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* Department of Dermatology, Charité, Humboldt University, Berlin, Germany;
Department of Pathology and Laboratory Medicine, University of Kentucky Medical Center, Lexington, Kentucky 0536–0084; USA; and
Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
2Correspondence: Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Martinstrasse 52, D-20246 Hamburg, Germany. E-mail: paus{at}uke.uni-hamburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: p75NTR • kerotinocyte catagen • NGF • NT-3 • BDNF • alopecia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. This cyclic activity of the HF is governed by
epithelial–mesenchymal interactions between keratinocytes (KC) of the
hair bulb and fibroblasts of the dermal papilla (DP), and every hair
cycle stage is regulated by the balance of numerous growth stimulatory
and inhibitory factors (2
3
4
5)
.
The hair follicle transformation from active growth to regression
(anagen–catagen transition) is characterized by a sudden decline in
the DP secretion of growth factors for hair matrix KC, leading to the
dramatic reduction of their proliferative activity, termination of hair
shaft production, and shortening of the HF length up to 70% due to
massive apoptosis in the proximal HF epithelium (6
7
8
9)
.
The molecular control of HF regression has long fascinated
developmental biologists and dermatologists, and important roles for
such growth factors as fibroblast growth factor 5, insulin-like growth
factor 1, transforming growth factor ß, and PTHrp in catagen
development have been shown during the last decade (3
, 5
, 10
11
12
13
14
15
16)
.
Recently we have demonstrated that neurotrophins, a family of
structurally and functionally related polypeptides that includes nerve
growth factor (NGF), neurotrophin 3 (NT-3), brain-derived neurotrophic
factor (BDNF), and neurotrophin 4 (NT-4), are also intimately involved
in the control of HF anagen–catagen transformation (17
, 18)
. Neurotrophins exert their biological effects via
interaction with high-affinity receptors (TrkA is a specific receptor
for NGF, TrkB, and TrkC—receptors for BDNF/NT-4 and NT-3,
respectively), as well as via low affinity binding to the p75
neurotrophin receptor (p75NTR) (cf. 19–21). NT-3 is able to interact
with low affinity with TrkA and TrkB, but binds with high affinity to
p75NTR (21
, 22)
.
The importance of neurotrophins for the anagen–catagen
transformation of the HF in mice was demonstrated in different models:
expression of NT-3, BDNF, and NT-4 is up-regulated in the regressing HF
compartments during catagen in C57BL/6 mice; mouse mutants with
overexpression or deletion of neurotrophins show alterations in catagen
development and in hair shaft length; finally, NT-3, BDNF, and
NT-4-stimulate catagen in murine skin organ culture (17
, 18)
.
Although both types of neurotrophin receptors (high-affinity receptors
of Trk family and low affinity p75NTR) are expressed in the regressing
HF compartments during catagen (9
, 17
, 18)
, their precise
roles in catagen control remain to be elucidated. However, an
involvement of p75NTR in the control of KC apoptosis during HF
regression is suggested because p75NTR is known to modulate cell death
in different models (23
24
25
26
27
28
29)
and HF regression is the
essentially apoptosis-driven process (8
, 9
, 30
31
32)
.
p75NTR is a member of a family of structurally related
cytokine receptors (TNF type I and II receptors, Fas-Apo1 receptor,
CD40, CD30, CD27), characterized by an extracellular domain with
repeated cysteine clusters and by an intracellular domain containing a
serine/threonine-X-valine carboxy-terminal motif similar to that of Fas
and TNFRI, referred to as a ‘death domain’ (reviewed in refs
33
34
35
36
). Proapoptotic signaling effects of p75NTR occur
when p75NTR is expressed alone, without coexpression with high-affinity
neurotrophin receptors TrkA, TrkB, or TrkC. In contrast, apoptotic cell
death mediated via p75NTR signaling does not occur if p75NTR is
coexpressed with high-affinity Trk receptors (35
36
37
38
39)
. In
this case, p75NTR promotes signaling transduction through Trk receptors
and hence cell survival. Thus, the pro- or anti-apoptotic action of
neurotrophins is strongly dependent on the relative expression of
p75NTR and Trk receptors on target cell populations.
To explore the role for p75NTR in HF regression, we studied the
expression and coexpression patterns of p75NTR, Trk receptors, and
apoptotic markers in the regressing HF compartments during catagen,
using a C57BL/6 mouse model (40
, 41)
with semiquantitative
reverse transcriptase-polymerase chain reaction (RT-PCR), double
and triple immunofluorescence techniques, and in situ end
labeling (TUNEL). The dynamics of catagen development were assessed
histomorphometrically in p75NTR knockout (-/-) mice and in
NGF-overexpressing mice. In addition, the effects of neurotrophins on
HF regression were compared in the organ-cultured skin of C57BL/6 and
p75NTR null mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Homozygous p75NTR knockout (-/-) mice were generated using
conventional gene targeting techniques under C57BL/6 background, as
described previously (42)
. NGF-overexpressing transgenic
mice were generated using K14 as promoter, which ensures strong
expression of NGF in the basal epidermal KC and KC of the HF outer root
sheath (43)
. Genotyping of mutant animals was performed
using PCR protocols for the mutated alleles and slot blot analysis of
isolated tail DNA (44)
. Both mouse strains are viable and
fertile and display no obvious, macroscopically visible hair growth
abnormalities.
Active hair growth (anagen) was induced as described
(40)
in the back skin of 6- to 9-wk-old C57BL/6 female
mice or in 8-wk-old p75NTR knockout (-/-) and their corresponding
age-matched wild-type mice in the telogen phase of the hair cycle. In
both mouse strains, HF anagen–catagen transformation was studied,
using at least four mice per time point: anagen VI (12 days after
anagen induction by depilation [12 days p.d.]), catagen II, IV,
VI-VIII (17–19 days p.d.) (1
, 6
, 40
, 41)
.
For the analysis of spontaneous hair follicle cycling in infantile
p75NTR knockout (-/-), NGF-overexpressing, and the corresponding
wild-type mice, skin was harvested 14–20 days after birth (P14-P20);
four to seven mice of every strain were studied. In all experiments,
the neck region of the back skin was harvested parallel to the
vertebral line and embedded using a special technique for obtaining
longitudinal cryosections through the HF from one defined site
(45)
.
RT-PCR
Total RNA was isolated from full-thickness back skin
samples (homogenized in liquid nitrogen), using a single-step guanidine
thiocyanate-phenol-chloroform method using RNeasy-total-RNA-kit
(Quiagen, Hilden, Germany). Skin samples included the subcutaneous
skeletal (panniculus carnosus) muscle layer. cDNA was synthesized by
reverse transcription of 3 µg total RNA, using a cDNA synthesis kit
(Invitrogen, San Diego, Calif.). The following sets of oligonucleotide
primers were used to amplify specific c-DNA: ß-actin: 5'-GAA AAC GCA
GCT CAG TAA CAG TCC G and 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3';
p75NTR: 5'-GCAGGGCGGCTAAAAGGGCATCAAG-3' and
5'-CACATACTCAGACGAAGCCAACCACG-3' (46)
. Amplification was
performed using taq polymerase (Life Technologies, Inc., Grand Island,
N.Y.) over 34 cycles, using an automated thermal cycler (Perkin Elmer
Cetus, Norwalk, Conn.). Each cycle consisted of the following steps:
denaturating at 94°C (1 min), annealing at 60°C (45 s), and
extension at 72°C (45 s). PCR products were analyzed by gel
electrophoresis (2% agarose) and enzymatic digestion using standard
methods (17
, 18)
. For semiquantitative RT-PCR, linear
correlation of signal intensity for ß-actin was found between 24 and
27 cycles, and for the p75NTR between 30 and 35 cycles using a video
scanner system Scan Pack 2.0 (Biometra, Göttingen, Germany). This
allowed for the demonstration of clearly different signals, as compared
to control signals.
Immunohistochemistry
All antisera used are listed in Table 1
. For the double immunodetection of p75NTR-immunoreactivity (IR) on the
one hand, and TrkA-, TrkB-, or TrkC-IR on the other, the tyramide
amplification method was used (17
, 18)
. Briefly, after
blocking of endogenous peroxidase and nonspecific avidin/biotin
binding, sections were incubated in TNB buffer (0.1 M Tris-HCl, pH 7.5,
0.15 M NaCl, and 0.5% DuPont Blocking Reagent, DuPont NEN, Boston,
Mass.) for 30 min. Then rat monoclonal antibody against mouse p75NTR
was applied overnight (1:1000), followed by application of the
biotinylated goat anti-rat antiserum diluted in TNB blocking buffer
(DuPont NEN, 1:200, 30 min), and a tyramide amplification kit (DuPont
NEN). Subsequently, sections were incubated in streptavidin-horseradish
peroxidase (1:100 in TNB, 30 min). Three washes with TNT buffer (0.1 M
Tris-HCl, pH 7.6, 0.15M NaCl, 0.05% Tween) were followed by a 10 min
application of TRITC-tyramide (1:50 in Amplification Diluent, DuPont
NEN). After blocking nonspecific binding by 10% of normal goat serum,
sections were incubated with rabbit antisera against TrkA, TrkB, or
TrkC (1:50, overnight at room temperature), washed in TBS (3x5 min),
followed by incubation with Cy2-conjugated goat anti-rabbit secondary
antibody (Jackson ImmunoResearch, West Grove, Pa., 1:20, 37°C,
1 h). Finally, sections were washed three times with Tris buffer
and counterstained by Hoechst 33342 for identification of cell nuclei
(9
, 47)
.
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Double immunovisualization of p75NTR- and Bcl-2-IR was performed
as described previously (9)
, using rat monoclonal antibody
against p75NTR and hamster monoclonal antibody against murine Bcl-2
(see Table 1
). For all antisera, incubation of skin sections without
primary antisera and skin cryosections from p75NTR, TrkA, TrkB, and
TrkC knockout mice were used as negative control. In addition, for
antisera against Trk receptors, the preabsorption of primary antisera
with 100 µg/ml of corresponding antigenic peptides
(37°C, 60 min) was used as negative control. Cryostat
sections of the whole-mount mouse embryos at E18.5 with differential
distribution of p75NTR and Trk receptors served a positive control for
immunostaining. All sections were examined under a Zeiss Axioscope
microscope and photo-documented with the help of a digital image
analysis system (ISIS MetaSystem, Altlussheim, Germany).
Double immunodetection of TUNEL-positive cells and p75NTR-IR
To evaluate apoptotic cells, we used an established,
commercially available TUNEL kit (ApopTag, Oncor, Gaithersburg, Md.) as
described before (9
, 47)
. For double immunofluorescence
detection of TUNEL-positive cells and p75NTR-IR, sections were
incubated with digoxigenin-dUTP in the presence of TdT, followed by
incubation with rabbit-anti-TrkB antiserum. Subsequently,
TUNEL-positive cells were visualized by anti-digoxigenin
FITC-conjugated F(ab)2 fragments, p75NTR-IR was
detected by a tyramide amplification kit using biotinylated
goat-anti-rat antibody, streptavidin-horseradish peroxidase, and
TRITC-tyramide as described above, and sections were counterstained by
Hoechst 33342 (9
, 47)
. Negative controls for the TUNEL
staining were made by omitting TdT, according to the manufacturer’s
protocol. Positive TUNEL controls were run, as described
(47)
, by comparison with tissue sections from the thymus
of young mice, which display a high degree of spontaneous thymocyte
apoptosis (9).
Skin organ culture
Four millimeter punch biopsies were prepared under sterile
conditions from either neonatal C57BL/6 mice or neonatal p75NTR
knockout mice at P17, i.e., at the beginning of the anagen–catagen
transformation of the HF (48)
, following previously
described protocols (49
, 50)
with some modifications.
Eight to 10 randomized skin punches derived from the back skin of three
different mice per experimental group were placed (dermis down) on
gelatin sponges (Gelfoam, Upjohn Co., Kalamazoo, Mich.) in 35 mm Petri
dishes containing 5 ml Dulbecco’s modified Eagle’s medium, 10% fetal
bovine serum, 50 mg/ml L-glutamine, and antibiotic/antimycotic mixture
(Life Technologies, Inc.). After addition of 250 ng/ml of mouse 2.5S
NGF (Boehringer Mannheim, Mannheim, Germany), 50 ng/ml of human
recombinant NT-3 (Promega, Madison, Wis.), or 5 ng/ml of human
recombinant BDNF (Promega), skin fragments were incubated at the
air-liquid interphase for 48 h at 37°C, in 5%
CO2 and 100% humidity. At the end of incubation,
all skin fragments were washed repeatedly in phosphate-buffered saline
buffer at 4°C, fixed in 4% paraformaldehyde, and embedded in
paraffin for routine histology and histomorphometry.
Histomorphometry and statistical analysis
In adolescent skin, IR patterns were scrutinized by studying at
least 50 different HF per mouse; five mice were assessed per hair cycle
stage. For each stage of HF cycling, the major IR patterns were
recorded in previously prepared, computer-generated schematic
representations of murine HF cycling, which allow a standardized,
easily reproducible, and systematic comparison of different follicular
IR patterns (14)
. For precise identification of the
defined stages of HF cycling, histochemical detection of endogenous
alkaline phosphatase activity was used as described, since this allows
one to visualize the morphology of the dermal papilla as a useful
morphological marker for staging HF cycling (51)
.
The percentage of HF in different stages of growth (anagen),
regression (catagen), or resting (telogen) was assessed and calculated
in p75NTR knockout (-/-) and NGF-overexpressing mice at P14-P20,
respectively, as well as in their corresponding age-matched wild-type
mice. During these days of postnatal development, the hair follicle,
after completion of morphogenesis, begins its life-long cycle of
regression, resting, and growth by spontaneous entry into the first
catagen stage (2
, 4)
. In normally cycling mice this occurs
around P17, and at P20–22 all hair follicles in back skin reach the
resting (telogen). All evaluations were performed on the basis of
accepted morphological criteria of HF classification (1
2
3
4
, 6
, 52)
. Only every tenth cryosection was used for analysis in order
to exclude the repetitive evaluation of the same HF, and 2–3
cryosections were assessed from each animal. All together, 200–350
follicles in 50–60 microscopic fields, derived from four to seven
animals (
50–60 follicles per animal) of distinct age, were analyzed
and compared to that of a corresponding number of HF from the
appropriate, age-matched wild-type mice. In skin organ culture, the
percentage of HF in defined catagen stages was calculated in 8–10
biopsies per group at magnification x400 under a Zeiss Axioscope
microscope, following accepted morphological criteria of classifying
defined stages of catagen development (6
, 41)
.
For quantitative assessment of the dynamics of catagen development (D)
during in vivo and in situ experiments, a
modified formula, described previously (53)
, was used:
D = CIx1 + CIIx2 + CIIIx3 + CIVx4 + CVx5 + CVIx6 + CVIIx7 +
CVIIIx8, where CI-CVIII = percentage of HF at distinct catagen
stages. Then the percentage of the acceleration or retardation of
catagen development was evaluated, taking a D value from the
corresponding control experiment as 100%.
The distance between the stratum corneum and the subcutis/panniculus carnosus border was measured for assessing the skin thickness in p75NTR knockout, NGF-overexpressing, and corresponding wild-type animals. In total, 40–50 such measurements were performed in 50–60 microscopic fields derived from three to five animals per mutant and wild-type group.
The number of TUNEL-positive cells was assessed in catagen VI-VII HF of p75NTR knockout, NGF-overexpressing, and corresponding wild-type animals at P17-P20. In total, 40–50 such measurements were performed in 50–60 microscopic fields derived from three to five animals per mutant and wild-type group. All sections were analyzed at x200–400 magnification, and means and SE were calculated from pooled data. Differences were judged as significant if the P value was lower than 0.05, as determined by the independent Student’s t test for unpaired samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. High steady-state
levels of p75NTR transcripts were detected in skin with all HF at the
anagen VI stage (Fig. 1
). Although during spontaneously developed regression between days 17
and 18 after depilation the p75NTR mRNA PCR-product was reduced vs.
anagen VI levels, steady-state levels of p75NTR mRNA were clearly
present in catagen skin (Fig. 1)
.
|
To define those cell populations in the catagen HF that express p75NTR
alone and therefore represent potential targets for the p75NTR-mediated
cell death cf. (22
, 37
, 38
, 54)
, coexpression of p75NTR
and Trk receptors was studied during spontaneous anagen–catagen
transformation in the back skin of adolescent C57BL/6 mice at days
12–19 of induced hair cycle (40)
, using a double
immunovisualization technique. p75NTR-, TrkA-, TrkB-, and TrkC-IR
patterns were each assessed with two distinct antisera (Table 1)
, since
antibody specificity in discriminating between neurotrophin receptors
may be unsatisfactory. However, both antisera against every distinct
receptor gave highly similar results in all staining protocols, as well
as in the positive and negative controls (not shown). For more
extensive analyses, a rat monoclonal antibody against murine p75NTR and
antisera against Trk receptors whose expression patterns in murine skin
were proven previously by in situ hybridization (indicated
by asterisks in Table 1
(18
, 55)
were selected, because
they provided the best signal/noise ratio. The observed expression and
coexpression patterns of p75NTR- and Trk receptor-IR are documented by
the representative examples in Fig. 2
and are schematically summarized in Fig. 3
.
|
|
In general, the follicular patterns of expression for p75NTR, TrkA,
TrkB, and TrkC receptors during anagen–catagen transformation defined
here are in line with those described in our previous studies (9
, 17
, 18
, 56)
. We found that in anagen VI HF (day 12 p.d.),
p75NTR-IR was expressed by KC of the proximal and central outer root
sheath (ORS), where its coexpression with TrkA-, TrkB-, and TrkC-IR was
seen (Fig. 2A
, B
, C
, Fig. 3
). However, using a more
sensitive immunodetection assay than before (9)
, the
expression of p75NTR-IR was also detected among the melanin granules in
the keratogenous zone of the hair matrix, just above and around the
most distal part of the dermal papilla (Fig. 2A
, B
, C
). Here, a colocalization of p75NTR- and
TrkA/TrkB-IR was seen (Fig. 2A
). No isolated p75NTR-IR
alone, i.e., without its coexpression with Trk receptors, was observed
in the anagen VI HF, whereas all Trk receptors were expressed in the
follicular inner root sheath (IRS) and hair matrix; relatively weak
TrkB- and TrkC-IR expression was detected in the dermal papilla (Fig. 2A
, B
, C
).
During the onset of HF regression (catagen II, day 17 p.d.), the
coexpression of p75NTR- and TrkA-, TrkB-, or TrkC-IR was still visible
in the central and proximal ORS; also, coexpression of p75NTR- and
TrkA-IR was seen in the keratogenous zone of the hair matrix (Fig. 2D
, E
, F
, Fig. 3
). In addition, expression of all
Trk receptors was observed in the IRS and hair matrix, and weak TrkB-IR
was detected in the dermal papilla (Fig. 2D
, E
, F
).
During further progression of catagen development (catagen IV-VI, days
18–19 p.d.), disappearance of TrkA and TrkB receptors and a
substantial decline of TrkC were observed in the regressing ORS, where
prominent p75NTR-IR, expressed alone, was still visible (Fig. 2G
, H
, I
, Fig. 3
). However, coexpression of p75NTR-
and TrkB-/TrkC-IR was found in the secondary hair germ of the catagen
VI HF (Fig. 2H
, 2I
). No p75NTR-IR was observed in
the epithelial strand, whereas prominent TrkA-IR expression was seen in
single cells located there (Fig. 2G
). TrkB- and TrkC-IR
expressed alone were found in the epithelial strand, secondary hair
germ, and IRS of the catagen VI HF (Fig. 2H
, 2I
).
To define whether or not cells of the regressing HF compartments that
express p75NTR are colocalized with apoptotic markers during catagen, a
double visualization of p75NTR-IR and TUNEL or p75NTR and Bcl-2 was
performed. Only cells in the regressing ORS expressed p75NTR alone in
catagen VI HF and showed colocalization of p75NTR and TUNEL (Fig. 2J
); no coexpression of p75NTR- and Bcl-2-IR was
seen. In the secondary hair germ, where p75NTR was coexpressed with
TrkB and TrkC (Fig. 2H
, 2I
; Fig. 3
),
colocalization of p75NTR- and Bcl-2-IR was found (Fig. 2L
)
and no coexpression of p75NTR and TUNEL was seen (Fig. 2K
).
Strong expression of Bcl-2-IR was observed in the dermal papilla,
whereas numerous TUNEL-positive cells were visible in the epithelial
strand, an HF compartment that showed no p75NTR-IR and only relatively
weak Bcl-2-IR during catagen (Fig. 2K
, 2L
).
Taken together, these morphological data suggest that p75NTR-related
signaling plays a role in the control of apoptosis-driven HF
regression, consistent with recent reports that NGF and BDNF can induce
apoptosis via p75NTR in other model systems (23
24
25
, 28)
.
Deletion of p75NTR leads to the retardation of hair follicle
regression
To determine whether p75NTR deletion alters spontaneous catagen
development, adolescent p75NTR knockout (-/-) mice (42)
were compared for the speed of spontaneous HF regression after hair
cycle induction by depilation, using age-matched wild-type mice as a
controls. In addition, the dynamics of the first catagen development
was assessed in mutant and wild-type mice at P18-P20, as described
previously (17
, 18)
.
During the depilation-induced hair cycle, p75NTR knockout mice showed a
significant (P<0.01) retardation of catagen development by
42.2% compared to wild-type controls. At day 19 postdepilation, more
than half of the HF in p75NTR null skin were still in catagen II-IV,
whereas exclusively catagen VI-VIII HF were seen in wild-type skin
(Fig. 4A
, C
, D
). In addition, skin thickness,
an important indicator of advanced catagen development (57
, 58)
, was significantly reduced (P<0.001) in
wild-type mice compared to p75NTR mutants (Fig. 4B
).
|
Similar differences in the dynamics of catagen development were found
between neonatal p75NTR knockout and wild-type mice at P18-P20 (data
not shown). Catagen VI HF in neonatal p75NTR knockouts, compared to
their age-matched wild-type mice, had a significantly reduced
(P<0.05) number of TUNEL-positive cells in the regressing
ORS (Fig. 4D
, E
, F
), whereas the number of
TUNEL-positive cells in the epithelial strand and IRS appeared to be
unaltered (Fig. 4G
, 4H
).
This suggests that deletion of p75NTR is associated with a retardation
of catagen development and a reduction of cells undergoing apoptosis in
the regressing ORS. This is the same compartment in which p75NTR
colocalizes with TUNEL and is not coexpressed with Trk receptors or
Bcl-2 during late catagen (Fig. 2G
, H
, I
, J
, L
).
Overexpression of NGF is associated with acceleration of catagen
development
NGF was the first member of the neurotrophin family described to
induce apoptosis via interaction with p75NTR (23
24
25)
. We
therefore tried to determine whether constitutive NGF overexpression
alters spontaneous catagen development. Neonatal transgenic mice with
NGF overexpression were compared to age-matched wild-type mice with
regard to the onset and speed of spontaneous HF regression.
NGF-overexpressing mice were generated using K14 promoter
(43)
. This is an attractive model for studying the
influence of NGF on the anagen–catagen transformation, since K14
targets NGF expression to the follicular ORS KC as well as to KC of the
epithelial strand, secondary hair germ, and the base of the club hair
(during catagen) (59
60
61)
.
After completion of HF morphogenesis, the HF begins its life-long cycle
of regression, resting, and growth by spontaneous entry into the first
catagen stage (2
3
4)
. In normally cycling mice, this
occurs around the postnatal day 17 (P17), and by P20-P22 almost all HF
are already in the resting stage of the cycle (telogen). At P14,
80–90% of HF in the skin of NGF-overexpressing mice already displayed
the morphological characteristics of catagen II follicles, whereas
exclusively anagen VI HF were found in the skin of wild-type controls
at this time (Fig. 5A
, B
). At P17, wild-type control mice still showed
no HF in late catagen (stages VII-VIII) (Fig. 5C
, 5D
), whereas the HF of NGF-overexpressing mice were already
almost all in late catagen (VI-VIII) stages (Fig. 5C
, 5E
) and showed acceleration of catagen development by 66.2%
compared to wild-type mice. At P20, only telogen HF were found in the
skin of NGF-overexpressing mice, whereas 10–20% of follicles in
wild-type mice were still in catagen VII-VIII (not shown).
|
In addition, compared to wild-type mice, the skin thickness was
significantly (P<0.01) lower in NGF-overexpressing mice at
P17 (Fig. 5D
, E
, F
). Since murine skin thickness is
strictly coupled to synchronized HF cycling and since catagen and
telogen skin is much thinner than anagen skin (57
, 58)
,
this indicated that the catagen-telogen HF transition in
NGF-overexpressing mice had almost been completed at this time point.
Furthermore, the number of TUNEL-positive cells in the regressing ORS
of the catagen VII HF was significantly enhanced in NGF transgenics
compared to wild-type HF (Fig. 5G
, H
, I
).
This suggests that intraepithelial overexpression of the p75NTR ligand
NGF accelerates the initiation and/or development of catagen.
Neurotrophins fail to stimulate spontaneous catagen development in
organ-cultured p75NTR null skin
Taking into consideration that alterations in HF catagen
development observed in p75NTR knockout mice and in NGF transgenics
might be connected to the consequences of p75NTR deletion or NGF
hyperexpression, such as differences in the skin and hair follicle
innervation (42
, 43
, 62
63
64)
, the effects of neurotrophins
on the anagen–catagen transformation in organ culture (i.e., in the
absence of functional skin nerves) were compared between wild-type and
p75NTR null skin. Organ culture of murine back skin was selected as an
established model for studying of neurotrophin action on hair follicle
regression in vitro in the absence of functional skin nerves
(17
, 18
, 42
, 50)
. NGF, BDNF, or NT-3 were added to
organ-cultured skin with most HF in the process of initiating the
anagen VI–catagen transformation of the hair cycle. For this purpose,
biopsies were taken from neonatal wild-type or p75NTR null mice at P17
(48)
and cultured at the air-liquid interphase on gelatin
gels (49
, 50)
for 48 h in the presence of those
concentrations of BDNF or NT-3 that showed maximal catagen-promoting
effects in the previous studies (17
, 18)
or in the
presence of 250 ng/ml of NGF.
We found that all neurotrophins tested significantly promoted
spontaneous catagen development in the organ-cultured biopsies of the
neonatal wild-type skin (Fig. 6A
) compared to the corresponding vehicle control. This was in
line with our data obtained previously on adolescent mouse skin during
the depilation-induced hair cycle (17
, 18)
. However, NGF,
BDNF, and NT-3 failed to induce significant acceleration of HF catagen
development in the biopsies of p75NTR null skin compared to vehicle
controls (Fig. 6B
). In contrast to wild-type skin, no
significant differences in the percentage of HF at distinct catagen
stages were seen between biopsies treated with NGF, NT-3, or BDNF and
control biopsies taken from p75NTR knockout mice (Fig. 6A
, 6B
). However, BDNF showed a slight retardation of catagen by
11.2% in p75NTR null skin, indicating the potential to suppress
catagen even in the absence of p75NTR. Instead, NT-3 showed a
relatively weak ability to promote catagen by 6.3% in p75NTR knockout
skin organ culture, hinting at the possibility of indirect pathways in
the NT-3-induced acceleration of catagen (Fig. 6B
).
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
3
4)
. Apoptosis is an essential component of
HF regression (4)
, and dysregulation of apoptosis during
catagen leads to alopecia or baldness in experimental and natural
models (2
, 9
, 65
66
67
68)
. Each anatomic compartment in the
cycling portion of HF is characterized during catagen by distinct
expression patterns of growth factor receptors implicated in apoptotic
cell death (9)
. These findings suggest distinct mechanisms
of apoptosis regulation in every HF compartment.
We show by multiple criteria that p75NTR signaling is involved in
apoptosis in the regressing follicular ORS during catagen: high
steady-state levels of p75NTR mRNA are present in skin during the
anagen–catagen transition of the HF (Fig. 1)
; p75NTR is not
coexpressed with Trk receptors or Bcl-2, but instead colocalizes with
TUNEL (Fig. 2G
, H
, I
, J
; Fig. 3
); p75NTR knockout mice
have retarded catagen development and a reduced number of apoptotic
cells in the regressing ORS (Fig. 4)
; overexpression of the prototypic
p75NTR ligand, NGF, in the follicular ORS is associated with
acceleration of catagen (Fig. 5)
; and in contrast to wild-type skin,
neurotrophins fail to promote catagen in the organ-cultured skin of
p75NTR null mice (Fig. 6)
.
Neurotrophins have been reported to induce apoptosis only in cells that
express p75NTR alone and do not coexpress Trk receptors (29
, 35
36
37
38
39)
. Consistent with these reports, we observed a
p75NTR-positive and Trk-negative cell population in the follicular ORS
as a potential target for p75NTR-mediated cell death. Indeed, only in
the ORS did p75NTR-positive cells coexpress TUNEL, indicative of
apoptosis, whereas p75NTR- and TrkB/TrkC-double-positive cells in the
secondary hair germ were TUNEL negative, yet positive for Bcl-2, an
anti-apoptotic protein (Fig. 2J
, K
, L
). NGF, NT-3,
and BDNF are also prominently expressed in the regressing ORS during
catagen (17
, 18
, 56)
and thus may induce apoptosis in
cells expressing p75NTR in paracrine/autocrine manner.
Participation of the p75NTR receptor in apoptosis does not exclude its
simultaneous participation in other HF cells in Trk receptor signaling,
such as in the secondary hair germ leading to neurotrophin-mediated
cell survival. Indeed, in the secondary hair germ, the HF compartment
characterized by predominance of Bcl-2 over Bax during catagen
(9)
, p75NTR colocalizes with TrkB/TrkC receptors and Bcl-2
(Fig. 2H
, 2I
, 2L
), but not with TUNEL
(Fig. 2K
).
Since deletion of p75NTR in vivo leads to the retardation of
HF regression (Fig. 4)
, this suggests that proapoptotic signaling
through this receptor predominates over any possible survival effects
during catagen. The abolishment of p75NTR signaling more effectively
retards the late steps of catagen development than early catagen
stages. This is consistent with the disappearance of Trk receptors from
the regressing ORS during late catagen (Fig. 2)
rendering cells
vulnerable to activation of p75NTR alone and hence to proapoptotic
signaling through p75NTR.
The precise molecular mechanisms of the control of Trk- and p75NTR gene
and protein expression, and therefore their ratio, during hair follicle
involution remain to be elucidated. However, it was shown in other
models that the expression of Trk receptors and p75NTR is specifically
controlled by Brn-3a and Sp-1 transcription factors, respectively
(69
, 70)
. Recently we demonstrated the up-regulation of
p75NTR during hair follicle morphogenesis under constitutive deletion
of noggin, an antagonist of bone morphogenetic proteins 2 and 4
(71)
. This indicates that the upstream effectors of Brn-3a
and Sp-1 transcription factors, as well as bone morphogenetic proteins
2/4, might serve as a candidate molecules controlling Trk/p75NTR ratio
during the anagen–catagen transition of the HF.
We show here that overexpression of NGF levels in vivo
accelerates HF regression (Fig. 5)
, which is in line with previous
observations that of catagen is stimulated in NT-3 and BDNF transgenic
mice (17
, 18)
. Although NGF transgenic mice show
substantial alterations in cutaneous innervation (43)
, our
data demonstrating enhanced HF regression in skin organ cultures
lacking functional skin nerves (Fig. 6)
suggest that catagen
stimulatory action of NGF is largely nerve independent.
Compared to other neurotrophins, NGF promoted HF catagen development in
organ culture only in relatively high concentrations (Fig. 6)
. One
possible explanation for this disparity is the greater affinity of NGF
for TrkA. If, compared to NT-3 and BDNF, NGF preferentially bound
p75NTR in a coordinate manner with TrkA receptors, the effects of NGF
would favor survival of cells expressing both receptors rather than
apoptosis of cells expressing p75NTR alone. Indeed, the capacity of NGF
to suppress KC apoptosis via TrkA stimulation has been shown previously
(72)
.
Since the ability of NGF to stimulate apoptotic cell death via
interaction with TrkA was also shown previously (73
, 74)
and neurotrophins could theoretically promote catagen via stimulation
of Trk receptors abundantly expressed in the regressing HF compartments
during catagen (17
, 18)
, followed by the release of other
proapoptotic molecules like TNF-alpha, Fas-ligand, or TGF-ß from
Trk-positive cell populations, one can also envision p75NTR-independent
pathways in the catagen stimulatory action of neurotrophins. However,
our data that NGF, BDNF, or NT-3 fail to stimulate HF regression in
p75NTR null skin (Fig. 6)
clearly suggest an involvement of p75NTR in
the realization of the catagen stimulatory activity of neurotrophins.
Only slight catagen stimulatory effects of NT-3 were observed in
organ-cultured p75NTR null skin (Fig. 6)
. This suggests that
p75NTR-independent pathways appear to be of relatively little
importance compared to direct effects of neurotrophins in p75NTR.
In additional experiments, we have observed that blockade of p75NTR signaling by a synthetic antagonist significantly retards HF catagen development in organ culture of C57BL/6 mouse skin and blocks catagen stimulatory effects of neurotrophins. Furthermore, the p75NTR antagonist failed to retard catagen in the organ-cultured skin of p75NTR knockout mice (Botchkarev et al., unpublished results), which is consistent with the data presented here.
Taken together, our data suggest that cell death during catagen is regulated differentially in every distinct HF compartment and that p75NTR signaling is critically important for apoptosis in the regressing ORS and, therefore, for its shortening during catagen. This raises the possibility of pharmacological manipulation of p75NTR signaling in the treatment of hair disorders that display premature entry into catagen such as telogen effluvium, androgenetic alopecia, and alopecia areata (2).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication November 18, 1999.
Revision received March 30, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
epidermal and upper dermal innervation on neurotrophins, trk receptors and p75 LNGFR. Dev. Biol. 198,57-81[Medline]
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