Targeted Expression of Human Vitamin D Receptor in the Skin Promotes the Initiation of the Postnatal Hair Follicle Cycle and Rescues the Alopecia in Vitamin D Receptor Null Mice
2002; Elsevier BV; Volume: 118; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2002.01727.x
ISSN1523-1747
AutoresJuan Kong, Yan Chun Li, Xiao Jian Li, Donna Gavin, Yulei Jiang,
Tópico(s)Skin Protection and Aging
ResumoAlopecia is a predominant feature of vitamin D receptor inactivation in mice and humans. To determine the role of vitamin D receptor in the regulation of hair growth directly, we used the human keratin 14 promoter to target human vitamin D receptor expression to the skin of transgenic mice, and generated vitamin D receptor null mice that express the human vitamin D receptor transgene. Parallel studies were carried out in littermates of wild-type, vitamin D receptor null, transgenic, and human vitamin D receptor-expressing null mice in two transgenic lines. The transgenic mice were grossly normal. The vitamin D receptor null and vitamin D receptor null/human vitamin D receptor mice were growth retarded and developed hypocalcemia, secondary hyperparathyroidism, and rickets. In contrast to the vitamin D receptor null mice that developed alopecia, however, the vitamin D receptor null/human vitamin D receptor mice displayed a normal hair coat, and their hair shaft and skin histology were indistinguishable from those of the wild-type mice. Immunohistochemical analyses revealed that the human vitamin D receptor was highly expressed in the basal layer of the epidermis and outer root sheath of the hair follicle. During follicular morphogenesis, no major histologic differences were seen in the skin of wild-type, vitamin D receptor null, transgenic, and vitamin D receptor null/human vitamin D receptor littermates. When anagen was induced by hair depilation at day 20 after birth, the vitamin D receptor null mice failed to initiate the hair cycle, whereas the vitamin D receptor null/human vitamin D receptor mice displayed the same pattern of anagen follicle formation as the wild-type mice. Interestingly, the transgenic mice initiated the follicular cycle earlier than the wild-type and vitamin D receptor null/human vitamin D receptor mice in a gene concentration-dependent manner. Taken together, these data provide direct evidence that vitamin D receptor is required for the initiation of the postnatal hair follicular cycle in mice. Alopecia is a predominant feature of vitamin D receptor inactivation in mice and humans. To determine the role of vitamin D receptor in the regulation of hair growth directly, we used the human keratin 14 promoter to target human vitamin D receptor expression to the skin of transgenic mice, and generated vitamin D receptor null mice that express the human vitamin D receptor transgene. Parallel studies were carried out in littermates of wild-type, vitamin D receptor null, transgenic, and human vitamin D receptor-expressing null mice in two transgenic lines. The transgenic mice were grossly normal. The vitamin D receptor null and vitamin D receptor null/human vitamin D receptor mice were growth retarded and developed hypocalcemia, secondary hyperparathyroidism, and rickets. In contrast to the vitamin D receptor null mice that developed alopecia, however, the vitamin D receptor null/human vitamin D receptor mice displayed a normal hair coat, and their hair shaft and skin histology were indistinguishable from those of the wild-type mice. Immunohistochemical analyses revealed that the human vitamin D receptor was highly expressed in the basal layer of the epidermis and outer root sheath of the hair follicle. During follicular morphogenesis, no major histologic differences were seen in the skin of wild-type, vitamin D receptor null, transgenic, and vitamin D receptor null/human vitamin D receptor littermates. When anagen was induced by hair depilation at day 20 after birth, the vitamin D receptor null mice failed to initiate the hair cycle, whereas the vitamin D receptor null/human vitamin D receptor mice displayed the same pattern of anagen follicle formation as the wild-type mice. Interestingly, the transgenic mice initiated the follicular cycle earlier than the wild-type and vitamin D receptor null/human vitamin D receptor mice in a gene concentration-dependent manner. Taken together, these data provide direct evidence that vitamin D receptor is required for the initiation of the postnatal hair follicular cycle in mice. Hair growth consists of two phases: the follicular morphogenesis and the postnatal hair cycle. Follicles form from the embryonic epidermis as a result of signals arising in both the primitive epithelium and the underlying mesoderm. In the process, the epithelium grows down into the dermis and joins at its proximal end a mesenchymal condensation called dermal papilla, which is thought to play an important part in follicular morphogenesis and cycling (Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1119) Google Scholar). In mice, follicular morphogenesis extends into the third week after birth, which is followed by the first genuine follicular cycle (Muller-Rover et al., 2001Muller-Rover S. Handjiski B. van der Veen C. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15https://doi.org/10.1046/j.0022-202x.2001.01377.xCrossref PubMed Google Scholar). The follicular cycle is a regeneration process in the adult tissue, through which new hair shafts are generated to replace the old ones. Each cycle consists of anagen (the growth stage), catagen (the regression stage), and telogen (the quiescence stage) (Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (805) Google Scholar;Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1119) Google Scholar), and the distinct stages of the murine hair cycle have been well characterized histologically (Muller-Rover et al., 2001Muller-Rover S. Handjiski B. van der Veen C. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15https://doi.org/10.1046/j.0022-202x.2001.01377.xCrossref PubMed Google Scholar). Although follicular morphogenesis and cycling are different processes, both are controlled by the interaction between the epidermis and the underlying mesenchyme that involves multiple gene families (Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1119) Google Scholar). The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily that mediates the action of 1,25-dihydroxy-vitamin D3[1,25(OH)2D3], the most active metabolite of vitamin D (Haussler et al., 1998Haussler M.R. Whitfield G.K. Haussler C.A. et al.The nuclear vitamin D receptor: biological and molecular regulatory properties revealed.J Bone Miner Res. 1998; 13: 325-349Crossref PubMed Scopus (1219) Google Scholar). The primary function of the vitamin D endocrine system is to regulate calcium homeostasis in the body (DeLuca, 1988DeLuca H.F. The vitamin D story: a collaborative effort of basic science and clinical medicine.FASEB J. 1988; 2: 224-236Crossref PubMed Scopus (479) Google Scholar). The notion of VDR playing multiple parts is implicated by its wide expression in various tissues involved in calcium metabolism as well as other functions. Genetic inactivation of the VDR gene in mice resulted in hypocalcemia, secondary hyperparathyroidism, osteomalacia, rickets, and alopecia (Li et al., 1997Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.Proc Natl Acad Sci U S A. 1997; 94: 9831-9835Crossref PubMed Scopus (810) Google Scholar;Yoshizawa et al., 1997Yoshizawa T. Handa Y. Uematsu Y. et al.Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.Nature Genet. 1997; 16: 391-396Crossref PubMed Scopus (975) Google Scholar), phenotypes closely resembling the symptoms of patients with hereditary hypocalcemic vitamin D-resistant rickets (HVDRR) who bear mutations in the VDR gene (Malloy et al., 1999Malloy P.J. Pike J.W. Feldman D. The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets.Endocr Rev. 1999; 20: 156-188Crossref PubMed Scopus (352) Google Scholar). The parathyroid and skeletal abnormalities in VDR null (Ko) mice were prevented by a diet with high calcium and phosphorus content, which normalized their blood ionized calcium levels, but alopecia persisted in the mutant mice with the normal calcium status (Li et al., 1998Li Y.C. Amling M. Pirro A.E. et al.Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice.Endocrinology. 1998; 139: 4391-4396Crossref PubMed Scopus (393) Google Scholar). Similar results, including alopecia, were recently observed in VDR/retinoid X receptor (RXR)γ double mull mutant mice (Yagishita et al., 2001Yagishita N. Yamamoto Y. Yoshizawa T. et al.Aberrant growth plate development in VDR/RXRgamma double null mutant mice.Endocrinology. 2001; 142: 5332-5341Crossref PubMed Scopus (40) Google Scholar); however, alopecia was not seen in mice ablated for the 25-hydroxyvitamin D 1α-hydroxylase gene or in patients with vitamin D-dependent rickets (VDDR) who carry a mutant 1α-hydroxylase gene (Fu et al., 1997Fu G.K. Lin D. Zhang M.Y.H. Bikle D.D. Shackleton C.H.L. Miller W.L. Portale A.A. Cloning of human 25-hydroxyvitamin D-1α-hydroxylase and mutations causing vitamin D-dependent rickets type I.Mol Endocrinol. 1997; 11: 1961-1970Crossref PubMed Google Scholar;St-Arnaud et al., 1997St-Arnaud R. Messerlian S. Moir J.M. Omdahl J.L. Glorieux F.H. The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus.J Bone Miner Res. 1997; 12: 1552-1559Crossref PubMed Scopus (265) Google Scholar;Takeyama et al., 1997Takeyama K. Kitanaka S. Sato T. Kobori M. Yanagisawa J. Kato S. 25-Hydroxyvitamin D3, 1alpha-hydroxylase and vitamin D synthesis.Science. 1997; 277: 1827-1830Crossref PubMed Scopus (454) Google Scholar;Panda et al., 2001Panda D.K. Miao D. Tremblay M.L. Sirois J. Farookhi R. Hendy G.N. Goltzman D. Targeted ablation of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction.Proc Natl Acad Sci USA. 2001; 98: 7498-7503Crossref PubMed Scopus (530) Google Scholar). These results suggest that VDR may function in the regulation of hair growth by a ligand-independent mechanism. Most recent studies suggest that alopecia in Ko mice is due to a defect in epithelial–mesenchymal communication required for anagen initiation (Sakai and Demay, 2000Sakai Y. Demay M.B. Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice.Endocrinology. 2000; 141: 2043-2049Crossref PubMed Scopus (81) Google Scholar;Sakai et al., 2001Sakai Y. Kishimoto J. Demay M.B. Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice.J Clin Invest. 2001; 107: 961-966Crossref PubMed Scopus (122) Google Scholar). In the hair follicle, VDR is highly expressed in the outer root sheath (ORS) and dermal papilla (Reichrath et al., 1994Reichrath J. Schilli M. Kerber A. Bahmer F.A. Czarnetzki B.M. Paus R. Hair follicle expression of 1,25-dihydroxyvitamin D3 receptor during the murine hair cycle.Br J Dermatol. 1994; 131: 477-482Crossref PubMed Scopus (78) Google Scholar), but the exact role of VDR in the epithelial and mesenchymal follicular compartments remains unclear. To assess the role of VDR in hair follicle homeostasis directly, we have used the human keratin 14 (K14) promoter to target human VDR (hVDR) expression to the skin of transgenic (Tg) mice and generated VDR knockout mice expressing hVDR (Ko/hVDR) through breeding. Through parallel analyses of wild-type (Wt), Tg, Ko, and Ko/hVDR mice, we provide direct evidence that VDR is required for the initiation of postnatal hair follicular cycling. 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 β-globin cassette (Saitou et al., 1995Saitou M. Sugai S. Tanaka T. Shimouchi K. Fuchs E. Narumiya S. Kakizuka A. Inhibition of skin development by targeted expression of a dominant- negative retinoic acid receptor [see comments].Nature. 1995; 374: 159-162Crossref PubMed Scopus (161) Google Scholar) (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., 1989Vassar R. Rosenberg M. Ross S. Tyner A. Fuchs E. Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.Proc Natl Acad Sci USA. 1989; 86: 1563-1567Crossref PubMed Scopus (298) Google Scholar;Wang et al., 1997Wang X. Zinkel S. Polonsky K. Fuchs E. Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy.Proc Natl Acad Sci USA. 1997; 94: 219-226Crossref PubMed Scopus (150) Google Scholar). 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., 1986Hogan B. Costantini F. Lacy E. Manipulating the Mouse Embryo. a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). 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+/–). The generation and characterization of Ko–/– mice have been described elsewhere (Li et al., 1997Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.Proc Natl Acad Sci U S A. 1997; 94: 9831-9835Crossref PubMed Scopus (810) Google Scholar). 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. Mouse tail genomic DNA was prepared as described (Hogan et al., 1986Hogan B. Costantini F. Lacy E. Manipulating the Mouse Embryo. a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). 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 (Li et al., 2001Li M. Chiba H. Warot X. Messaddeq N. Gerard C. Chambon P. Metzger D. RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations.Development. 2001; 128: 675-688Crossref PubMed Google Scholar). Freshly dissected tissues were placed in ice-cold Laemmli sample buffer (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar) and processed as described previously (Li et al., 2001Li M. Chiba H. Warot X. Messaddeq N. Gerard C. Chambon P. Metzger D. RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations.Development. 2001; 128: 675-688Crossref PubMed Google Scholar). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis according to Laemmli (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar), and western blot analyses were performed using an antibody against VDR (Santa Cruz Biotechnology) as described before (Li et al., 2001Li M. Chiba H. Warot X. Messaddeq N. Gerard C. Chambon P. Metzger D. RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations.Development. 2001; 128: 675-688Crossref PubMed Google Scholar). 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). 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. 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., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 122: 777-784Crossref PubMed Scopus (220) Google Scholar). Skin biopsies were taken from the mid-dorsum at days 4, 7, and 18 after depilation for histologic evaluations. Skin specimen were fixed in 4% formaldehyde in phosphate-buffered saline (pH 7.2) overnight, processed, embedded in paraffin wax, and cut into 6 µm 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. 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 µm 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. 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. 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., 1989Vassar R. Rosenberg M. Ross S. Tyner A. Fuchs E. Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.Proc Natl Acad Sci USA. 1989; 86: 1563-1567Crossref PubMed Scopus (298) Google Scholar;Wang et al., 1997Wang X. Zinkel S. Polonsky K. Fuchs E. Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy.Proc Natl Acad Sci USA. 1997; 94: 219-226Crossref PubMed Scopus (150) Google Scholar), where a high level of VDR expression was seen previously (Stumpf et al., 1984Stumpf W.E. Clark S.A. Sar M. DeLuca H.F. Topographical and developmental studies on target sites of 1,25(OH)2 vitamin D3 in skin.Cell Tissue Res. 1984; 238: 489-496Crossref PubMed Scopus (65) Google Scholar;Reichrath et al., 1994Reichrath J. Schilli M. Kerber A. Bahmer F.A. Czarnetzki B.M. Paus R. Hair follicle expression of 1,25-dihydroxyvitamin D3 receptor during the murine hair cycle.Br J Dermatol. 1994; 131: 477-482Crossref PubMed Scopus (78) Google Scholar). 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., 1997Wang X. Zinkel S. Polonsky K. Fuchs E. Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy.Proc Natl Acad Sci USA. 1997; 94: 219-226Crossref PubMed Scopus (150) Google Scholar), 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. 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., 1997Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.Proc Natl Acad Sci U S A. 1997; 94: 9831-9835Crossref PubMed Scopus (810) Google Scholar), 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. 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). 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. 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, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1119) Google Scholar), 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 byMuller-Rover et al., 2001Muller-Rover S. Handjiski B. van der Veen C. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15https://doi.org/10.1046/j.0022-202x.2001.01377.xCrossref PubMed Google Scholar, 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., 2001Muller-Rover S. Handjiski B. van der Veen C. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15https://doi.org/10.1046/j.0022-202x.2001.01377.xCrossref PubMed Google Scholar). 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). We then used BrdU labeling to confirm cell p
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