texto atual

7/27/2019 texto atual

1/3

novel strategies for several diseases including cancer progression

and cystic fibrosis (8, 2024). Here, we provide evidence for the

first time that the inhibition of the UPS increases the stabiliza-

tion 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 mecha-

nism involved in the proper localization of DP and might sug-

gest a novel therapy approach for diseases characterized bymislocalization of DP.

AcknowledgementsSL 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.

Conflict of interestThe authors state no conflict of interest.

References1 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. Sci-

ence 2010: 329: 805

810.9 Schwartz A L, Trausch J S, Ciechanover A et al.

Proc Natl Acad Sci U S A 1992: 89: 55425546.

10 Pasdar M, Nelson W J. J Cell Biol 1988: 106:677685.

11 Green K J, Simpson C L. J Invest Dermatol2007: 127: 24992515.

12 Franke W W. Cold Spring Harb Perspect Biol2009: 1: a003061.

13 Green K J, Stappenbeck T S, Parry D A et al.JDermatol 1992: 19: 765769.

14 Kartenbeck J, Schwechheimer K, Moll R et al.JCell Biol 1984: 98: 10721081.

15 Watt F M, Mattey D L, Garrod D R. J Cell Biol1984: 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 Biol1987: 105: 23272334.

19 Wallis S, Lloyd S, Wise I et al.Mol Biol Cell2000: 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 ONE2011: 6: e25888.

22 Hishinuma S, Komazaki H, Fukui H et al.J Neu-rochem 2010: 113: 9901001.

23 Citri A, Soler-Llavina G, Bhattacharyya S et al.Eur J Neurosci 2009: 30: 14431450.

24 Loffek S, Woll S, Hohfeld J et al. Hum Mutat2010: 31: 466476.

Supporting InformationAdditional Supporting Information may be found inthe online version of this article:

Figure S1. Primary keratinocytes were cultured inKGM (Ctr) or in high calcium [1.8 mM] (HCa) or inthe presence of MG132 [50lM] for 3 h. (a) Cells werelysed and subsequently separated into detergent-solubleand insoluble fractions. Equal amounts of protein wereseparated by SDS-PAGE. Figure shows representativedesmoplakin (DP) and plakoglobin (PG) immunoblotsof the detergent-insoluble and the soluble fraction.

(b) Cells were cultured in KGM and treated withMG132 and (upper panel) nocodazole (Noc) or (lowerpanel) cytochalasin D (Cyto. D). Cells were fixed andstained with indicated antibodies. Arrow heads indicatethe MG132-induced re-localization of DP to cell-cellcontacts even in the presence of depolymerized tubulincytoskeleton. 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. Cellswere 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 ofMG132 [50lM] or in high calcium [2 mM] for 3 h.

Thereafter, cells were washed with PBS and incubatedwith 2.4 U/ml dispase for 20 min at 37 C. Pictureswere taken at indicated time-points after applying dis-pase.

Table S1. Table provides the details of the antibodiesused in this study.

Data S1. Dispase assay.

Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than miss-ing material) should be directed to the correspondingauthor for the article.

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

Letter to the Editor

Conditioned media obtained from human outer root sheathfollicular keratinocyte culture activates signalling pathways thatcontribute to maintenance of hair-inducing capacity and increasestrichogenicity 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

1Department 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: [email protected] 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

2012 John Wiley & Sons A/SExperimental Dermatology, 2012, 21, 783801 793



2/3

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 datasuggest 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

BackgroundFollicular 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 significantly 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 regen-eration 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,

identification of cell culture supplements and/or culture condi-

tions that enable dermal cells to maintain trichogenicity is an

important matter. Maintenance of hair-inductive potential of cul-

tured dermal cells by addition of conditioned media obtained

from rodent epidermal cells or human skin epidermal keratino-

cytes 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 trichogenici-

ty 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.

Questions addressedIn 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 kerati-

nocyteconditioned 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 theactivation of signalling pathways that contribute to maintenance

of trichogenicity.

Experimental designHuman ORS keratinocytes were prepared using a previously

described method, with minor modifications (7). For preparation

of FKCM, culture medium was switched to DMEM supplemented

with 10% FBS when cells at passage 2 reached 80~90% confluency.

Harvest of the medium was performed after 4 days, followed by

centrifugation, and filtration through a 0.20-lm membrane filter

(Fig. S1). Isolation and cultivation of rat vibrissa DP were con-

ducted 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 modifications

(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 ofproteins in conditioned medium.

ResultsTo 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.

FKCM + FKCM

* FKCM + FKCM

(a) (b) (c)

(d)

FKCM + FKCM

(e) (f)

0 (0/5)

60 (3/5)

100(5/5)

0

20

40

60

80

100

120

Ha

irfollicleinduction

ratio(%)

FKCM + FKCM Fresh

dermal

cells

(g)

Figure 1. Increment of hair follicle induction in follicular keratinocyteconditionedmedia (FKCM)-treated mouse dermal cells and rat dermal papilla cells.Representative data showing hair follicles induced by mouse dermal cells culturedin 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 ratdermal papilla cells cultured in the absence (d) or presence (e) of FKCM. Close-upimage of the boxed region of e is shown in (f). Values represent the ratio of hairfollicle induction from five independent experiments (g).

794 2012 John Wiley & Sons A/S

Experimental Dermatology, 2012, 21, 783801



3/3

Three of five 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 five of five (100%) positive control

experiments, which involved implantation of freshly isolated

dermal cells and epidermal cells together. The number of hair fol-

licles 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

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 immunofluorescence staining,

however, did not show a significant increase in markersrepresenting intrinsically defined 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).

ConclusionsIn this study, we isolated human ORS keratinocytes to generate con-

ditioned medium, FKCM. Results of both patch assays and chamber

grafts showed increased trichogenicity of both mouse neonatal der-

mal 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 enrichmentof proteins, including IGFBP-7, IL-8, uPA and OPG in FKCM. To

expand on findings from array analysis, we are currently performing

grafting assays using various concentrations and combinations of

these proteins for identification of proteins responsible for trichoge-

nicity. Identification of proteins and adjustment of optimal concen-

tration 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.

AcknowledgementsThis work was supported by the Korea Research Foundation (KRF) grant funded

by the Korea government (MEST) (2012-001047). M.H. Lee and S. Im per-

formed 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.

Conflict of interestsThe authors have declared no conflicting interests.

References1 Teumer J, Cooley J. Semin Plas Surg 2005: 19:

193200.2 Ohyama M, Zheng Y, Paus R et al. Exp Derma-

tol 2010: 19: 8999.

3 Yang C C, Cotsarelis G. J Dermatol Sci 2010:57: 211.

4 Inamatsu M, Matsuzaki T, Iwanari H et al. JInvest Dermatol 1998: 111: 767775.

5 Qiao J, Zawadzka A, Philips E et al. Regen Med2009: 4: 667676.

6 Inoue K, Aoi N, Yamauchi Y et al. J Cell MolMed 2009: 13: 46434656.

7 Kwack M H, Sung Y K, Chung E J et al. JInvest Dermatol 2008: 128: 262269.

8 Messenger A G, Senior H J, Bleehen S S. Br JDermatol 1986: 114: 425430.

9 Liang Y, Silva K A, Kennedy V et al. Exp Der-matol 2011: 20: 10111015.

10 Zheng Y, Du X, Wang W et al. J Invest Derma-tol 2005: 124: 867876.

11 Lichti U, Weinberg W C, Goodman L et al. JInvest Dermatol 1993: 101: 124129.

12 Kishimoto J, Burgeson R E, Morgan B A. GeneDev 2000: 14: 11811185.

13 Shimizu H, Morgan B A. J Invest Dermatol2004: 122: 239245.

14 Rendl M, Polak L, Fuchs E. Genes Dev 2008:22: 543557.

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

Supporting Information

Additional Supporting Information may be found inthe 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 wereformed in each chamber grafting assay.

Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than miss-ing material) should be directed to the correspondingauthor for the article.

(a)

(b)

FKCM + FKCM

0 10 30 60 0 10 30 60 (min)

pSMAD 1/5/8

-catenin

Actin

IGFBP -7

IL-8

uPA

Osteoprotegerin

+FKCM FKCM

IGFBP -7

IL-8

Osteoprotegerin

uPA

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

2012 John Wiley & Sons A/SExperimental Dermatology, 2012, 21, 783801 795


Documents

texto atual