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Dominant Bcl-2 expression during telogen–anagen transition phase in human hair
Journal of Dermatological Science (2004) 36, 183—185
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Dominant Bcl-2 expression during telogen—anagen transition phase in human hair KEYWORDS Apoptosis, Bulge, Hair cycle, Secondary hair germ, Stem cells
Apoptosis plays important roles both in morphogenesis and cycling of mammalian hair follicles. The proto-oncogene bcl-2 works as an anti-apoptotic molecule in these processes [1]. During morphogenesis of human skin, Bcl-2 is distributed both in the epithelial portion and follicular papilla of the developing hair follicles [2]. In adult human skin, Bcl-2 is detected in the basal keratinocytes, melanocytes, dermal papilla and eccrine sweat gland [2,3]. Bcl-2 protein was also predominantly expressed in the outermost cell layer of outer root sheath at bulge level in anagen hair follicles [4]. Expression pattern of Bcl-2 in mouse hair cycling was reported [5] but still obscure in human cycling. Here, we report the localization of Bcl-2 at three different stages of the human hair cycle. Human scalp skin tissues were obtained from plastic surgery under the informed consent. AntiBcl-2 antibodies (clone 100) purchased from Zymed Laboratories Inc. (South San Francisco, CA) were used for immnohistochemial staining using paraffin sections. Heat-induced epitope retrieval was performed in 10 mM citrate buffer (pH 6.0) for the unmasking of Bcl-2 antigen. In late anagen hair follicles, dermal papilla is strongly positive for Bcl-2 immunostaining (Fig. 1A), as shown in a previous report [4]. Bulb matrix cells are negative for Bcl-2 immunoreactivity at this phase. The dermal papilla keeps strong Bcl-2 expression in catagen and telogen hair follicles (Fig. 1B and C). During telogen—anagen transition,
the follicular epithelium of the secondary hair germ is positive for Bcl-2 immunostaining (Fig. 1D). The round and condensed dermal papilla which indicate re-entry into the activated anagen stage show intense Bcl-2 immunoreactivity. Several cells are intensely stained in the follicular epithelium that is initiated downward growth into subcutaneous tissues (arrows in Fig. 1D). These cells are probably progeny of stem cells, or melanomcytes because melanomcytes express higher levels of Bcl-2 proteins than the basal keratinocytes in the epidermal basal layer [3]. The outermost cell layer of the follicular epithelium in the bulge area shows more intense Bcl-2 immunoreactivity equivalent to the dermal papilla (Fig. 1D). In contrast, Bcl-2 is not detectable in the inner layers of the follicular epithelium including the club hair. Bcl-2 immunoreactivity localized at the bulge level was detected in each hair cycling stage, anagen, catagen, and telogen (Fig. 1A—C). The level of Bcl-2 expression in each stage was almost identical in human hair follicles although Bcl-2 is down regulated in telogen hair follicles in mice [5]. The secretory portion of eccrine sweat gland is positive for Bcl-2 immunostaining. Some of the basal cells of secretory portion show intense Bcl-2 immunoreactivity (arrow heads in Fig. 1D). This staining pattern is similar to basal cells. Bcl-2 immunoreactivity was observed in the dermal papilla of anagen, catagen, telogen and telogen—anagen transition stages suggesting that Bcl-2 is expressed in the dermal papilla throughout mature human hair cycle as seen in mouse hair follicles [5]. Apparently, dermal papilla cells are resistant to the apoptotic forces in the hair cycling process by dominant Bcl-2 expression. We also detected the intense Bcl-2 immunoreactivity in the outermost cell layer of the follicular epithelium at the bulge level in all three stages. This is in accordance with previous observations that epithelial stem cells reside in this region [6]. Moreover, the secondary hair germ maintaining Bcl-2 expression seemed to be derived from the bulge stem cells. In contrast to the bulge, minimal expression of Bcl-2 was detected in the bulb matrix of cycling anagen
0923-1811/$30.00 # 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.09.003
184
Letter to the Editor
cant difference was observed in bcl-2 expression between the areas of the scalp that were clinically affected and unaffected androgenetic alopecia [7]. Bcl-2 expression was almost lost in the dermal papilla and the bulge area of alopecia areata [4]. Bcl-2 may be an important key player of hair loss in human, although bcl-2 deficient mice showed the delay of hair morphogenesis, the hypopigmentation of hair coats, but not hair loss [8]. This may be due to the plasticity in embryogenesis. Since the anagen entry was retarded in the depilation-induced hair cycle of adult bcl-2 deficient mice [9], the future investigation using aged bcl-2 knockout mice will provide valuable information about roles of Bcl-2 in the hair disorder.
Acknowledgment The authors would like to thank Dr. Tetsuo Ezaki for his cooperation in obtaining materials.
References
Fig. 1 Expression pattern of Bcl-2protein in human hair follicles. Formalin-fixed human scalp skin sections were stained with anti-Bcl-2 monoclonal antibodies. (A—C) Bcl-2 was dominantly expressed in the dermal papilla and bulge at anagen, catagen, and telogne pahses. (D) During telogen—anagen transition phase, the outermost cell layer of the follicular epithelium at the bulge area showed intense Bcl-2 immunoreactivity. The secondary hair germ above the dermal papilla is also strongly positive for Bcl-2 immunostaining. Several cells showed intense Bcl-2 immunoreactivity in the secondary hair germ (arrows). Arrowheads show the basal cells of eccrine sweat gland. DP, dermal papilla; SG, secondary hair germ. Scale bars: 50 mm.
hair follicles. These findings suggest that Bcl-2 expression is important for the maintenance of the follicular epithelial stem cells, and the formation of the secondary hair germ. Recently, signifi-
[1] Muller-Rover S, Rossiter H, Lindner G, Peters EM, Kupper TS, Paus R. Hair follicle apoptosis and Bcl-2. J Investig Dermatol Symp Proc 1999;4:272—7. [2] Sellheyer K, Krahl D, Ratech H. Distribution of Bcl-2 and Bax in embryonic and fetal human skin: antiapoptotic and proapoptotic proteins are differentially expressed in developing skin. Am J Dermatopathol 2001;23:1—7. [3] Rodriguez-Villanueva J, Colome MI, Brisbay S, McDonnell TJ. The expression and localization of bcl-2 protein in normal skin and in non-melanoma skin cancers. Pathol Res Pract 1995;191:391—8. [4] Ito K, Nakamura A, Ito M. Evaluation of PCNA, cyclin B and bcl2 protein in normal anagen hair follicles and hair follicles of alopecia areata. In: van Neste D, Randall VA, editors. Hair Research for the Next Millennium. Amsterdam: Elsever Science B.V.; 1996. p. 213—6. [5] Stenn KS, Lawrence L, Veis D, Korsmeyer S, Seiberg M. Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle. J Invest Dermatol 1994;103:107—11. [6] Sun TT, Cotsarelis G, Lavker RM. Hair follicular stem cells: the bulge-activation hypothesis. J Invest Dermatol 1991;96:77S— 8S. [7] Morgan MB, Rose P. An investigation of apoptosis in androgenetic alopecia. Ann Clin Lab Sci 2003;33:107—12. [8] Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 1993;75:229— 40.
Letter to the Editor
[9] Yamamura K, Kamada S, Ito S, Nakagawa K, Ichihashi M, Tsujimoto Y. Accelerated disappearance of melanocytes in bcl-2-deficient mice. Cancer Res 1996;56:3546—50.
Tsutomu Soma Toshihiko Hibino Shiseido Life Science Research Center 2-12-1 Fukuura, Kanazawa-ku Yokohama 236-8643, Japan
185
Corresponding author. Tel.: +81 45 788 7291 fax: +81 45 788 7277 E-mail address: [email protected] (T. Soma) 19 April 2004
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Dominant Bcl-2 expression during telogen—anagen transition phase in human hair KEYWORDS Apoptosis, Bulge, Hair cycle, Secondary hair germ, Stem cells
Apoptosis plays important roles both in morphogenesis and cycling of mammalian hair follicles. The proto-oncogene bcl-2 works as an anti-apoptotic molecule in these processes [1]. During morphogenesis of human skin, Bcl-2 is distributed both in the epithelial portion and follicular papilla of the developing hair follicles [2]. In adult human skin, Bcl-2 is detected in the basal keratinocytes, melanocytes, dermal papilla and eccrine sweat gland [2,3]. Bcl-2 protein was also predominantly expressed in the outermost cell layer of outer root sheath at bulge level in anagen hair follicles [4]. Expression pattern of Bcl-2 in mouse hair cycling was reported [5] but still obscure in human cycling. Here, we report the localization of Bcl-2 at three different stages of the human hair cycle. Human scalp skin tissues were obtained from plastic surgery under the informed consent. AntiBcl-2 antibodies (clone 100) purchased from Zymed Laboratories Inc. (South San Francisco, CA) were used for immnohistochemial staining using paraffin sections. Heat-induced epitope retrieval was performed in 10 mM citrate buffer (pH 6.0) for the unmasking of Bcl-2 antigen. In late anagen hair follicles, dermal papilla is strongly positive for Bcl-2 immunostaining (Fig. 1A), as shown in a previous report [4]. Bulb matrix cells are negative for Bcl-2 immunoreactivity at this phase. The dermal papilla keeps strong Bcl-2 expression in catagen and telogen hair follicles (Fig. 1B and C). During telogen—anagen transition,
the follicular epithelium of the secondary hair germ is positive for Bcl-2 immunostaining (Fig. 1D). The round and condensed dermal papilla which indicate re-entry into the activated anagen stage show intense Bcl-2 immunoreactivity. Several cells are intensely stained in the follicular epithelium that is initiated downward growth into subcutaneous tissues (arrows in Fig. 1D). These cells are probably progeny of stem cells, or melanomcytes because melanomcytes express higher levels of Bcl-2 proteins than the basal keratinocytes in the epidermal basal layer [3]. The outermost cell layer of the follicular epithelium in the bulge area shows more intense Bcl-2 immunoreactivity equivalent to the dermal papilla (Fig. 1D). In contrast, Bcl-2 is not detectable in the inner layers of the follicular epithelium including the club hair. Bcl-2 immunoreactivity localized at the bulge level was detected in each hair cycling stage, anagen, catagen, and telogen (Fig. 1A—C). The level of Bcl-2 expression in each stage was almost identical in human hair follicles although Bcl-2 is down regulated in telogen hair follicles in mice [5]. The secretory portion of eccrine sweat gland is positive for Bcl-2 immunostaining. Some of the basal cells of secretory portion show intense Bcl-2 immunoreactivity (arrow heads in Fig. 1D). This staining pattern is similar to basal cells. Bcl-2 immunoreactivity was observed in the dermal papilla of anagen, catagen, telogen and telogen—anagen transition stages suggesting that Bcl-2 is expressed in the dermal papilla throughout mature human hair cycle as seen in mouse hair follicles [5]. Apparently, dermal papilla cells are resistant to the apoptotic forces in the hair cycling process by dominant Bcl-2 expression. We also detected the intense Bcl-2 immunoreactivity in the outermost cell layer of the follicular epithelium at the bulge level in all three stages. This is in accordance with previous observations that epithelial stem cells reside in this region [6]. Moreover, the secondary hair germ maintaining Bcl-2 expression seemed to be derived from the bulge stem cells. In contrast to the bulge, minimal expression of Bcl-2 was detected in the bulb matrix of cycling anagen
0923-1811/$30.00 # 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.09.003
184
Letter to the Editor
cant difference was observed in bcl-2 expression between the areas of the scalp that were clinically affected and unaffected androgenetic alopecia [7]. Bcl-2 expression was almost lost in the dermal papilla and the bulge area of alopecia areata [4]. Bcl-2 may be an important key player of hair loss in human, although bcl-2 deficient mice showed the delay of hair morphogenesis, the hypopigmentation of hair coats, but not hair loss [8]. This may be due to the plasticity in embryogenesis. Since the anagen entry was retarded in the depilation-induced hair cycle of adult bcl-2 deficient mice [9], the future investigation using aged bcl-2 knockout mice will provide valuable information about roles of Bcl-2 in the hair disorder.
Acknowledgment The authors would like to thank Dr. Tetsuo Ezaki for his cooperation in obtaining materials.
References
Fig. 1 Expression pattern of Bcl-2protein in human hair follicles. Formalin-fixed human scalp skin sections were stained with anti-Bcl-2 monoclonal antibodies. (A—C) Bcl-2 was dominantly expressed in the dermal papilla and bulge at anagen, catagen, and telogne pahses. (D) During telogen—anagen transition phase, the outermost cell layer of the follicular epithelium at the bulge area showed intense Bcl-2 immunoreactivity. The secondary hair germ above the dermal papilla is also strongly positive for Bcl-2 immunostaining. Several cells showed intense Bcl-2 immunoreactivity in the secondary hair germ (arrows). Arrowheads show the basal cells of eccrine sweat gland. DP, dermal papilla; SG, secondary hair germ. Scale bars: 50 mm.
hair follicles. These findings suggest that Bcl-2 expression is important for the maintenance of the follicular epithelial stem cells, and the formation of the secondary hair germ. Recently, signifi-
[1] Muller-Rover S, Rossiter H, Lindner G, Peters EM, Kupper TS, Paus R. Hair follicle apoptosis and Bcl-2. J Investig Dermatol Symp Proc 1999;4:272—7. [2] Sellheyer K, Krahl D, Ratech H. Distribution of Bcl-2 and Bax in embryonic and fetal human skin: antiapoptotic and proapoptotic proteins are differentially expressed in developing skin. Am J Dermatopathol 2001;23:1—7. [3] Rodriguez-Villanueva J, Colome MI, Brisbay S, McDonnell TJ. The expression and localization of bcl-2 protein in normal skin and in non-melanoma skin cancers. Pathol Res Pract 1995;191:391—8. [4] Ito K, Nakamura A, Ito M. Evaluation of PCNA, cyclin B and bcl2 protein in normal anagen hair follicles and hair follicles of alopecia areata. In: van Neste D, Randall VA, editors. Hair Research for the Next Millennium. Amsterdam: Elsever Science B.V.; 1996. p. 213—6. [5] Stenn KS, Lawrence L, Veis D, Korsmeyer S, Seiberg M. Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle. J Invest Dermatol 1994;103:107—11. [6] Sun TT, Cotsarelis G, Lavker RM. Hair follicular stem cells: the bulge-activation hypothesis. J Invest Dermatol 1991;96:77S— 8S. [7] Morgan MB, Rose P. An investigation of apoptosis in androgenetic alopecia. Ann Clin Lab Sci 2003;33:107—12. [8] Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 1993;75:229— 40.
Letter to the Editor
[9] Yamamura K, Kamada S, Ito S, Nakagawa K, Ichihashi M, Tsujimoto Y. Accelerated disappearance of melanocytes in bcl-2-deficient mice. Cancer Res 1996;56:3546—50.
Tsutomu Soma Toshihiko Hibino Shiseido Life Science Research Center 2-12-1 Fukuura, Kanazawa-ku Yokohama 236-8643, Japan
185
Corresponding author. Tel.: +81 45 788 7291 fax: +81 45 788 7277 E-mail address: [email protected] (T. Soma) 19 April 2004