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. Author manuscript; available in PMC: 2011 Jul 18.
Published in final edited form as: J Invest Dermatol. 2010 Sep 30;131(1):257–260. doi: 10.1038/jid.2010.279

Matrix metalloproteinase-9 is involved in regulation of the hair canal formation

Andrey A Sharov 1,*, Mandy Schroeder 1,2, Tatyana Y Sharova 1, Andrei N Mardaryev 3, Eva MJ Peters 2, Desmond J Tobin 3, Vladimir A Botchkarev 1,3,*
PMCID: PMC3138469  NIHMSID: NIHMS302250  PMID: 20882038

To the Editor:

Hair follicle (HF) morphogenesis is governed by a series of signals exchanged between the epidermal keratinocytes committed to HF-specific differentiation and the mesenchymal cells forming the follicular papilla (Fuchs, 2007; Schmidt-Ullrich and Paus, 2005). These interactions lead to the construction of the hair bulb, in which keratinocytes rapidly proliferate and differentiate into several cell populations forming the medulla, cortex and cuticle of the hair shaft, as well as the cuticle, Huxley and Henle layers of the inner root sheath. During the final steps of development, the HF elongates up to its maximal length and the differentiating hair shaft emerges through the epidermis via the hair canal that is formed at the distal portion of the HF epithelium (Schmidt-Ullrich and Paus, 2005).

Morphologically, the hair or pilary canal is considered as a continuation of the hair germ upward into the epidermis and its formation begins at stage 3 of HF morphogenesis, when the epidermal cells above the HF become oriented perpendicularly to the surface of the skin (Pinkus, 1958). During later stages of HF development (stage 4–6), these cells keratinized and form a central core surrounded by a tubular layer of epithelial cells inside of the epidermis and HF infundibulum (Hashimoto, 1970). Finally, the centre of the keratinized tissue in a lumen of the hair canal is eliminated by the tip of the emerging hair (stages 7–8 of development) (Breathnach, 1971; Sengel, 1976).

Despite recent advances in identification of the molecular signaling/transcription networks that govern the development of the skin and HF (Fuchs, 2007), the mechanisms controlling the epithelial tissue remodeling during hair canal formation remain unclear. It has been long appreciated that matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in tissue remodeling during embryonic and postnatal development (Ortega et al., 2003; Werb and Chin, 1998). MMP-9 (gelatinase B) is involved in the degradation of extracellular matrix and/or modulation of growth factor signaling through its degrading activity during development (Mott and Werb, 2004). MMP-9 activity is present in the culture medium containing explants of developing HFs (Obana et al., 1996), as well as in normal adult mouse skin with actively growing (anagen) HFs (Paus et al., 1994). In human anagen HFs, MMP-9 is expressed in the Henle’s layer of the inner root sheath (Jarrousse et al., 2001).

To elucidate a role for MMP-9 in the control of HF development and hair canal formation, its expression was studied by immunohistochemistry in cryosections of embryonic and postnatal skin of C57BL/6 mice (Botchkarev et al., 2001; Botchkarev et al., 1999; Sharov et al., 2003). Immunohistochamical data were correlated to the micro-anatomy of the developing hair canal depicted by high-resolution light microscopy (HRLM), as described previously (Magerl et al., 2001). MMP-9 expression was not seen in the epidermis and developing HF placodes (Fig. 1 A, B). Starting from E17.5, single MMP-9 expressing cells became visible in the central part of the developing intra-epidermal hair canal (stage 3 of HF morphogenesis; Fig. 1 C, D). In stage 4–6 HFs, the number of MMP-9 expressing cells increased in central portion of the hair canal, which walls showed appearance of the trichohyalin granules, while the other HF compartments (hair bulb, sebaceous gland, connective tissue sheath) did not show any detectable MMP-9 immunoreactivity (Fig. 1 E, F). During final stages of HF development (stage 7 and 8), MMP9+ cells disappeared from the hair canal and were visible only in the dermis (Fig. 1 G, H).

Figure 1. MMP-9 is expressed in the hair canal of developing hair follicles.

Figure 1

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Samples of embryonic and early postnatal mouse skin were prepared for high resolution light microscopy, as described previously (Magerl et al., 2001). Cryosections of embryonic and early postnatal mouse back skin were immunostained with primary antibodies against MMP-9 (1:100, Chemicon) and secondary TRITC-labeled goat-anti-rabbit IgG (1:200, Jackson ImmunoResearch). Border between the epidermis or HF and dermis in A–F is depicted by the dotted line. Nuclei were counterstained by DAPI (B, D, F, H).

A, B – E16.5 (Stage 1 of HF morphogenesis). Cells in the suprabasal layer of the epidermis above the follicular placode show no spaces between each other (A, inset, arrows). Lack of MMP-9 expression in the epidermis and hair follicle placode (B).

C, D – E17.5 (Stage 3 of HF morphogenesis). Formation of spaces between the cells located in the intra-epidermal part of the developing hair canal show (C, inset, arrows) and appearance of single MMP-9 positive cells in the central part of the hair canal (D, inset, arrow).

E, F – P0.5 (Stage 6 of HF morphogenesis). Appearance of trichohyalin granules in the lateral parts of the developing hair canal and lumen in its central portion (E, inset, arrow). Increase of MMP-9 immunoreactivity in the central part of the hair canal (F, inset, arrow).

G, H – P4.5 (Stage 8 of morphogenesis). Hair shaft in the fully developed hair canal (G, inset, arrow). Lack of MMP-9 immunoreactivity in the walls of the hair canal (H, large arrow). Single MMP9+ cells are seen in the dermis (H, small arrow).

To further define the functions of MMP-9 in hair canal formation, the dynamics of HF development were compared between wild-type (n=5) and MMP-9 knockout (−/−) mice (n=7) at postnatal day 4.5 (P4.5). It was shown previously that MMP-9 knockout mice are characterized by the defects in ossification and angiogenesis, delayed wound healing, and are resistant to experimentally-induced bullous pemphigoid (Kyriakides et al., 2009; Liu et al., 1998; Vu et al., 1998). Consistently with the expression pattern for MMP-9 in the developing skin (Fig. 1), histo-morphometric analyses of the early postnatal skin showed significant (p<0.05) retardation of the process of hair shaft emergence through the epidermis in MMP-9 −/− mice versus wild-type controls (Fig. 2 A). Despite apparently normal earlier stages of HF development and skin morphology, MMP-9 −/− mice showed significant (p<0.05) increase in a number of HFs at stage 7 of morphogenesis in which hair shaft was retained inside of the hair canal versus the age-matched wild-type mice that showed the increased number of stage 8 HFs and more rapid hair shaft appearance at the skin surface (Fig. 2 B, C). In addition to these differences, the width of the hair canal determined as a distance between the internal surfaces of its walls and used as an important parameter of the dynamics of its formation was significantly (p<0.05) decreased in MMP9 −/− mice compared to wild-type mice (Fig. 2 D–F).

Figure 2. Retardation of the hair shaft emergence through the epidermis in MMP-9 knockout mice.

Figure 2

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Dorsal skin of MMP-9 knockout (−/−) and wild-type mice was harvested at P4.5 and processed for histo-enzymatic detection of alkaline phosphatase and histo-morphometry. At least 400 hair follicles were analyzed in skin cryosections of wild-type mice (n=5) and MMP-9 knockout mice (n=7).

A – Decrease in number of stage 8 hair follicles and increase of stage 7 hair follicles in the skin of MMP-9 knockout versus wild-type mice (* p<0.05, Student’s t-test).

B, C – Appearance of the hair fibers at the skin surface in wild-type mice (B, arrows) and retention of the hair shafts in the follicular infundibulum in MMP-9 knockout mice (C, arrows). Insets show high magnification of the labeled areas.

D – Decrease of the width of the hair canal in MMP-9 −/− mice versus wild-type mice (p<0.05, Student’s t-test). Width of the hair canal was determined as a distance between two trichohylain layers at the internal surfaces of its walls at the level of the angle between the epidermis and follicular infundibulum.

E, F – Hair shaft inside of the fully developed hair canal in wild-type skin (E, large and small arrows, respectively). Retention of the hair shaft at the intra-infundibular part of the hair canal and decrease of the hair canal width in MMP-9 −/− mice (F, large and small arrows, respectively).

These data suggest that MMP-9 plays an important role in the extracellular matrix remodelling during hair canal formation. MMP-9 degrades the denatured collagens (gelatin), as well as can cleave native type IV, V and XI collagens, elastin and a number of other molecules (Nothnick, 2008). MMP-9 is expressed in the central part of the hair canal (Fig. 1 D, F) containing keratinized “core cells”, which later desquamate and form the initial lumen of the hair canal prior to the appearance of the hair tip (Hashimoto, 1970). By active extracellular matrix remodeling, MMP-9 may promote the formation of the initial lumen, thus preparing the hair canal for the hair shaft emergence and preventing its walls from a damage induced by the growing hair fiber.

However, it is unclear whether MMP-9 and/or other metalloproteinases play a role in the control of hair canal formation during morphogenesis of the human HFs. Furthermore, mechanisms controlling the expression of MMP-9 during hair canal formation in mice also remain unclear. In other cell types (lymphocytes, fibroblasts, osteoblasts, isolated keratinocytes, etc.), MMP-9 expression is regulated by the Wnt, TGF-beta/BMP, HGF and EGF signaling pathways (McCawley et al., 1998; Nothnick, 2008; Wu et al., 2007). These signaling pathways also serve as key regulators of HF development (Fuchs, 2007; Schmidt-Ullrich and Paus, 2005), which suggest a possibility for their involvement in the control of MMP-9 expression in a subset of keratinocytes that specify the hair canal areas in the epidermis. Future research in this direction will help in further understanding the roles of MMP-9 and other metalloproteinases in the control of extracellular matrix remodeling in the developing skin, as well as in establishing the molecular signaling networks that underlie the complex programs of skin appendage morphogenesis.

Acknowledgements

Authors thank Prof. Z. Werb (UCSF) for providing the MMP-9 knockout mice. This study was supported in part by the NIH grants to AAS (AR056771-01) and VAB (AR049778-07) and by a DFG grant to EP (PE 890/4-1).

Abbreviations

HF

Hair follicle

MMP-9

matrix metalloproteinase 9

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