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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0247fje. |
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* Department of Dermatology, University Hospital Hamburg-Eppendorf, University of Hamburg, Hamburg, Germany;
Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan;
Department of Pathology, Molecular and Cellular Biology, and Molecular and Human Genetics, Baylor College of Medicine, Texas, USA;
Memorec Stoffel GmbH, 50829 Cologne, Germany;
|| Deutsches Rheumaforschungszentrum, Berlin, Germany; and
Institute of Cell Biology, Swiss Federal Institute of Technology, Zürich, Switzerland
2Correspondence: Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail: paus{at}uke.uni-hamburg.de
SPECIFIC AIMS
Hair follicle (HF) morphogenesis and hair cycling are controlled by complex bidirectional ectodermal–mesenchymal interactions and are regulated by a complex balance between a wide variety of stimulatory and inhibitory growth factors. We explored the role of TGF/BMP family member, activin, and its antagonist, follistatin, in hair follicle development and cycling.
PRINCIPAL FINDINGS
1. Follistatin mRNA is expressed in the hair follicle epithelium whereas activin ßA expression predominates in the hair follicle mesenchyme
To ascertain whether follistatin and activin are involved in the control of hair follicle development, we first characterized the expression of follistatin and activin ßA mRNA in the back skin of mice at embryonic day (E) E18.5 by in situ hybridization. Follistatin transcripts were detected in the epithelial hair placode, hair matrix, outer root sheath keratinocytes, and interfollicular epidermis whereas activin ßA message was mainly visible in developing dermal papilla cells.
2. Activin receptors IA, IB, II, II B show a distinct follicular expression pattern
To identify potential target cells for activin bioactivity in developing mouse HFs, the expression of all activin receptor subtypes was studied in the developing skin of E18.5 mice using immunohistochemistry. Activin receptor IA immunoreactivity was seen in outer root sheath, inner root sheath and interfollicular epidermal keratinocytes as well as in dermal papilla fibroblasts. Activin receptor IB protein was expressed in similar regions. ActRII immunoreactivity was seen in hair placodes, outer root sheath, inner root sheath, and interfollicular keratinocytes. ActRIIB expression was found in hair placodes, outer and inner root sheath keratinocytes, and dermal papilla fibroblasts.
3. Hair follicle morphogenesis is retarded in follistatin-deficient mice
To explore the functional role for follistatin during HF development, we studied the dynamics of HF morphogenesis in homozygous follistatin knockout mice by quantitative histomorphometry.
Back skin from E18.5 follistatin knockout mice displayed a higher percentage of HFs at early stages (stages 1-2) and a lower number of HFs in more advanced stages of development (stage 3-4) than those from age-matched wild-type littermates (Fig. 1
)A–C. Correspondingly, morphogenesis staging scores, which offer an excellent cumulative marker for differences in the speed of HF development between mutant and wild-type mice, were significantly lower for follistatin knockout mice than those of age-matched wild-type mice at E18.5. These in vivo data suggest that follistatin is indeed functionally involved in the control of HF morphogenesis and that follistatin transduces stimulatory signals in early murine HF development.
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4. Intrafollicular keratinocytes of follistatin-deficient mice show reduced proliferative activity
To elucidate the effect of follistatin on keratinocyte proliferation and apoptosis in situ during early HF development, we compared the proliferative activity of intrafollicular keratinocytes in stage 1 and 2 hair follicles and the number of apoptotic cells, using Ki-67/TUNEL as proliferation/apoptosis markers. Quantitative histomorphometry revealed a significant reduction in the percentage of Ki67+ keratinocytes in follistatin knockout mice compared with wild-type controls, but no significant difference in the number of TUNEL keratinocytes.
5. Hair follicle morphogenesis is delayed in activin ßA transgenic mice
To test whether activin A, whose activity is antagonized by follistatin, plays a functional role in hair follicle development, transgenic mice that overexpress the activin ßA subunit under the control of the keratin 14 promoter were screened at E18 for differences in their rate of hair follicle morphogenesis from wild-type littermates. Samples of E18 back skin from activin ßA transgenic mice displayed a higher percentage of HFs in the earliest stages (stages 1 + 2) and a lower number of HFs in more advanced stages of development (stages 3 + 4) than those from age-matched wild-type littermates. Morphogenesis staging scores for E18 activin ßA transgenic mice were also significantly lower than those of wild-type littermates.
6. Follistatin stimulates hair follicle development in vitro
The above results suggest that both follistatin and activin are functionally important during murine pelage hair follicle development. To further probe this concept and to directly test the hair growth modulatory effects of follistatin and activin, recombinant human follistatin was added to organ-cultured normal mouse embryonic skin with and without coadministration of human activin A. Skin biopsies were taken from the back skin of E16.5 C57BL/6 mice and cultured for 48 h in the presence of recombinant human follistatin with or without activin A addition. As shown in Fig. 2
A, C, 100 ng/mL of recombinant human follistatin significantly accelerated murine hair follicle development in skin organ culture (Fig. 2A-C
). This effect was inhibited by the addition of 30 ng/mL of recombinant human activin A, suggesting that the stimulatory effects of follistatin on hair follicle development can be antagonized by activin A.
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7. Hair follicle regression (catagen) is retarded in activin ßA-overexpressing mice
To determine the functional significance of activin A in mature HF, the dynamics of spontaneous, apoptosis-driven catagen development were examined in activin ßA transgenic mice. Compared with their age-matched wild-type controls, activin ßA transgenic mice showed a significant catagen retardation during the first catagen entry.
8. Activin transgenic mice show down-regulation of BMP-2 and up-regulation of matrix GLA protein (MGP) expression
To further explore potential follicular target genes of activin bioregulation with activin receptor stimulation in murine skin, comparative microarray analyses were performed. A microarray approach and semiquantitative RT-PCR showed up-regulation of the expression of bone morphogenetic protein 2 (BMP-2) gene and down-regulation of the BMP-2 inhibitory MGP gene in activin ßA transgenic mice.
CONCLUSIONS
With in situ hybridization and immunohistochemistry, we show that activin ßA is expressed mainly in the hair follicle mesenchyme whereas activin receptors IA, RIB, RII, RIIB, and the activin interaction partner follistatin are expressed mainly in the HF epithelium. Both activin ßA transgenic mice and follistatin-deficient mice reveal a retardation of hair follicle development (Fig. 1)
, and follistatin stimulates HF development in skin organ culture (Fig. 2)
.
Taken together, this suggests the following hypothetical scenario of activin-follistatin interactions during HF morphogenesis (Fig. 3
A). Activin A is secreted by specialized inductive fibroblasts of the dermal papilla and binds to activin receptors in the developing HF epithelium as a "brake" on hair follicle development. Follistatin in turn may bind to secreted activins in the follicle epithelium, thus preventing activins from binding to their cognate receptors, resulting in disinhibition of activin’s activity on hair follicle morphogenesis (Fig. 3A
). This scenario pictures activin–follistatin as another interaction pair in the complex molecular controls of hair follicle induction and morphogenesis and further strengthens the concept that the controls of HF development may be dominated by inhibition–disinhibition regulatory systems.
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Activin inhibits HF development and catagen development. The apparently contradictory effects of the same hair growth-modulatory factor during hair follicle development vs. hair follicle cycling are not unusual, since we had also observed contradictory effects of neurotrophin-3 (NT-3) on HF morphogenesis and catagen induction in mice (both are stimulated by NT-3). This only illustrates once again that the same molecular player can unfold apparently opposite growth modulatory activities during different windows of skin development.
To identify genes regulated by activin in skin, we performed microarray and semiquantitative RT-PCR and show that BMP-2 is down-regulated and the BMP-2 inhibitor MGP is up-regulated in activin ßA transgenic mice at P17.
BMP-2 mRNA is indeed expressed in the precortical hair follicle matrix. Mice overexpressing the key BMP inhibitor noggin exhibited severe impairment in differentiation of the hair shaft. Application of noggin protein to telogen skin accelerates telogen-anagen transformation. These results have revealed that BMP-2 plays an important role in postnatal hair follicle cycling. Therefore, the retardation of catagen entry seen in the current study in activin ßA transgenic mice might result at least in part from a down-regulation in BMP-2 expression.
We have also shown up-regulation of MGP gene expression in activin transgenic mice. MGP protein binds to BMP-2 protein and thereby down-modulates BMP-2 activity. Overexpression of activin ßA in murine skin not only down-regulates the BMP-2 gene, but also up-regulates the expression of its inhibitor, thus maximizing the counter-regulatory effect of activin on this potent suppressor of hair growth. Therefore, this dual regulation of both BMP-2 and its inhibitor (MGP) by activin may underlie the hair cycle modulatory effects of activins identified in the current study (Fig. 3
).
Our study encourages exploitation of activin and follistatin for manipulating hair growth. Clinically, our study promises to facilitate the bioengineering of hair follicles from stem cells by appropriate manipulation with follistatin/activin in vitro, whereas locally administered activin and/or follistatin may become exploited for treating unwanted hair loss (alopecia) or unwanted hair growth (hirsutism).
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0247fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0247fje 
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