-
The FASEB Journal express article 10.1096/fj.03-0244fje.
Published online December 4, 2003.
Thymosin 4 increases hair growth by activation of hair follicle
stem cells Deborah Philp, Mychi Nguyen, Brooke Scheremeta, Sharleen
St-Surin, Ana M. Villa, Adam Orgel, Hynda K. Kleinman, and Michael
Elkin Cell Biology Section, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda,
Maryland 20892
Corresponding author: Hynda K. Kleinman, Ph.D., Cell Biology
Section, NIH, NIDCR, Building 30, Room 433, 30 Convent Dr. MSC
4370, Bethesda, MD 20892. E-mail: [email protected]
ABSTRACT
Thymosin 4, a 43-amino acid polypeptide that is an important
mediator of cell migration and differentiation, also promotes
angiogenesis and wound healing. Here, we report that thymosin 4
stimulates hair growth in normal rats and mice. A specific subset
of hair follicular keratinocytes in mouse skin expresses thymosin 4
in a highly coordinated manner during the hair growth cycle. These
keratinocytes originate in the hair follicle bulge region, a niche
for skin stem cells. Rat vibrissa follicle clonogenic
keratinocytes, closely related, if not identical, to the
bulge-residing stem cells, were isolated and their migration and
differentiation increased in the presence of nanomolar
concentrations of thymosin 4. Expression and secretion of the
extracellular matrix-degrading enzyme matrix metalloproteinase-2
were increased by thymosin 4. Thus, thymosin 4 accelerates hair
growth, in part, due to its effect on critical events in the active
phase of the hair follicle cycle, including promoting the migration
of stem cells and their immediate progeny to the base of the
follicle, differentiation, and extracellular matrix remodeling.
Key words: matrix metalloproteinase-2 clonogenic
keratinocytes
he mature hair follicle is a complex miniorgan that has a
tightly regulated growth cycle. During postnatal development, the
follicle undergoes successive phases of active hair shaft
production (anagen), apoptosis-driven regression (catagen), and a
quiescent phase
(telogen) (1). During the anagen phase, active hair growth
involves cell proliferation in the proximal follicular epithelium,
followed by invasion of the elongating follicle into the
subcutaneous tissue, differentiation of the epithelium at the base
of the follicle, and formation of hair matrix cells, which
proliferate and generate a new hair shaft. Then a regression phase
(catagen) of the hair growth cycle ensues, during which the lower
part of the follicle undergoes programmed cell death and involution
(13). At this point, the follicle enters telogen, the resting
period. The cycle is then repeated. The ability of hair follicles
to constantly renew is ensured by the presence of the multipotent
stem cells, which, upon division, generate two types of daughter
cells. Some of the daughter cells retain the same multipotent
phenotype, while others become rapidly dividing transit-amplifying
(TA) cells, which provide differentiated progeny for the
T
-
regeneration of the lower follicle at the onset of each new
cycle (4, 57). The bulge region of the follicle, located close to
the insertion of the arrector pili muscle, has been identified as a
stem cell niche (510). At the onset of anagen, bulge-localized,
multipotent stem cells or their daughter TA cells migrate to the
base of the follicle to further differentiate to matrix cells and
to produce a new hair shaft (6, 7, 9, 10). Interestingly, cells
emanating from the bulge region migrate downward to repopulate the
hair matrix and also migrate upwards to replenish the skin
epithelium and may therefore contribute to wound healing processes
(9).
Thymosin 4, a ubiquitous 4.9-kDa polypeptide originally isolated
from bovine thymus, is a potent mediator of cell migration and
differentiation (1116). It was identified as a gene up-regulated
four- to sixfold during early endothelial cell tube formation and
found to promote angiogenesis. It is present in wound fluid (17),
and when added topically or given systemically, it promotes
angiogenesis and wound healing (13). Thymosin 4 elicits cell
migration through a specific interaction with actin (18, 19).
Recently, we demonstrated that a central 7-amino acid (LKKTETQ)
actin binding domain has both angiogenic and wound healing activity
while other domains are inactive (20). In angiogenesis and in wound
healing, thymosin 4 acts by accelerating the migration of
endothelial cells and keratinocytes and increasing the production
of extracellular matrix-degrading enzymes (14, 19). Thymosin 4 also
has anti-inflammatory activity (11, 21).
The process of hair growth utilizes many cellular and molecular
mechanisms common to angiogenesis and wound healing, namely
migration, differentiation, and remodeling of the extracellular
matrix (911, 2225). In the present study, we investigated the role
of thymosin 4 in hair growth in different in vitro and in vivo
experimental models. Thymosin 4 promotes hair growth in normal rats
and mice. A specific subset of follicular keratinocytes in the
mouse skin, which originates at the bulge region, expresses
thymosin 4. The temporal and spatial distribution of these
keratinocytes parallel the pattern reported for the stem cells and
their daughter TA cells at the different stages of the hair cycle
(9, 10). We isolated clonogenic keratinocytes from the bulge
compartment of the rat vibrissa follicle, further characterized
them as an immediate progeny of the stem cells, and found that
these cells express high levels of thymosin 4 when cultured in
vitro. We show that thymosin 4 promotes hair clonogenic
keratinocyte cell migration, as well as secretion of the
extracellular matrix-degrading enzyme matrix metalloproteinase 2
(MMP-2). We also found that thymosin 4 initiated early
differentiation of these cells based on reduction in the expression
of keratin 15, a specific marker of epidermal stem cells (26).
Taken together, our results suggest that in addition to its known
angiogenic and wound healing effects, thymosin 4 is a naturally
occurring modulator of hair growth that acts by stimulation of stem
cell migration, protease production, and differentiation.
METHODS
Hair growth
Thymosin 4 was prepared by the FDA (Bethesda, MD). Thymosin 4
and the peptides at 0.05% (w/v) in 0.2% polyacrylic acid hydrogel
(from Mahnaz Badamchian, George Washington University School of
Medicine) were applied topically to the dorsal lateral areas of
shaved rats. For the rat studies, thymosin 4 (0.05%) was applied to
a dorsal/lateral area of the skin and the
-
control vehicle was applied to an opposing adjacent area on the
same animal (3-5 rats/dose). For the mouse studies, 8-wk-old
wild-type C57BL6 mice (2 mice/dose) with their hair in telogen
phase, as identified by their pink backskin color (27), were shaved
and treated separately with either thymosin 4 (0.02%) or control
vehicle. The skin samples were fixed in 4% paraformaldehyde,
embedded in paraffin, and 5 m sections were stained with
Masson-TriChrome. The number of hair follicles in the histological
sections was counted by two different blinded observers with at
least three sections of different fields counted per rat (3-5
rats/data point). These experiments were repeated twice with
similar results. Microphotos were taken at x32 for the rat skin and
x66 for the mouse skin with a Zeiss Stem SVII dissecting scope.
Induction of hair cycle
Depilation was used to induce hair growth in resting follicles,
as described previously by Paus et al. (28). The dorsal skin of
8-wk-old female C57BL/6 mice at the telogen phase (as identified by
their pink skin color) was depilated using hair remover wax strip
kit (Del Laboratories, Farmingdale, NY), leading to synchronized
development of anagen hair follicles. Skin tissue samples were
collected at day 0 (telogen), day 4 postdepilation (early anagen),
and day 9 postdepilation (late anagen; 5 mice/time point). The
samples were fixed in 4% paraformaldehyde and processed for
histological examination and immunostaining. This experiment was
done twice with similar results.
Immunohistochemistry
Immunohistochemical staining was performed on 5 m paraffin
sections using a polyclonal rabbit antibody raised against
full-length thymosin 4 peptide sequence (from Allan Goldstein,
George Washington University School of Medicine). Primary antibody
was detected using Dako EnVision Kit (DakoCytomation, Denmark), and
counterstaining was performed using hematoxylin.
Immunofluorescence
After culture on glass coverslips (12 mm; Carolina Biological
Supply Company, NC), cells were fixed with 4% paraformaldehyde in
PBS with 5% sucrose. The cells were incubated with antibody raised
against thymosin 4 peptide, followed by staining with secondary
CY3-conjugated donkey anti-rabbit antibody, diluted 1:200. Samples
were mounted with GEL/MOUNTTM (Biomeda Corp., Foster City, CA) and
visualized with a Zeiss LM 510 confocal microscope.
Isolation of clonogenic keratinocytes (hair follicle stem
cells)
Clonogenic keratinocytes were isolated from Fisher 344 rat
vibrissa follicles as described previously by Kobayashi et al.
(29). After the animals were killed, the upper lip containing the
vibrissal pad was cut, and its inner surface was exposed. While the
animals were under a dissecting microscope, vibrissa follicles were
dissected and plucked from the pad. A fragment of the follicle
containing the bulge region was cut off and incubated for 30 min in
collagenase/dispase solution (1 mg/ml; Roche Molecular
Biochemicals) at 37C. The epithelial core was detached from the
collagen capsule and further incubated in 0.05%
-
trypsin/collagenase/dispase solution (30 min, 37C) to facilitate
the dissociation of epithelial cells. Isolated cells were cultured
in keratinocyte-SFM medium, containing EGF (2.5 g/500 ml), bovine
pituitary extract (25 mg/500 ml; Invitrogen, Carlsbad, CA), and 10%
FCS. The seeding density was 1000 cells/35 mm plate, and 60-80
colonies per plate were formed. For the proliferation assay, 5000
cells were seeded per well in 96-well plates, and proliferation was
assessed using the CellTiter AQueous Cell Proliferation Assay Kit
(Promega, Madison, WI). This experiment was repeated three times
with similar results. Western blot Cells were lysed by addition of
RIPA buffer, and equal protein aliquots of cell lysates were
separated on 4-12% Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA).
Proteins were transferred to a nitrocellulose membrane (Invitrogen)
and detected using polyclonal antibodies against mouse keratins
(Covance Research Products, Richmond, CA). K15 antibodies were
obtained from Covance and NeoMarkers (Fremont CA). The membranes
were stripped and reprobed with anti-GAPDH antibody (Research
Diagnostics, Flanders, NJ), and densitometry measurements were
taken using NIH Image Software with keratin 15 normalized using
GAPDH.
Migration assays
Migration was studied in 48-well Boyden chambers using 8-m pore
polycarbonate, PVPF, membranes (Poretics, Livermore, CA) coated
with 50 g/ml of collagen type 1 (BD Biosciences, Bedford, MA)
diluted in keratinocyte-SFM that contained 72 mM HEPES buffer.
Cultured clonogenic keratinocytes were harvested using trypsin and
resuspended in keratinocyte-SFM containing 1% bovine serum albumin
factor-V and 25 mM HEPES-buffer. The bottom chamber was filled with
increasing amounts of thymosin 4.. Fibroblast-conditioned medium
was the positive control. Keratinocytes, 30,000 cells/well, were
added to the upper chambers and were incubated at 37C with 5% CO2
for 4.5 h. The membranes were then fixed and stained with Diff-Quik
(VWR, Bridgeport, NJ). Cell migration was quantitated in three
random microscopic fields of triplicate wells. Cells were acquired
at x10 magnification using a Nikon Optiphot-2 microscope for
counting. The assay was repeated twice.
Motility
Clonogenic keratinocytes were plated on 35-mm dishes. Migration
was monitored for 20 h using a Zeiss inverted microscope. Digital
images were collected using a CCD camera (model 2400; Hamamatsu
Photonics) at 10-min intervals, stored as image stacks, converted
to QuickTime movies, and analyzed using MetaMorph Group 3.5
software (Universal Imaging Corp., London, UK). This experiment was
repeated twice, and six cells were tracked in each experiment.
Zymography
Clonogenic keratinocytes were cultured for 9 days. After 16 h
serum deprivation, the cells were incubated for 6 h in the presence
of increasing concentrations of thymosin 4. Aliquots of the cell
lysate and resulting conditioned medium were analyzed for
gelatinolytic activity, using Novex Zymogram Gels (Invitrogen). The
band intensity was determined by densitometry measurements using
NIH Image Software.
-
Statistics
The InStat program was used to determine P values. All data are
means SD.
RESULTS
Thymosin 4 promotes hair growth in rats and mice
While studying wound healing in rat skin, we unexpectedly
observed visually and at the histological level increased hair
growth at the wound margins 7 days after topical treatment with
thymosin 4 (unpublished observation). In this study, we have shaved
the skin of healthy rats and applied thymosin 4 topically on one
side of the shaved area and the control vehicle on the opposing
lateral side of the same animal. After 7 days of treatment, we
observed an increased number of anagen-phase hair follicles in the
skin areas treated with thymosin 4 (Fig. 1a and d). The number of
anagen follicles was approximately twofold greater than in rats
treated with vehicle alone. The increased number of hairs in anagen
phase was retained with continued tri-weekly treatment over 30
days. Within 14 days of treatment cessation, the number of active
hair follicles decreased to control levels. We next tested whether
thymosin 4 would promote hair growth in 8-wk-old C57BL6 wild-type
mice. Animals used in this experiment have all of their hair
follicles in the telogen stage as judged by their pink skin color
(27). The mice were shaved and thymosin 4 was applied topically on
the shaved area as described in Methods. Control animals were
treated with vehicle alone. As shown in Fig. 1c and f, thymosin
4-treated (but not control) animals displayed quick hair regrowth.
Histological examination confirmed the thymosin 4-induced
activation of the hair follicles (Fig. 1b and e).
Thymosin 4 protein expression by a subset of hair follicle stem
cells
We first explored the spatial and temporal pattern of endogenous
thymosin 4 expression in hair follicles during depilation-induced,
synchronized adult hair cycling in C57BL/6J mice. We wanted to
correlate the observed effects of thymosin 4 administration with
possible functional involvement of endogenous thymosin 4 in hair
growth. Low levels of thymosin 4 protein were observed in follicles
at the telogen (resting) phase, before depilation (Fig. 2). In
these follicles, thymosin 4 expression was confined to a small
number of cells residing in the bulge region, at the level of the
insertion of the arrector pili muscle. Hair follicle transition to
early anagen (day 4 after depilation) was associated with an
increased number of thymosin 4-expressing cells in the bulge region
(Fig. 2). Moreover, some thymosin 4-positive-stained cells were
detected in the lower part of the follicle, between the bulge and
bulb area (Fig. 2, arrowhead). At late anagen (day 9 after
depilation), a significant number of the cells located in the lower
follicle (matrix-surrounding part of the outer root sheath)
expressed thymosin 4 both in the nucleus and cytoplasm. The
sebaceous gland was stained at all stages, due to nonspecific
absorption as found by others with several different antibodies.
Thus, with the progression of the hair growth cycle, thymosin
4-positive cells, initially detected only in the bulge, were
observed at the bulb area, suggesting that they are migrating from
the bulge region. These data show that the temporal and spatial
distribution of thymosin 4-expressing cells was similar to the
pattern proposed for the hair follicle stem cells and their
daughter TA cells, i.e., emanating from the bulge and migrating
downward to give rise to matrix cells that subsequently generate
the hair shaft (9, 10).
-
Cultured rat vibrissa clonogenic keratinocytes express thymosin
4
We studied rat vibrissae follicle keratinocytes from the bulge
region, representing the stem cell population (10, 30), to
determine if isolated stem cells express thymosin 4. Previously,
hair follicle stem cells have been identified as bulge-residing
keratinocytes with a high in vitro clonogenic potential (58, 10,
29-31). Although hair follicle stem cells are not fully
characterized in terms of specific markers, they preferentially
express keratin 15 (K15) (26). We isolated clonogenic keratinocytes
from the rat vibrissa bulge region and found that the isolated
cells were highly clonogenic (Fig. 3a). These cells were positive
for the stem cell marker keratin 15 as well as for keratins 5, 6,
and 14 (Fig. 3c), also known to be expressed by bulge stem cells
(32). Furthermore, these cells lacked keratin 10 (Fig. 3c), a known
early marker of terminal keratinocyte differentiation (4, 31).
Moreover, when cultured in vitro, these cells were able to move
with an average velocity of 0.43 m/min, a typical mobility rate
reported for epidermal stem cells and their daughter TA cells (33).
Based on these characteristics and on previous reports by Kobayashi
et al., (29) and Oshima et al., (10), we conclude that the obtained
cell population represents the immediate progeny of hair follicle
stem cells. Using RT-PCR and immunofuorescent staining approaches,
we found that these cells expressed thymosin 4 mRNA (not shown) and
protein (Fig. 3b) after 7-10 days of culturing in vitro.
Interestingly, treatment of the clonogenic keratinocytes with
exogenous thymosin 4 caused a dose-dependent decrease in the
expression levels of the multipotent undifferentiated stem cell
marker K15 (Fig. 4). A decline in K15 is associated with stem cell
differentiation. Further, we found that thymosin 4 had no effect on
stem cell proliferation (data not shown). These data indicate that
the clonogenic keratinocytes isolated from rat vibrissa bulge
represent the stem cell population and suggest that thymosin 4 is
important for early stem cell differentiation.
Thymosin 4 promotes the migration of hair clonogenic
keratinocytes in vitro
Thymosin 4 has been previously shown to promote endothelial cell
migration (14). Here, we found that cultured clonogenic
keratinocytes migrate to thymosin 4 after 4.5 h in Boyden chamber
assays. In the presence of thymosin 4, cell migration was increased
almost twofold (69.07.1 vs. 113.35.5 cell number per field, P0.001)
at 1 ng over migration in the presence of medium containing vehicle
alone (negative control). The effect of thymosin 4 on cell
migration was greatest at 1 ng/ml and at 100 and 1000 ng/ml
migration was decreased. Previously, we found that 1 ng/ml was very
potent for endothelial and keratinocyte migration (13, 14).
Thymosin 4 augments the production and secretion of MMP-2 by
clonogenic keratinocytes
Enzymatic degradation and remodeling of extracellular matrix,
and particularly the basement membrane that separates the
epithelial (i.e., outer root sheath) and stromal (i.e., dermal
sheath, dermal papilla) compartments of the follicle, are necessary
steps in normal hair development and growth (22, 23, 25, 34). Here,
we have examined the effect of thymosin 4 on the enzymatic activity
of MMPs, enzymes responsible for degradation of major extracellular
matrix proteins and, therefore, matrix and basement membrane
remodeling in many biological processes, including hair follicle
development and growth (22, 25). Treatment of the clonogenic
keratinocytes with exogenous thymosin 4 caused a dose-dependent
increase in the levels of secreted and cell-derived enzyme MMP-2
(Fig. 5). MMP-2 degrades collagen type IV and
-
laminin, key proteins of the basement membrane. This effect on
MMP-2 was specific, since the level of another collagen
IV-degrading enzyme, MMP-9, was unchanged during thymosin 4
treatment (Fig. 5). Since MMP-2 plays a role in both hair
growth-associated extracellular matrix remodeling and cell
migration (2225, 3537), our data suggest that this enzyme may be a
downstream effector through which thymosin 4 exerts its effect on
hair growth.
DISCUSSION
Thymosin 4 is a small 4.9-kDa molecule that functions as a major
actin-sequestering protein in cells and has many biological
activities. It is upregulated during endothelial cell
differentiation, and when added exogenously, it promotes
endothelial cell differentiation and migration (14, 15). In vivo,
it promotes wound repair and is a potent anti-inflammatory agent
(1113, 21). Thymosin 4 is overexpressed in metastatic tumors, and
its up-regulation results in increased tumor cell motility and
metastatic potential (18, 38). A related family member, thymosin
15, is also important in the metastasis of certain tumor types (39,
40). It was reported that thymosin 4 exerts its effects on cell
locomotion through specific interactions with actin that regulate
cytoskeletal organization (18, 19). Here, we show that exogenously
delivered thymosin 4 promotes hair growth in normal rats and mice.
When examining the distribution of endogenous thymosin 4 through
sequential phases of depilation-induced hair growth, we found that
in the resting (telogen) follicle it is expressed in the small
number of cells originating in the bulge region of the outer root
sheath. As the follicles enter the active growth phase (anagen),
the subset of thymosin 4-expressing cells in the outer root sheath
is expanded toward the base of the follicle. At the peak of anagen,
a significant number of thymosin 4-expressing cells are found in
the bulb area, both in the outer root sheath and among the hair
matrix cells. Furthermore, isolated clonogenic hair follicle
keratinocytes, closely related, if not identical, to the hair
follicle stem cells (10, 30), produce thymosin 4 when cultured in
vitro for 7-10 days. In addition, the presence of exogenous
thymosin 4 caused a dose-dependent decrease in the expression of
the stem cell marker K15 by clonogenic keratinocytes, suggesting
that thymosin 4 may promote early stem cell differentiation (i.e.,
transition to the TA phenotype). Most important, treatment of the
bulge-derived clonogenic keratinocytes with exogenous thymosin 4
increased their migration and production of MMP-2.
A critical step in the hair growth cycle, at the transition
between telogen and anagen, is the movement of some of the
bulge-residing stem cells downward, where their differentiated
progeny contribute to complete regrowth or regeneration of the
lower, cycling portion of the follicle (Fig. 2; refs 1, 57, 9, 10).
Our data indicate that thymosin 4 facilitates this movement of the
stem cells and their immediate progeny and, thus, exerts its
promoting effect on hair growth. Apart from its direct role in cell
locomotion, mediated by interaction with actin (18, 19), the effect
of thymosin 4 on MMP-2 expression appears to play an important role
in this system. MMP-2 was previously shown to contribute to
cellular migration, both by means of degrading extracellular matrix
barriers for cell movement and through direct effects on cell
locomotion in vitro (37). In addition, MMP-2 is involved in
hair-cycle-associated remodeling of the basement membrane, the
specialized extracellular matrix structure surrounding the
epithelial core of the follicle. Basement membrane remodeling is
necessary for signaling between epithelial and stromal elements of
the growing follicle and for elongation and invasion of the lower
follicle into subcutaneous tissue during the anagen phase (2325,
35, 36).
-
Hair growth acceleration by thymosin 4 may also be attributed,
in addition to its effects on stem cells, to pro-angiogenic and
other previously described biological activities of this molecule.
It was recently reported that VEGF promotes hair follicle
development, presumably due to its angiogenic activity (41).
Thymosin 4 is angiogenic, like VEGF, and the activity of thymosin 4
may, in addition to its effects on stem cells, be due to its
angiogenic activity. Recently, another angiogenic molecule,
hepatocyte growth factor, has been identified in hair follicles and
found to promote hair growth (34, 4244). Hepatocyte growth factor
up-regulates thymosin 4 expression (44) and may be acting by
increasing thymosin 4 and/or synergizing with it. Furthermore,
steroids have been used successfully to treat certain types of hair
loss (46). Thymosin 4 is the anti-inflammatory molecule identified
as increased in steroid-treated monocytes (21). Thus, treatment
with steroids may also involve the activity of thymosin 4 on the
hair growth.
Taken together, our results suggest that thymosin 4 exerts a
profound hair-promoting effect through a combined action on several
critical events in hair follicle growth, such as stem cell progeny
migration, ECM-degrading enzyme production, and differentiation.
Increased angiogenesis likely also contributes to the hair
growth-promoting effects of thymosin 4.
REFERENCES
1. Paus, R., and Cotsarelis, G. (1999) The biology of hair
follicles. N. Engl. J. Med. 341, 491497
2. Cotsarelis, G. (1997) The hair follicle: dying for attention.
Am. J. Pathol. 151, 1505 1509
3. Stenn, K. S., and Paus, R. (2001) Controls of hair follicle
cycling. Physiol. Rev. 81, 449494
4. Janes, S. M., Lowell, S., and Hutter, C. (2002) Epidermal
stem cells. J. Pathol. 197, 479491
5. Sun, T. T., Cotsarelis, G., and Lavker, R. M. (1991) Hair
follicular stem cells: the bulge-activation hypothesis. J. Invest.
Dermatol. 96, 77S78S
6. Wilson, C., Cotsarelis, G., Wei, Z. G., Fryer, E.,
Margolis-Fryer, J., Ostead, M., Tokarek, R., Sun, T. T., and
Lavker, R. M. (1994) Cells within the bulge region of mouse hair
follicle transiently proliferate during early anagen: heterogeneity
and functional differences of various hair cycles. Differentiation
55, 127136
7. Wilson, C. L., Sun, T. T., and Lavker, R. M. (1994) Cells in
the bulge of the mouse telogen follicle give rise to the lower
anagen follicle. Skin Pharmacol. 7, 811
8. Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E.,
Albelda, S., and Cotsarelis, G. (1999) Human hair follicle bulge
cells are biochemically distinct and possess an epithelial stem
cell phenotype. J. Investig. Dermatol. Symp. Proc. 4, 296301
9. Taylor, G., Lehrer, M. S., Jensen, P. J., Sun, T. T., and
Lavker, R. M. (2000) Involvement of follicular stem cells in
forming not only the follicle but also the epidermis. Cell 102,
451461
-
10. Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K., and
Barrandon, Y. (2001) Morphogenesis and renewal of hair follicles
from adult multipotent stem cells. Cell 104, 233245
11. Sosne, G., Szliter, E. A., Barrett, R., Kernacki, K. A.,
Kleinman, H., and Hazlett, L. D. (2002) Thymosin beta 4 promotes
corneal wound healing and decreases inflammation in vivo following
alkali injury. Exp. Eye Res. 74, 293299
12. Sosne, G., Hafeez, S., Greenberry, A. L., II, and
Kurpakus-Wheater, M. (2002) Thymosin beta4 promotes human
conjunctival epithelial cell migration. Curr. Eye Res. 24,
268273
13. Malinda, K. M., Sidhu, G. S., Mani, H., Banaudha, K.,
Maheshwari, R. K., Goldstein, A. L., and Kleinman, H. K. (1999)
Thymosin beta4 accelerates wound healing. J. Invest. Dermatol. 113,
364368
14. Malinda, K. M., Goldstein, A. L., and Kleinman, H. K. (1997)
Thymosin beta 4 stimulates directional migration of human umbilical
vein endothelial cells. FASEB J. 11, 474481
15. Grant, D. S., Kinsella, J. L., Kibbey, M. C., LaFlamme, S.,
Burbelo, P. D., Goldstein, A. L., and Kleinman, H. K. (1995)
Matrigel induces thymosin beta 4 gene in differentiating
endothelial cells. J. Cell Sci. 108, 36853694
16. Low, T. L., Hu, S. K., and Goldstein, A. L. (1981) Complete
amino acid sequence of bovine thymosin beta 4: a thymic hormone
that induces terminal deoxynucleotidyl transferase activity in
thymocyte populations. Proc. Natl. Acad. Sci. USA 78, 11621166
17. Frohm, M., Gunne, H., Bergman, A. C., Agerberth, B.,
Bergman, T., Boman, A., Liden, S., Jornvall, H., and Boman, H. G.
(1996) Biochemical and antibacterial analysis of human wound and
blister fluid. Eur. J. Biochem. 237, 8692
18. Kobayashi, T., Okada, F., Fujii, N., Tomita, N., Ito, S.,
Tazawa, H., Aoyama, T., Choi, S. K., Shibata, T., Fujita, H., et
al. (2002) Thymosin-beta4 regulates motility and metastasis of
malignant mouse fibrosarcoma cells. Am. J. Pathol. 160, 869882
19. Roy, P., Rajfur, Z., Jones, D., Marriott, G., Loew, L., and
Jacobson, K. (2001) Local photorelease of caged thymosin beta4 in
locomoting keratocytes causes cell turning. J. Cell Biol. 153,
10351048
20. Philp, D., Badamchian, M., Scheremeta, B., Nguyen, M.,
Goldstein, A. L., and Kleinman, H. K. (2003) Thymosin beta 4 and a
synthetic peptide containing its actin-binding domain promote
dermal wound repair in db/db diabetic mice and in aged mice. Wound
Repair Regen. 11, 1924
21. Young, J. D., Lawrence, A. J., MacLean, A. G., Leung, B. P.,
McInnes, I. B., Canas, B., Pappin, D. J., and Stevenson, R. D.
(1999) Thymosin beta 4 sulfoxide is an anti-inflammatory agent
generated by monocytes in the presence of glucocorticoids. Nat.
Med. 5, 14241427
-
22. Karelina, T. V., Bannikov, G. A., and Eisen, A. Z. (2000)
Basement membrane zone remodeling during appendageal development in
human fetal skin. The absence of type VII collagen is associated
with gelatinase-A (MMP2) activity. J. Invest. Dermatol. 114,
371375
23. Jahoda, C. A., Mauger, A., Bard, S., and Sengel, P. (1992)
Changes in fibronectin, laminin and type IV collagen distribution
relate to basement membrane restructuring during the rat vibrissa
follicle hair growth cycle. J. Anat. 181, 4760
24. Yuspa, S. H., Wang, Q., Weinberg, W. C., Goodman, L.,
Ledbetter, S., Dooley, T., and Lichti, U. (1993) Regulation of hair
follicle development: an in vitro model for hair follicle invasion
of dermis and associated connective tissue remodeling. J. Invest.
Dermatol. 101, 27S32S
25. Scandurro, A. B., Wang, Q., Goodman, L., Ledbetter, S.,
Dooley, T. P., Yuspa, S. H., and Lichti, U. (1995) Immortalized rat
whisker dermal papilla cells cooperate with mouse immature hair
follicle buds to activate type IV procollagenases in collagen
matrix coculture: correlation with ability to promote hair follicle
development in nude mouse grafts. J. Invest. Dermatol. 105,
177183
26. Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E.,
Albelda, S., and Cotsarelis, G. (1998) The C8/144B monoclonal
antibody recognizes cytokeratin 15 and defines the location of
human hair follicle stem cells. J. Cell Sci. 111, 31793188
27. Paus, R., Stenn, K. S., and Link, R. E. (1989) The induction
of anagen hair growth in telogen mouse skin by cyclosporine A
administration. Lab. Invest. 60, 365369
28. Paus, R., Stenn, K. S., and Link, R. E. (1990) Telogen skin
contains an inhibitor of hair growth. Br. J. Dermatol. 122,
777784
29. Kobayashi, K., Rochat, A., and Barrandon, Y. (1993)
Segregation of keratinocyte colony-forming cells in the bulge of
the rat vibrissa. Proc. Natl. Acad. Sci. USA 90, 73917395
30. Barrandon, Y., and Green, H. (1987) Three clonal types of
keratinocyte with different capacities for multiplication. Proc.
Natl. Acad. Sci. USA 84, 23022306
31. Rochat, A., Kobayashi, K., and Barrandon, Y. (1994) Location
of stem cells of human hair follicles by clonal analysis. Cell 76,
10631073
32. Fuchs, E. (1995) Keratins and the skin. Annu. Rev. Cell Dev.
Biol. 11, 123153
33. Jensen, U. B., Lowell, S., and Watt, F. M. (1999) The
spatial relationship between stem cells and their progeny in the
basal layer of human epidermis: a new view based on whole-mount
labelling and lineage analysis. Development 126, 24092418
34. Yamazaki, M., Tsuboi, R., Lee, Y. R., Ishidoh, K., Mitsui,
S., and Ogawa, H. (1999) Hair cycle-dependent expression of
hepatocyte growth factor (HGF) activator, other proteinases, and
proteinase inhibitors correlates with the expression of HGF in rat
hair follicles. J. Investig. Dermatol. Symp. Proc. 4, 312315
-
35. Link, R. E., Paus, R., Stenn, K. S., Kuklinska, E., and
Moellmann, G. (1990) Epithelial growth by rat vibrissae follicles
in vitro requires mesenchymal contact via native extracellular
matrix. J. Invest. Dermatol. 95, 202207
36. Weinberg, W. C., Brown, P. D., Stetler-Stevenson, W. G., and
Yuspa, S. H. (1990) Growth factors specifically alter hair follicle
cell proliferation and collagenolytic activity alone or in
combination. Differentiation 45, 168178
37. Stetler-Stevenson, W. G., and Yu, A. E. (2001) Proteases in
invasion: matrix metalloproteinases. Semin. Cancer Biol. 11,
143152
38. Clark, E. A., Golub, T. R., Lander, E. S., and Hynes, R. O.
(2000) Genomic analysis of metastasis reveals an essential role for
RhoC. Nature 406, 532535
39. Bao, L., Loda, M., Janmey, P. A., Stewart, R., Anand-Apte,
B., and Zetter, B. R. (1996) Thymosin beta 15: a novel regulator of
tumor cell motility upregulated in metastatic prostate cancer. Nat.
Med. 2, 13221328
40. Bao, L., Loda, M., and Zetter, B. R. (1998) Thymosin beta15
expression in tumor cell lines with varying metastatic potential.
Clin. Exp. Metastasis 16, 227233
41. Yano, K., Brown, L. F., and Detmar, M. (2001) Control of
hair growth and follicle size by VEGF-mediated angiogenesis. J.
Clin. Invest. 107, 409417
42. Grant, D. S., Kleinman, H. K., Goldberg, I. D., Bhargava, M.
M., Nickoloff, B. J., Kinsella, J. L., Polverini, P., and Rosen, E.
M. (1993) Scatter factor induces blood vessel formation in vivo.
Proc. Natl. Acad. Sci. USA 90, 19371941
43. Lee, Y. R., Yamazaki, M., Mitsui, S., Tsuboi, R., and Ogawa,
H. (2001) Hepatocyte growth factor (HGF) activator expressed in
hair follicles is involved in in vitro HGF-dependent hair follicle
elongation. J. Dermatol. Sci. 25, 156163
44. Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker,
P., Handjiski, B., Roloff, B., and Paus, R. (2000) Involvement of
hepatocyte growth factor/scatter factor and met receptor signaling
in hair follicle morphogenesis and cycling. FASEB J. 14, 319332
45. Oh, I. S., So, S. S., Jahng, K. Y., and Kim, H. G. (2002)
Hepatocyte growth factor upregulates thymosin beta4 in human
umbilical vein endothelial cells. Biochem. Biophys. Res. Commun.
296, 401405
46. Shapiro, J., and Price, V. H. (1998) Hair regrowth.
Therapeutic agents. Dermatol. Clin. 16, 341356
Received April 8, 2003; accepted October 21, 2003.
-
Fig. 1
Figure 1. Histological and gross appearance of skin from control
and thymosin 4-treated rats and mice. For the rat studies, animals
(3-5 per group) were treated on one side of the animal with vehicle
and on the other side of the same animal with thymosin 4 and for
the mouse studies (2 per group), separate mice were used for each
treatment. All sections are stained with Massons Trichrome.
Microphotographs of the rat and mouse histological sections were
made at x32 and x66, respectively. a) Control vehicle-treated rat
skin after 7 days. b) Control vehicle-treated mouse skin after 28
days. c) Gross appearance of control vehicle-treated mouse skin
after 28 days. d) Rat skin after 7 days of thymosin 4 treatment. e)
Mouse skin after 28 days of thymosin 4 treatment. f) Gross
appearance of mice after 28 days of thymosin 4 treatment.
-
Fig. 2
Figure 2. Temporal and spatial distribution of thymosin
4-expressing cells during depilation-induced hair growth cycle.
C57BL/6 mouse dorsal skin of defined hair cycle stages (telogen,
unmanipulated skin; early anagen, 4 days postdepilation; late
anagen, 9 days postdepilation) was harvested and processed for
H&E staining (lower panels, magnification x50) and
immunohistochemical analysis of thymosin 4 expression (upper
panels, magnification x320). Telogen skin: few thymosin
4-positive-stained cells (arrowhead) are localized in the bulge
region, at the site of the insertion of arrector pili muscle. Early
anagen skin: thymosin 4 expression in cells emanating from the
bulge region (arrowhead). Late anagen skin: longitudinal and
transverse sections through the hair follicle basis are shown,
thymosin 4-expressing cells could be easily detected at the basis
of the follicle, in the outer root sheath, in the portion
surrounding the matrix cells (arrowheads). Sebaceous gland (S);
arrector pili muscle (AP).
-
Fig. 3
Figure 3. Isolation, culture, and keratin and thymosin 4
expression by clonogenic keratinocytes from rat vibrissa bulge. a)
Culture of bulge region clonogenic keratinocytes 9 days after
isolation, as described in Methods. Two clonogenic populations are
shown. Magnification: x100. b) Fluorescent immunostaining of
clonogenic keratinocytes with anti-thymosin 4 antibody. Cells were
incubated with primary rabbit antibody raised against full-length
thymosin 4 peptide sequence, followed by staining with secondary
CY3- conjugated donkey anti-rabbit antibody, as described in
Methods. Magnification x630. Control samples were processed without
primary antibody, and no significant background staining was
detected (not shown). c) Pattern of keratin expression by
clonogenic keratinocytes. Cells were washed with PBS and lysed in
RIPA buffer. Lysates were analyzed by Western blot with antibodies
against keratin (K) 5, 6, 10, 14, and 15, as described in Methods.
Note the presence of epidermal stem cell marker K15 and absence of
K10, which is an early marker of terminal differentiation.
-
Fig. 4
Figure 4. Exogenous thymosin 4 decreases expression of keratin
15 in clonogenic keratinocytes. a) Western blot analysis with
anti-K15 antibody. Rat vibrissa clonogenic keratinocytes isolated
from the bulge region were kept in the serum-free medium for 3 h
and then incubated for 8 h in the absence or presence of various
concentrations of thymosin 4. Cells were washed with PBS and lysed
in RIPA buffer. Protein concentration was determined and lysates
normalized for equal protein were analyzed by Western blot with
antibodies against keratin 15. The same membranes were reprobed
with anti-GAPDH antibody. b) Quantitation of the K15 signal was
normalized to GAPDH after densitometry measurements using NIH
software.
-
Fig. 5
Figure 5. Effect of thymosin 4 on MMP-2 production and secretion
by clonogenic keratinocytes. Cells were incubated (16 h) in the
absence or presence of increasing concentrations of thymosin 4 in
serum-free medium. Cell lysates and aliquots of the incubation
media were normalized for equal cell protein and applied for
zymography, as described in Methods. Intensity of the gelatinolytic
activity bands was quantitated by densitometry. a) Zymographic
analysis of secreted material (incubation medium). b and c)
Quantitation of MMP-9 (b) and MMP-2 (c) gelatinolytic activity,
secreted by clonogenic keratinocytes in the absence or presence of
thymosin 4. Intensity of the gelatinolytic activity bands
(quantitated by densitometry) is presented (meanSD, P=0.0368, ANOVA
test).