Inhibition of glycogen synthase kinase-3 enhances the expression of ...

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Feb 24, 2009 - of alkaline phosphatase and insulin-like growth factor-1 in human primary dermal papilla cell culture and maintains mouse hair bulbs in organ ...
Arch Dermatol Res (2009) 301:357–365 DOI 10.1007/s00403-009-0929-7

ORIGINAL PAPER

Inhibition of glycogen synthase kinase-3 enhances the expression of alkaline phosphatase and insulin-like growth factor-1 in human primary dermal papilla cell culture and maintains mouse hair bulbs in organ culture Koichi Yamauchi · Akira Kurosaka

Received: 2 September 2008 / Revised: 6 January 2009 / Accepted: 27 January 2009 / Published online: 24 February 2009 © Springer-Verlag 2009

Abstract Dermal papilla (DP) at the hair follicle base is important for hair growth. Recent studies demonstrated that mouse vibrissa DP cells can be cultured in the presence of Wbroblast growth factor-2 (FGF-2), but lose expression of versican and their follicle-inducing activity during the culture, and that activation of the Wnt signal, which is inhibited by glycogen synthase kinase-3 (GSK-3), in the DP cells promotes hair growth activity. We therefore investigated the inXuence of a GSK-3 inhibitor, (2⬘Z,3⬘E)-6bromoindirubin-3⬘-oxime (BIO), on the growth of human DP cells and mouse vibrissa follicles in culture. We Wrst demonstrated that, similarly to mouse DP cells, human DP cells were able to be cultured up to 15 passages in the presence of FGF-2, and lost the expression of alkaline phosphatase (ALP). When human DP cells later than ten passages were treated with BIO, the expression of ALP as well as insulin-like growth factor-1 (IGF-1), another DP marker, was signiWcantly elevated. Nuclear and perinuclear translocation of -catenin was also observed. We then cultured mouse vibrissa follicles. In the presence of BIO, the follicles could be maintained for at least 3 days without detectable regression of the hair bulbs. The morphology and ALP expression were well preserved. BIO successfully retrieved the expression of DP marker molecules, such as ALP and IGF-1 in cultured human DP cells, and maintained mouse

hair bulbs. Thus, treatment with BIO may be useful to prepare DP cells with hair follicle-inducing activity. Keywords Dermal papilla · Hair follicle · Glycogen synthase kinase-3 inhibitor · Alkaline phosphatase · Insulin-like growth factor-1 Abbreviations ALP Alkaline phosphatase BIO (2⬘Z,3⬘E)-6-bromoindirubin-3⬘-oxime GSK-3 Glycogen synthase kinase-3 DMEM Dulbecco’s modiWed Eagle’s medium DP Dermal papilla EDTA Ethylenediaminetetraacetic acid FBS Fetal bovine serum FGF-2 Fibroblast growth factor-2 FGF-7 Fibroblast growth factor-7 FGFR-1 Fibroblast growth factor receptor-1 G3PDH Glyceraldehyde-3-phosphate dehydrogenase IGF-1 Insulin-like growth factor-1 PBS Phosphate buVered saline RT-PCR Reverse transcription polymerase chain reaction SCF Stem cell factor TUNEL Terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling

Introduction K. Yamauchi Hair Clinic Reve-21 Corporation, 2-1-61 Shiromi, Chuo-ku, Osaka 540-6122, Japan A. Kurosaka (&) Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan e-mail: [email protected]

A hair follicle is part of the skin that consists of mesenchymal and epithelial tissues, and interactions between dermal and epidermal cells are important for the development and regeneration of hair follicles. Under physiological conditions, adult hair follicles undergo dynamic changes during the hair cycle. They have three phases, hair growing phase

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(anagen), regression (catagen), and quiescent (telogen). The hair cycle is maintained by interactions among skin cells. For example, in the anagen, a cluster of mesenchymal cells, known as dermal papilla (DP) cells, interacts with hair matrix keratinocytes, and activates them to diVerentiate into several types of hair follicle cells. Transplanted DP cells can induce hair follicle formation in hairless skin of mice by reconstructing interactions with epidermal cells in the tissue [17]. DP cells are therefore regarded as an important component to maintain and regenerate the hair growth cycle. Dermal papilla cells express alkaline phosphatase (ALP), a hydrolase that releases phosphate groups in a mild alkaline environment, and ALP activity in mouse vibrissa follicle DP cells has been examined to study the relationship between their activity and control of the hair cycle. The ALP level is related to the activity to induce follicle formation; DP cells with higher ALP activity induce hair follicles more eYciently when they are grafted onto an ear skin wound of recipients [14]. Thus, ALP activity is regarded as a useful marker to estimate the activity of follicle mesenchymal cells to induce hair growth [14]. Recently, Iida et al. [5] demonstrated that ALP activity in mouse vibrissa follicles changes in relation to the hair cycle; activity is strong at the anagen, and weak at the catagen. Besides ALP, DP cells express several characteristic proteins, some of which are recognized as marker molecules, and insulin-like growth factor-1 (IGF-1) is one of the proteins expressed in DP cells [8]. IGF-1 has various important biological activities, such as the promotion of growth and diVerentiation of various cell types and, in hair follicles, it is involved in promoting hair growth [24]. In transgenic mice overexpressing IGF-1 in the skin, hair follicles developed earlier than in control mice [1], indicating the hair inductive abilities of IGF-1. Interestingly, an inhibitor of glycogen synthase kinase-3 (GSK-3), designated 603287-31-8, increases ALP expression in mouse mesenchymal cells [13], as well as IGF-1 expression in rat renal mesangial cells [21]. GSK-3, a multifunctional serine/threonine kinase, is a key regulator of numerous signaling pathways and inhibits the Wnt signaling pathway by phosphorylating a Wnt signal transducer, -catenin, which is ubiquitinated after phosphorylation and degraded in the cytoplasm. GSK-3 is active in cells without the Wnt signal and is regulated through inhibition of its activity. GSK-3 inhibitors increase -catenin in the cytoplasm [20], leading to its translocation into the nucleus and the transcription of a set of downstream genes. In mouse hair follicles, strong nuclear -catenin expression in the DP is detected during anagen [18], and Wnt signaling maintains the hair-inducing activity of the DP [11]. However, the role of GSK-3 in the growth of human DP cells still remains to be elucidated. In this study, we investigated the eVects of (2⬘Z, 3⬘E)-6bromoindirubin-3⬘-oxime (BIO), a GSK-3 inhibitor, on

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cultured DP cells from human scalp hairs and organ cultures of mouse vibrissa follicles. We found that cultured human DP cells lost ALP activity in early passages, and that when cells were treated with the GSK-3 inhibitor, they recovered ALP activity and also exhibited enhanced expression of IGF-1. The mouse vibrissa hair bulbs also retained high ALP expression when treated with BIO. The inhibition of GSK-3 therefore would be useful to prepare DP cells with follicle-inducing activity.

Materials and methods Biochemicals A GSK-3 inhibitor IX, 361550, BIO was purchased from Calbiochem (La Jolla, CA, USA). All other reagents were of analytical grade. Tissues and DP culture Human DP was prepared from a plucked male hair. Approximately 0.5-1% hairs plucked from scalp had follicles with DP. Five-week-old female C3H/He mice were sacriWced painlessly, and vibrissa follicles were obtained by dissecting under a microscope. DP cells were established from human hair follicles and mouse vibrissa follicles according to the method of Messenger [15]. Obtained DP was treated with 0.1% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA) for 15 min at 37°C, transferred into a well of a collagen I-coated 12-well plate, and then cultured in Dulbecco’s modiWed Eagle’s medium (DMEM; Wako Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 10 ng/ml human or mouse Wbroblast growth factor-2 (FGF-2) (Bio Vision, Mountain View, CA, USA and R&D Systems, Minneapolis, MN, USA, respectively), and 100 U/ml penicillin-100 ng/ml streptomycin. The culture medium was changed every 3 days. After 10– 15 days, DP cells were harvested by treating with 0.1% trypsin-0.02% EDTA, and transferred to a well of the collagen I-coated dish. DP cells were subcultured at subconXuence. In this study, DP cells of passages 8–11 were used for experiments. To investigate the inXuence of GSK-3 inhibition, BIO was added to the culture medium of DP cells. The culture was incubated at 37°C under a 5% CO2 atmosphere. Organ culture of mouse vibrissa follicles For organ culture of mouse vibrissa follicles, mid- or late-anagen-phase follicles were chosen. Vibrissa follicles were plated on cell culture inserts (BD Biosciences Clontech, Palo Alto, CA, USA), cultured in DMEM supplemented with 100 U/ml penicillin-100 ng/ml streptomycin, and BIO was added to the

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culture medium. Vibrissa follicles were incubated over 3 days and then used for the experiments. The culture was incubated at 37°C and under a 5% CO2 atmosphere. Determination of cell growth and detection of alkaline phosphatase activity Dermal papilla cells were trypsinized and the cells were cultured in 96-well plates at the cell density of 2,000/well in 10% FBS–DMEM containing various concentrations (from 0.01 to 10 M) of BIO for 72 h. For each concentration of BIO, three wells of cell culture were used. To determine the cell number, 10 l of Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan) was added to each well, and the plate was incubated at 37°C for 2 h. The cell number was determined by measuring absorbance at 450 nm, and then the reagent was removed, the well was rinsed with 100 l of phosphate-buVered saline (PBS) twice, and the plate was subjected to three freeze (¡80°C)/thaw cycles [13]. Two hundred microliters of 1 mg/ml 4-nitrophenylphosphate bisalt (Sigma, St Louis, MO, USA) in 1 M diethanolamine buVer (pH 9.8) were added to the well, and the plate was incubated at 37°C for 30 min and read at 405 nm to determine ALP activity. For ALP staining of vibrissa follicles, mouse follicles were embedded in Optimal Cutting Temperature compound (Sakura Finetechnical, Tokyo, Japan), frozen, sectioned at 6 m thickness, and placed on a glass slide. For ALP staining in DP cells, the cells were cultured on a coverslip. Frozen sections or cultured cells on the glass slide or coverslip were Wxed with acetone and methanol (1:1, v/v) for 10 min. Samples were then washed with PBS, and exposed to ALP Fast Blue RR substrate (Sigma) in solution (10 mg of Fast Blue RR, 10 ml of substrate buVer, and 1.5 ml of naphthol AS–MX phosphate concentrate) at pH 8.1 for 1 h.

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transcriptase (Invitrogen) according to the manufacturer’s instructions. The cDNA (» 50 ng) thus obtained was used as a template for reverse transcriptase-polymerase chain reaction (RT-PCR), which was carried out with AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) for 35 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 2 min using the primers listed below for ampliWcation. Real-time PCR was performed using SYBR green Q-PCR master mix (Invitrogen) on the AB 7500 Real Time PCR System (Applied Biosystems) under the following conditions: 45 cycles of 95°C for 15 s, and 60°C for 1 min. The sequences of the PCR primer sets used in this study are as follows. ALP (NM_000478.3): (F) 1,034– 1,056 5⬘-CAAACCGAGATACAAGCACTCCC-3⬘, and (R) 1,128–1,104 5⬘-CGAAGAGACCCAATAGGTAGTC CAC-3⬘. IGF-1 (NM_000618.3): (F) 246–269 5⬘-TGTCCT CCTCGCATCTCTTCTACC-3⬘, and (R) 381–358 5⬘-TAA AAGCCCCTGTCTCCACACACG-3⬘. Versican (NM_00 4385.3): (F) 669–692 5⬘-GGGATTGAAGACACACAAG ACACG-3⬘, and (R) 817–794 5⬘-TGCTCTGGAGTTGC TATGACTGCC-3⬘. FGF-7 (NM_002009.2): (F) 765–784 5⬘-TTGTGGCAATCAAAGGGGTG-3⬘, and (R) 927-905 5⬘-CCTCCGTTGTGTGTCCATTTAGC-3⬘. Fibroblast growth factor receptor-1 (FGFR-1) (NM_023110.2): (F) 1,589–1,610 5⬘-TAATGGACTCTGTGGTGCCCTC-3⬘, and (R) 1,669–1,650 5⬘-ATGTGTGGTTGATGCTGCCG3⬘. Stem cell factor (SCF) (NM_003994.4): (F) 635–658 5⬘-TC AAGGACTTTGTAGTGGCATCTG-3⬘, and (R) 728–709 5⬘-TCTCCAGGGGGATTTTTGGC-3⬘. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (NM_00 2046): (F) 597–620 5⬘-TGACAACTTTGGTATCGTGGAA GG-3⬘, and (R) 726–705 5⬘-AGGGATGATGTTCTGG AGAGCC-3⬘. Apoptosis assay

Immunocytochemistry Cultured DP cells on the coverslips were Wxed with acetone and methanol (1:1, v/v) for 10 min, blocked with 3% bovine serum albumin–PBS for 30 min, and then treated with the rabbit monoclonal anti--catenin antibody (1:500, Epitomics, Burlingame, CA, USA) for 1 h at room temperature. Samples were then washed with PBS, and reacted with Alexa 488-conjugated secondary antibodies (Molecular Probe, Carlsbad, CA, USA) for 30 min. Samples were washed with PBS and the cell nuclei were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA, USA). Reverse transcriptase-PCR and real-time PCR One microgram of total RNA from cultured human DP cells was prepared using Sepasol RNA (Nacalai Tesque) and reverse-transcribed with Superscript III reverse

To determine the levels of apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL (In Situ Cell Death Detection Kit, Roche, Basel, Switzerland), was used for immunostaining cryostat sections of vibrissa follicles cultured for 24, 48, and 72 h.

Results Culture of human and mouse DP cells Human hairs were obtained from an adult man, and those with an intact hair bulb at the bottom of the follicle and in the anagen phase were used for the following experiments (Fig. 1a). DP cells were prepared according the method of Messenger [15] (Fig. 1b). We Wrst cultivated the isolated DP cells in 10% FBS–DMEM in a collagen I-coated dish,

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Fig. 1 Dermal papilla cell culture. a A human hair follicle with DP at the bottom. b Isolated human DP. c Isolated human DP cultured in 10% FBS–DMEM in the presence of 10 ng/ml FGF-2 for 8 days. d Human DP cells at passage 8 cultured in 10% FBS–DMEM containing 10 ng/ml FGF-2 in the absence of 1.5 M BIO. e, f indicate human DP cells at passage 10 cultured in 10% FBS–DMEM without and with 1.5 M BIO, respectively. For (f), photographs were taken 72 h after the addition of BIO. Scale bar = 100 m

but they failed to grow. Osada et al. [16] recently reported that FGF-2 is eVective for long-term culture of mouse vibrissa DP cells. We therefore cultured human scalp DP cells in the presence of 10 ng/ml FGF-2 (Fig. 1c, d). For the initial few days, the DP cells did not grow, but about 1 week after the cell culture began, they started to proliferate (Fig. 1c), reaching conXuence on day 15. For the continuous cell culture, we subcultured the subconXuent DP cells (Fig. 1d). Under these conditions, we succeeded in culturing the DP cells up to 15 passages without an apparent decrease in the growth rate, and obtained the total number of » 1 £ 109 cells at the 8th passage; however, after passage 15, cell division gradually slowed, and eventually the cells stopped dividing. GSK-3 inhibition We treated the human DP cells with 1.5 M of BIO, and cultured in 10% FBS–DMEM to investigate the relationship between the -catenin signal pathway and hair growth. When cells at passage 10 or later, which exhibited a thin, Wbroblast-like appearance (Fig. 1e), were treated with BIO, we observed morphological alterations of the cells, which were Xatter and rounder in shape (Fig. 1f). Immunostaining of -catenin using a speciWc antibody demonstrated that in control cells, -catenin was almost exclusively associated with the cell membrane with a very small amount in the cytoplasm (Fig. 2a–c), but when the cells were treated with BIO, -catenin was translocated in the nuclear and perinuclear region in DP cells (Fig. 2d–f). Similarly, in the mouse vibrissa DP cells, which were prepared according the method of Osada et al. [16] and treated with 5 M BIO, we found nuclear and perinuclear

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accumulation and translocation of -catenin as well (data not shown). Gene expression analysis of DP cells treated with BIO There is a reported decrease in the expression of versican [12] in cultured mouse vibrissa DP cells. We examined the expression of six proteins that are highly expressed in the DP in vivo [4, 6, 14, 19, 22, 24] using RT-PCR and realtime PCR (Fig. 3). For this purpose, total RNA was prepared from human DP cells treated with and without BIO for 3 days, and cDNA obtained by reverse-transcription was used as a template for PCR. Figure 3a shows that the expressions of ALP and IGF-1 were weak in control cells, but apparently their expressions were markedly increased in BIO-treated DP cells. Real-time PCR indicated the most signiWcant elevation of ALP expression with a 5.1-fold increase as well as about a twofold increase in IGF-1 expression (Fig. 3b). Contrary to this, the other four genes (versican, FGF-7, FGFR-1, and SCF) were expressed in DP cells at passage 10 at a high level, and their expressions also remained high in BIO-treated cells. ALP is considered as a DP marker, the activity of which is related to follicleinducing activity [14], and IGF-1 is also involved in promoting hair growth [24]. The treatment of DP cells with the GSK-3 inhibitor was therefore eVective in preparing DP cells with high ALP and IGF-1 expression, which could retain their ability to induce hair growth. ALP activity in cultured DP cells We then examined ALP activity of cultured human DP cells. The DP cells at passage 10 also showed very weak

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Fig. 2 Translocation of -catenin in human DP cells treated with BIO. -Catenin and nuclei were stained with the speciWc antibody and Hoechst 33258, respectively, in cells cultured in the absence (a, b) and presence of 1.5 M BIO (d, e) for 48 h. c, f are merged images of (a, b) and (d, e), respectively. -Catenin was observed in the cytoplasm and plasma membrane in control cells (a–c). In cells treated with BIO, -catenin is translocated and accumulated in the nuclear and perinuclear region (d–f). Scale bar = 10 m

of human DP cells with 1.5 M BIO was useful for obtaining cells having high ALP activity. We also investigated the eVects of BIO on mouse vibrissa DP cells. Similarly to the human cells, mouse DP cells at passage 10 showed very weak ALP activity (Fig. 4d), but those treated with BIO recovered expression of ALP (Fig. 4e). Its increase was also dependent on the concentration of BIO, with the highest activity at 5 M, followed by a decrease at higher concentrations (data not shown). BIO modestly diminished the proliferation rate of DP cells below 5 M, with stronger growth inhibition above that concentration. Thus, the optimal concentration of BIO for mouse DP cells was 5 M. Fig. 3 Polymerase chain reaction analysis of gene expression in human DP cells. RT-PCR (a) and real-time PCR (b) were performed as described under Sect. ”Materials and methods” to examine the expression of genes expressed in DP cells. Total RNA was isolated from control DP cells (passage 10) and cells (passage 10) treated with 1.5 M BIO for 72 h, reverse-transcribed, and used as a template. The ordinate in (b) indicates the ratio of gene expression levels in the BIOtreated cells to those in the control. Data for real-time PCR are the mean values § SD of three separate experiments. Alkaline phosphatase (ALP), insulin-like growth factor-1 (IGF-1), Wbroblast growth factor-7 (FGF-7), Wbroblast growth factor receptor-1 (FGFR-1), stem cell factor (SCF), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH)

ALP activity, when ALP was cytochemically stained by incubating with substrates (Fig. 4a). In fact, the cells lost ALP activity quickly. The cells at passage 3 gave similar staining as in Fig. 4a (data not shown). Addition of the GSK-3 inhibitor, however, remarkably elevated ALP activity in DP cells. Its increase was dependent on the concentration of BIO, with the highest activity at 1.5 M, followed by a decrease at higher concentrations (Fig. 4c). On the other hand, BIO modestly diminished the proliferation rate of DP cells below 1.5 M, with stronger growth inhibition above that concentration. The staining of ALP in cells (passage 10) treated with 1.5 M of BIO demonstrated the recovered expression of ALP (Fig. 4b). Thus, the treatment

EVect of BIO on cultured mouse vibrissa follicles Finally, we investigated the eVects of BIO on the mouse hair follicle culture. Isolated mouse vibrissa follicles were cultured in DMEM in the absence or presence of BIO. Three days after organ culture, the shapes of follicles were compared. Figure 5a shows that hair bulbs at the bottom of the control follicle were degenerated and almost completely lost. In contrast, those treated with BIO seemed apparently unaVected, although regression was observed to some extent (Fig. 5b). Histochemical staining of ALP activity in cultured follicle sections showed that in control sections DP cells at day 0 exhibited high ALP activity (Fig. 5c), but after 3-day culture, only faint ALP activity was detected at the bottom of the follicle and the DP structure was almost completely impaired (Fig. 5d). DP cells treated with BIO, on the other hand, exhibited strong ALP activity (Fig. 5e), indicating the structural integrity of DP. We also examined apoptosis in the mouse hair bulbs, which occurs in the epithelial cells in the bulb and the outer root sheath during the catagen phase [9]. We used the TUNEL reaction, which preferentially labels DNA strand breaks generated during apoptosis. The analysis of mouse hair follicle culture showed signiWcantly more TUNEL-positive cells in the control, which were

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Fig. 4 EVects of BIO on ALP expression and growth of DP cells. Human DP cells at passage 10 were cultured in the absence (a) and presence (b) of 1.5 M of BIO for 72 h. The substrate for ALP was added to the culture medium and stained (a, b). ALP expression in the control cells was low (a), but that in BIOtreated cells recovered (b). ALP activity and the numbers of cells treated with varying concentrations of BIO for 72 h were determined as described in Sect. ”Materials and methods” (c). The growth was expressed as the relative increase in the cell number after the cell culture. Mouse DP cells at passage 10 were cultured in the absence (d) and presence (e) of 5 M of BIO for 72 h. Similarly to human cells, mouse DP cells treated with BIO retrieved the ALP expression. Scale bar = 100 m

located in the hair matrix and the dermal sheath (Fig. 6). Staining was most intense on day 1 and gradually lessened over the 3-day organ culture. By contrast, there was little, if any, TUNEL staining in BIO-treated follicles (Fig. 6). The mouse hair follicle culture experiments demonstrated that the GSK-3 inhibitor might be eVective in activating and maintaining DP cells in the organ culture.

Discussion Dermal papilla cells play pivotal roles in controlling the hair cycle as well as regenerating hair growth in the skin,

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and it is therefore important to establish culture conditions to grow human DP cells with hair follicle-inducing activity, especially for clinical treatment of hair growth disorders. A recent report demonstrated that DP cells from mouse vibrissa can be cultivated over 34 passages or more by adding FGF-2 to the culture medium [16]. We also succeeded in culturing mouse vibrissa DP cells over 30 passages using the same culture conditions as theirs. Furthermore, we investigated whether FGF-2 is also eVective for culturing human scalp DP cells. Similarly to mouse vibrissa DP cells, the human DP cells also required FGF-2 for their growth, but showed a less eYcient growth rate than mouse cells. They were able to proliferate in the presence of FGF-2 up

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to passage 15, but could not continue to grow well beyond that passage. This may reXect the diVerence in the species and origin of the hairs. In mice, it is reported that cycling of pelage follicles is diVerently controlled from that of

vibrissa [23]. It would be reasonable to speculate that DP cells from distinct species or those from diVerent regions of the same species would reside in a characteristic niche and they therefore require distinct conditions for growing in vitro. The growth of human scalp DP cells was less eYcient than mouse vibrissa cells, but still we were able to obtain » 1 £ 109 cells at passage 8, which was enough to investigate their proWles. -Catenin is a cytoplasmic protein and is phosphorylated by GSK-3 under resting conditions (i.e., in the absence of the Wnt signal) and the phosphorylated -catenin is degraded by the ubiquitin–proteasome system in the cytoplasm. When the Wnt signal is transmitted into the cytoplasm through activation of Dishevelled, a component of the Wnt receptor complex, GSK-3 is inhibited and then a pool of cytoplasmic -catenin stabilizes, leading to its translocation into the nucleus and the transcription of target genes. The inhibitors of GSK-3 hence are activators of the Wnt signaling pathway. Since the -catenin-dependent expression of genes responsive for the Wnt signal is detected in developing dermal condensates and in hair follicles [2] and strong nuclear -catenin expression in DP cells is detected during the anagen [18], the nuclear accumulation of -catenin in DP cells is important for the regeneration of follicles and hair growth. In this study, the GSK-3 inhibitor BIO was used to enhance the canonical Wnt signal

Fig. 6 TUNEL staining of mouse vibrissa follicles. Mouse hair follicles cultured in DMEM for 1, 2, or 3 days were subjected to Hoechst staining (blue) to detect nuclei, and TUNEL analysis (green) to local-

ize apoptotic nuclei in the follicles. Arrows and arrowheads indicate hair matrix and dermal sheath, respectively, dp dermal papilla. Scale bar = 100 m

Fig. 5 EVects of BIO on mouse hair follicle culture. Mouse vibrissa follicles were isolated and cultured in DMEM with and without BIO for 3 days. a, b Indicate images of mouse vibrissa follicles at day 0 and 3 cultured in the absence and presence of 25 M BIO, respectively. Sections were prepared from cultured vibrissa and exposed to the ALP substrate to localize ALP (c–e). In control hair follicles, after 3-day culture regression of the hair bulb and decreased ALP activity were observed (d). By contrast, in BIO-treated cells, the hair bulb structure was preserved and ALP activity was retained (e). Scale bar = 100 m

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through the activation of -catenin. In human cultured DP cells treated with BIO, -catenin exhibited the nuclear and perinuclear accumulation. Also, concomitant decrease in the membrane-localized -catenin, which is reported to bind to the cytoplasmic domain of E-cadherin [10], was observed. The decrease is consistent with the recent reports that GSK-3 inhibition downregulates E-cadherin both in vivo and in vitro [3, 25], and that decreased expression of E-cadherin promotes transcription directed by LEF1 [10], to which -catenin binds. Interestingly, BIO restored the expression of ALP and IGF-1 in DP cells, whose expression had decreased during cell culture. ALP activity is strong in anagen DP [5] and is considered as a marker for hair follicle-inductive abilities [14]. IGF-1 also has the activity to promote hair growth [24]. Thus, the regained activity of ALP and IGF-1 in DP cells by BIO may indicate restoration of the potency to induce hair follicles. Our observations on the organ culture of mouse vibrissa follicles also supported the idea that BIO elevates ALP activity and helps the hair bulb maintain its activity to promote hair growth. In spite of elevated ALP and IGF-1 activity in BIOtreated human DP cells, our preliminary experiments failed to induce de novo hair follicles in the back of athymic mice, in which cultured human DP cells treated with BIO were implanted subcutaneously together with mouse epidermal cells. Unlike human DP cells, several studies have reported hair-follicle formation using cultured murine DP cells. For example, Inamatsu et al. [7] demonstrated that rat DP cells cocultured with rat sole-derived keratinocytes retain hair follicle-inducing activity. More recently, Osada et al. [16] reported that cultured mouse vibrissal DP cells regain the hair follicle-inducing activity by letting them aggregate to form spheres which, when implanted with mouse keratinocytes, can induce hair follicles. Although mouse or rat DP cells can induce de novo hair follicles, to the best of our knowledge, no reports have so far demonstrated the hair follicle-inducing activity of cultured human DP cells using murine keratinocytes as partner cells to form hair follicles. Our approach to use BIO for obtaining active DP cells is easier than the sphere formation method and is seemingly a convincing method, judging from the gene expression proWles. We are currently trying to adjust the experimental conditions for the restoration assay using human DP cells treated with BIO.

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