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* Department of Dermatology, Charité, Humboldt-University, Berlin, Germany;
Research Laboratories of Schering AG, Berlin, Germany;
Growth Factors Groups, Department of Oncology, MRC Centre, Cambridge, U.K.; and
§ Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892-4255, USA; and
¶ Department of Dermatology, University Hospital Eppendorf, University of Hamburg, D-20246, Hamburg, Germany
2Correspondence: Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Martinistr. 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: apoptosis • hair growth • HGF/SF • keratinocytes • murine hepatocyte growth factor receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
.
HGF/SF is a pleiotropic factor isolated independently as a
growth-promoting agent for liver cells (4)
and as a
fibroblast-derived protein leading to dissociation and motility of
epithelial cells (scatter factor) (5
, 6)
. The factor is
synthesized as a 728 amino acid precursor that is processed by
different proteases—e.g., HGF activator, tPA, or urokinase—to
generate the mature growth factor consisting of a disulfide-linked 69
kDa 
chain and a 34 kDa ß chain (1
, 2)
.
Physiologically, HGF/SF has been described as a mesenchymally derived,
paracrine regulator of epithelial cells expressing the Met receptor.
However, autocrine activation of HGF/SF promotes tumorgenesis as
demonstrated by the fact that transgenic (TG) mice overexpressing
HGF/SF develop a remarkably broad array of histologically distinct
tumors of both mesenchymal and epithelial origin (7)
.
Recently, HGF/SF has been implicated in hair growth control, a
prototypical epithelial–mesenchymal interaction system. One intriguing
feature of the hair follicle (HF) is that after a prolonged period of
growth (anagen), it spontaneously enters into a phase of rapid,
apoptosis-driven, organ involution (catagen) until the follicle
reenters anagen via an interspersed resting phase (telogen)
(8
9
10)
. This process is controlled by a stringent cross
talk between mesenchymally derived dermal papilla (DP) fibroblasts and
epithelial keratinocytes. No other mammalian organ rhythmically
undergoes, during the entire life span of the organism, such dramatic,
but physiological, apoptosis and a rapid reconstruction of an entire
organ by remodeling the architecture of the follicle epithelium
(8
, 10
, 11)
.
Several families of signaling molecules have recently been implicated
in the control of HF development. After formation of the hair bud prior
to birth in sonic hedgehog (Shh) knockout mice, HF development appears
to be arrested due to a disrupted epithelial/mesenchymal communication
(12)
. In TG mice overexpressing constitutively active
ß-catenin, an effector of intercellular adhesion signaling, de
novo HF development occurs (13)
. Yet the exact role
in physiological HF induction remains to be clarified. However, HGF/SF
stimulation increases the synthetic rate of ß-catenin
(14)
and leads to nuclear accumulation of ß-catenin
(15)
in epithelial cells in vitro.
Hair follicle morphogenesis and the periodical growth and
regression of murine HF critically depend on precisely organized
mesenchymal–epithelial signaling (8
, 10
, 16
, 17)
. In this
context, HGF/SF and its receptor (Met) are of significance to hair
biologists, since this signaling system has been shown to stimulate
growth and motility of keratinocytes (18
19
20)
, to promote
melanogenesis of human melanocytes (19)
, is involved in
the control of angiogenesis in vivo (21
22
23
24)
,
and has the ability to dissociate epithelial cells by modulating the
expression of cell adhesion molecules such as cadherins (3
, 25
, 26)
.
Cultured dermal papilla cells of the HF were described to express
HGF/SF, which stimulates growth of human HF in vitro
(27)
. In addition, HGF/SF has been reported to stimulate
the growth of mouse vibrissae follicles in vitro
(20)
. A dose-dependent increase of DNA and protein
synthesis as well as an elongation of hair shafts could be measured by
adding human-derived HGF/SF to the culture medium (20)
.
The same group reported that human recombinant HGF/SF can delay murine
HF regression and promote the growth of pelage HF in vivo
(28)
, suggesting that exogenous HGF/SF can manipulate HF
cycling in mice.
We, therefore have explored the question of whether endogenous HGF/SF
and Met are involved in the regulation of hair follicle morphogenesis
and cycling (growth, regression, resting) using the C57BL/6 mouse model
(9
, 29
30
31
32
33
34
35)
. The immunohistological expression patterns of
HGF/SF and Met were characterized during all stages of HF morphogenesis
and cycling. This was complemented by an analysis of HGF/SF and Met
gene expression during synchronized HF cycling (semiquantitative
reverse transcriptase-polymerase chain reaction, or RT-PCR).
Furthermore, recombinant murine HGF/SF was administered to murine skin
organ culture and in vivo (34
, 35)
in order to
test its effects on spontaneous HF regression (catagen development).
Finally, the characteristics of HF morphogenesis and cycling were
compared by quantitative histomorphometry (12
, 32
, 35)
between wild-type (WT) and TG mice overexpressing HGF/SF (7
, 36)
. The results of these experiments demonstrate a major role
for HGF/SF and Met in murine HF morphogenesis and cycling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 32)
. After 17–19
days, these depilation-induced anagen follicles enter spontaneously
into catagen, as can be appreciated from the conversion of their skin
color from black to gray, and finally to pink hair shaft plucking. All
stages of the anagen/catagen/telogen transformation of the murine hair
cycle were studied (classification according to ref 29
).
For analysis of HF morphogenesis, embryonic and neonatal skin was
harvested at various time points of estimated gestational age
(embryonic days E12.5, 16.5, 17.5, and 18.5; E0.5 = the morning on
which a vaginal plug is found) or postnatal age (postnatal days
P0–P19) (16)
. Most non-tylotrich pelage HF in murine back
skin develop perinatally; even 1 day after birth (P1), all stages of HF
morphogenesis can still be found in mouse skin (33
, 37)
.
Stages of HF morphogenesis were assessed by morphological criteria, as
described in detail elsewhere (38)
.
The neck region of murine back skin was harvested parallel to the
vertebral line to obtain longitudinal sections through the hair
follicles (38
, 39)
The skin was deep frozen in liquid
nitrogen, covered with embedding medium, and processed for
immunohistochemistry and TUNEL staining (TdT-mediated dUTP-digoxigenin
nick end-labeling) as described below.
Transgenic mice
Inactivation of the HGF/SF gene (40)
and that of
met (41)
cause embryonic lethality in mice between E12.5
and E16.5, around the time when HF formation is initiated. This renders
conventional knockout mice unsuitable for studies of HF morphogenesis
and cycling. Therefore, the skin of HGF/SF overexpressing TG mice was
analyzed. These TG mice were generated by inserting a mouse HGF/SF cDNA
under the control of the mouse metallothionein (MT-I) promoter
(42)
, thus driving strong HGF/SF expression within the
skin (7)
. TG mice express a characteristic 2.4 kb RNA in
virtually all adult tissues at a level between 3- to 50-fold higher
than the major 6 kb endogenous HGF/SF transcript (43)
.
Mice were housed in the animal facilities of the National Cancer
Institute, Bethesda, Md. Neonatal skin fragments from three to five
different TG and age-matched WT mice as controls (postnatal days 3 and
17) were washed repeatedly in phosphate-buffered saline (PBS)
buffer, fixed in 4% paraformaldehyde, and embedded in
paraffin for routine histology (hematoxilin/eosin staining) and
quantitative histomorphometry (32
, 35
, 38)
.
RT-PCR
Steady-state levels of skin HGF/SF and Met gene expression were
studied by semiquantitative RT-PCR (35
, 44
, 45)
. Total RNA
was isolated from full-thickness back skin samples (homogenized in
liquid nitrogen), using a single-step guanidine
thiocyanate-phenol-chlorophorm method (RNeasy total RNA kit, Quiagen,
Hilden, Germany). Skin samples included the subcutaneous (s.c.)
skeletal muscle layer (panniculus carnosus). cDNA was synthesized by
reverse transcription of 3 µg total RNA, using a cDNA synthesis kit
(Invitrogen, San Diego, Calif.) (in the case of HGF/SF, the amount of
cDNA used for RT-PCR was 10-fold higher). The following sets of
oligonucleotide primers (TIP Molbiol, Berlin, Germany) 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' (45)
; HGF/SF:
5'-TT GGC CAT GAA TTT GAC CTC-3' 5'-AC ATC AGT CTC ATT CAC AGC-3'
(46)
; and Met: 5'-GAA TGT CGT CCT ACA CGG CC-3' and 5'-CAG
GGG CAT TTC CAT GTA GG-3' (46)
.
Amplification was performed using Taq polymerase (Life
Technologies, Inc., Grand Island, N.Y.) over 30 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) (44
, 45)
. For semiquantitative RT-PCR, linear correlation of signal
intensity for ß-actin was found between 24 and 27 cycles, and for the
other markers, between 30 and 35 cycles using computer-assisted video
scanner densitometry (ScanPack 2.0; Biometra, Göttingen,
Germany). This allowed relative comparison of the signals from
different samples.
Photographs and graphical presentations represent the results of three independent experiments derived from three different animals.
Immunohistochemistry
For simultaneous immunodetection of HGF/SF and Met within murine
skin, cryosections were processed by combining standardized
immunofluorescence (IF) labeling methods (9)
. Cryostat
sections (10 µm) were fixed in acetone (10 min at -20°C) and
preincubated with 10% donkey normal serum, followed by an incubation
with the primary sheep anti-mouse HGF/SF antibody overnight at 4°C
obtained after repeated immunizations with purified recombinant HGF/SF.
Sections were then incubated with FITC-conjugated donkey anti-sheep
secondary antibody (1:200, Jackson Laboratories, West Grove, Pa.) for
30 min at room temperature.
In the second part of this double-staining protocol, sections were blocked with 10% goat normal serum in PBS for 10 min at room temperature and incubated overnight with rabbit anti-Met antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) at 4°C. The secondary antibody (goat anti-rabbit TRITC-conjugated F(ab)2 fragments, 1:200, Jackson Laboratories) was incubated 30 min at room temperature.
Nuclei were covisualized by HOECHST 33342 (Sigma, Deisenhofen, Germany). Each step (except after blocking) was interspersed by washing with PBS. Finally, slides were mounted with VectorShield (Vector Laboratories, Burlingame, Calif.). Positive staining for HGF/SF and Met was identified by green and red fluorescence, respectively. As a negative control for Met, neutralizing peptide (1:50 in PBS; Santa Cruz Biotechnologies) was preincubated with the anti-Met antibody for 1 h at room temperature.
The distribution and intensity of HGF/SF- and Met-IR
(Met-immunoreactivity/immunoreactive) cells of at least 50 different HF
per mouse (n=5), were determined with a fluorescence
microscope (Zeiss, Jena, Germany) at magnifications from 100 to 400x.
All specific and reproducible IR-patterns were recorded and summarized
in computer-generated schematic representations (Designer 4.0,
Micrografx) designed to reflect the key features of murine hair
follicle anatomy and their changes during hair follicle development and
cycling as accurately as possible (33
, 38)
.
Photomicrographs were processed using a digital image analysis system
(ISIS Metasystems, Altusheim, Germany).
Combined immunolabeling with TUNEL/Hoechst 33242 staining
To evaluate apoptotic cells in colocalization with the IR
pattern of the Met receptor or Ki67, a key marker for proliferating
cells, we used a previously described combined TUNEL/Hoechst
33342/antibody triple-staining method (9
, 47)
. In brief,
10 µm cryostat sections of C57BL/6 back skin were freshly prepared
and fixed in formalin, postfixed in ethanol/acetic acid, and incubated
with digoxigenin-dUTP in the presence of TdT, followed by incubation
with either rabbit anti-Met (Santa Cruz Biotechnology) or rabbit
anti-Ki67 (Dianova, Hamburg, Germany) antiserum, respectively.
Subsequently, TUNEL-positive cells were visualized by anti-digoxigenin
FITC-conjugated F(ab)2 fragments, whereas Met- or
Ki67-IR was detected by a TRITC-labeled goat-anti-rabbit antibody
(Jackson Laboratories, West Grove, Pa.). Counterstaining with
HOECHST 33342 dye (10 µg ml-1 in PBS, Sigma, Deisenhofen,
Germany) was performed by a subsequent incubation step. Finally,
sections were mounted using VectaShield (Vector Laboratories). Positive
TUNEL controls were run, as described (48)
, by comparison
with tissue sections from the thymus of young mice, which display a
high degree of spontaneous thymocyte apoptosis. Negative controls for
the TUNEL staining were made by omitting terminal desoxynucleotidyl
transferase (TdT), according to the manufacturer’s protocol. Sections
were then examined under a Zeiss Axioscope microscope (Jena, Germany),
using the appropriate excitation-emission filter systems for studying
the fluorescence, induced by Hoechst 33342, FITC, or TRITC.
Photodocumentation was done with the help of a digital image analysis
system (ISIS Metasystems)
In vivo HGF/SF injection
Recombinant HGF/SF was produced in the NS0 mouse myeloma line
transfected with a full length mouse HGF/SF cDNA and purified from
culture supernatants with a combination of heparin-Sepharose6CL and
Mono S column chromatography (Amersham Pharmacia; M. Sharpe and E.
Gherardi, unpublished results).
To check the effect of HGF/SF on spontaneous catagen development (cf.
refs 35
, 47
), the dorsal back skin of 14-day-old female
C57BL/6 young mice (n=5) with all HF in the final stage of
HF morphogenesis was injected intradermally with recombinant HGF/SF 8
µg/20 g body weight in 100 µl PBS (0.1% bovine serum albumin, or
BSA) with consecutive injections on days 15 and 16. The animals were
killed at day 17 p.d. (postdepilation: day after anagen induction
by depilation). Note that around P17, murine pelage HF synchronously
enter into HF cycling by entry into the first spontaneous catagen
(16
, 33
, 38)
. In addition, 17-day-old mice
(n=5) with all HF in the anagen/catagen transformation were
injected intradermally with 1 µg/20 g body weight recombinant HGF/SF
in 100 µl PBS (0.1% BSA) with a consecutive injection at day 18 p.d. The mice were killed on day 19 p.d. All control mice were
injected with PBS (0.1% BSA) alone.
In both experiments, the neck region around the injection site of the dorsal skin was harvested and processed for subsequent TUNEL/Hoechst 33342/Ki67 staining as described above.
To demonstrate the concentration and gradient-dependent catagen retardation in HGF/SF-treated skin, four consecutive microscopic fields of a single representative skin section were taken and merged using a computer-aided digital image analysis system (ISIS Metasystems).
Skin organ culture
Four millimeter punch biopsies were prepared under sterile
conditions from adolescent C57BL/6 mouse back skin with all HF in the
late anagen VI or early catagen stage of the induced hair cycle (i.e.,
day 17 after depilation; ref 29
), following previously
described protocols (31
, 34
, 35
, 47
, 49)
with some
modifications. For each experimental group, eight to ten randomized
skin punches derived from the back skin of three different mice were
placed (dermis down) on gelatin sponges (Gelfoam, Upjohn Co.,
Kalamazoo, Mich.) in 35 mm petri dishes, containing 5 ml Williams E
supplemented with 50 mg/ml L-glutamine, 1% antibiotic/antimycotic
mixture (Life Technologies), 0.1% hydrocortisone, and 0.25% insulin.
After addition of 10 ng/ml recombinant mouse HGF/SF, organ cultures
were incubated at the air-liquid interphase for 48 h at 37°C in
5% CO2 and 100% humidity, with one change of
medium and the appropriate amount of growth factor after 24 h. At
the end of incubation, all skin fragments were washed repeatedly in
PBS, fixed in 4% paraformaldehyde, and embedded in paraffin for
routine histology and histomorphometry.
Quantitative histomorphometry and statistical analysis
IR patterns were scrutinized by studying at least 50
different HF per mouse and five mice were assessed per stage. For each
stage of HF morphogenesis and cycling, the major IR patterns were
recorded in previously prepared, computer-generated schematic
representations of murine HF morphogenesis and cycling, which allow a
standardized, easily reproducible, and systematic comparison of
different follicular IR patterns (33
, 38)
.
The number of hair follicles per unit length of epidermis (HF ostia)
was calculated in paraffin sections of HGF/SF transgenic skin
(n=3) at P3 and compared to that of age-matched wild-type
controls (n=3). The percentage of hair follicles in
different stages of morphogenesis was assessed and defined on the basis
of accepted morphological criteria (16
, 38)
. At least 60
longitudinally cut HF sections in >50 microscopic fields, derived from
three HGF/SF TG animals, were analyzed and compared to that of 
3
age-matched wild-type mice.
For counting the number of HF in HGF/SF-transgenic and
age-matched wild-type mice, horizontally sectioned tissue were screened
using established morphological criteria (16
, 38
, 51)
.
At least 100 measurements from 4 sections per animal were performed and compared to wild-type skin, using digital image analysis system (Zeiss KS400, Jena, Germany). Sections were analyzed at x200 or x400 magnification, and means and SE were calculated from pooled data. Differences were judged as significant if the P value was <0.05, as determined by the independent Student’s t test for unpaired samples.
Since differences in HF morphogenesis and cycling are associated with
distinct changes in the overall skin thickness (12
, 31
, 38
, 50
, 51)
, skin thickness was compared between TG and WT skin by
assessing the distance between the stratum corneum and the distal
border of the s.c. muscle layer (panniculus carnosus). In total, 40–50
such measurements were performed in a corresponding number of different
microscopic fields derived from three animals per mutant and wild-type
group. All sections were analyzed at x20 and x200 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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, 45)
. Although Met transcripts were
not detectable in mouse skin with all HF in the resting stage of the
hair cycle (telogen; Fig. 1A, B
, day 0), RNA extracts from full-thickness mouse skin
showed two peaks of steady-state levels of Met transcripts during early
(Fig. 1A, B
, day 1) and late anagen (Fig. 1A, B
,
day 12) as well as during the anagen-catagen transformation (Fig. 1A, B
, day 17). Only low Met mRNA steady-state levels were
seen in mid-anagen skin (Fig. 1A, B
, day 8). Low but
detectable steady-state levels of HGF/SF-mRNA were seen during early
and the late anagen (Fig. 1A, B
, days 3 and 12)
(note that 10-fold more concentrated cDNA had to be used for
the RT-PCR reaction compared to Met and ß-actin),
indicating that HGF/SF mRNA is transcribed at comparatively low levels
in murine skin.
|
The follicular expression of HGF/SF and Met is stage specific and
localized to the mesenchyme (HGF/SF) and epithelium (Met)
Next we analyzed the IR patterns for the corresponding proteins by
IF. Hair follicle morphogenesis was characterized by a developmentally
regulated HGF/SF-IR pattern that was restricted exclusively to
mesenchyme-derived dermal papilla fibroblasts and their precursor cells
whereas Met distribution was seen in the neighboring follicle
epithelium.
During all stages of hair follicle morphogenesis, most epidermal
keratinocytes (both in the basal and suprabasal layers) were Met-IR
(Fig. 2A, B
, red fluorescence; Fig. 3
0–8, red). During the visible onset of HF morphogenesis (stage 1), the
invaginating keratinocytes that later form the new hair plug were all
Met-IR (Fig. 2A
, asterisk; Fig. 3A
). Stage 2 is
characterized by formation of the hair peg, which was also
homogeneously positive for Met (Fig. 2B
, arrowhead; Fig. 3B
). During stages 3 and 4, Met-IR declined in the outer
cell layers of the distal hair peg but remained noticeable in the
developing proximal inner root sheath (IRS) (Fig. 2C
,
arrowhead; Fig. 3C
). Clustered dermal fibroblasts in close
vicinity to the proximal hair peg showed neither Met- nor HGF/SF-IR
during the early stages of HF morphogenesis (stages 1 to 3) (Fig. 2A, B
; Fig. 3A, B
). During stage 4, DP
fibroblasts were the first cells to become homogeneously HGF/SF-IR
(Fig. 2C
, green fluorescence, arrow; Fig. 3C
,
green). DP fibroblasts of the developing HF remained HGF/SF-IR until
the onset of catagen, by which stage the HF entered the first hair
cycle (Fig. 2D-F
, arrowheads; Fig. 3D-F
).
During stage 5, the distal portion of the HF epithelium, including the
gradually formed bulge/isthmus region, showed prominent Met-IR (Fig. 3D
), whereas the hair matrix became Met negative (Fig. 2D
; Fig. 3D
).
|
|
Whereas Met-IR declined from the proximal and central ORS and proximal
IRS during the subsequent stages 6–8 (Fig. 2E
; Fig. 3E
), DP fibroblasts remained strongly HGF/SF-IR (Fig. 2E
, arrowhead; Fig. 3E
).
With entry into the first adult hair cycle, which is initiated by HF
regression (10
, 38)
, catagen I and II were associated with
an increase of Met-IR in the regressing hair bulb, particularly in the
proximal IRS and ORS (Fig. 2F
, arrows; Fig. 3F
). HGF/SF-IR dramatically declined from the
shrinking DP (Fig. 2F
, arrowhead) and was absent within the
DP during the subsequent catagen stages (Fig. 2G
; Fig. 3G
). Throughout catagen, the central IRS and ORS were Met
negative whereas distal ORS keratinocytes, including the bulge/isthmus
region, displayed Met-IR during catagen stages I-VII (Fig. 3F-H
). The keratinocytes of the regressing hair
matrix directly above the DP became Met negative (Fig. 2G
,
arrowhead; Fig. 3G
), whereas the residual proximal IRS and
ORS of the regressing HF epithelium remained Met-IR for as long as they
existed during catagen (Fig. 2G
, arrow; Fig. 3G
).
In catagen VI and VII, Met was prominently expressed in the secondary
hair germ and the epithelial strand (Fig. 2H
, arrowheads;
Fig. 3H
), where massive apoptosis occurred
spontaneously (see Fig. 2M
, arrow) (9)
.
During the resting stage of the adolescent hair cycle (telogen), no
Met- or HGF/SF-IR were seen in murine hair follicles (Fig. 2I
; Fig. 3I
) (except in the cells of the
arrector pili muscle (Fig. 2I
, arrowhead), which were Met-IR
during the entire hair cycle (Fig. 3F-L
). Transient
amplifying cells in the follicle epithelium just above the DP
(52)
were the first to become Met-IR in early anagen I
(Fig. 2J
, arrowhead; Fig. 3J
).
Keratinocytes of the developing IRS and outer root sheath (ORS) began
to show substantial Met-IR in anagen II and III (Fig. 2K
,
arrow; Fig. 3K
). Anagen III was the first stage of
the adolescent hair cycle where HGF/SF-IR was again seen within the DP
(Fig. 2K
, arrowhead; Fig. 3K
). Note
that, morphologically, anagen III resembles to some extent stage 4 of
HF development (compare Fig. 2D, K
and Fig. 3D, K
) since anagen development recapitulates in part HF
morphogenesis (10
, 38
, 53
, 54)
. HGF/SF remained
consistently expressed in the DP during anagen III-catagen II (Fig. 2F, K, L
, arrowheads; Fig. 3F, K, L
). The
proximal and central IRS and the central and distal ORS were Met-IR in
anagen IV (Fig. 3
, anagen IV) and anagen V, whereas Met-IR disappeared
from the proximal hair matrix in late anagen VI (Fig. 2L
;
Fig. 3L
). Central IRS and ORS were prominently Met-IR (Fig. 2L
; arrows; Fig. 3L
) and the distal ORS including
the bulge/isthmus region remained Met positive (Fig. 3L
).
The IR patterns for Met and HGF/SF during the subsequent catagen phase
of the adolescent hair cycle were exactly as described above for the
first catagen development in infantile mice (not shown).
Since HGF/SF expression seen by immunohistochemistry was
restricted to the relatively small population of dermal papilla
fibroblasts, this may explain why only a very low HGF/SF transcript
number was detected by RT-PCR (Fig. 1A, B
). We failed to
demarcate convincing in situ hybridization (ISH) signals for
HGF/SF mRNA in this mouse model despite rigorous attempts and the use
of different in situ probes and ISH techniques following
previously successful ISH protocols for the murine system (46
, 55)
.
Expression of Met-IR and TUNEL positivity appeared to be mutually
exclusive
Based on observations that the cutaneous steady-state levels of
Met transcripts and the corresponding protein expression were
up-regulated during the anagen-catagen transformation (Figs. 1
2
3)
and
that Met-IR was expressed particularly strongly in selected
compartments of the regressing hair follicle epithelium during catagen
(Fig. 2H
, 2M
, arrowheads; Fig. 3H
), we next asked whether or not keratinocytes
undergoing catagen-associated apoptosis in the regressing epithelial
strand and the secondary hair germ of catagen HF coexpress Met-IR.
Using a technique for double immunovisualization of TUNEL-positive and
Met-IR cells in the regressing HF, we demonstrated that TUNEL+ (i.e.,
apoptotic cells) did not display Met-IR (Fig. 2M
, arrow).
This suggested that Met expression awards HF keratinocytes some degree
of protection from apoptosis and that administration of HGF/SF could
inhibit apoptosis-driven catagen (see below).
Injection of HGF/SF in vivo retards catagen
development
To probe this concept, recombinant mouse HGF/SF was injected
intradermally into the back skin of either 14- or 17-day-old mice,
whose back skin HF were all in anagen VI, a few days before the
spontaneous onset of catagen (P14) or that were about to undergo
catagen development (P17) (8
, 10
, 33
, 38)
.
As shown in Fig. 2N
and Fig. 4
, recombinant HGF/SF indeed
significantly retarded spontaneous catagen development in C57BL/6 mice
during both the early and late stages of HF regression. HGF/SF-injected
mice (first injection on day 14 p.d.) showed a gradually delayed
catagen development from the area of injection to the periphery (Fig. 2N
), as demonstrated by TUNEL/Hoechst 33342/Ki67
triple staining. On day 17, close to the injection site (open
arrowhead), late anagen VI HF that were about to undergo the
anagen-catagen transformation displayed almost no TUNEL+ cells, but
instead showed dense clusters of Ki67+ keratinocytes in the proximal
follicle epithelium (Fig. 2N
, arrows). Along the HGF/SF
gradient (i.e., in the periphery of the site of growth factor
injection), most HF already displayed the signs of far progressed
catagen development, as demonstrated by a dramatically increased amount
of TUNEL+ cells in the regressing epithelial strand and secondary hair
germ (Fig. 2N
, arrowheads) (9)
, along with a
lack of cells proliferating in the proximal HF (Fig. 2N
,
arrows). Complementary to this highly suggestive, qualitative
observation, the quantitative assessment of HF stages around the HGF/SF
injection site confirmed the presence of significantly higher numbers
of HF in earlier catagen stages compared to vehicle-treated control
mice, which displayed an increased amount of HF in very late catagen
and telogen (Fig. 4A
).
|
Quantitative histomorphometry of mice injected at day 17 of the first
adult hair cycle also revealed a retardation of catagen development in
HGF/SF-treated mice. On day 19, the majority of HF in vehicle-treated
mice were in catagen VIII or telogen stage (Fig. 4B
, black
bars), whereas skin sections of HGF/SF-treated mice showed a
significantly retarded catagen development, as evident from the
predominance of HF in late catagen (catagen stages VII and VIII) (Fig. 4B
, gray bars). This acceleration of catagen development by
one stage corresponded to a significant, morphologically easily
recognizable difference in skin thickness between HGF/SF-treated
(442.3+28.6 µm) and control skin (294.7+34.3
µm, P>0.05). Murine skin thickness is strictly
coupled to synchronized HF cycling and anagen VI skin is considerably
thicker than catagen skin, which again is thicker than telogen skin
(31
, 50
, 56)
, which indicated that the catagen-telogen
transition in vehicle-treated mice had almost been completed at this
time point, further attesting to the potent
catagen-inhibitory activity of HGF/SF in vivo.
HGF/SF overexpression increases the number of developing HF and
accelerates HF morphogenesis
Given the highly differential, developmentally controlled
expression of HGF/SF during HF morphogenesis (Figs. 2
and 3)
, we next
explored whether HGF/SF overexpression affects HF development. Neonatal
HGF/SF transgenic mice were compared with age-matched WT mice controls
for the number of HF that had developed as well as for the speed of HF
morphogenesis. In normal mice, almost all HF were in the early to mid
stages of HF morphogenesis (stages 4 to 6) around postnatal day 3 (P3).
In HGF/SF overexpressing mice, most HF were instead already in advanced
stages of HF morphogenesis (stages 7–8) (Fig. 5A, C
), whereas practically all HF of wild-type mice were in
earlier stages (5 and 6) at this time point (Fig. 5B, C
).
This relative acceleration of HF morphogenesis in HGF/SF transgenic
mice was independently confirmed by the fact that, the skin was
substantially thicker in HGF/SF-overexpressing mice
(735.2+61.2 µm) (P<0.05) at P3 compared to
wild-type mice (423.3+52.1 µm), as is easily visible in
the representative photomicrographs shown in Fig. 5A, B
.
Since murine skin thickness is strictly coupled to synchronized HF
cycling and skin thickness is dramatically increasing during
sequentially morphogenic stages (38)
, this strongly
supports the concept that HGF/SF overexpression accelerates HF
morphogenesis.
|
Moreover, a significantly increased total number (twofold) of hair
bulbs was counted at P3 in transgenic mice (Fig. 5D
; Fig. 5F
, white bar) compared to WT controls (Fig. 5E
;
Fig. 5F
, black bar). In addition, as an independent, even
more reliable parameter, the amount of HF that had developed by P3 was
also assessed by counting the number of detectable HF ostia per
millimeter epidermal length (16
, 38
, 51)
so as to avoid
the problem of counting individual HF more than once. This
supplementary quantitative histomorphometrical assessment also revealed
a highly significant increase (P<0.001) in the number of HF
ostia in HGF/SF overexpressing mice (Fig. 5G
, white bar)
compared to age-matched wild-type mice (Fig. 5G
, black bar).
These data underscore the potent HF morphogenic activity of HGF/SF.
HGF/SF overexpression retards catagen development
After the completion of HF morphogenesis (which is often
mislabeled as the ‘first hair cycle’), the hair
follicle begins its life-long cycle of regression, resting,
and growth by spontaneous entry into the first catagen stage
(10)
, which occurs around P17 (8
, 10
, 38)
.
Since HGF/SF-overexpressing TG mice showed a significant
acceleration of follicle development during neonatal morphogenesis on
P3, it was most intriguing that the HF in transgenic skin sections from
P17 showed signs of a significant retarded catagen development compared
to age-matched wild-type littermates (Fig. 6A-C
), even though their morphogenesis likely was completed
earlier than in control skin (Fig. 5A-C
). As documented by
representative photomicrographs and quantitative histomorphometry, the
majority of HF in HGF/SF TG mice at P17 were still in late anagen VI to
early catagen (Fig. 6A, C
, open circles), whereas the
majority of WT HF already were in late stages of catagen development
(Fig. 6B, C
, black squares) at this time.
|
Recombinant HGF/SF retards catagen development in skin organ
culture
To address the question of whether the observed alterations in HF
cycling in HGF/SF transgenic and HGF/SF-injected mice might be
connected to consequences of systemic or other secondary effects of
HGF/SF, such as an altered innervation or vascular supply, rather than
reflecting a direct role of HGF/SF in hair follicle control, HGF/SF was
added to organ-cultured murine skin (47
, 49)
. For this
purpose, biopsies were taken from normal C57BL/6 mouse skin 17 days
after anagen induction by depilation (31
, 32)
so that skin
contained homogeneous, well-defined HF populations about to undergo
spontaneous, apoptosis-driven regression (9
, 29
, 32)
and
were devoid of functional innervation or vascular supply. These skin
fragments were cultured at the air-liquid interphase on gelatin gels
for 48 h in the presence or absence of recombinant HGF/SF.
In line with the results of the in vivo studies
delineated above, quantitative histomorphometric analysis revealed that
HGF/SF-supplemented skin biopsies showed a significant retardation of
catagen development in skin organ culture. The majority of HF in skin
biopsies that had been cultured in the presence of 10 ng/ml of
recombinant HGF/SF displayed catagen stage VI (Fig. 7
, open circles), whereas most HF in vehicle-treated control skin were
already in catagen VII, VIII, or telogen (Fig. 7
, black squares). Thus,
HGF/SF inhibits catagen development by a local, intracutaneous effect
on HF cycling .
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Every
step in the extensive tissue remodeling events that constitute HF
morphogenesis and the subsequent HF cycling appears to be controlled by
changes in the local balance of stimulating and suppressing activities
of different growth factors and their corresponding receptors (8
, 10
, 17
, 47
, 57
, 58)
. Here we provide evidence that HGF/SF and
Met, an essential growth factor/receptor pair for the development and
function of a variety of mammalian organs (1
2
3
, 40
, 41
, 43
, 59)
, is one such factor. We report the first comprehensive expression map of HGF/SF and Met during murine hair follicle morphogenesis and cycle. The described follicular expression patterns of Met and HGF/SF demonstrate a striking developmental regulation and hair cycle dependence, and suggest that under physiological conditions, Met expression is restricted to the HF epithelium whereas its ligand is produced by the HF mesenchyme. This designates the HF an exquisite model system for further dissection of the role of HGF/SF in epithelial–mesenchymal interactions in general. The transient and strikingly localized mesenchymal appearance of HGF/SF in the DP (during morphogenesis and during anagen III to catagen II of the hair cycle), along with the substantial epithelial Met expression in the vicinity of HGF/SF-expressing DP fibroblasts, provided suggestive phenomenological evidence for an involvement of this growth factor-signaling system in hair growth control, both during morphogenesis and cyclic remodeling of this mini-organ.
HGF/SF and its receptor (Met) are involved in the control of
angiogenesis in vivo (21
22
23
24)
. Since
angiogenesis is closely coupled to HF anagen development
(60)
, this might be an additional argument for the
significance of HGF/SF and Met to hair biology.
Extracellular proteolytic cleavage by urokinase is required for
activation of HGF/SF, and nexin-1 as well as PA inhibitor type 1 is a
potential inhibitor of urokinase (61)
. Nexin-1 is also an
inhibitor of thrombin and t-PA, and has been reported to be an
important activity-limiting factor HGF/SF (1
, 61)
. Nexin-1
is expressed exclusively in rat DP fibroblasts during anagen, whereas
it is absent during catagen (62)
. This corresponds well to
the observation that HGF/SF-IR is most prominent during anagen (see
Fig. 3
), and suggests that an antiproteolytic system (nexin-1) operates
in the HF, thus regulating HGF/SF activity.
Anagen induction likely requires multiple interconnected signals
that initiate Met expression to achieve competency for HGF/SF binding.
This might be accomplished by key regulatory factors implicated in the
control of HF formation during morphogenesis such as Shh, ß-catenin,
Bmp family members, and their antagonists (12
, 13
, 16
, 58)
. The strong morphogenic potential of the HGF/SF/Met
signaling system within murine skin is supported by the finding that
HGF/SF-overexpressing transgenic mice displayed a twofold increase in
the number of HF and a significantly accelerated HF morphogenesis.
In addition, we show that HGF/SF overexpression retards HF
regression in vivo and that HGF/SF is capable of reproducing
this effect in denervated skin organ culture and in vivo,
suggesting that HGF/SF/Met signaling rank among the elusive molecular
controls of catagen (8
, 10
, 63)
. DP-derived HGF/SF may be
important for supporting epithelial cell growth and for suppressing
follicle keratinocyte apoptosis during anagen development and
maintenance. Since no Met-IR was seen in TUNEL+ cells, while the level
of Met transcripts was highest during early catagen, selected
Met-expressing cells in the regressing epithelial strand may be rescued
from apoptosis during catagen by successfully competing for the
declining level of HGF/SF produced by DP cells (Fig. 2)
. Thus, the
HGF/SF/Met signaling system may contribute to control of
apoptosis-driven (9)
catagen development by the induction
of keratinocyte apoptosis via HGF/SF deprivation, as has been
described for several other model systems (64
65
66)
. HGF/SF
has been reported to be a potent cell survival factor that suppresses
epithelial cell apoptosis (67
68
69)
. BAG-1, a functional
binding partner of the apoptosis inhibitory protein bcl-2, can
associate with Met, thereby linking the HGF/SF/Met signaling pathway
with the anti-apoptotic machinery (67
, 70)
.
It is unclear whether HGF/SF is also involved in anagen induction.
However, we recently noted that cyclosporin A, a potent trigger for
hair growth induction (30
, 71)
, causes a premature
expression of HGF/SF in dermal papilla fibroblasts (G. Lindner and R.
Paus, unpublished results). Therefore, cyclosporin A may induce anagen,
at least in part, by the up-regulation of intrinsic HGF/SF, thus
triggering premature keratinocyte proliferation and differentiation in
the surrounding HF epithelium.
In conclusion, our study implicates the HGF/SF and Met system in hair
growth control and indicates that Met agonists and antagonists should
be considered as novel agents for therapeutic hair growth control (cf.
refs. 8
, 63
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 30, 1999. Accepted for publication October 6, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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