Letter to the Editor

novel strategies for several diseases including cancer progression and cystic brosis (8, 2024). Here, we provide evidence for the rst time that the inhibition of the UPS increases the stabilization of cellcell contacts in human keratinocytes, which might be mediated by the maintenance of DP at desmosomes. Therefore, these data increase the understanding of the molecular mechanism involved in the proper localization of DP and might suggest a novel therapy approach for diseases characterized by mislocalization of DP.

Acknowledgements
SL designed the study, performed the research, analysed the data and wrote the paper. TMM designed the study. LBT and TMM revised the paper. This work was partially supported by DFG MA-1316/11, Bonner Forum Biomedizin and TRM Leipzig.

Conict of interest
The authors state no conict of interest.

References
1 Jonkman M F, Pasmooij A M, Pasmans S G et al. Am J Hum Genet 2005: 77: 653660. 2 Armstrong D K, McKenna K E, Purkis P E et al. Hum Mol Genet 1999: 8: 143148. 3 Whittock N V, Wan H, Morley S M et al. J Invest Dermatol 2002: 118: 232238. 4 Mahoney M G, Sadowski S, Brennan D et al. J Invest Dermatol 2010: 130: 968978. 5 Vasioukhin V, Bauer C, Degenstein L et al.Cell 2001: 104: 605617. 6 Urbe S. Essays Biochem 2005: 41: 8198. 7 Haglund K, Sigismund S, Polo S et al.. Nat Cell Biol 2003: 5: 461466. 8 Okiyoneda T, Barriere H, Bagdany M et al. Science 2010: 329: 805810. 9 Schwartz A L, Trausch J S, Ciechanover A et al. Proc Natl Acad Sci U S A 1992: 89: 5542 5546. 10 Pasdar M, Nelson W J. J Cell Biol 1988: 106: 677685. 11 Green K J, Simpson C L. J Invest Dermatol 2007: 127: 24992515. 12 Franke W W. Cold Spring Harb Perspect Biol 2009: 1: a003061. 13 Green K J, Stappenbeck T S, Parry D A et al.J Dermatol 1992: 19: 765769. 14 Kartenbeck J, Schwechheimer K, Moll R et al.J Cell Biol 1984: 98: 10721081. 15 Watt F M, Mattey D L, Garrod D R. J Cell Biol 1984: 99: 22112215. 16 Jones J C, Goldman R D. J Cell Biol 1985: 101: 506517. 17 Mattey D L, Garrod D R. J Cell Sci 1986: 85: 95111. 18 Penn E J, Burdett I D, Hobson C et al.J Cell Biol 1987: 105: 23272334. 19 Wallis S, Lloyd S, Wise I et al.Mol Biol Cell 2000: 11: 10771092. 20 Henry L, Lavabre-Bertrand T, Douche T et al. Exp Dermatol 2010: 19: 10541059. 21 Palmieri G, Bergamo P, Luini A et al. PLoS ONE 2011: 6: e25888. 22 Hishinuma S, Komazaki H, Fukui H et al.J Neurochem 2010: 113: 9901001. 23 Citri A, Soler-Llavina G, Bhattacharyya S et al. Eur J Neurosci 2009: 30: 14431450. ffek S, Wo ll S, Ho hfeld J et al. Hum Mutat 24 Lo 2010: 31: 466476. (b) Cells were cultured in KGM and treated with MG132 and (upper panel) nocodazole (Noc) or (lower panel) cytochalasin D (Cyto. D). Cells were xed and stained with indicated antibodies. Arrow heads indicate the MG132-induced re-localization of DP to cell-cell contacts even in the presence of depolymerized tubulin cytoskeleton. Scale bar, 10 m. Figure S2. (a) Cells were cultured in LCa or in HCa, or in the presence of ALLN [100 M] for 3 h. Cells were stained with K5 (in green) and desmoplakin (DP; in red) antibodies. Nuclei were stained with DAPI. Arrows indicate the presence of DP at cell-cell contacts. (b) Cells were cultured in LCa or in the presence of MG132 [50lM] or in high calcium [2 mM] for 3 h. Thereafter, cells were washed with PBS and incubated with 2.4 U/ml dispase for 20 min at 37 C. Pictures were taken at indicated time-points after applying dispase. Table S1. Table provides the details of the antibodies used in this study. Data S1. Dispase assay. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Primary keratinocytes were cultured in KGM (Ctr) or in high calcium [1.8 mM] (HCa) or in the presence of MG132 [50lM] for 3 h. (a) Cells were lysed and subsequently separated into detergent-soluble and insoluble fractions. Equal amounts of protein were separated by SDS-PAGE. Figure shows representative desmoplakin (DP) and plakoglobin (PG) immunoblots of the detergent-insoluble and the soluble fraction.

DOI: 10.1111/j.1600-0625.2012.01570.x www.blackwellpublishing.com/EXD

Letter to the Editor

Conditioned media obtained from human outer root sheath follicular keratinocyte culture activates signalling pathways that contribute to maintenance of hair-inducing capacity and increases trichogenicity of cultured dermal cells
Mi Hye Lee1*, Sanguk Im1*, Seung Hyun Shin1, Mi Hee Kwack1, Sang-Eun Jun1,2, Moon Kyu Kim1, Jung Chul Kim1 and Young Kwan Sung1
1 Department of Immunology, School of Medicine, Kyungpook National University, Daegu, Korea Correspondence: Young Kwan Sung, Department of Immunology, School of Medicine, Kyungpook National University, 101 Dong-In-Dong, Jung-Gu, Daegu, 700-422, Korea, Tel.: 82-53-420-4874, Fax: 82-53-423-4628, e-mail: ysung@knu.ac.kr 2 Present address: College of Nursing, Keimyung University, Daegu, Korea *These authors contributed equally to this work.

Abstract: Findings from recent studies have demonstrated that hair-inducing capacity (trichogenicity) of cultured dermal cells can be maintained by addition of conditioned media obtained

from culture of epidermal keratinocytes. In this study, we investigated the question of whether treatment with human follicular keratinocyteconditioned media (FKCM) can result in

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Letter to the Editor

activation of signalling pathways that contribute to trichogenicity and increase the trichogenicity of cultured dermal cells. Through conduct of hair reconstitution assays, we observed that treatment of cells with FKCM resulted in induction of a greater number of hair follicles, compared with control cells. Treatment of dermal cells with FKCM resulted in the activation of BMP and b-catenin signalling pathways. In addition, higher levels of IGFBP-7, IL-8, OPG and uPA were observed in FKCM. Altogether, our data suggest that a patients own FKCM would be ideal for expansion of the patients own follicular dermal cells for cell therapy for treatment of hair loss.

Abbreviations: DP, dermal papilla; FKCM, follicular keratinocyte conditioned media; IGFBP-7, insulin-like growth factor binding protein-7; IL-8, interleukin-8; OPG, osteoprotegerin; uPA, urokinase plasminogen activator; ORS, outer root sheath. Key words: follicular cell implantation hair reconstitution trichogenicity

Accepted for publication 30 June 2012

Background
Follicular cell implantation (FCI) for treatment of hair loss is believed to offer the possibility of permanent hair transplantation (1). Neogenesis of the hair follicle through FCI is believed to depend signicantly on the ability to reproducibly expand hair-inductive dermal cells in vitro. Dermal papilla (DP) cells and dermal sheath cells are believed to be the best dermal cells for hair regeneration (2,3); thus, there have been many attempts at regeneration of hair follicles by transplantation of these cells. However, the hair-inductive capacities of these cells are lost during in vitro cultivation. Therefore, to achieve successful hair follicle neogenesis, identication of cell culture supplements and/or culture conditions that enable dermal cells to maintain trichogenicity is an important matter. Maintenance of hair-inductive potential of cultured dermal cells by addition of conditioned media obtained from rodent epidermal cells or human skin epidermal keratinocytes has been reported in recent studies. Inamatsu et al. (4) reported on sustained inductive ability of cultured rat DP cells at a level comparable to that of intact DP by addition of conditioned medium harvested from keratinocytes of sole skin. In addition, Qiao et al. (5) recently demonstrated maintenance of trichogenicity of cultured human DP cells cultured in medium conditioned with keratinocytes of newborn foreskin. In addition, Inoue et al. (6) reported that use of conditioned media obtained from culture of human facial skin keratinocytes resulted in preservation of the trichogenicity of human DP cells.

(Fig. S1). Isolation and cultivation of rat vibrissa DP were conducted according to a previously described method (8). Among a few in vivo assays for measurement of trichogenicity of a dermal or an epidermal cell population (9), the patch assay (10) and the chamber grafts (11) were performed with minor modications (legend of Fig. S2). The human antibody array I kit (Ray Biotech, Norcross, MN, U.S.A.) was used according to the manufacturers instructions for simultaneous detection of expression levels of proteins in conditioned medium.

Results
To investigate the question of whether treatment of cells with FKCM could result in induction of more hair follicles, we adopted both the patch assay and chamber assay (Fig. S2). We observed that, compared with control cells, newborn dermal cells treated with FKCM showed greater induction of hair follicles (Fig. 1a,b). Induction of 119 48 hair follicles was observed in dermal cells treated with FKCM, while induction of 76 19 hair follicles was observed in control dermal cells (Fig. 1c). For the chamber assay, cultured rat vibrissa DP cells in the presence or absence of 50% FKCM were mixed with fresh isolated newborn epidermal cells.

(a)
FKCM

(b)
+ FKCM

(c)
*

Questions addressed
In this study, we hypothesized that conditioned media from hair follicular keratinocytes of patients would be ideal for expansion of patients dermal cells. Therefore, using hair reconstitution assays, we investigated the question of whether treatment of cultured dermal cells with human outer root sheath (ORS) follicular keratinocyteconditioned media (FKCM) can result in increased hair-inducing capacity. We also investigated the question of whether treatment of dermal cells with FKCM can result in the activation of signalling pathways that contribute to maintenance of trichogenicity.
FKCM + FKCM

(d)

(e)

(f)

(g)
Hair follicle induction ratio (%)

120 100 80 60 40 20 0
0 (0/5) 60 (3/5) 100(5/5)

FKCM

+ FKCM

FKCM + FKCM

Experimental design
Human ORS keratinocytes were prepared using a previously described method, with minor modications (7). For preparation of FKCM, culture medium was switched to DMEM supplemented with 10% FBS when cells at passage 2 reached 80~90% conuency. Harvest of the medium was performed after 4 days, followed by centrifugation, and ltration through a 0.20-lm membrane lter

Fresh dermal cells

Figure 1. Increment of hair follicle induction in follicular keratinocyteconditioned media (FKCM)-treated mouse dermal cells and rat dermal papilla cells. Representative data showing hair follicles induced by mouse dermal cells cultured in the absence (a) or presence (b) of FKCM. Box-and-whisker plots (c): mid-line, median; box, 25th to 75th percentiles; and whiskers, minimum and maximum. *P < 0.05). Representative photographs showing hair follicles induced by rat dermal papilla cells cultured in the absence (d) or presence (e) of FKCM. Close-up image of the boxed region of e is shown in (f). Values represent the ratio of hair follicle induction from ve independent experiments (g).

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Letter to the Editor

(a)
0

FKCM 10 30 60 0

+ FKCM 10 30 60 (min) pSMAD 1/5/8 -catenin Actin

(b)
FKCM +FKCM

level of b-catenin showed an increase in the presence of FKCM. We also investigated the question of whether treatment with FKCM could result in the activation of the BMP signalling pathway, which has also been implicated in enhanced hair follicle inductive capacity (14). An increase in the level of p-SMAD 1/5/8 (p-SMAD) was observed after treatment with FKCM (Fig. 2a). Results of real-time PCR and immunouorescence staining, however, did not show a signicant increase in markers representing intrinsically dened trichogenic markers (15), such as Sox2 and Corin, in cells treated with FKCM. Results of protein antibody array analysis showed higher levels of IGFBP-7, IL-8, OPG and uPA in FKCM (Fig. 2b).

Conclusions
IGFBP -7 IL -8 IGFBP -7 IL -8

Osteoprotegerin

Osteoprotegerin

uPA

uPA

Figure 2. Activation of BMP and b-catenin signalling pathways by follicular keratinocyteconditioned media (FKCM) treatment and detection of enriched proteins in FKCM. Mouse dermal cells were cultured for two days in the presence or absence of FKCM, and immunoblotting (a) was performed. Cells were treated with either 10% DMEM or FKCM for the indicated times, and total cell lysates were probed with antibodies against pSMAD1.5.8 (top panel), b-catenin (middle panel) and actin (bottom panel). For detection of enriched proteins in FKCM (b), following biotinylation of the primary amine of proteins in cell culture conditioned medium, the biotin-labelled sample was placed on an array membrane and incubated at 4C overnight. Following incubation with HRP-streptavidin, the signal was visualized by chemiluminescence. Expression levels of proteins in FKCM (right panel) were compared to those of control 10% DMEM media (left panel); enriched proteins in FKCM are boxed in red.

Three of ve mice (60%) implanted with FKCM-treated DP cells showed hair neogenesis, while no hair formation was observed in mice (0/5) having control DP cells (Fig. 1dg). Formation of hair follicles was observed in ve of ve (100%) positive control experiments, which involved implantation of freshly isolated dermal cells and epidermal cells together. The number of hair follicles that were formed in each chamber grafting assay is shown in Table S1. Consistent with the results of the hair reconstitution assay, treatment with FKCM resulted in the activation of the b-catenin signalling pathway, which contributes to maintenance of trichogenicity of dermal cells (12,13). As shown in Fig. 2a, the

In this study, we isolated human ORS keratinocytes to generate conditioned medium, FKCM. Results of both patch assays and chamber grafts showed increased trichogenicity of both mouse neonatal dermal cells and rat vibrissa DP cells when FKCM was used as a media supplement. We also observed that FKCM activated b-catenin and BMP signalling pathways. This result is consistent with results of the hair reconstitution assay and provides information on certain underlying regulatory mechanisms of enhanced hair induction by FKCM. Findings of the present study also demonstrated enrichment of proteins, including IGFBP-7, IL-8, uPA and OPG in FKCM. To expand on ndings from array analysis, we are currently performing grafting assays using various concentrations and combinations of these proteins for identication of proteins responsible for trichogenicity. Identication of proteins and adjustment of optimal concentration of proteins during expansion of dermal cell culture would be very exciting and would be of importance for future cell therapy for treatment of hair loss. In conclusion, in this study, our data provide a strong indication that FKCM has potential for use in research on hair reconstitution and that it may be applied for expansion of the patients own dermal cells for use in cell-based therapy for treatment of hair loss.

Acknowledgements
This work was supported by the Korea Research Foundation (KRF) grant funded by the Korea government (MEST) (2012-001047). M.H. Lee and S. Im performed the research. S.H. Shin, M.H. Kwack and S. Jun contributed essential reagents or tools. Y.K. Sung, M.K. Kim and J.C. Kim designed the research study. Y.K. Sung and M.H. Lee performed data analysis and wrote the paper.

Conict of interests
The authors have declared no conicting interests.

References
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15 Driskell R R, Juneja V R, Connelly J T et al. J Invest Dermatol 2012: 132: 10841093.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Procedure for collection of FKCM. Figure S2. Schematic of the hair reconstitution assay. Table S1. The number of hair follicles that were formed in each chamber grafting assay. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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