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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 4, 2003 as doi:10.1096/fj.03-0244fje. |
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Cell Biology Section, NIDCR, National Institutes of Health (NIH), Bethesda, Maryland, USA;
* Drexel University, Philadelphia, Pennsylvania, USA;
NIAMS, NIH, Bethesda, Maryland, USA;
10123 Prince Place, Upper Marlboro, Maryland, USA;
University of Maryland, Baltimore, Maryland, USA;
** University of Maryland, Rockville, Maryland, USA; and
Tumor Biology Unit, Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120 Israel
2Correspondence: Cell Biology Section, NIH, NIDCR, Bldg. 30, Room 433, 30 Convent Dr., MSC 4370, Bethesda, MD 20892, USA. E-mail: hkleinman{at}dir.nidcr.nih.gov
SPECIFIC AIMS
We have explored the ability of thymosin ß4 to promote hair growth in normal rats and mice and determined the mechanism by which thymosin ß4 acts to promote hair growth by examining its effects on follicle stem cell growth, migration, differentiation, and protease production.
PRINCIPAL FINDINGS
1. Thymosin ß4 promotes hair growth in normal rats and mice
While studying wound healing in rat skin, we unexpectedly observed at the histological level increased hair growth at the wound margins 7 days after topical treatment with thymosin ß4. We shaved the skin of healthy rats and applied thymosin ß4 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 skin areas treated with thymosin ß4 (Fig. 1
a, d). The number of anagen follicles was 
twofold greater than in rats treated with vehicle alone. The increased number of hairs in anagen phase was retained with triweekly 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 ß4 would promote hair growth in 8-wk-old C57BL6 wild-type mice. Mice used in this experiment had all their hair follicles in the telogen stage as judged by the pink skin color. The mice were shaved and thymosin ß4 was applied topically on the shaved area. Control animals were treated with vehicle alone. As shown in Fig. 1b, e
, thymosin ß4-treated (but not control) animals displayed quick hair regrowth. Histological examination confirmed thymosin ß4-induced activation of the hair follicles (Fig. 1b, e
). Hair was grossly visible in the treated mice (Fig. 1c, f
).
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2. Thymosin ß4 protein expression by a subset of hair follicle stem cells
We first explored the spatial and temporal pattern of endogenous thymosin ß4 expression in hair follicles during depilation-induced, synchronized adult hair cycling in C57BL/6J mice. We wanted to correlate the observed effects of thymosin ß4 administration with possible functional involvement of endogenous thymosin ß4 in hair growth. Low levels of thymosin ß4 protein were observed in follicles at the telogen (resting) phase before depilation. Thymosin ß4 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 ß4-expressing cells in the bulge region. At late anagen (day 9 after depilation), a significant number of cells located in the lower follicle (matrix surrounding part of the outer root sheath) expressed thymosin ß4. The sebaceous gland was stained at all stages due to nonspecific absorption, as found by others with different antibodies. Thus, with the progression of hair growth cycle, thymosin ß4-positive cells initially detected only in the bulge were observed at the bulb area, suggesting they are migrating from the bulge region. These data show that the temporal and spatial distribution of thymosin ß4-expressing cells was similar to the pattern proposed for the hair follicle stem cells and their daughter cells, i.e., emanating from the bulge and migrating downward to give rise to matrix cells that subsequently generate the hair shaft.
3. Cultured rat vibrissa clonogenic keratinocytes express thymosin ß4
We studied rat vibrissae follicle keratinocytes from the bulge region to determine whether isolated stem cells express thymosin ß4. Hair follicle stem cells have been identified as bulge-residing keratinocytes with a high in vitro clonogenic potential. Although hair follicle stem cells are not fully characterized, they preferentially express keratin 15 (K15). We isolated clonogenic keratinocytes from the rat vibrissa bulge region and found that the isolated cells were highly clonogenic. These cells were positive for the stem cell marker keratin 15 as well as for keratins 5, 6, and 14, known to be expressed by bulge stem cells (data not shown). These cells lacked keratin 10, an early marker of terminal keratinocyte differentiation (data not shown). When cultured in vitro, these cells were able to move with an average velocity of 0.43 µm/min. We conclude that the obtained cell population represents the immediate progeny of hair follicle stem cells. We found that these cells expressed thymosin ß4 after 7–10 days of culturing in vitro. Treatment of the clonogenic keratinocytes with exogenous thymosin ß4 caused a dose-dependent decrease in the expression levels of the multipotent undifferentiated stem cell marker K15 (Fig. 2
). A decline in K15 is associated with stem cell differentiation. We found that thymosin ß4 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 ß4 is important for early stem cell differentiation.
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4. Thymosin ß4 promotes the migration of hair clonogenic keratinocytes in vitro
Thymosin ß4 has been shown to promote endothelial cell migration. We found that cultured clonogenic keratinocytes migrate to thymosin ß4 after 4.5 h in Boyden chamber assays. In the presence of thymosin ß4, cell migration was increased almost twofold (69.0±7.1 vs. 113.3±5.5 cell number/field, P
0.001) at 1 ng over migration in the presence of medium containing vehicle alone (negative control). The effect of thymosin ß4 on cell migration was greatest at 1 ng/mL; at 100 and 1000 ng/mL migration was decreased. Earlier we found that 1 ng/mL was potent for endothelial and keratinocyte migration.
5. Thymosin ß4 augments the production and secretion of matrix metalloproteinase 2 (MMP-2) by clonogenic keratinocytes
Enzymatic degradation and remodeling of the extracellular matrix are necessary in normal hair development and growth.We have examined the effect of thymosin ß4 on the enzymatic activity of matrix metalloproteinases (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. Treatment of the clonogenic keratinocytes with exogenous thymosin ß4 caused almost a twofold increase in MMP-2. The increase in the levels of secreted and cell-derived enzyme MMP-2 was dose dependent. 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 ß4 treatment. Since MMP-2 plays a role in hair growth-associated extracellular matrix remodeling and cell migration, our data suggest that this enzyme may be a downstream effector through which thymosin ß4 exerts its effect on hair growth.
CONCLUSIONS AND SIGNIFICANCE
Thymosin ß4 is a 4.9 kDa molecule that functions as a major actin-sequestering protein in cells. It is up-regulated during endothelial cell differentiation; when added exogenously, it promotes endothelial cell differentiation and migration. In vivo, it promotes wound repair and is a potent anti-inflammatory agent. A related family member, thymosin ß15, is also important in the metastasis of certain tumor types. It was reported that thymosin ß4 exerts its effects on cell locomotion through specific interactions with actin that regulate cytoskeletal organization. We show that exogenously delivered thymosin ß4 promotes hair growth in normal rats and mice. When examining the distribution of endogenous thymosin ß4 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 active growth phase (anagen), the subset of thymosin ß4-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 ß4-expressing cells are found in the bulb area, in the outer root sheath, and among the hair matrix cells. Isolated clonogenic hair follicle keratinocytes, closely related (if not identical) to the hair follicle stem cells, produce thymosin ß4 when cultured in vitro for 7–10 days. The presence of exogenous thymosin ß4 caused a dose-dependent decrease in the expression of the stem cell marker K15 by clonogenic keratinocytes, suggesting that thymosin ß4 may promote early stem cell differentiation (i.e., transition to the transi-amplifying cell phenotype). Most important, treatment of the bulge-derived clonogenic keratinocytes with exogenous thymosin ß4 increased migration and production of MMP-2.
A critical step in the hair growth cycle 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. Our data indicate that thymosin ß4 facilitates this movement of the stem cells and their immediate progeny and, thus, exerts its promoting effect on hair growth (Fig. 3
). The effect of thymosin ß4 on MMP-2 expression appears to play an important role in this system. MMP-2 has been shown to contribute to cellular migration by degrading extracellular matrix barriers for cell movement and through direct effects on cell locomotion in vitro. 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.
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Hair growth acceleration by thymosin ß4 may also be attributed to proangiogenic and other biological activities of this molecule. It was recently reported that VEGF promotes hair follicle development presumably due to its angiogenic activity. Thymosin ß4 is angiogenic (like VEGF), and the activity of thymosin ß4 may be due to its angiogenic activity. Another angiogenic molecule, hepatocyte growth factor, has been found to promote hair growth. Hepatocyte growth factor up-regulates thymosin ß4 expression and may be acting by increasing thymosin ß4 and/or synergizing with it. Steroids have been used to treat certain types of hair loss. Thymosin ß4 is the anti-inflammatory molecule identified as increased in steroid-treated monocytes. Thus, treatment with steroids may involve the activity of thymosin ß4 on hair growth.
Taken together, our results suggest that thymosin ß4 exerts a profound hair-promoting effect through a combined action on several critical events in hair follicle growth such as stem cell migration, ECM-degrading enzyme production, and differentiation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0244fje 
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