermal cysts clearly seen in the hairless regions at 5 mo of age. The morphology or fiber pattern of the hair shaft of both Ko/hVDR lines, as revealed by scanning electron microscopic analysis, was the same as that of Wt and Tg littermates (Figure 4b), indicating that the rescued hair fiber structure was normal. Histologic analyses demonstrated that the skin microscopic structure of the Ko/hVDR mice in both lines was indistinguishable from that of the Wt mice, whereas the Ko mice skin already displayed dilated piliary canals and large dermal cysts by 75 d (Figure 4c). Of particular note is a complete rescue of the alopecia in line 11 Tg^+/–VDR^–/– mice that only expressed hVDR at about half the endogenous mVDR level (Figure 1). The interfollicular epidermis of the Ko mice is generally one to two cell layers thicker than that of the Wt mice, which was not seen in the Ko/hVDR mice either (Figure 4d). Normal skin histology was also seen in the two Tg lines (Figures 4c, d). Similar histologic results were observed in mice at 90 d of age (data not shown).

Figure 4.

The hVDR transgene rescues the alopecia of VDR knockout mice.(A) Four month old Wt, Ko, Tg, and Ko/hVDR littermates in line 11 and line 14. (B) Scanning electron micrographs of the back hair shaft of these mice. L14, line 14; L11, line 11. Scale bar: 10 m. (C) Hematoxylin and eosin staining of back skin obtained from 75 d old Wt, Ko, Tg, and Ko/hVDR littermates in both Tg lines. DC, dermal cyst. Arrow indicates the dilated piliary canal. Scale bar: 100 m. (D) High magnification of the epidermal region of the skin. Arrow points to the epidermis of Ko mice. Scale bar: 25 m.

Full figure and legend (112K)

To localize the expression of the hVDR transgene in the skin specifically, immunohistochemical staining with an anti-VDR antibody was used to examine the Ko/hVDR mouse skin, in which no interference of the endogenous mVDR was expected. As shown in Figure 5, a high level of hVDR expression was mainly detected in the nuclei of the basal cells of the epidermis (Figure 5a) and the ORS cells of the anagen hair follicle (Figure 5b–d), but no expression was seen in the dermal papilla (Figure 5c), or in the ORS of Ko mice (Figure 5e). These results demonstrated that targeted expression of hVDR in the ORS and/or in the basal keratinocytes effectively rescued the alopecia.

Figure 5.

The hVDR transgene is expressed in keratinocytes in the basal layer of the epidermis and the ORS. Dorsal skins of 7 d old Tg^+/–VDR^–/– (a–d) and VDR^–/– (e) mice were analyzed by immunohistochemical staining with an anti-VDR antibody. (a) Interfollicular epidermal region. Arrows indicate positive keratinocytes in the basal layer and distal ORS. (b, c) Proximal hair follicle. Arrows indicate the positive cells in the ORS. DP, dermal papilla; M, melanin; MC, matrix cells. (d) Middle and distal hair follicle. Arrows point to the positive cells in the ORS. (e) Ko mouse hair follicle. Arrows indicate the negative ORS. Scale bar: 25 m.

Full figure and legend (234K)

Stimulation of hair cycle initiation by the hVDR transgene

To define further the role of VDR in the regulation of hair cycle, we performed detailed histologic analyses of the skins from Wt, Ko, Tg, and Ko/hVDR littermates in both lines at different stages of the follicular cycle. At 3 and 8 d after birth, when the hair follicles were still in the morphogenesis phase (^{Stenn and Paus, 2001}), anagen follicles with the same morphologic pattern were seen in the skins of Wt, Ko, Tg, and Ko/hVDR pups (data not shown), suggesting that VDR is dispensable during follicular morphogenesis. This observation reflected the fact that Ko mice developed a normal hair coat after birth; however, after hair depilation at 20 d of age, Ko mice failed to initiate the follicular cycle, whereas the Ko/hVDR mice displayed approximately the same pattern of anagen follicle induction as the Wt mice, and most strikingly, the Tg mice initiated the follicular cycle earlier than the Wt and Ko/hVDR littermates. In our examination of multiple litters in both Tg lines, a clear gene concentration-dependent effect of VDR was observed on the induction of the follicular cycle, so that in each litter the Tg^+/+VDR^+/+ littermate always displayed the most advanced anagen follicles at the initiation phase. As shown in Figure 6(a, b), Figure 4d after depilation, no anagen follicle formation was seen in the skin of VDR^–/– mice, whereas anagen II, IIIa, or IIIb follicles, as defined recently by^{Muller-Rover et al (2001)}, were seen in Tg^+/–VDR^–/–, VDR^+/–, or Tg^+/–VDR^+/– littermates, and Tg^+/+VDR^+/– and Tg^+/+VDR^+/+ littermates already formed anagen IIIc or IV follicles, characterized by longer follicles extending deep into the subcutis, mass accumulation of melanin in the follicle bulbs, and thicker skin (^{Muller-Rover et al, 2001}). Similar results were observed at day 7 after depilation (data not shown). Consistent with these observations, the Tg^+/+VDR^+/+ mice had the longest hair shaft as determined at day 17 after depilation (data not shown).

Figure 6.

The hVDR transgene stimulates the initiation of the follicular cycle by a gene concentration-dependent fashion. (A, B) Dorsal skin biopsies were taken from each litter 4 d after depilation at day 20 after birth and stained with hematoxylin and eosin. The genotype of each mouse is shown on the top of each panel. Scale bar: 50 m. (A) Female littermates from line 11. (B) Female littermates from line 14. Note the gene dose or the VDR content in the skin increases from panel a to e in line 11 or from a to d in line 14, which is correlated with the advance of the anagen stages. Arrows point to melanin in anagen IIIc and IV follicles. (C, D) Skin biopsies were taken from each litter after depilation at day 20 after birth and stained for BrdU incorporation. The genotype of each mouse is shown on the top of each panel. Scale bar: 25 m. (C) Line 11 littermates at day 7 after depilation. (D) Line 14 littermates at day 4 after depilation. Arrows point to some BrdU-positive cells. MC, matrix cells; DP, dermal papilla.

Full figure and legend (113K)

We then used BrdU labeling to confirm cell proliferation during anagen induction. As shown in Figure 6(c, d), Figure 4 or 7 d after depilation massive BrdU incorporation was seen in the hair matrix cells of Ko/hVDR, Wt, and Tg mice, which are the hair-forming progenitor cells (^{Oshima et al, 2001}). In contrast, little BrdU labeling was detected in the hair follicles of Ko mice; instead, most of the BrdU was incorporated into interfollicular keratinocytes and dermal fibroblasts. These data indicated that the hVDR transgene was required for the restoration of matrix cell proliferation in the anagen follicles.

Discussion

The data presented in this study provide direct evidence that VDR is required for the progression of the postnatal follicular cycle in mice. This conclusion is based on several lines of evidence: (i) genetic ablation of the VDR gene does not affect prenatal and postnatal hair morphogenesis, but abrogates the initiation of the following hair cycle; (ii) targeted expression of hVDR in the skin of Ko mice specifically rescues the alopecia and other skin abnormalities, but does not prevent the development of other nonskin phenotypes; (iii) the hVDR transgene is targeted to the keratinocytes of the basal layer and the ORS, with little or no expression in other tissues; and (iv) the hVDR transgene restores the initiation of the hair cycle and anagen hair matrix cell proliferation in Ko mice, and overexpression of the transgene in Tg mice accelerates the formation of anagen follicles. These results suggest that VDR is required either for the initiation of the postnatal follicular cycle or for the orderly progress of catagen/telogen in the follicular morphogenesis phase so that the next anagen can be initiated properly. In either situation, the genetic inactivation of VDR should lead to a blockade of the onset of the postnatal anagen.

That VDR is dispensable during the morphogenesis of hair follicles but is crucial for its postnatal initiation is consistent with the fact that Ko mice develop a normal hair coat after birth. Their hair loss is, therefore, due to the failure of postnatal follicular cycle initiation. Follicular morphogenesis arises from a primitive epidermis, and follicular cycle is generated from adult tissues. Although these two processes share many features, they are regulated by different factors (^{Stenn and Paus, 2001}). The exact expression pattern of VDR in follicular morphogenesis is not known, but the lack of VDR function may well be compensated by the redundant pathways known to exist in follicular morphogenesis. Indeed, so far no gene has been found absolutely crucial to hair follicle morphogenesis (^{Stenn and Paus, 2001}).

Hair follicle formation is a result of cross-talks between the epithelial and mesenchymal compartments (^{Hardy, 1992;Stenn and Paus, 2001}), and molecules expressed in both compartments have been shown to be important for follicular morphogenesis and cycling (^{Stenn and Paus, 2001}). It is known that VDR is expressed in both ORS keratinocytes and dermal papillae, the key mesenchymal structure of the hair follicle (^{Reichrath et al, 1994}); however, it is unclear whether the presence of VDR in the epithelial or in the mesenchymal component is important to follicle formation. We show here that hVDR expression in the ORS can fully restore hair growth in Ko mice, indicating that the presence of VDR in the epithelial, rather than the mesenchymal compartment, is sufficient to initiate hair follicle cycle. This conclusion is consistent with the recent finding that alopecia in Ko mice is due to defects in keratinocytes rather than in dermal papilla cells (^{Sakai et al, 2001}). Indeed, ORS, the keratinocyte layer lining the hair follicle, has been shown to be crucial for hair growth. ORS contains the multipotent stem cells required for the formation of hair follicles (^{Oshima et al, 2001}). Increasing the survival rate of the ORS cells significantly shortens the anagen phase and prolongs the telogen phase, leading to shorter hairs (^{Pena et al, 1999}), and inactivation of fibroblast growth factor-5 in ORS cells results in long hairs (^{Hebert et al, 1994}). At present, the coupling between VDR expression in the ORS and the initiation of follicle formation is not known. One possibility can be that VDR, as a transcriptional factor, may be required for the production of some soluble paracrine molecules by the ORS that diffuse to the hair matrix or dermal papilla to facilitate the cross-talks between the epithelial and mesenchymal compartments. It is also plausible that VDR is required for the stem cells to respond to morphogenetic signals to generate hair follicles (^{Oshima et al, 2001}).

Although many genes are known to be involved in hair morphogenesis (^{Stenn and Paus, 2001}), factors proved to be required for postnatal hair follicle initiation are few. Localized transient expression of Sonic hedgehog has been shown to induce postnatal anagen and hair growth in mice (^{Sato et al, 1999}). Skin specific inactivation of RXR, the heterodimeric partner of VDR, results in impaired anagen initiation after hair depilation (^{M Li et al, 2001}). Here we demonstrate that targeted expression of hVDR in the skin not only restores the follicular cycle without altering other abnormalities in Ko mice, but also accelerates the induction of anagen follicle formation when it is overexpressed. These data directly prove that VDR plays an indispensable and stimulatory role in postnatal hair cycle initiation.

Alopecia is a predominant feature of the HVDRR patients and VDR knockout mice (^{Li et al, 1997;Malloy et al, 1999}), but is not seen in mice genetically ablated for the 1-hydroxylase gene or in VDDR patients (^{Fu et al, 1997;St-Arnaud et al, 1997;Takeyama et al, 1997;Panda et al, 2001}), suggesting that VDR regulates hair growth by a 1,25(OH)₂D₃-independent mechanism. Previous studies have shown that alopecia is independent of hypocalcemia, secondary hyperparathyroidism or the high level of vitamin D metabolites associated with VDR inactivation (^{Li et al, 1998;Sakai et al, 2001}), but whether VDR inactivation indeed directly causes the alopecia still needs to be further proved. Our data presented in this study unequivocally demonstrate that it is VDR inactivation per se that directly causes the hair loss. Therefore, VDR is among the growing list of transcriptional factors (^{Stenn et al, 1996;Stenn and Paus, 2001}) that are indispensable for the maintenance of normal hair follicle cycle. VDR heterodimerizes with RXR, but only RXR inactivation leads to alopecia, whereas RXR knockout mice show no overt abnormalities, and the skin phenotype of VDR/RXR double knockout mice is the same as VDR knockout mice (^{Krezel et al, 1996;M Li et al, 2001;Yagishita et al, 2001}). Furthermore, it is of interest to note that hair loss resulted from the inactivation of VDR, RXR and hairless genes in mice shares some common features, including the apparent normal hair morphogenesis and similar skin histologic abnormalities such as dilation of piliary canals and formation of dermal cysts (^{Sundberg, 1994;YC Li et al, 1998;M Li et al, 2001}). These observations strongly suggest that the formation of VDR-RXR heterodimer is required for hair follicle cycling (^{Li et al, 2001}); however, the relationship between VDR and hairless gene remains to be elucidated.

In summary, by targeting hVDR expression to the skin, we have demonstrated that the hVDR transgene rescues the alopecia of Ko mice, and accelerates anagen follicle formation when overexpressed in Tg mice. Our data indicate that VDR is required for the initiation of the postnatal hair follicle cycle.

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Acknowledgments

We thank Merry Bolt and Michael Sitrin for technical assistance and helpful discussions. We are also grateful to Elaine Fuchs for providing the K14 promoter, to Linda Degenstein for microinjection of the DNA construct and to Edward Williamson for scanning electron microscopic analysis of the hair shaft. This work was supported by a start-up fund from the University of Chicago, a grant from the National Alopecia Areata Foundation and a NIH grant DK59327 (to Y.C.L.).