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Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Marie B. Demay, M.D., Endocrine Unit, Wellman 501, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: demay{at}helix.mgh.harvard.edu
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Introduction |
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-hydroxylase mutations.
The development of alopecia in mice and humans with VDR mutations but
not with ligand deficiency remains unexplained. 1,25(OH)2D3 has been shown to be a potent inhibitor of keratinocyte proliferation. 1,25(OH)2D3 also stimulates keratinocyte differentiation in a concentration-dependent manner as evidenced by enhanced formation of cornified envelopes and induction of marker gene expression (4, 5).
The hair follicle consists of mesenchymal cells and keratinocytes that form the bulb region, deep in the hypodermal fat (6). The outermost keratinocytes give rise to the outer root sheath, the inner root sheath and the hair shaft. Mice are born without hair although histological examination of the skin at the time of birth reveals immature hair follicles. Numerous anagen follicles are evident within the first 6 days of life and by 15 days the hair cycle enters the catagen phase, progressing to the telogen phase. Subsequently, the follicle undergoes cycles of growth (anagen), regression (catagen), and rest (telogen). In vivo, VDR expression in the outer root sheath keratinocytes correlates with decreased keratinocyte proliferation and increased differentiation in late anagen and catagen (7). We hypothesized that ablation of the VDR might alter the proliferation and differentiation of keratinocytes, and thereby lead to the alopecia observed in humans and mice with VDR mutations. Primary keratinocytes were, therefore, isolated from VDR null mice and control littermates to examine their proliferation rate and expression of keratinocyte differentiation markers. We also examined the expression of two candidate genes which, when misexpressed, lead to alopecia: PTH-related protein (PTH-rP) and hairless (hr).
PTH-rP, initially discovered as the cause of humoral hypercalcemia of malignancy (8), is expressed in a wide variety of normal cells, including epidermal keratinocytes and has been implicated in keratinocyte differentiation (9). Forced overexpression of PTH-rP in keratinocytes of normal mice interferes with normal hair follicle development (10). Consistent with these observations, bPTH (7–34), an antagonist of the PTH/PTH-rP receptor, has been shown to increase the number and length of hair shafts in SKH-1 hairless mice (11). Because the expression of PTH-rP in human keratinocytes is suppressed by 1,25(OH)2D3, it is possible that overexpression of this hormone by the keratinocytes of the VDR knockout mice contributes to the alopecia observed.
The second potential candidate gene we examined was the hairless gene. Like the VDR knockout mice, hairless (hr/hr) mice have a normal first hair coat, however, they develop alopecia at approximately 2 weeks of age (12). Furthermore, like the VDR knockout mice, the alopecia is accompanied by the presence of large dermal cysts. Mutations of this gene in humans have been shown to be the cause of congenital atrichia in several families (13).
Although many genes implicated in hair follicle development are expressed in the mature hair follicle, it is thought that factors that control folliculogenesis are distinct from the factors responsible for the regulation of the hair cycle. The first coat of hair is dependent on factors that control development, whereas subsequent coats are dependent on normal cycling of hair follicles. Therefore, we also performed studies to examine whether the alopecia in the VDR-ablated mice was secondary to a hair cycle defect.
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Materials and Methods |
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-irradiated test diet
(TD96348, Teklad, Madison, WI, containing 2% calcium, 1.25%
phosphorus, and 20% lactose supplemented with 2.2 IU vitamin D/g)
which has been shown to prevent abnormalities in mineral ion
homeostasis in VDR-ablated mice (2).
Cell culture
Primary keratinocytes were isolated from 2- to 3-day-old
receptor-ablated mice and control littermates by a trypsin floating
procedure as previously described (14). Briefly, the skin was isolated
and floated on 0.25% trypsin (Life Technologies, Inc.,
Grand Island, NY) at 4 C overnight. The epidermis was then separated
from the dermis, minced with scissors and stirred in MEM with 4%
Chelex-treated FCS (HyClone Laboratories, Inc., Logan,
UT), epidermal growth factor (EGF; 10 ng/ml; Collaborative Research, Inc., Cambridge, MA) and 0.05 mM CaCl2
(low calcium medium) for 1 h at 4 C. The cell suspension derived
from two mice of the same genotype was filtered through three layers of
gauze and plated in low calcium medium in collagen (Vitrogen 100, Palo
Alto, CA)-coated 100-mm dishes and incubated at 34 C, 8%
CO2. After achieving 80% confluence, these cells
were reseeded at 2.5 x 105 cells/well of a
6-well plate in low calcium medium and grown to 80% confluence before
addition of 1,25(OH)2D3
(10-8 M) and/or inducing
differentiation by increasing the calcium concentration to 2.0
mM. Forty hours later, total RNA was prepared for northern
analysis.
[3H]thymidine incorporation
Keratinocytes were plated at 5.0 x
104 cells/well of a 24-well plate and grown to
80% confluence in keratinocyte growth medium containing 0.05
mM calcium. Cells were then treated with high calcium
and/or 10-8 M
1,25(OH)2D3. After 24
h, cells were labeled for 12 h with
[3H]thymidine (NEN Life Science Products, Boston, MA) and radionucleotide incorporation was
assessed as previously described (15). The
[3H]thymidine counts/min (cpm) were corrected
for protein concentration.
BrdU incorporation
Mice were injected ip with 5-bromo-2'-deoxyuridine (Brdu) (250
mg/kg; Sigma, St. Louis, MO) and 5-fluoro-2'-deoxyuridine
(Fdu) (30 mg/kg; Sigma). Animals were killed 2 h
after Brdu/Fdu injection. Skin specimens were obtained from the
middorsum of VDR-ablated mice and control littermates. After fixation
for 3 h in 4% formaldehyde in PBS (pH 7.2), specimens were
processed, embedded in paraffin, and cut into 6-µm sections with a
Leica Corp. RM 2025 microtome (Leica Corp.,
Deerfield, IL). Brdu staining was performed using a Brdu Staining Kit
(Zymed Laboratories, Inc., South San Francisco, CA)
following the manufacturer’s instructions.
RNA isolation and Northern blot analysis
Total RNA was isolated from cultured keratinocytes and mouse
skin using TRI Reagent (Sigma) according to the
manufacturer’s instructions. For Northern analysis, total RNA (3–20
µg) was electrophoresed through 1% agarose-formaldehyde gels and
transferred to Biotrans nylon membranes (ICN Pharmaceuticals, Inc., Irvine, CA) in 20 x SSC. The membranes were
hybridized with complementary DNA (cDNA) probes labeled with
[-32P]dATP (NEN Life Science Products) or with a -32P-labeled
antisense oligonucleotide probe for 18S ribosomal RNA. The cDNA probes
used were a 0.7- kb EcoRI-XhoI fragment of mouse
keratin-1, a 2.1-kb EcoRI-XhoI fragment of mouse
involucrin, a 1.4-kb EcoRI-XhoI fragment of mouse
loricrin (from American Type Culture Collection, Manassas,
VA), a 0.4- kb AvrII-SmaI fragment of mouse PTH-rP and a
0.9-kb BamHI-HindIII fragment of mouse hairless
(gift from Dr. Jonathan P. Stoye, National Institute for Medical
Research, Mill Hill, London, NW71AA, UK). Hybridization was carried out
at 68 C in QuikHyb (Stratagene, La Jolla, CA). These
probes all generated single transcripts of the appropriate size. The
intensity of the messenger RNA (mRNA) bands was assessed by a Cyclone
Storage Phosphor System (Packard Instrument Company, Meriden, CT) using
Opti Quant software. 18S ribosomal RNA signals were used to normalize
for variations in RNA loading.
Anagen induction
Under avertin-induced general anesthesia, 18-day-old receptor-
ablated mice and control littermates were subjected to depilation
of their dorsal hair using Wax Strips (Del Laboratories, Farmingdale,
NY) following the manufacturer’s instructions. Histological
examination was performed 6 and 10 days following this procedure.
Statistical analyses
Data are presented as the mean ± SEM.
Student’s unpaired t test was used to identify significant
differences (P < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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), follicle, or
interfollicular region (data not shown). These data confirmed the
in vitro findings that the proliferation rate of
keratinocytes lacking functional VDRs did not differ significantly from
that of wild-type keratinocytes.
The expression of differentiation markers in VDR null and wild-type
keratinocytes was then examined. Keratin 1 (K1) is an early marker of
differentiation that is expressed in the spinous layer of the
epidermis. Involucrin, a component of the cornified envelope, is a
suprabasal marker of keratinocyte differentiation that is expressed in
the late spinous layer and throughout the granular layer. Loricrin is a
marker of the granular and cornified layers (17). All three
differentiation markers were expressed equally in the VDR null and
wild-type keratinocytes under both proliferative (0.05 mM
CaCl2) and differentiating (2 mM
CaCl2) conditions (Fig. 2A). Although the proliferation rate of
the keratinocytes decreased with addition of high CaCl2 media, the only
keratinocyte differentiation marker that changed significantly was
involucrin. Although the circulating levels of 1,25-dihydroxyvitamin D
in suckling mice are not suppressed (3; and data not shown), it is
possible that high intracellular levels may be present in
keratinocytes, which may lead to differential expression of hormonally
regulated genes in vivo. To ascertain that the observations
in primary keratinocytes reflected the in vivo state, we
examined expression of these markers in skin mRNA isolated from
VDR-ablated mice and wild-type littermates. Northern analyses revealed
similar levels of expression of keratin 1, involucrin and loricrin at 4
days of age (Fig. 2B).
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). The suppression of involucrin
expression by 1,25(OH)2D3
is a novel observation and may be a species-specific phenomenon.
Previous studies in human keratinocytes have demonstrated induction of
this gene by 1,25(OH)2D3,
correlating with the differentiation promoting effects of this steroid
hormone (4).
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).
The expression of PTH-rP in wild-type keratinocytes, but not
receptor-ablated cells, was suppressed by 10-8
M 1,25(OH)2D3
under proliferating (0.05 mM CaCl2 not shown) and
differentiating (2.0 mM CaCl2) conditions, respectively
(Fig. 3B). Therefore, although repression of PTH-rP mRNA by
1,25(OH)2D3 is
VDR-dependent, the basal expression of this hormone is not altered in
VDR null keratinocytes.
Mutation of Hr (hairless) has been shown to cause alopecia in both mice
(12) and humans (13). Like the VDR knockout mice, hairless (hr/hr) mice
have a normal first coat of hair then develop alopecia accompanied by
the presence of large dermal cysts. It is possible, therefore, that
reduced expression of this gene by the keratinocytes of the VDR null
mice could be responsible for the alopecia observed. However, the
expression of hr was found to be normal in both cultured keratinocytes
and skin isolated from VDR null mice (Fig. 4).
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). Control sections obtained from skin
not subjected to depilation did not reveal induction of anagen
follicles or skin thickening (data not shown). In contrast, rare Brdu
positive cells were seen among the hair follicle keratinocytes of the
VDR null mice (Fig. 5B). Also notable at this stage is the absence of
skin thickening and lack of anagen follicles in the receptor-ablated
animals. Even 10 days post depilation, a time when hair shafts are
being formed in the wild-type littermates (Fig. 5C, arrows),
there was no clinical or histological evidence of anagen induction in
the VDR null mice (Fig. 5D). This data suggests that the alopecia in
the VDR null mice is secondary to a defect in initiation of the hair
cycle. Although clinically, alopecia totalis is not observed in the
receptor-ablated mice until approximately 100 days of age, these
studies demonstrate that the defect is present at 18 days of age. It is
of note that the wild-type littermates have no evidence of having been
subjected to depilation after 3 weeks, whereas the VDR-ablated mice
remain profoundly alopecic 13 weeks post depilation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hair follicle development during embryogenesis requires a series of reciprocal interactions between the epithelium and the underlying mesenchymal cells. Initially, the dermal mesenchyme signals the epithelium to form the epidermal placode. The epithelium then sends a message to the underlying mesenchyme to initiate mesenchymal condensation. In response to signals from the condensed mesenchyme, hair elongation is observed. Recent studies have identified fibroblast growth factors, bone morphogenic proteins, and sonic hedgehog as epithelial-derived signaling molecules in the early stages of hair follicle morphogenesis. Postnatally, the maintenance of normal hair is dependent on the integrity of the dermis, epidermis, and normal hair cycles. Each hair follicle perpetually goes through three stages: growth (anagen), involution (catagen), and rest (telogen). Normal cycling of hair follicles is dependent on the interaction of the follicular keratinocytes with the mesenchymal dermal papilla cells. At the initiation of anagen, signals, thought to originate from the dermal papilla cells, induce the epithelial cells of the hair follicle to proliferate resulting in the full-length anagen follicle. These cells then differentiate to form the mature hair follicle, which includes the outer root sheath, the inner root sheath, and the hair shaft. The anagen follicle subsequently receives a signal that results in the initiation of catagen, characterized by apoptosis of the lower part of the hair follicle (20). The follicle then goes through the telogen, or resting, phase until the initiation of the following anagen by factors thought to emanate from the dermal papilla cells.
The protein products of several genes important for hair morphogenesis, such as insulin-like growth factor 1 and fibroblast growth factor 7, are also expressed at different stages of the hair cycle in adults, suggesting that they may play a role, not only in the development of the initial hair follicles but also in hair cycling. Whether the same proteins and signaling pathways are responsible for both folliculogenesis in utero and the onset of anagen after the first hair coat is not known. Like the VDR knockout mice, hairless (hr/hr) mice have normal hair morphogenesis; however, the hairless mice develop alopecia totalis by approximately 3 weeks of age (12). The onset of this alopecia coincident with the telogen phase of the first hair coat supports the hypothesis that factors that regulate the hair cycle postnatally are largely distinct from those responsible for hair follicle morphogenesis.
The association of VDR gene mutations with alopecia in both humans with
hereditary 1,25-dihydroxyvitamin D- resistant rickets (HVDRR) (21)
and mice (22, 3), combined with the observation that VDR-ablated mice
develop alopecia regardless of their mineral ion status [2 and Fig. 5], suggests that mutation of the VDR per se is responsible
for the hair loss. The absence of alopecia in profound vitamin D
deficiency and in patients with 25-hydroxyvitamin
D3 1-hydroxylase mutations, further supports
the hypothesis that the pathogenesis of the alopecia is a consequence
of impaired receptor function rather than ligand deficiency. It is
possible, however, that even in the absence of low or undetectable
levels of circulating vitamin D metabolites, the skin is able to
produce a vitamin D metabolite capable of mediating physiological
effects. Therefore, absence, rather than deficiency, of ligand or
functional receptor may be required for alopecia to be observed.
We have demonstrated that VDR ablation does not have significant effects on keratinocyte proliferation or differentiation in vitro or in vivo in neonatal mice. VDR ablation could, however, lead to alopecia by alternative means, including ligand independent effects of the VDR (23), hormone toxicity or interactions with an alternative receptor.
Because 1,25(OH)2D3
down-regulates its own biosynthesis, by repressing the
25-hydroxyvitamin D-1-hydroxylase gene (24, 25) and increases its
metabolism by up-regulating the 24-hydroxylase gene through
VDR-dependent actions (26, 27), VDR-ablated cells, which possess these
enzymes may have very high intracellular levels of
1,25(OH)2D3 even in the
setting of normal mineral ion homeostasis. This hormone or its
metabolites produced locally in the skin may have a toxic effect on
hair growth by interacting with a membrane receptor or second nuclear
receptor. The receptor mediating this toxic effect could be specific
for vitamin D metabolites, or alternatively, bind an unrelated ligand
under normal physiological conditions. Precedent for this latter
suggestion has been provided by studies examining the interaction of
progesterone with the oxytocin receptor (28). In these studies, the
effect of progesterone on uterine sensitivity to oxytocin was shown to
involve direct, nongenomic actions of progesterone on the rat oxytocin
receptor. Like progesterone, vitamin D or its metabolites may mediate
effects by interacting with other receptors. One would not expect these
effects to be evident in the intact animal with a functional VDR, since
the duration and degree of
1,25(OH)2D3 toxicity
required would be anticipated to result in fatal
hypercalcemia.
Elucidation of the molecular basis for the alopecia in the VDR-ablated mice will provide insight, not only into factors that regulate hair follicle homeostasis, but is also expected to clarify novel actions of this nuclear receptor in the skin.
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Acknowledgments |
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Footnotes |
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Received October 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase and vitamin D synthesis. Science 277:1827–1830-hydroxylase gene maps to the
pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone
Miner Res 12:1552–1559[CrossRef][Medline]
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