1 Cancer Research UK, Cell Adhesion and Disease Laboratory, Richard Dimbleby
Department of Cancer Research, St Thomas' Hospital, London SE1 7EH, UK
2 2nd Floor, South Wing, Block 7, St John's Institute of Dermatology, St Thomas'
Hospital, London SE1 7EH, UK
* Author for correspondence (e-mail: kairbaan.hodivala-dilke{at}cancer.org.uk)
Accepted 10 March 2003
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Summary |
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Key words: 3-integrin, F-actin, Hair follicle abnormalities
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Introduction |
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Hair follicles consist of an outermost layer called the outer root sheath
(ORS), which is contiguous with the basal epidermal layer and sebaceous gland,
a lining of inner root sheath keratinocytes and an innermost hair shaft. The
outer root sheath also contains the bulge region, which is at least one of the
sites at which epidermal stem cells reside. The base of the hair follicle, or
bulb, contains both specialised keratinocytes (matrix) and mesenchymally
derived dermal papillae cells. In postnatal life the upper portion of the hair
follicle (including the bulge), sebaceous glands and dermal papilla are
permanent, but the remainder of the hair follicle undergoes cycles of growth
(anagen), regression (catagen) and rest (telogen)
(Hardy, 1992;
Fuchs et al., 2001
). These
cycles are dependent on inductive signals between the bulge and the base of
the hair follicle for new hair development
(Cotsarelis et al.,
1990
; Fuchs et al.,
2001
).
Hair follicle and interfollicular epidermal (IFE) keratinocytes adhere to
the basement membrane, which contains collagen type IV (Coll IV), laminin 5
(Lm-5), entactin (ent) and fibronectin
(Timpl, 1989;
Carter et al., 1991
;
Mosher et al., 1992
) through
cell-matrix adhesion molecules known as integrins. Integrins are heterodimeric
transmembrane glycoproteins consisting of an
and ß subunit, and
the heterodimer composition confers ligand specificity
(Hynes, 1992
). Basal epidermal
keratinocytes express several integrins, including
2ß1-,
3ß1-,
5ß1-,
9ß1-,
vß5- and
6ß4-integrins (Watt,
2002
; Palmer et al.,
1993
); in particular,
3ß1- and
6ß4-integrins are abundantly expressed by epidermal keratinocytes
and are predominantly receptors for Lm-5.
6ß4 localises to
hemidesmosomes, which are specialised adhesion structures that connect Lm-5 to
the keratin cytoskeleton (Carter et al.,
1990
). Hemidesmosomes play an important role in maintaining
structural integrity at the dermal-epidermal junction (DEJ), and genetic
ablation of the
6- or ß4-integrin subunits results in severe skin
blistering owing to a failure of epidermal keratinocytes to adhere to the
basement membrane (Georges-Labouesse et
al., 1996
; van der Neut et
al., 1996
).
Conversely, 3ß1-integrin is localised in vivo between
hemidesmosomes and links the basement membrane to the actin cytoskeleton. Mice
in which the
3-integrin gene has been ablated die perinatally
and present several developmental abnormalities in kidneys, lungs
(Kreidberg et al., 1996
) and
brain (Anton et al., 1999
).
Newborn
3-integrin-deficient mice also display epidermal-dermal
microblisters on footpads, which are reminiscent of a human blistering disease
called junctional epidermolysis bullosa. Analysis of the basement membrane of
these mice indicates that deposition of Lm-5 is altered, and it was suggested
that this may be the cause of blistering
(DiPersio et al., 1997
). These
data indicate that in newborn skin,
3ß1-integrin plays important
functions in regulating the maintenance of the epidermal basement membrane.
However, these studies could not address the role of
3-integrin in the
development of adult epidermis and its appendages.
Since ß1-integrin-deficient mice have an embryonic lethal phenotype
(Stephens et al.,
1995), conditional mutant models were required to
investigate the roles of ß1-integrins in postnatal skin development. Mice
in which ablation of the ß1 gene is restricted to the basal
layer of epidermal keratinocytes present skin blistering at the DEJ, impaired
downgrowth of hair follicles, probably because of poor basement membrane
organisation (Raghavan et al.,
2000
), and eventual hair loss
(Brakebusch et al., 2000
). It
has been postulated that because ß1-integrin-deficient hair follicles are
not regenerated this may reflect an important role of ß1-integrins in
regulating the epidermal stem cell compartment. However, this severe phenotype
reflects the cumulative deficiency of all the ß1-integrins expressed in
keratinocytes, that is,
2-,
3-,
5- and
9ß1-integrins, and does not address the specific roles of
individual
-subunits in postnatal epidermal morphogenesis.
The expression levels of 3ß1-integrin change during development
and in many pathological conditions (Kreidberg et al., 2000). However, it is
unknown whether these changes are causal or consequential, and it is therefore
important to understand the roles that
3ß1-integrin plays during
normal tissue development. Therefore, to investigate the precise roles of
3ß1-integrin in postnatal follicular and interfollicular epidermal
development, we developed a method to graft full-thickness newborn mouse skin,
comprising both dermis and epidermis, onto adult ICRF nu/nu athymic mice. We
report that
3-deficient skin develops fully, but that maintenance of
hair follicle morphology is significantly compromised after the first hair
cycle. The abnormalities include stunted hair follicle growth, defective
differentiation of hair follicle keratinocytes and severe aberrations in
filamentous actin (F-actin) organisation and the outer root sheath. The
morphology and differentiation of interfollicular epidermis was not affected
by
3 deficiency, implying that loss of a single integrin subunit can
have distinct effects in the interfollicular and follicular skin compartments.
Our results indicate novel roles for
3-integrin in hair follicle
morphogenesis that could not have been predicted based on prior in vitro and
in vivo studies and show for the first time that a single
-integrin
subunit can regulate adult hair follicle development.
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Materials and Methods |
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Skin grafting
Six-week-old male ICRF nu/nu mice (ICRF nu/nu) were anaesthetised with an
intraperitoneal injection of Avertin, and a full thickness skin disc (8 mm in
diameter) was excised to expose the underlying fascia. Litters of newborn mice
including 3-integrin-deficient (Kriedberg et al., 1996) (also available
from Jackson laboratories as the Itgatm1Jak mouse) and wild-type
(WT) littermates, outbred between C57Bl6/J and 129Sv/ter, were sacrificed by
decapitation, and the tails were collected for genotyping
(DiPersio et al., 1997
).
Transpore tape (Southern Syringe Service, London, UK) was used to support the
pup skin while removing it from the carcass. Using an 8mm biopsy punch
(Stiefel Labs, Buckinghamshire, UK) a disc of donor skin was cut and applied
onto the recipient fascia. Steri-strips and circular plasters (Southern
Syringe Service, London, UK) were applied to secure the grafts in place.
Grafts were then dressed with gauze bandage, and the recipient mice were
administered analgesic. Dressings were removed after seven days and grafts
processed as described below. In total, over 600 skin grafts were prepared for
this study. All mice were killed by CO2 inhalation at either 14
days or 45 days post grafting.
Immunohistochemistry and apoptosis detection
Skin grafts were shaved, excised and bisected along the anterior-posterior
axis. Half the graft was then embedded in OCT compound and snap-frozen in
isopentane chilled in liquid nitrogen. The other half was fixed in 4%
formaldehyde solution in PBS for 24 hours and then embedded in paraffin. All
sections were cut longitudinally through the skin. This was established for
each sample, firstly by orientating the skin sample perpendicularly to the
plane of the cut, further by always observing longitudinal sections through
hair follicles in each section and finally by verifying that the thickness of
the interfollicular epidermis was no more than two to three layers of
keratinocytes. No tangential sections were used in the analysis. For
morphological analysis, paraffin-embedded skin sections were dewaxed in
xylene, rehydrated in decreasing concentrations of ethanol and finally stained
with hematoxilin and eosin.
To examine apoptosis, paraffin-embedded sections were dewaxed and rehydrated as described above and apoptosis-related DNA fragmentation was detected using the TUNEL detection kit (Intergen company, Oxford, UK).
Immunohistochemistry of 6 µm cryosections was carried out as follows:
sections were fixed in 4% formaldehyde in PBS for 20 minutes at room
temperature and blocked in 0.1% BSA, 0.2% Triton X-100, 0.1% glycine in PBS
for 1 hour prior to a 40 minute incubation with primary antibodies. For
detection of entactin, keratin-1, keratin-6, keratin-14 and Hb2, sections were
incubated with a primary antibody diluted in blocking solution for 40 minutes
at room temperature, washed in PBS and incubated with FITC-conjugated
secondary antibody diluted in blocking solution for 40 minutes. Alternatively,
antibodies to loricrin, collagen type IV and 3-integrins were followed
by incubation with a biotin-conjugated secondary antibody, washed in PBS and
then incubated with FITC-conjugated streptavidin for 40 minutes. Finally,
sections were washed in distilled water and mounted in Gelvatol supplemented
with DABCO antifading agent. Representative fields were photographed using a
Hamamatsu Digital Camera (Improvision, London, UK) on a Zeiss Axioplan
microscope (Zeiss, HERTS, UK), with the exception of F-actin images, which
were acquired using a Zeiss LSM-10 confocal microscope.
Electron microscopy
Skin grafts were harvested as described above, and prepared for electron
microscopy as follows: small pieces of tissue were fixed with 2.5%
gluteraldehyde in 0.1 M Sörensens phosphate buffer for 1 hour at room
temperature, washed in Sörensens buffer, postfixed in 1% osmium tetroxide
in 0.5 M Sörensens buffer for 30 minutes at room temperature, washed in
Sörensens buffer and then finally washed in distilled water. Samples were
then dehydrated in ethanol and propylene oxide, infiltrated with
araldite/propylene oxide 1:1 overnight at room temperature, infiltrated with
araldite, embedded in resin and polymerised at 60°C overnight. Ultrathin
(80 nm) sections were cut, mounted on grids, stained with uranyl acetate in
lead cytrate and washed in distilled water. Sections were viewed using a JEOL
1010 Transmission Electron Microscope.
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Results |
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Morphological analysis of the effect of 3-integrin deficiency in
skin was carried out by examining hematoxilin- and eosin (H&E)-stained
sections from WT and
3-integrin-deficient skin grafts.
3ß1
is a major ß1-integrin in keratinocytes, and ß1-integrins have been
proposed to be important in regulating keratinocyte differentiation
(Adams and Watt, 1989
).
However, at 14 days the morphology and thickness of
3-integrin-deficient interfollicular epidermis appeared normal when
compared to WT skin grafts (Fig.
1C,D), and both genotypes had evenly spaced, anagen-phase hair
follicles. Immunohistochemistry with antibodies directed against
3-integrin showed that in WT skin grafts,
3-integrin was
expressed in the basal layer of the interfollicular epidermis and in the ORS
of hair follicles (Fig. 1E),
but was undetectable in
3-integrin-deficient skin grafts
(Fig. 1F). These data indicate
that
3ß1, despite being a major Lm5 receptor in skin, is not
required for the initial postnatal development of mouse skin and hair.
Abnormal hair follicle morphogenesis in adult
3-integrin-deficient skin
To investigate the effect of 3 deficiency on adult skin development,
we analysed
3-integrin-deficient and WT skin grafts 45 days post
grafting. Morphological analysis of H&E-stained sections revealed that
interfollicular epidermis appeared normal in both WT and
3-integrin-deficient skin grafts and was two to three layers thick
(Fig. 2A,B), establishing that
3ß1 is not important for the regulation of adult interfollicular
epidermal morphology. However, several hair follicle abnormalities were
observed in adult
3-integrin-deficient skin grafts. WT hair follicles
were distributed evenly, and a significant proportion was in the anagen growth
phase (Fig. 2C). In contrast,
most
3-integrin-deficient hair follicles appeared stunted and abnormal
(Fig. 2D). Quantification
revealed that in adult
3-integrin-deficient skin the average number of
hair follicles in the catagen or telogen phases of the hair cycle was
increased when compared with age-matched WT skin grafts (50%, WT verses 86%,
3-integrin deficient). This suggested that
3-integrin deficiency
affects hair cycle progression.
|
In addition, other abnormalities were observed in
3-integrin-deficient skin grafts. The most striking involved the
aggregation of multiple hair follicles in clusters
(Fig. 2E,H) often with
misplaced sebaceous glands (data not shown). It is important to note that
these clustered hair follicles were observed in longitudinally cut sections
through the skin and were not caused by oblique sectioning (see Materials and
Methods). Indeed, clusters could be detected directly beside normal
longitudinally cut hair follicles (Fig.
2D). The majority of WT hair follicles contained a single hair
shaft, and follicles containing a maximum of two hair shafts could only be
found occasionally. In contrast, in
3-integrin-deficient skin, the
number of hair shafts per cluster unit varied and the most severely abnormal
clusters included up to 11 hair shafts. The percentage of abnormal hair
follicles was quantified (as the percentage of all hair follicles that formed
clustered units) and showed that, although there was no change in the total
number of hair follicles between genotypes (data not shown), adult
3-integrin-deficient skin grafts had a significantly higher number of
abnormal hair follicles when compared with WT controls
(Fig. 2H).
Another frequently observed abnormality in adult
3-integrin-deficient skin grafts was the presence of pigment deposits,
which were found in different locations: inside the hair follicle bulb,
detached from hair follicles deeper in the dermis or at the base of club hairs
(Fig. 2F). Histochemical
analysis using Masson fontana staining showed that these pigment deposits
contained melanin (data not shown). We counted the number of deposits in WT
and
3-integrin-deficient skin grafts, and although they were never be
found in 14 day (data not shown) or in adult WT skin grafts, they were
abundant in adult
3-integrin-deficient follicles
(Fig. 2I). Pigment deposits are
often indicative of deteriorating hair follicles and may reflect an increase
in hair follicle fragility in
3-integrin-deficient skin.
A third abnormality observed in adult 3-integrin-deficient skin
grafts was an aberrant distribution of hair follicles, which were often
grouped together, with large areas of interfollicular epidermis between groups
(Fig. 2G). Frequently,
different abnormalities coexisted in the same skin graft. Although the gross
appearance of adult WT and
3-integrin-deficient skin was comparable,
and there was no significant loss of hair at this or older time points (3 and
6 months post grafting), the results establish that the maintenance of hair
follicle morphology is compromised in the absence of
3-integrin.
Hair follicle differentiation is abnormal in
3-integrin-deficient skin.
Because ß1-integrins have been implicated in controlling keratinocyte
differentiation, and adult 3-integrin deficient hair follicles were
morphologically abnormal but interfollicular epidermis in the same samples
appeared normal, we wished to examine keratinocyte differentiation in these
two epidermal compartments. Immunohistochemical analysis showed that ß1
expression levels were reduced, and the distribution of
6-, ß4-,
5- and
v-integrin subunits were unaltered in
3-integrin-deficient skin (data not shown). K14 (a basal keratinocyte
marker) was confined to basal keratinocytes in the interfollicular epidermis
and was also expressed in the hair follicle ORS, with no difference in
expression between WT and
3-integrin-deficient skin grafts
(Fig. 3A-D). Markers of
epidermal differentiation include K1, loricrin and filaggrin. In both WT and
3-integrin-deficient skin grafts, K1 was expressed in the suprabasal
epidermal layers (Fig. 3E,F),
and loricrin and filaggrin were expressed in the cornified layers of the
epidermis (Fig. 3G,H and data
not shown). K6 is normally confined to hair follicles and its expression in
3-integrin-deficient skin was similar to that found in WT hair
follicles (Fig. 3I,J). However,
in contrast, hair-specific keratin Hb2
(Langbein et al., 2001
) was
expressed in the cuticle of WT hair follicles
(Fig. 3K), but never found in
hair follicle clusters (Fig.
3L). These findings indicate that
3-integrin deficiency
does not affect differentiation of the interfollicular epidermis but does
affect differentiation of hair follicle keratinocytes.
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Reduced proliferation and apoptosis in adult
3-integrin-deficient skin grafts
Hair follicle keratinocytes undergo coordinated cycles of proliferation and
apoptosis that control hair follicle structure and morphology. Because both
these processes are thought to be affected by integrin expression, we wished
to test if alterations in proliferation and/or apoptosis correlate with the
formation of abnormal, poorly differentiated hair follicles in
3-integrin-deficient skin.
We analysed the expression of the proliferation marker Ki67 in
3-integrin-deficient and WT skin grafts. Ki67-positive cells were found
in the interfollicular epidermis, hair matrix, ORS and occasionally in
sebaceous glands. No significant difference in the number of cells expressing
Ki67 was found in the interfollicular epidermis of
3-integrin-deficient
skin grafts when compared with WT control grafts
(Fig. 4A,B). However, in
3-integrin-deficient hair follicles, the number of Ki67 positive cells
was significantly lower than in WT hair follicles
(Fig. 4C and D). Furthermore,
hair follicle clusters observed in
3-integrin deficient skin grafts had
few or no Ki67-positive cells (Fig.
4E), and quantitation of the percentage of hair follicle
keratinocytes that were positive for Ki67 showed that it was significantly
lower in
3-integrin-deficient samples than in WT controls
(Fig. 4F). No significant
difference in the number of proliferating keratinocytes was detected in
14-day-old skin grafts (data not shown). The proliferation studies were
confirmed by BrdU incorporation experiments (data not shown).
|
Apoptosis has been reported to occur normally in the IRS, bulge and bulb of
regressing hair follicles (Lindner et al.,
1997). We analysed apoptosis in WT and
3-integrin-deficient
skin by using TUNEL staining for the presence of positive nuclei. No apoptotic
cells were detected in the interfollicular epidermis of WT
(Fig. 5A) or
3-integrin-deficient (Fig.
5B) grafts. In contrast, in WT hair follicles apoptotic profiles
were normal (Fig. 5C), but were
surprisingly low in
3-integrin-deficient follicles
(Fig. 5D). In addition, very
few apoptotic nuclei could be detected in
3-integrin-deficient hair
follicle clusters (Fig. 5E).
Calculation of the percentage of TUNEL-positive cells confirmed that
3-integrin-deficient hair follicles had significantly fewer apoptotic
nuclei when compared with WT controls (Fig.
5F).
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These results may reflect the difference in hair morphology in WT and
3-integrin-deficient skin and suggest that
3-integrin is
important in maintaining normal proliferative and apoptotic profiles in adult
hair follicles.
Laminin 5 and entactin are disorganised in adult
3-integrin-deficient interfollicular but not follicular epidermis
Since keratinocyte interaction with the underlying basement membrane is
important in regulating epidermal morphology, we examined the distribution of
basement membrane proteins in adult 3-integrin-deficient and WT skin
grafts. Adult
3-integrin-deficient and WT skin were analysed by
immunofluorescence with antibodies to different basement membrane proteins,
including laminin 5 (Lm-5), entactin (ent), collagen type IV (Coll IV) and the
extracellular matrix proteins fibronectin (Fn) and collagen type I (data not
shown). In WT skin grafts Lm-5, entactin and Coll IV were expressed in the
basement membrane zones of interfollicular epidermis and hair follicles
(Fig. 6A,D,G). In contrast, in
the interfollicular epidermis of
3-integrin-deficient skin grafts, Lm-5
and entactin were often deposited in multiple layers below the plane of the
basement membrane but appeared to be intact around hair follicles
(Fig. 6B,E) and hair follicle
clusters (Fig. 6C,F). In some
conditions, such as psoriasis, disruption of the basement membrane may be
associated with mast cell infiltration. However, we did not observe any
changes in dermal cell infiltrate in the mutant skin grafts (data not shown).
In WT and
3-integrin-deficient skin grafts Fn was expressed at the
dermal-epidermal junction and throughout the dermis, with no significant
difference between genotypes. Sometimes, in areas of
3-integrin-deficient interfollicular epidermis where Lm-5 expression
was disrupted, a dense accumulation of Fn was observed. In addition, the
distribution of Fn appeared less organised around
3-integrin-deficient
hair follicles compared with WT controls (data not shown). In contrast, Coll
IV (Fig. 6G,H,I) and Coll I
(data not shown) were distributed normally in
3-integrin-deficient skin
grafts.
|
The lamina densa is disrupted in 3-integrin-deficient
interfollicular epidermis
Since the morphology and differentiation of interfollicular epidermis in
3-integrin-deficient skin grafts appeared normal by histological and
immunohistochemical analyses, but Lm-5 and entactin distribution was
disrupted, we went on to study adult
3-integrin-deficient skin at the
ultrastructural level in order to examine better the keratinocyte structure
and basement membrane integrity. The morphology of the basal, spinous and
cornified layers was comparable in the interfollicular epidermis of WT and
3-integrin-deficient samples (Fig.
6J,K), and there was no significant difference in the number of
hemidesmosomes expressed by
3-integrin-deficient grafts when compared
with WT skin. However, in
3-integrin-deficient interfollicular
epidermis the lamina densa often appeared discontinuous in between
hemidesmosomes (Fig. 6L),
confirming that the integrity of the interfollicular basal lamina was
compromised in adult
3-integrin-deficient skin.
Severe fragility of 3-deficient hair follicle keratinocytes
and lamina densa disorganisation
To better understand the cause of 3-integrin-deficient hair follicle
defects we examined the ultrastructure of hair follicles in adult WT and
3-integrin-deficient skin grafts. Follicular keratinocytes normally
assemble hemidesmosomes in the regions of the follicle proximal to the
epidermis. In WT skin grafts, the lamina densa was intact regardless of
whether hemidesmosomes were present or not
(Fig. 7A,C). In contrast, we
observed that maintenance of the basal lamina in
3-integrin-deficient
hair follicles differed depending on whether keratinocytes assembled
hemidesmosomes or not. Where hemidesmosomes were present, the lamina densa was
disorganised and keratinocytes appeared to retract from it, adhering poorly
with finger-like projections, giving the cells a ruffled appearance
(Fig. 7B). In the areas lacking
hemidesmosomes the lamina densa appeared to be deposited in multiple layers
(Fig. 7D). In addition, dermal
fibroblasts accumulated in dense layers close to the lamina densa in
3-integrin-deficient hair follicles
(Fig. 7D,F), and this was not
observed in WT samples. Thus, although by immunofluorescence detection of
basement membrane molecules no defects were apparent in the basement membrane
of
3-integrin-deficient hair follicles, ultrastructural analysis
revealed severe abnormalities in the lamina densa organisation.
|
The presence of pigment deposits and stunted hair follicles in
3-integrin-deficient skin strongly suggest that
3-integrin
deficiency causes increased hair follicle fragility. At the ultrastructural
level WT hair follicle keratinocytes in the outer and inner root sheaths were
well organised and had good intercellular contacts
(Fig. 7E). In contrast, cells
in the ORS of
3-integrin-deficient hair follicles were severely
ruptured, indicating an increase in cellular fragility, and keratinocytes of
both the ORS and IRS presented large intra- and intercellular spaces, in which
the IRS cells extended numerous filopodial processes
(Fig. 7F,G). Thus, the severe
ultrastructural abnormalities in the hair follicle keratinocytes most probably
contributes significantly to the stunted and fragile nature of
3-integrin-deficient hair follicles.
F-actin is both disorganised and arranged in prominent bundles in
3-integrin-deficient hair follicles
Since the assembly of actin filaments profoundly affects the structural
organisation of cells within tissues and their adhesive behaviour, we wondered
whether changes in F-actin distribution correlated with the morphological
abnormalities and cellular fragility observed in adult
3-integrin-deficient hair follicles. Cryosections of adult WT and
3-integrin-deficient skin were stained with rhodamine-conjugated
phalloidin. In both WT and
3-integrin-deficient interfollicular
epidermis, F-actin was distributed in a pericellular fashion in all
keratinocyte layers with no significant difference between genotypes
(Fig. 8A,B). However,
comparison of F-actin distribution in WT and
3-integrin-deficient hair
follicles gave a strikingly different result. F-actin was distributed
subcortically in WT hair follicle keratinocytes
(Fig. 8C). In contrast, in
3-integrin-deficient hair follicles it appeared either severely
disorganised (Fig. 8D,E) or
formed prominent actin bundles, especially at the base cells, within ORS and
IRS keratinocytes (Fig. 8F).
These data establish that in adult skin,
3-integrin deficiency
specifically affects the organisation of filamentous actin in follicular but
not interfollicular epidermal keratinocytes and that this correlates with hair
follicle fragility in
3-integrin-deficient skin.
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Discussion |
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3ß1 is important for structural integrity of hair
follicles via regulation of the cytoskeleton
Our observations show that 3-integrin-deficient skin begins to
develop normally and that abnormalities in hair follicle morphology were not
observed in 14-day-old grafts. At this early stage hair follicles in both
genotypes appeared to be in the first anagen growth phase. Between 14 and 45
days post grafting, the WT hair follicles regressed, through catagen and
telogen, and entered the second anagen phase (data not shown). However, in
adult
3-integrin-deficient skin, the majority of hair follicles
remained in catagen or telogen, implying that
3 deficiency may inhibit
entrance into the second anagen phase. Clusters of hair follicles in the
3-integrin-deficient skin may represent multiple unsuccessful attempts
of a hair follicle to enter anagen. Furthermore, the general level of reduced
proliferation and apoptosis in the
3-integrin-deficient hair follicles
suggests that these follicles may be in an extended resting phase. These
results indicate that
3-integrin plays a crucial role in regulating
hair follicle morphology specifically during the catagen phase of the hair
cycle.
Since 3ß1 is one of the most highly expressed epidermal
ß1-integrins, one might expect that deficiency in
3- or
ß1-subunits would have overlapping or similar effects on hair follicle
cycling. The epidermis-specific, ß1-integrin-deficient mice have severely
compromised survival (Brakebusch et al.,
2000
; Raghavan et al.,
2000
) and hair follicles either do not invaginate properly
(Raghavan et al., 2000
) or are
lost by 16 days after birth implying that ß1-integrins are essential in
matrix formation (Brakebusch et al.,
2000
). In short, both
3- and ß1-integrins affect hair
follicle growth, but they do so at different stages of the hair cycle. In
addition, we observed that in adult
3-integrin-deficient skin grafts,
stunted hair follicles often appeared to associate with pigment deposits or
casts in the dermis. Such pigment deposits are regarded to reflect an increase
in hair follicle fragility. Together with an increase in cell fragility in the
ORS and a decrease in hair-specific keratin 2 (Hb2) expression, our data imply
that
3ß1, unlike the combination of all ß1-integrins, is
important in maintaining the structural integrity of hair follicles.
What could cause increased hair follicle fragility in
3-integrin-deficient adult skin? Changes in F-actin organisation within
cells has been shown to alter both cell-cell and cell-matrix adhesion
(Vasioukhin et al., 2000
;
Vasioukhin et al., 2001). In
3-integrin-deficient hair follicles
F-actin was both severely disorganised and formed prominent actin bundles.
Neither of these patterns of F-actin distribution were found in WT hair
follicles. This indicates that, in adult skin,
3-integrin is important
in maintaining the correct organisation of the actin cytoskeleton in hair
follicles and suggests that F-actin disorganisation causes the observed cell
fragility. Consistent with these observations, F-actin is organised into
prominent bundles in
3-integrin-deficient newborn pad skin and
keratinocytes (Hodivala-Dilke et al.,
1998
). The reason for the apparent recovery of F-actin
organisation in adult interfollicular epidermis is unclear. One explanation
may be that the adult skin grafts are essentially from thin, back skin, and
the newborn phenotype was described for thick, pad skin that developed
blisters. Blistering is a type of wound, and it has been reported previously
that keratinocytes in a wound have bundles of F-actin on their basal face.
Furthermore since newborn epidermis is less quiescent than adult
interfollicular epidermis and no blisters were evident in the adult thin skin,
this may explain why the F-actin appears normal in adult thin interfollicular
epidermis. Normally, activation of Rho GTPases leads to changes in the actin
cytoskeleton, thus enhancing integrin clustering and ECM organisation
(Wennerberg et al., 1996
;
Nguyen et al., 2001
). One
might speculate that in the absence of
3ß1 abnormal activation of
Rho results in disorganised F-actin and an increase cell fragility. Taken
together, these symptoms represent a likely cause for both the stunted and
clustered abnormal hair follicle growth in
3-integrin-deficient
skin.
The role of 3-integrin in follicular versus interfollicular
epidermal development
As 3ß1-integrin is expressed in both IFE keratinocytes and hair
follicles, why does its deficiency affect follicular and not inter-follicular
epidermal morphogenesis and differentiation? One difference between hair
follicles and IFE is that although hemidesmosomes (and thus
6ß4)
are expressed throughout the IFE, they are present only in the distal part of
hair follicles (Nutbrown and Randall,
1995
). Consequently, in adult
3-integrin-deficient skin,
the lower part of the hair follicle is doubly-deficient in both
3ß1- and
6ß4-integrins. Hence, this combined loss of
Lm-5 receptors, although not necessarily the only reason, might contribute to
the severe loss of cytoskeletal (both F-actin and hair specific keratin 2)
organisation and the extreme cellular fragility of
3-integrin-deficient
hair follicles.
Another difference between hair follicles and IFEs is that hair follicles
undergo cycles of massive proliferation and apoptosis, corresponding to the
anagen and catagen phases of the hair cycle, respectively. This is unlike the
relatively low rates of proliferation and apoptosis in adult quiescent IFEs.
Since ß1-integrins have been implicated in the control of proliferation
and apoptosis (Gonzales et al.,
1999; DiPersio et al.,
2000
; De Arcangelis and
Georges-Labouesse, 2000
;
Frisch and Screaton, 2001
), it
is perhaps not surprising that
3 deficiency alters the proliferative
and apoptotic potential of keratinocytes predominantly in hair follicles.
These changes, in turn, probably contribute to the increase in telogen phase
and morphological abnormalities observed in
3-integrin-deficient hair
follicles.
3-integrin is required for correct maintenance of the basement
membrane in adult skin
Basement membrane disruption is associated with dermal-epidermal blistering
in 3-integrin-deficient newborn pads
(DiPersio et al., 1997
;
Hodivala-Dilke et al., 1998
),
and, importantly, the blistering was confined to hairless areas of thick skin.
Interestingly, in adult
3-integrin-deficient skin, the lack of basement
membrane integrity in the IFE does not cause skin blistering. How could this
be explained? One possibility is that in
3-integrin-deficient skin
grafts the presence of hair provides the skin with increased mechanical
resistance, thereby preventing skin blisters. In addition, because blistering
is often a skin-site-associated problem, our results may simply indicate that
3-integrin deficiency in thick skin causes blistering, whereas in thin
skin it does not. Although the follicular basal lamina was disrupted in
ultrastructural experiments, immunohistochemistry revealed that entactin and
Lm-5 appeared intact, indicating that the basement membrane is less severely
disorganised in follicular than in interfollicular epidermis. One reason for
this may be that cycling of hair follicles induces a constant regeneration of
the surrounding basement membrane, thereby preventing the accumulation of
disorganised ECM proteins around hair follicles and thus protecting the skin
from sheer stress effects.
A possible role for 3-integrin in maintenance of the epidermal
stem cell compartment?
Epidermal stem cells express high levels of 2ß1- and
3ß1-integrins (Jones and Watt,
1993
; Jones et al.,
1995
; Bagutti et al.,
1996
; Moles and Watt,
1997
; Jensen et al.,
1999
; Zhu et al.,
1999
; Watt, 2001
).
An important question is whether these integrins are simply markers of stem
cells or whether they are vital for the functional maintenance of the stem
cell compartment in vivo (Levy et al.,
2000
; Raghavan et al.,
2000
). Previous studies using mice either totally deficient in
3-integrin (Kreidberg et al.,
1996
; DiPersio et al.,
1997
) or epidermis-specific ß1-integrin-deficient mice
(Raghavan et al., 2000
;
Brakebusch et al., 2000
) have
demonstrated that the epidermis can develop in the absence of these integrins,
at least until birth. In the ß1 mutant mice, hair follicles disappear
after the first hair cycle, implying that ß1-integrin might be involved
in the maintenance of an epithelial stem cell compartment or might play a role
in the activation of stem cells at the onset of anagen
(Brakebusch et al., 2000
).
However, immune cell infiltration and hair follicle breakdown at this stage
make it difficult to state categorically whether hair follicles are not
regenerated because of an intrinsic stem cell defect and/or because an immune
response has eradicated all follicular stem cells. The approach that we have
used overcame the majority of these immune-response problems since
3-integrin-deficient skin was grafted onto immunocompromised mice. Our
observations, that adult
3-deficient skin can develop fully and form
hair follicles and sebaceous glands indicate that
3ß1 probably is
not essential for the maintenance of the stem cell compartment.
In conclusion, we present strong evidence that adult
3-integrin-deficient skin can develop fully but, surprisingly, it has
hair follicle and not interfollicular abnormalities. These abnormalities are
probably caused by a combination of cytoskeletal aberrations, loss of basement
membrane integrity and a resultant increase in cell fragility. Taken together
our data establish that
3ß1-integrin is important in maintaining
hair follicle architecture during the hair cycle and provides the first
evidence for the role of a single
-subunit in regulating adult hair
follicle morphology.
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Acknowledgments |
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