Alteration of Skin Wound Healing in Keratinocyte-Specific Mediator Complex Subunit 1 Null Mice
Itami S (2014) Alteration of Skin Wound Healing in Keratinocyte-Specific Mediator Complex Subunit 1 Null
Mice. PLoS ONE 9(8): e102271. doi:10.1371/journal.pone.0102271
Alteration of Skin Wound Healing in Keratinocyte- Specific Mediator Complex Subunit 1 Null Mice
Fumihito Noguchi 0
Takeshi Nakajima 0
Shigeki Inui 0
Janardan K. Reddy 0
Satoshi Itami 0
Richard L. Eckert, University of Maryland School of Medicine, United States of America
0 1 Department of Regenerative Dermatology, Graduate School of Medicine, Osaka University , Osaka , Japan , 2 Department of Dermatology, Osaka General Medical Center, Osaka, Japan, 3 Department of Pathology, Feinberg School of Medicine, Northwestern University , Chicago, Illinois , United States of America
MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1epi2/2) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1epi2/2 and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1epi2/2 mice compared with age-matched wildtype mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1epi2/2 mice, whereas wound contraction and the area of a-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1epi2/2 keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1epi2/2 keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1epi2/2 keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1epi2/2 keratinocytes compared with wild-type keratinocytes. These findings indicate that the activinfollistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1epi2/2 mice. On the other hand, skin wound healing in 6-month-old Med1epi2/2 mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wildtype mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1epi2/2 mice, indicating a decreased contribution of hair follicle stem cells to epidermal regeneration after wounding in 6-month-old Med1epi2/2 mice. This study sheds light on the novel function of MED1 in keratinocytes and suggests a possible new therapeutic approach for skin wound healing and aging.
. These two authors contributed equally to this work.
The wound healing process is divided into three phases: an
inflammatory phase, a proliferative phase and a remodeling phase
[1,2]. The inflammatory phase occurs immediately after injury.
Tissue damage initially causes the disruption of vascular vessels
and extravasation, followed by the production of a temporary
platelet plug and a fibrin clot which stops bleeding and supplies a
transient anchorage for subsequently infiltrating inflammatory
cells. Next, during the proliferative phase, which occurs several
days after tissue damage, keratinocytes and endothelial cells
proliferate and migrate to the wound, resulting in
re-epithelialization and angiogenesis. Finally, in the remodeling phase, some
fibroblasts are stimulated by macrophages to differentiate into
myofibroblasts, causing wound contraction. During this phase,
production of the extracellular matrix, including collagen,
proteoglycan and fibronectin, is increased, which results in the
formation of a mature scar [3,4]. All of these events require the
orchestrated efforts of different types of cells. Failure in any of
these phases of the wound healing process can lead to chronic
wounds, hypertrophic scars and/or wound-related tumor
Mediator complex subunit 1 (MED1) is integrated into the
Mediator complex as a coactivator of various transcription factors,
including nuclear receptors, p53 and BRCA1 [6,7]. MED1 has
also been reported to play critical roles in regulating hair cycling
and epidermal proliferation . Previously, we established
keratinocyte-specific MED1-null (Med1epi2/2) mice and
characterized the roles of MED1 in regulating the proliferation of
keratinocytes and the maintenance of hair follicle bulge stem cells
. In this study, we investigated the process of wound healing in
Med1epi2/2 skin and analyzed the underlying mechanisms,
including the activin-follistatin system and epithelial stem cells.
To study the effect of MED1 depletion in keratinocytes on the
skin wound healing process, we created full-thickness circular
excisional wounds on the backs of 8-week-old Med1epi2/2 mice
and wild-type (Med1+/+) mice and observed the healing process
through days 1 to 7 after injury (Figure 1A). Macroscopic
evaluation revealed that wound closure of Med1epi2/2 mice was
significantly accelerated on day 3 after injury compared with
wildtype mice (Figure 1B, p,0.05). Next, we performed skin biopsies
at these wound sites on days 1, 3 and 5 after injury and evaluated
the skin wound healing process microscopically (Figure 1C).
Hematoxylin and eosin (H&E) staining of wound sites indicated
that re-epithelialization after wounding was significantly enhanced
in Med1epi2/2 mice on days 3 (p,0.01) and 5 (p,0.05) compared
with wild-type mice (Figure 1D).
Migrating epithelial tongues are elongated and the
proliferation of keratinocytes is accelerated in 8-week-old
To investigate the mechanism(s) underlying the accelerated
wound healing in 8-week-old Med1epi2/2 mice, we next compared
the lengths of migrating epithelial tongues and observed a
significant elongation in Med1epi2/2 mice on days 1 (p,0.01)
and 3 (p,0.01) after injury (Figure 2A). Moreover, Ki67
immunostaining in the aforementioned period clearly showed
that the number of Ki67-positive keratinocytes was increased at
the transitional epidermis and the epithelial tongues were longer in
8-week-old Med1epi2/2 mice on days 1 (p,0.01) and 3 (p,0.05)
after injury compared with those in age-matched wild-type mice
(Figure 2B and C), indicating the acceleration of keratinocyte
proliferation by Med1 knockout. We have previously reported that
Ki67-positive proliferating keratinocytes in unwounded skin of
8week-old Med1epi2/2 mice were 1.57 times more frequently
observed than in wild-type mice . The number of Ki67-positive
proliferating keratinocytes in the wounded epidermis was
increased by 2.56-fold in 8-week-old Med1epi2/2 mice compared
with wild-type mice on day 1 after injury (Figure 2C). These
findings suggest that enhanced keratinocyte migration and
Figure 1. Skin wound healing is accelerated in 8-week-old Med1epi2/2 mice. A: Representative macroscopic views of skin wounds on days 1,
3, 5 and 7 after wounding in 8 week old wild-type and Med1epi2/2 mice. Full-thickness wounds (4 mm in diameter) were made on the middle of the
backs of mice to synchronize tension and wound healing was monitored by taking digital photographs. Note the acceleration of wound healing in
Med1epi2/2 mice. B: Evaluation of wound closure by morphometrical analysis of the wound areas. The % of the wound area to the initial area was
calculated from the photographs. N = number of mice; n = number of measurements. Bars = means 6 SE. *P,0.05. C: Representative histological view
of skin wound healing on day 3. Arrowheads and arrows indicate original wound edges and re-epithelialized leading edges, respectively. Scale
bar = 500 mm. D: Time-course of changes of the re-epithelialization ratio after wounding in wild-type and Med1epi2/2 mice. The % re-epithelialization
was calculated by measuring the distance between the leading edges and the width between original wound edges as described in the Materials and
Methods. N = number of mice; n = number of sections. Bars = means 6 SE. *P,0.05, **P,0.01.
Figure 2. Migrating epithelial tongues are elongated and the proliferation of keratinocytes is enhanced in 8-week-old Med1epi2/2
mice. A: The lengths of migrating epithelial tongues were measured on days 1 and 3 after injury in 8-week-old wild-type and Med1epi2/2 mice.
N = number of mice; n = number of measurements. Bars = means 6 SE. **P,0.01. B: Analysis of keratinocyte proliferation at the re-epithelialized
leading edges in 8-week-old wild-type and Med1epi2/2 mice. Images show representative high-power fields of immunohistochemistry for Ki67 in
epidermal cells in the transitional epidermis and in the migrating epithelial tongues on days 1, 3 and 5 after injury. Scale bar = 50 mm. C:
Quantification of proliferating cells on days 1, 3 and 5 after injury. Ki67-positive cells were counted in the transitional epidermis and the epithelial
tongues  of wound sites in 8-week-old wild-type and Med1epi2/2 mice and were related to the area of the same part of the epidermis. N = number
of mice; n = number of measurements. Bars = means 6 SE. *P,0.05, **P,0.01. D: Evaluation of distance between the original wound edges in
8week-old wild-type and Med1epi2/2 mice on days 1, 3 and 5 after injury. N = number of mice; n = number of sections. Bars = means 6 SE. N.S., not
significant. E: Immunohistochemistry of a-SMA for the detection of myofibroblasts in the granulation tissue on days 5 and 7 after wounding. F: The
area stained with a-SMA was determined by planimetric image analysis using ImageJ software. N = number of mice; n = number of sections.
Bars = means 6 SE. N.S., not significant.
proliferation contribute to the acceleration of skin wound healing
in 8-week-old Med1epi2/2 mice.
As wound contraction also significantly contributes to the
wound healing process, the distance between the original wound
edges was microscopically measured to precisely evaluate the
contraction of wounds in Med1epi2/2 skin. The original wound
edges were determined as the start sites of re-epithelialization. As
shown in Figure 2D, there was no significant difference in wound
contraction between Med1epi2/2 and wild-type mice. Further,
because myofibroblasts play pivotal roles in granulation and scar
formation as well as in wound contraction, we investigated dermal
myofibroblasts in the wound sites on days 5 and 7 after wounding
(Figure 2E). Myofibroblasts were identified by staining for a-SMA.
a-SMA-positive myofibroblasts were similarly distributed in
8week-old Med1epi2/2 and wild-type mice (Figure 2F), suggesting
that granulation and scar formation were not affected in
8-weekold Med1epi2/2 mice.
Follistatin expression is decreased and the MAPK
pathway is activated in Med1epi2/2 keratinocytes in vitro
Our previous microarray study comparing gene expression
profiles between Med1epi2/2 and wild-type keratinocytes ,
which is deposited in the GEO repository (http://www.ncbi.nlm.
nih.gov/geo) under the accession number GSE35406, revealed
that the expression of follistatin is significantly suppressed in
Med1epi2/2 keratinocytes, while the expression of activin, a target
of follistatin, as well as activin receptors is not altered. Consistent
with this previous data, the expression of follistatin in Med1epi2/2
keratinocytes was significantly decreased compared with wild-type
keratinocytes (Figure 3A). In the activin-follistatin system crucial
for wound repair [10,11], follistatin sequesters and inhibits activin.
On the other hand, activin secreted from keratinocytes and
fibroblasts during the wound healing process [12,13] activates the
MAPK pathway in keratinocytes, influencing their proliferation as
well as their migration . These facts prompted us to study
whether the MAPK pathway is activated in Med1epi2/2
keratinocytes. The phosphorylation of JNK as well as ERK was enhanced
in Med1epi2/2 -derived keratinocytes (Figure 3A) compared with
wild-type keratinocytes but the phosphorylation of p38 was not
apparently enhanced in Med1epi2/2 keratinocytes (data not
shown). These results indicated that endogenous activin secreted
from keratinocytes in vitro can robustly activate the MAPK
pathway in an autocrine manner in Med1epi2/2 keratinocytes,
where follistatin expression was decreased.
Migration is enhanced in Med1epi2/2 keratinocytes in
Next, to test the effect of endogenous activin secreted from
keratinocytes on the migration of Med1epi2/2 keratinocytes, we
performed an in vitro wound healing assay in medium without
growth factors (Figure 3B). Under these conditions, Med1epi2/2
keratinocytes showed enhanced motility at 24 h, 48 h and 72 h
after wounding compared with wild-type keratinocytes
(Figure 3C), indicating that endogenous activin secreted from
keratinocytes in vitro may activate migration more intensely in
Med1epi2/2 keratinocytes, conceivably because their expression of
JNK phosphorylation in Med1epi2/2 keratinocytes is
augmented by exogenous activin A in vitro
It has been reported that activin A is mainly secreted from
dermal fibroblasts and acts on keratinocytes in a paracrine
manner, contributing to skin homeostasis, wound healing and
hair cycling . Accordingly, several reports have suggested that
exogenous activin can enhance the proliferation and migration of
keratinocytes by activating the MAPK pathway .
Therefore, we next examined the activation of JNK in Med1epi2/2
keratinocytes by exogenous activin A. As shown in Figure 3D,
activin A caused an immediate and transient JNK
phosphorylation, which was detectable at 10 min after the treatment and was
reduced to the basal level at 30 min in Med1epi2/2 and in
wildtype keratinocytes. The peak level as well as the basal level of
phosphorylation of JNK in Med1epi2/2 keratinocytes was
Figure 4. Skin wound healing is delayed in 6 month old Med1epi2/2 mice. A: Representative macroscopic views of skin wounds on days 1, 3, 5
and 7 after wounding in 6-month-old wild-type and Med1epi2/2 mice. Full-thickness wounds (4 mm in diameter) were made on the middle of the
back skins of mice and wound healing was monitored by taking digital photographs. Note that wound healing in 6-month-old Med1epi2/2 mice was
significantly delayed compared with the age-matched wild-type mice. B: Evaluation of wound closure by morphometrical analysis of the wound areas
in 6-month-old wild-type and Med1epi2/2 mice. The % of the wound area to the initial area was calculated from the photographs. N = number of mice;
n = number of measurements. Bars = means 6 SE. **P,0.01. C: Re-epithelialization ratio on days 1, 3 and 5 after wounding in 6-month-old wild-type
and Med1epi2/2 mice. The % re-epithelialization was calculated as mentioned above. N = number of mice; n = number of sections. Bars = means 6 SE.
*P,0.05, **P,0.01. D: The lengths of migrating epithelial tongues were measured on days 1, 3 and 5 after injury in 6-month-old wild-type and
Med1epi2/2 mice. N = number of mice; n = number of measurements. Bars = means 6 SE. *P,0.05, **P,0.01. E: Evaluation of distance between
original wound edges in 6-month-old wild-type and Med1epi2/2 mice on days 1, 3 and 5 after injury. N = number of mice; n = number of sections.
Bars = means 6 SE. N.S., not significant.
augmented compared with wild-type keratinocytes (Figure 3E),
suggesting that exogenous as well as endogenous activin A
enhances JNK phosphorylation in Med1epi2/2 keratinocytes
because of their decreased expression of follistatin.
Exogenous activin A increases the percentage of
Med1epi2/2 keratinocytes in S-phase
We next asked if exogenous activin A also influences the cell
cycle of Med1epi2/2 keratinocytes. To optimize the readout, the
cells were cultured in KBM and starved for 24 h, were
subsequently treated with activin A for 24 h and then were finally
subjected to cell cycle analysis. The results showed that the
Sphase percentage of activin A-treated Med1epi2/2 keratinocytes
cultured in KBM was significantly higher than activin A-treated
wild-type keratinocytes (Figure 3F, left, p,0.01). On the other
hand, cell cycle phases were similar in activin A-treated Med1epi2/
2 and wild-type keratinocytes when cultured in keratinocyte
growth medium (KGM) containing numerous growth promoters
(Figure 3F, right). This is probably because the excess growth
promoters in the KGM masked the endogenous activin A effect.
Together these data suggest that the wound healing acceleration
in 8-week-old Med1epi2/2 mice could be ascribed to the alteration
of follistatin-activin balance in the wound sites, which activates
MAPK signaling and keratinocyte proliferation and migration.
Skin wound healing in old Med1epi2/2 mice is delayed
Next, to assess the skin wound healing process in older
Med1epi2/2 mice, we performed wound healing assays in
6month-old wild-type and Med1epi2/2 mice. As demonstrated in
Figure 4A and 4B, the wound healing process was significantly
delayed on days 1 (p,0.01), 3 (p,0.05) and 5 (p,0.01) after
injury in 6-month-old Med1epi2/2 mice, compared with wild-type
mice. In line with this observation, the re-epithelialization ratio
was significantly decreased on days 1 (p,0.05) and 3 (p,0.01)
after injury in 6-month-old Med1epi2/2 mice (Figure 4C). The
lengths of migrating epithelial tongues were correspondingly
decreased on days 1 (p,0.05) and 3 (p,0.05) after injury in
6month-old Med1epi2/2 mice (Figure 4D), while no significant
6 SE. **P,0.01. C: BrdU-positive slow-cycling label retaining cells in hair follicles in 6-month-old wild-type and Med1epi2/2 mice were detected on day
difference was observed in the wound contraction between
6month-old wild-type and Med1epi2/2 mice (Figure 4E). Compared
with age-matched wild-type mice, Ki67-positive proliferating
keratinocytes were decreased in 6-month-old Med1epi2/2 mice
per area of transitional epidermis and the epithelial tongue in
wound sites on days 1 (p,0.01), 3 (p,0.01) and 5 (p,0.01) after
the injury (Figure 5A and B). Immunohistochemical staining for
follistatin revealed no difference in the expression of follistatin
between 8-week-old and 6-month-old Med1epi2/2 mice (data not
shown), suggesting that a mechanism(s) other than the
activinfollistatin system could influence this impediment of wound
healing in 6-month-old Med1epi2/2 mice.
There is less contribution of BrdU-positive label retaining
cells in hair follicles to cutaneous wound healing in old
It has been reported that, after epidermal injury, hair follicle
stem cells give rise to short-lived transient amplifying cells, which
migrate into the wound epithelium and promote the epidermal
regeneration . In our previous study, CD34-positive and
keratin 15-positive hair follicle bulge stem cells decreased in
Med1epi2/2 mice after several months of age, resulting in sparse
hair in older Med1epi2/2 mice . Therefore, we hypothesized
that the delay of skin wound healing in the older Med1epi2/2 mice
can be attributed to the possible reduction of hair follicle stem
cells. To investigate the contribution of hair follicle stem cells to
skin wound healing in old Med1epi2/2 mice, we performed a BrdU
pulse-labeling experiment in 6-month-old Med1epi2/2 mice and
age-matched wild-type mice, 2 months before the wound creation.
The analysis of BrdU label retaining cells in hair follicles adjacent
to the wounds demonstrated that the number of BrdU-positive
follicular slow-cycling cells was decreased in 6-month-old
Med1epi2/2 mice compared with age-matched wild-type mice
(Figure 5C, p,0.01), indicating a significant depletion of hair
follicle bulge stem cells in 6-month-old Med1epi2/2 mice, which
corresponds to our previous study . Furthermore, BrdU-positive
label retaining cells that had migrated into the epidermis adjacent
to the wounds were detected in hair follicles of 6-month-old
wildtype mice, while no such migrating cells were detected in
6-monthold Med1epi2/2 mice (Figure 5C). This observation suggested that
there is a distinct contribution of hair follicle bulge stem cells to the
epidermal regenerative process in 6-month-old wild-type mice, but
not in 6-month-old Med1epi2/2 mice. On the other hand, the
number of label retaining cells in hair follicles in 8-week-old
Med1epi2/2 mice was comparable with age-matched wild-type
mice (Figure 5D).
In the present study, we investigated the effects of MED1
depletion in the epidermis on cutaneous wound healing in
Med1epi2/2 mice. Our results provide the first evidence that
cutaneous wound healing is accelerated in 8-week-old Med1epi2/2
mice compared with age-matched wild-type mice (Figure 1). The
8-week-old Med1epi2/2 mice demonstrated a rapid
re-epithelialization due to enhanced epidermal proliferation as well as
migration in the wound sites but that was not due to the wound
contraction (Figure 2). Although MED1 is known to function as a
co-activator of nuclear receptors, such as PPAR, RXR and VDR,
there has been no report demonstrating the accelerated wound
healing phenotype in PPAR-KO mice, RXR-KO mice or
VDRKO mice . Interestingly, Med1epi2/2 keratinocytes show a
significantly decreased expression of follistatin, a potent inhibitor
of activin, with significantly increased MAPK activity compared
with wild-type keratinocytes (Figure 3).
Activins, members of the TGF-b superfamily, are
disulfidelinked dimeric proteins comprised of two b subunits. Three
different forms of activin, homodimeric activin A (bAbA),
homodimeric activin B (bBbB) and heterodimeric activin AB
(bAbB), have been identified. Activins bind to heteromeric
complexes of transmembrane receptor serine/threonine kinases,
type I (ACVR1, 1B and 1C) and type II (ACVR2A and 2B) activin
receptors , mediating their biological roles including the
regulation of proliferation, differentiation, apoptosis, metabolism,
homeostasis, immune function, endocrine function and wound
repair in many tissues . Follistatins, antagonists of activins, are
soluble extracellular proteins consisting of varying molecular
weight isoforms due to alternative splicing at the 39 end of the
mRNA [13,3335]. The most common isoforms of follistatin
consist of 288 and 315 amino acids (FS288 and FS315,
respectively). Follistatins have a higher affinity to the activin b
subunits than the activin receptors  and inhibit the action
of activins by two distinct mechanisms, as follows: 1)
Membranebound follistatin FS288 has a high affinity to cell surface bound
heparin sulfate, which causes the follistatin/activin complex to be
internalized and subjected to lysosomal degradation. 2) The
circulatory form of follistatin FS315, which contains a C-terminal
acid tail, binds to activin and prevents binding to its receptors .
Although the precise distribution of the components of activin
signaling in normal skin is uncertain, it is likely that the activinbA
subunit is expressed in dermal cells while the activinbB subunit is
expressed in proliferating keratinocytes at the wound edge and in
the migrating epithelial tongue after injury . On the other
hand, follistatin mRNA is expressed mainly in the dermis and at
low levels in the epidermis [12,13].
Several studies using transgenic and knockout mice have clearly
suggested the critical involvement of activins and follistatins during
cutaneous wound healing. It has been reported that transgenic
mice over-expressing the activinbA chain in keratinocytes showed
an acceleration of the skin wound healing process with increased
keratinocyte proliferation, hyperthickening of the tongue
epithelium and excessive scar formation after skin injury [39,40].
Additionally, it has been reported that mice without follistatin
expression in keratinocytes (Fst null mice) show enhanced
keratinocyte proliferation in the tail epidermis resulting in a
thicker epithelium at the wound edge without excessive scarring
after skin injury .
Previously it was reported that activins mediate wound repair
after injury through the MAPK signaling pathway . It has
been reported that the blockade of JNK signaling by a
JNKspecific inhibitor significantly suppresses keratinocyte proliferation
at the wound site and subsequently delays wound closure [15,16].
In our model, Med1epi2/2 keratinocytes exhibit decreased
follistatin expression and an increased activity of the MAPK
pathway with or without the existence of exogenous activin A in
vitro. Moreover, migration is enhanced in Med1epi2/2
keratinocytes in vitro without exogenous activin A, while exogenous activin
A elicits an increase in the percentage of Med1epi2/2 keratinocytes
in S-phase (Figure 3). After injury, 8-week-old Med1epi2/2 mice
show accelerated cutaneous wound healing without excessive
granulation tissue formation (Figure 2). These findings suggest that
in Med1epi2/2 mice, the loss of follistatin expression in
keratinocytes enhances the biological activity of activin secreted from
keratinocytes and/or dermal fibroblasts and thus constitutively
activates the MAPK signaling pathway in the epidermis, resulting
in rapid wound healing just like in Fst null mice.
Several reports have suggested that MED1 and nuclear
receptors are involved in regulating follistatin expression in various
tissues. Necela et al. reported that activation of PPARc
downregulates the expression of follistatin mRNA through dimerization
with RXR in intestinal epithelial cells . Matsumoto et al.
reported that follistatin shows higher levels of expression in normal
livers after partial hepatectomy but not in MED1-deficient livers,
using transgenic mice . Therefore, it is conceivable that
MED1 depletion in keratinocytes has a direct and/or indirect
effect on the expression of follistatin in our model, although the
details are yet to be elucidated.
Accumulating evidence indicates that there are multiple
populations of epithelial stem cells locating in different parts of
the epidermis [19,20,23,24,4457]. They maintain normal skin
homeostasis by regenerating the distinct epithelial cell lineages in
the distinct parts of the epidermis as well as contribute to wound
healing upon injury by recruiting undifferentiated progenitor cells
to the wounded epidermis [21,2326]. Hair follicle stem cells do
not normally contribute to epidermal homeostasis. However, after
epidermal injury, hair follicle stem cells give rise to short-lived
transient amplifying cells which are recruited into the wounded
epidermis, facilitating the epidermal regeneration . Hair
follicles contain several populations of epithelial stem cells
characterized by distinct expression patterns of stem cell markers,
including CD34 and keratin 15 [19,20]. Previously, we reported
that the numbers of hair follicle stem cells which express CD34
and keratin 15 are reduced in Med1epi2/2 mice from a few months
to one year after birth, which suggests that MED1 plays a distinct
role in the maintenance of hair follicle stem cells .
Correspondingly, skin wound healing in 6-month-old Med1epi2/2 mice is
significantly delayed with decreased numbers of Ki67-positive
proliferating keratinocytes compared with age-matched wild-type
mice (Figures 4, 5). The analysis of BrdU-positive label retaining
cells further showed decreased numbers of hair follicle bulge stem
cells migrating into the epidermis adjacent to the wound sites in
6month-old Med1epi2/2 mice, while no apparent change in
8-weekold Med1epi2/2 mice, compared with age-matched wild-type mice
(Figure 5). These findings indicate an impaired skin wound healing
process due to the lack of CD34-positive and/or keratin
15positive epithelial stem cells, which counteracts the positive effect
of follistatin down-regulation on the wound healing in
6-monthold Med1epi2/2 mice (shown schematically in Figure 6). To our
knowledge, such a phenotype, in which cutaneous wound healing
is accelerated in adolescence and is retarded in the elderly due to
depletion of hair follicle stem cells, has not been previously
reported and therefore is specific.
Although the precise mechanism by which MED1 depletion
participates in the activin-follistatin system in keratinocytes and in
hair follicle stem cell maintenance remains unclear, and the
possibility that other factors involved in the wound healing process
of Med1epi2/2 skin can not be excluded, our findings shed light on
a novel function of MED1 and offer possible new therapeutic
approaches to target MED1 in the epidermis for cutaneous wound
healing and aging.
Materials and Methods
The generation of Med1epi2/2 mice, in which Med1 is disrupted
under control of the keratin 5 promoter, was described elsewhere
. All animal studies were performed according to protocols
approved by the Institutional Animal Care and Use Committee at
Osaka University. Mice that were used for wound healing study
were housed appropriately as previously described . Briefly,
Mice were raised under light/dark (12-h/12-h) cycles and fed
adlibitum amount of standard chow and water according to the
Institutional Animal Care and Use Committee at Osaka
University. Mice were observed daily by the investigators and treated
Wound Creation and Macroscopic Examination
Full-thickness wounds were made using a sterile biopsy punch
with a diameter of 4 mm (NIPRO, Osaka, Japan) on the middle
dorsal shaved telogen skin of Med1epi2/2 mice and wild-type
(Med1+/+) littermates at either 8 weeks or 6 months of age. Mice
were administered sodium pentobarbital with or without
sevoflurane anesthesia before wounding. The wounds were left
uncovered and the animals were housed in separate cages. Wound
healing was macroscopically monitored by digital photography at
the indicated time points. The wound areas (percentage of wound
area relative to the original wound) were calculated using the
following formula: Relative open wound area (%) = [Open area on
the indicated time point/Original wound area]6100.
Immunohistochemical staining was performed as previously
described . In brief, 5 mm thick paraffin sections were
deparaffinized and autoclaved in 10 mM sodium citrate (pH 6.0)
for 15 min at 121uC to retrieve epitope structures. After washing
in TBS-T (Tris-buffered saline with 1% Tween 20), the sections
were treated with H2O2 and endogenous peroxidase activity was
blocked. Specimens were then blocked with Protein Block
SerumFree (Dako, Glostrup, Denmark), incubated with rabbit polyclonal
anti-Ki67 IgG (1:500; Leica Microsystems, Buffalo Grove, IL), and
mouse monoclonal anti-a-SMA IgG (1:100; Dako) overnight at
4uC followed by incubation and visualization with a ChemMate
ENVISION/HRP kit (Dako). Immunohistochemical staining for
BrdU was performed using a BrdU In-Situ Detection kit (BD
Bioscience, New Jersey, US) according to the manufacturers
Analysis of Re-Epithelialization and Wound Contraction
The width of each wound and the distance of the traversed
epithelium were measured in H&E-stained sections at the
indicated time points. The percentage of re-epithelialization was
calculated according to the following formula: [distance of the
minor axis covered by epithelium]/[distance of the minor axis
between original wound edges]6100. The original wound edges
were determined as the start sites of re-epithelialization (See
Figure 1C). Wound contraction was estimated by measuring the
distance of the minor axis between the original wound edges.
Analysis of Cell Proliferation and Granulation Tissue
Ki67-positive cells were counted in the transitional epidermis
and the epithelial tongue  of wounds and were related to the
area of the same part of epidermis. The area of wound epidermis
was determined using ImageJ software (National Institutes of
Health, Bethesda, MD, USA). Myofibroblasts were identified by
immunostaining of a-SMA in the granulation tissue and the
stained area was determined by planimetric image analysis using
Isolation and Culture of Keratinocytes
Isolation and culture of keratinocytes was performed as
previously described . In brief, skins of newborn mice were
derived after the mice had been sacrificed with excess anesthesia.
Derived newborn mice skins were then treated with dispase and
trypsin to separate the epidermis from the dermis. Isolated
keratinocytes were then seeded on type I collagen coated dishes,
and were cultured in CnT07 conditioned culture medium (KGM,
CELLnTEC, Bern, Switzerland). For each experiment,
keratinocytes were used as a primary culture or after one passage.
Immunoblotting was performed as previously described .
Keratinocytes were cultured in keratinocyte basal medium (KBM,
COSMO BIO, Tokyo, Japan) containing 0.03 mM calcium for
24 h and were washed and lysed in protein extraction buffer
containing pH 7.2, 20 nmol/L HEPES with 1% Nonidet P-40,
0.4 M NaCl and aprotinin. Total protein extracts (10 mg/lane)
were mixed with 26 SDS-PAGE buffer, and were heat denatured
with 5% mercaptoethanol for 5 min at 80uC before loaded onto 4
to 12% gradient Tris-glycine gels (Invitrogen, Carlsbad, CA).
After electrophoresis and transfer of proteins to nitrocellulose
membranes, membranes were blocked in 5% milk in TBS-T
buffer for 1 h, followed by 1 h incubation with rabbit polyclonal
anti-follistatin IgG (sc-30194) (1:200; Santa Cruz Biotechnology,
Santa Cruz, CA), rabbit polyclonal anti-phospho-SAPK/JNK
IgG (#9251) (1:1000; Cell Signaling, Danvers, MA), rabbit
polyclonal anti-SAPK/JNK IgG (#9252) (1:1000; Cell
Signaling), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2)
(Thr202/Tyr204) IgG (#9101) (1:1000; Cell Signaling), rabbit
polyclonal anti-p44/42 MAPK (Erk1/2) IgG (#9102) (1:1000;
Cell Signaling) or anti-bb-actin. After washing 3 times with
TBS-T, membranes were incubated for 1 h with horseradish
peroxidase conjugated secondary antibody. Visualization of the
blots was performed using the ECL Plus Western Blotting
Detection System (GE Healthcare, Buckinghamshire, UK). To
investigate the phosphorylation of JNK and ERK by activin A
treatment, after 24 h of supplement depletion, cultured
keratinocytes were treated with or without 5 ng/mL recombinant
human/mouse/rat activin A (R&D Systems, Minneapolis, MN)
in KBM and were then harvested at 10, 30 and 60 min after the
treatment. Quantification and densitometric analysis was
performed using ImageJ software.
Keratinocyte migration assay
For a wound healing assay, keratinocytes derived from skins of
newborn Med1epi2/2 mice and wild-type (Med1+/+) littermates
were cultured in KBM and allowed to form confluent monolayers.
After serum starvation for 24 h, keratinocytes were incubated with
an S-phase cell cycle blocker mitomycin C (0.5 mg/ml) for 2 h.
After straight scratch wounds were made with a p200 pipette tip,
the suspended cells were removed washing with PBS and then
incubated in KBM for 72 h. The number of cells which migrated
into each wounded space was counted microscopically at the noted
time intervals and related to the wounded area.
Cell cycle analysis
For cell cycle analysis, keratinocytes derived from skins of
newborn Med1epi2/2 mice and wild-type (Med1+/+) littermates
were seeded (2.56105) and cultured in KBM for 24 h to
synchronize the cell cycles. Then, keratinocytes were treated with
activin A (5 ng/mL) and BrdU (10 mM) and cultured in KBM for
24 h and harvested. The cell cycle of keratinocytes was analyzed
by FACS CantoII(BD Biosciences) using a BD Pharmingen BrdU
Flow kit (BD Biosciences) according to the manufacturers
BrdU labeling procedures
BrdU labeling of slow-cycling cells was performed as previously
described . Briefly, for BrdU labeling in 6-month-old mice,
4month-old Med1epi2/2 mice and wild-type (Med1+/+) littermates
were intraperitoneally injected with BrdU (50 mg per g body
weight) twice daily for 5 d and then conventional club hair
plucking was performed. Eight weeks after plucking, wound
creation and subsequent skin biopsy were performed and followed
by immunohistochemistry for BrdU as described above. The
number of BrdU-positive cells was counted in hair follicle sections
with bulge region in 6-month-old Med1epi2/2 mice and
agematched wild-type mice. For BrdU labeling in 8-week-old mice,
neonatal mice were subcutaneously injected with BrdU (50 mg per
g body weight) twice daily for 3 d from the third day after birth.
After eight weeks, conventional club hair plucking and subsequent
skin biopsy were performed and followed by
immunohistochemistry for BrdU. The number of BrdU-positive cells was counted in
hair follicle sections with bulge region in 8-week-old Med1epi2/2
mice and age-matched wild-type mice.
An unpaired t-test was used to determine statistical significance
when the values were normally distributed. An F-test was used to
test if the variances are equal. When variances were significantly
different according to the F-test, an unpaired t-test with Welchs
correction was used.
We greatly appreciate Ms. Ayako Sato for technical assistance.
Conceived and designed the experiments: FN TN S. Inui S. Itami.
Performed the experiments: FN. Analyzed the data: FN. Contributed
reagents/materials/analysis tools: FN. Wrote the paper: FN. Contributed
to creation of MED1-KO mice: JKR.
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