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Published as doi: 10.1096/fj.07-8628com.
 (The FASEB Journal. 2007;21:3346-3354.)
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Human hair follicles are an extrarenal source and a nonhematopoietic target of erythropoietin

Eniko Bodó*,1, Arno Kromminga{dagger},1, Wolfgang Funk{ddagger}, Magdalena Laugsch§, Ute Duske{dagger}, Wolfgang Jelkmann§ and Ralf Paus*,2

* Department of Dermatology, University of Lübeck, Lübeck, Germany;

{dagger} Institute for Immunology, Clinical Pathology, Molecular Medicine, Hamburg, Germany;

{ddagger} Klinik Dr. Kozlowski, Munich, Germany; and

§ Department of Physiology, University of Lübeck, Lübeck, Germany

2Correspondence: Department of Dermatology, University Hospital Schleswig-Holstein, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: ralf.paus{at}uk-sh.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Erythropoietin primarily serves as an essential growth factor for erythrocyte precursor cells. However, there is increasing evidence that erythropoietin (EPO)/EPO receptor (EPO-R) signaling operates as a potential tissue-protective system outside the bone marrow. Arguing that growing hair follicles (HF) are among the most rapidly proliferating tissues, we have here explored whether human HFs are sources of EPO and targets of EPO-R-mediated signaling. Human scalp skin and microdissected HFs were assessed for EPO and EPO-R expression, and the effects of EPO on organ-cultured HFs were assessed in the presence/absence of a classical apoptosis-inducing chemotherapeutic agent. Here, we show that human scalp HFs express EPO on the mRNA and protein level in situ, up-regulate EPO transcription under hypoxic conditions, and express transcripts for EPO-R and the EPO-stimulatory transcriptional cofactor hypoxia-inducible factor-1{alpha}. Although EPO does not significantly alter human hair growth in vitro, it significantly down-regulates chemotherapy-induced intrafollicular apoptosis and changes the gene expression program of the HFs. The current study points to intriguing targets of EPO beyond the erythropoietic system: human HFs are an extrarenal site of EPO production and an extrahematopoietic site of EPO-R expression. They may recruit EPO/EPO-R signaling e.g., for modulating HF apoptosis under conditions of hypoxia and chemotherapy-induced stress.—Bodó, E., Kromminga, A., Funk, W., Laugsch, M, Duske, U., Jelkmann, W., Paus, R. Human hair follicles are an extrarenal source and a non-hematopoietic target of erythropoietin.


Key Words: anagen hair follicles • tissue protection • hemoglobin alpha-1 • kinesin light chain 3 • aminase oxidase • calmegin • RAS-like family 10


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE GLYCOPROTEIN HORMONE ERYTHROPOIETIN (EPO) serves as an essential viability and growth factor for erythrocyte precursor cells (1 2 3 4 5) . EPO is the main regulator of maintenance of the blood cell mass by stimulating the proliferation and differentiation of precursor cells and by inhibiting apoptosis of erythroid cells in the bone marrow (1 2 3 4 5) .

The classical site of EPO synthesis is peritubular interstitial cells of the kidney when stimulated by hypoxia (1 , 2 , 6 , 7) . A primary mediator of hypoxia-induced EPO gene expression is the hypoxia-inducible dimeric transcription factor 1 (HIF-1 {alpha}/ß; refs. 1 , 8 , 9 ). However, HIF-1{alpha} and HIF-1ß mRNAs are continuously produced and remain essentially unaltered by the induction of hypoxia (1 , 10 , 11) . Instead, oxygen sensing and activation of the EPO gene are initiated by post-translational modifications of the HIF-1{alpha} protein (1) . EPO activates a specific receptor [erythropoietin receptor (EPO-R)] of the cytokine receptor gene family, the (mainly Jak2 and STAT5-mediated) signaling of which is thought to chiefly induce changes in gene expression that result in inhibition of the proapoptotic machinery of EPO-R expressing target cells, thereby suppressing apoptosis (1 , 2 , 5 6 7) .

Recently, however, it has surfaced that the apoptosis-inhibiting property of EPO is not restricted to erythropoietic cells and that apoptosis in several other tissues can be suppressed by EPO. Apparently, EPO serves as a potent paracrine-autocrine cell-protective factor protecting from, for example, ischemia-induced apoptosis (1 , 3 , 4) . One of the best studied fields is the nervous system (1 , 3 , 4) : functional EPO-R have been demonstrated on neuronal cells (1 , 12 , 13) , and application of EPO has neuroprotective effects in in vivo animal studies (14 15 16 17) via regulation of, for example, bcl-xL (18) , an antiapoptotic factor. EPO even may reduce infarct size and may improve recovery of stroke patients (16 , 19) . Also, human recombinant EPO was reported to prevent lipopolysaccharide-induced apoptosis in bovine pulmonary artery endothelial cells (20) and to reduce necrosis and apoptosis during experimental injury in the kidney in vivo (21) . Furthermore, human fetal liver, placenta, retina, and adrenal cortex all have been reported to express EPO, respectively, EPO-R (22) .

Whether or not EPO/EPO-R signaling plays a role in human skin biology and pathology is as yet unknown. Recombinant, subcutaneously administered human EPO has been applied successfully to enhance wound healing in the skin of C57BL/6 mice, where EPO increased significantly the area of reepithelialization and accelerated the wound closure in deep-dermal second burn (23) . Selzer et al. (24) reported EPO-R expression in transformed human melanocytes in vitro but not in normal neonatal and adult primary human melanocytes. In contrast, Kumar et al. (25) have described highly expressed EPO-R and very weak, if any, EPO signals by Western blotting and immunocytochemistry in cultured human primary melanocytes. Note, however, that the non-specific C-20 antibody against EPO-R (26) was used for the latter study. Recently, an antibody that reportedly recognizes the soluble form EPO-R was claimed to demarcate immunoreactivity in cutaneous mast cells (27) . Very recently, LeBaron et al. (28) demonstrated that hair follicle (HF) dermal papilla fibroblasts are EPO target cells and respond to EPO treatment by an activation of EPO-R signaling. Thus, the cutaneous targets of EPO-induced signaling and whether mammalian skin expresses specific EPO-R and/or its ligand in situ remain unclear.

Arguing that growing (anagen) HFs are among the most rapidly proliferating and most damage-sensitive tissues in the human body (29 , 30) , we have here explored whether human HFs are sources of EPO expression and targets of EPO-R-mediated signaling. Matrix keratinocytes of human anagen VI HFs are characterized by a very high proliferative potential (29) and must be perfused entirely via the HF mesenchyme. Therefore, they may continuously experience conditions of relative hypoxia and have even been claimed to primarily resort to anaerobic glycolysis to meet their energy requirements (31) .

To investigate the EPO/EPO-R system in the skin and HFs has become particularly important since human skin and human pilosebaceous units operate as endocrine organs that synthesize classical (neuro)endocrine messengers (TSH, CRH, ACTH, {alpha}-MSH, prolactin, and melatonin; refs. 32 33 34 35 36 37 38 39 ) and display a fully functional peripheral equivalent of the central hypothalamic-pituitary-adrenal stress response axis (36 , 40) .

Here, we show that normal human skin joins the growing list of tissues that recruit the EPO/EPO-R system for cell protection and that healthy human HFs are an extrarenal site of EPO and an extrahematopoietic site of EPO-R expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
HF organ culture and hypoxia treatment
Anagen VI HFs were isolated from normal human scalp skin obtained after written patient consent from healthy females undergoing routine face-lift surgery for cosmetic purposes as described previously (41) , adhering to Helsinki guidelines. HFs were microdissected and organ-cultured as described previously (39 , 41 , 42) . Isolated HFs were maintained in supplemented, serum-free Williams’ E medium (Biochrom, Cambridge, UK) supplemented with 2 mmol/l L-glutamine (Invitrogen, Paisley, UK), 10 ng/ml hydrocortisone (Sigma-Aldrich, Taufkirchen, Germany), 10 µg/ml insulin (Sigma-Aldrich), and 1% antibiotic/antimycotic mixture (100x, Life Technologies, Germany, Karlsruhe). HFs were first incubated overnight, and the next day medium was exchanged and vehicle (culture medium)/EPO (100 U/ml, Epoetin beta; Roche/Boehringer Mannheim, Mannheim, Germany) was added to each well. HFs were treated over 9 days, and hair shaft length measurements were performed every second day on each individual HF, using a Zeiss inverted binocular microscope with an eyepiece measuring graticule.

In selected experiments, microdissected HFs were first cultured under normoxic conditions (humidified atmosphere at 37°C, 5% CO2, and 95% air; ref. 43 ) for 12 h. Then, HFs were placed in an InvivO2 400 hypoxia workstation (Ruskinn Technology, Leeds, United Kingdom) with a gas mixture of 3% O2, 5% CO2, and balance N2 for additional 24 h to induce conditions of relative hypoxia (43) .

Apoptosis induction in human HFs by chemotherapy
To explore potential cytoprotective effects of EPO, HFs were first incubated overnight without any treatment, and the next day (day 1) medium was exchanged and vehicle (culture medium)/EPO (100 U/ml) was added to each well. After 24 h preincubation, HFs were cotreated with the key toxic metabolite of the potent alopecia-inducing cytostatic agent cyclophosphamide [4-hydroperoxycyclophosphamide (4-HC), Niomec, Bielefeld; refs 44 , 45 ] for 4 days. In this assay system, we had previously shown that keratinocyte growth factor (FGF7) operates as a potent cytoprotective agent (45) .

Immunohistochemistry and quantitative immunohistology
For the detection of EPO in human scalp skin, the highly sensitive EnVision (Dako, Glostrup, Danmark) technique (46) was performed. The staining specificity was verified using human kidney cryosections as positive control. Acetone-fixed, 8 µm thick cryoslides of human scalp skin were preincubated with 10% normal goat serum (in TBS, Dako) for 20 min and then incubated with a rabbit anti-human EPO antiserum (1:50 in TBS, generated by hyperimmunization of rabbits with recombinant human EPO) overnight at 4°C. Slides were then treated with the EnVision secondary antibody solution (Dako, 45 min) against rabbit/mouse immunoglobulin followed by a counterstaining with hematoxylin (Sigma-Aldrich). Negative control experiments were performed by omitting primary antibody and by the absence of EPO immunoreactivity in sections showing human kidney epithelium. Instead, specific immunoreactivity in peritubular/interstitial kidney fibroblasts was used as a positive control (see Fig. 1 A).


Figure 1
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  		Figure 1. Normal human skin and hair follicles express EPO protein in situ. EPO immunoreactivity (EnVision technique) is localized to central outer root sheath (B) keratinocytes (ORS) of growing hair follicles and blood vessels (BV) of skin (D). All cell types of hair bulb (C) and epidermis were negative (A). Frozen sections of human kidney (E) served as positive (peritubular interstitial cells, open arrows) and negative (epithelial cells, arrow) control. NC = negative control (by omitting primary antibody); DP = dermal papilla; MK = matrix keratinocytes; HS = hair shaft; scale bars = 50 µM.

For detection of experimentally induced apoptosis, the terminal dUTP nick end-labeling (TUNEL) staining was performed, using murine spleen sections as positive control (47) . Cryostat sections were fixed in paraformaldehyde and ethanol-acetic acid (2:1) and were labeled with digoxigenin-deoxyUTP (ApopTag Fluorescein In Situ Apoptosis detection kit; Intergen, Purchase, NY, USA, 60 min, 37°C). The enzyme reaction was stopped (stop buffer, ApopTag Fluorescein In Situ Apoptosis Detection Kit), and slides were labeled with a FITC-conjugated antidigoxigenin antibody (Apoptosis Kit). Negative controls were performed by omitting TdT enzyme.

For a simultaneous labeling of proliferating and apoptotic cells, the Ki-67/TUNEL double-staining was applied as described earlier (42) . Quantitative immunohistomorphometry was performed as desribed previously (39) : Ki-67, TUNEL, or DAPI+ cells were counted in a previously defined reference region of the HF matrix, and the percentage of Ki-67+/TUNEL+ cells was determined.

Quantitative real-time polymerase chain reaction
Total RNA was extracted from HF cultured under normoxic and hypoxic conditions, respectively, using the RNA easy kit (Qiagen, Hilden, Germany) and was reverse-transcribed using random hexamers as primers and Transcriptor Reverse Transcriptase (Roche, Mannheim, Germany). A specific EPO cDNA fragment of 158 bp covering the region 537–694 according to the most recent accession number of the GenBank DNA databank was amplified by real-time polymerase chain reaction (PCR; 95°C for 10 min; 35 cycles of 95°C for 10 s, 68°C for 10 s, 72°C for 16 s) using undisclosed primer pairs from LC Search (Heidelberg, Germany). SybrGreen was used for staining of the PCR products. The amount of cDNA in all samples was standardized by the amplification of the housekeeping gene GAPDH (Roche, Mannheim, Germany). The specificity of the amplification reactions was assessed by a melting curve analysis. The second derivative maximum analysis of the relative quantification software package, version 1.0 (Roche), was used for a relative quantification of specific EPO PCR products. The PCR products were also visualized using a 0.7% agarose gel with ethidium bromide.

ELISA analysis of secreted EPO in culture medium
EPO in the supernatant of the cultured HFs was analyzed using a commercial sandwich ELISA from R&D Systems (Minneapolis, MN, USA). This ELISA makes use of an immobilized monoclonal murine catching antibody that binds EPO. The second polyclonal (rabbit) detecting antibody is conjugated with horse-radish peroxidase and binds immobilized EPO. Before the analysis, HF culture supernatant was up-concentrated 10-fold using centrifugal Microcon filter unit YM-10 (cut-off 10 kDa; Millipore, Billerica, MA, USA). YM-10 filters were centrifuged at 14,000 g for 10 min at room temperature.

Microarray analysis of selected candidate genes
Gene expression analysis of HFs from two different individuals (both female) using Human Whole Genome Oligo Microarray (44 K) was performed as a commercial service by Miltenyi Biotech GmbH (Cologne, Germany). Twenty HFs per group were treated with vehicle/EPO (100 U/ml) for 6 h, and total RNA was isolated according to standard protocols (Trizol, Sigma-Aldrich). Quality of total RNA was controlled via the Agilent 2100 Bioanalyzer System. Linear amplification of RNA and hybridization of whole genome oligo microarray were performed according to Agilent’s standard protocols. All compared test and control HFs were derived from one defined scalp skin region of the same donor, and the gene expression profiles of two donors were compared independently.

"Differentially regulated" candidate genes were selected according the following rigid selection criteria: only those genes were further evaluated whose transcription had changed >1.5-fold and with a P value of < 0.01, and this in an equidirectional manner (i.e., the transcription was significantly up- and down-regulated, respectively, in both individuals). In a separate analysis, those genes were selected whose transcription was substantially modulated (i.e., >5-fold, P<0.01) in HF RNA extracts of only one of the two examined patients (Table 1 ).


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  		Table 1. EPO administration causes differential gene expression changes


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
First, we asked, whether human scalp skin transcribes and translates EPO. As shown in Fig. 1 , with the use of a specific antibody and human kidney sections as positive and negative control (Fig. 1A ), EPO-like specific immunoreactivity was mainly found in the HF epithelium and in large blood vessels (Fig. 1A-D ): epidermal/follicular melanocytes did not seem to express EPO. The most intensive staining was seen in central outer root sheath keratinocytes (Fig. 1B ).

Moreover, EPO was secreted into the culture medium as demonstrated by ELISA (data not shown). These microdissected human scalp HFs < ~50 cell layers the central ORS/ follicle, 2.5x105 cells/experiment) secreted 2.4 mU/ml EPO protein into the medium after 48 h of organ culture under serum-free conditions (in comparison, the normal EPO concentration in human serum is between 3–17 mU/ml; ref. 48 ).

By quantitative, real-time reverse transcriptase (RT)-PCR, both EPO and EPO-R transcripts were detected in freshly microdissected hair bulbs of human scalp HFs in the growth phase of the hair cycle (anagen VI; ref. 29 ; Fig. 2 A, B). We could also detect transcripts for HIF-1{alpha}, a main mediator of EPO production, in microdissected, organ-cultured human scalp HFs (Fig. 2 ; see Supplemental Fig. S1). Since commercially available anti-EPO-R antibodies cannot reliably detect EPO-R antigen because of their low specificity and affinity (26) , we could not investigate the precise localization of EPO-R protein expression in the HFs.


Figure 2
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  		Figure 2. Detection of EPO, EPO-R, and HIF-1{alpha} mRNA by real-time PCR in extract isolated from microdissected hair follicles. A) Freshly microdissected and cultured HFs (n=30/group) were analyzed by quantitative real-time RT-PCR for EPO, EPO-R, and HIF-1{alpha}. Organ cultured hair follicles up-regulate EPO but not EPO-R and HIF-1{alpha} mRNA under hypoxic conditions (3% O2). *P < 0.05; mean ± SE. B) PCR products for EPO and EPO-R were visualized on a 0.7% agarose gel with ethidium bromide (note that these PCR products are obtained at the very end of the PCR reactions (after 35 cycles). At that time point, concentration differences can not be observed.) M = marker; NOX = normoxia; HOX = hypoxia.

Since renal EPO production is primarily stimulated by hypoxia (1) , we cultured HFs under hypoxic conditions and compared the EPO, EPO-R, and HIF-1{alpha} steady-state transcript levels to the normoxic ones by quantitative real time PCR. Both HIF-1 and EPO-R steady-state transcript levels were unchanged under hypoxic conditions (Fig. 2 ; see Supplemental Fig. S1). However, there was a significant up-regulation of EPO mRNA in hypoxia-treated HFs (Fig. 2 ; Supplemental Fig. S1), suggesting that, just as in the kidney, EPO production in human HFs is inducible by hypoxia.

Next, we investigated how EPO-R activation influences key HF parameters, such as hair shaft elongation or proliferation/apoptosis of human hair matrix keratinocytes in situ under normoxic standard organ-culture conditions. However, HFs treated with EPO for 9 days failed to show significant changes in hair shaft elongation (Fig. 3 ). Also, proliferation/apoptosis of hair matrix keratinocytes under standard culture conditions remained essentially unaltered after EPO administration (Fig. 3) .


Figure 3
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  		Figure 3. EPO does not significantly influence hair shaft elongation and proliferation/apoptosis of matrix keratinocytes in vitro. A) Hair follicles were treated with vehicle/EPO (100 U/ml) and hair shaft length was measured every second day. P > 0.05; mean ± SE. B, C) Cryosections of cultured, vehicle/EPO treated hair follicles were double labeled with Ki-67(red)/TUNEL(green) staining. Ki-67/TUNEL positive cells were counted below Auber's line (indicated in white) and percentage of positive cells was compared between vehicle and EPO-treated follicles. P > 0.05, mean ± SE; scale bars = 50 µm.

In contrast, chemotherapy-induced intrafollicular apoptosis in situ was inhibited by prior and concomitant EPO administration (Fig. 4 ): When organ-cultured human anagen HFs were exposed to a key toxic metabolite of the potent alopecia-inducing cytostatic agent cyclophosphamide [4-hydroperoxy-cyclophosphamide (4-HC); 44 , 45 ], the massive apoptosis induced in the HF epithelium by 4-HC was significantly reduced (from 6.6±2.4 to 3.0±1.3) by EPO (as measured by quantitative immunohistomorphometry of TUNEL+ cells in the hair matrix; ref 39 ; Fig. 4 ). In view of the unavailability of sufficiently specific antibodies for the immunohistological detection of EPO-R protein, this finding also serves as indirect evidence that the detected EPO-R transcripts are translated into functionally active EPO-R.


Figure 4
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  		Figure 4. EPO (100 U/ml) reduces the massive apoptosis induced by the cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC). HFs were incubated overnight without any treatment, the next day (day 1) medium was exchanged and vehicle (culture medium)/EPO (100 U/ml) was added to each well. After 24 h preincubation, HFs were cotreated with key toxic metabolite of the potent alopecia-inducing cytostatic agent 4-HC for 4 days. Apoptotic cells were detected by TUNEL immunostaining (green). EPO reduced number of apoptotic cells after 4-HC treatment. Scale bars = 50 µm; n = 10 HFs/group.

To obtain further evidence of EPO-R functionality and to search for previously unknown target genes for extraerythropoietic EPO-R-mediated signaling, we, finally, subjected two independent sets of organ-cultured human scalp HFs (derived from 2 different, healthy female face lift patients) to DNA microarray after stimulation with EPO (100 U/ml; Fig. 5 ). This was performed as a commercial service (Human Whole Genome Oligo Microarray, Miltenyi), and rigid selection criteria were employed to single-out "differentially expressed" genes.


Figure 5
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  		Figure 5. EPO administration causes differential gene expression changes. HFs of 2 different female donors (HF1 and HF2) were treated with vehicle or EPO (100 U/ml) and microarray analysis (Human Whole Genome Microarray, Miltenyi; expression differences visualized by Agilent technique) was performed. When only genes with equidirectional changes in both individuals were included, where, in addition, P value was <0.01 and fold-changes were >1.5, five differentially regulated genes were identified: hemoglobin alpha-1 (HBA1), kinesin light chain 3 (KLC3), aminase oxidase (copper containing 2, AOC2), calmegin (CLGN), and RAS-like family 10, member B (RASL10B). The genes whose transcription changed substantially (5–27-fold, P<0.01) in only one patient are listed separately in Table 1 .

When only equidirectional expression changes in HF RNA extracts from both individuals with a P value of <0.01, and fold changes of >1.5 were accepted as strong indications for "differential gene expression," only two up-regulated and three down-regulated genes were identified: hemoglobin alpha-1 (HBA1), kinesin light chain 3 (KLC3), aminase oxidase (copper containing 2, AOC2), calmegin (CLGN), and RAS-like family 10, member B (RASL10B), respectively (Fig. 5) . The complete list of genes whose expression changed markedly (5–27-fold) in at least one of two examined patients is shown in Table 1 .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we provide the first evidence that normal human skin expresses EPO and functional EPO-Rs in situ and that human scalp HFs are important extrarenal sources and extrahematopoietic targets of EPO-induced signaling. We show that EPO protein is exclusively produced by outer root sheath keratinocytes of human anagen VI HFs and is secreted into the organ culture medium. In contrast to what has been reported for cultured melanocytes and melanoma cells (24 , 25) , neither epidermal nor follicular melanocytes in normal human skin in situ showed convincing, specific immunoreactivity for EPO protein with the antibody employed here.

As a prototypic neuroectodermal-mesodermal interaction system (29) , human HF organ culture offers itself as an instructive and physiologically relevant, novel research tool for dissecting the intriguing but still unclear extraerythropoietic functions of EPO in peripheral tissues in the human system. Similar to its effect on kidney, liver, and brain (1 , 49) , hypoxia stimulated EPO production in human HFs in situ, suggesting that the latter utilize a classical hypoxia-sensing system employed elsewhere in the human body (7) . Since we also show that HFs transcribe the main "oxygen sensor" HIF-1{alpha}, these cutaneous miniorgans may be able to detect insufficient O2 levels via the oxygen-dependent level of HIF-1{alpha}. This may represent an important mechanism for regulating the metabolic balance of the extremely fast-renewing and proliferating cell population of hair matrix keratinocytes. The regulation of HIF-1{alpha} generally occurs by post-translational modifications; hypoxic cells show an increase in HIF-1{alpha} protein levels, while the HIF-1{alpha} mRNA levels remain unchanged at low pO2 (1 , 50) . We have here shown that, as described elsewhere (1 , 50) , HIF-1{alpha} transcription was not changed by hypoxia in organ cultured HFs at mRNA levels. This suggests that intrafollicular HIF-1{alpha} production is regulated at the post-transcriptional level, as well.

Although the precise role of intrafollicular EPO production for human HF biology remains to be fully explored, due to its potent antiapoptotic properties (3 , 4 , 6) , EPO may serve as an endogenous HF cytoprotectant, similar to what has been reported for keratinocyte growth factor (45) or melatonin (38) . It now deserves to be assessed whether EPO can be exploited clinically as an anti–hair loss agent, for example, in chemotherapy-induced alopecia. Our data provide further evidence for the emerging concept that EPO may play a role as a general protective factor (wound healing, chemotherapy-induced apoptosis) for the skin (2 , 3 , 23 , 45) .

Furthermore, in the light of the previously reported wound healing-promoting effect of EPO (23) , it is intriguing to ask whether HF-derived EPO may also serve to facilitate wound healing. Not the least in view of the fact that HF-derived keratinocytes are a major cell source for reepithelization during wound healing (51) and that the HF connective tissue sheath is a likely source for granulation tissue formation (52) , this possibility deserves systematic further analysis.

As shown by microarray analysis, EPO administration changed the gene expression (hemoglobin alpha-1, kinesin light chain 3, aminase oxidase, calmegin, and RAS-like family 10) program of human HFs. Although the newly identified candidates as EPO-target genes and their functional significance need to be confirmed and clarified in further experiments, our results invite one to speculate on the potential involvement of some of these novel EPO-target genes in skin or hair biology. For example, kinesin molecules are involved in melanosomal movement along melanocyte dendrites (obligate event of the maintenance of the skin color) (53) . In addition, the down-regulation of RAS10B, a new member of the Ras superfamily with tumor suppressor potential (54) , may be related to the antiapoptotic features of EPO. Furthermore, here we provide the first evidence that, beyond expressing the beta globin gene (55) , HFs, unexpectedly, transcribe the alpha chain of hemoglobin and that this transcription is upregulated by EPO.

When the genes whose transcription was markedly modulated in only one of the two examined patients (Table 1) are included as candidate EPO-target genes in human HFs, our results point to several genes coding for enzymes or receptors with relevance to skin neuroendocrinology. One of the most interesting is tryptophan hydroxylase 2 (TPH2). TPH (TPH1) plays a pivotal role in the synthesis of serotonin as catalyzer of the rate-limiting step of the serotonin synthesis in the skin [as reviewed by Slominski et al. (56) ]. TPH and also serotonin and serotonin receptor were described to be expressed in whole human, C57BL/6 mice and hamster skin and several cultured skin cells (HaCaT cells, melanocytes, and melanoma cell lines; refs. 56 57 58 ). Furthermore, serotonin is involved in human skin physiology as regulator of cell proliferation, vasoactive agent and immune modulator (56) . TPH 2 has been described to localize to the central nervous system and not to the skin (56) . Here we provide the first evidence that TRH 2 is translated by human HFs and its expression is inhibited by EPO. This raises the intriguing question of whether, for example, EPO influences the serotoninergic system via modulation of TPH 2 transcription.

Naturally, these analyses do not allow one to conclude whether EPO modulated the intrafollicular expression of these genes directly or indirectly, for example, by changing the secretion or surface expression of molecules, which then implemented the observed differential gene expression. Nevertheless, these data clearly demonstrate that the stimulation of normal, EPO-R-expressing human scalp HFs with EPO elicits defined and reproducible gene expression changes, thus providing further evidence for the functional activity of HF EPO-R. Last but not least, these initial microarray data indicate that human HF organ culture offers an excellent discovery tool for identifying novel EPO target genes in peripheral tissue biology, as a key element in the ongoing endeavor to explore nonclassical EPO functions in the human system.


   ACKNOWLEDGMENTS
 
The authors are grateful to A. Becker for excellent technical assistance. This work was supported in part by a grant from Deutsche Forschungsgemeinschaft (DFG) to R. Paus (Pa 345/11–2).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication March 22, 2007. Accepted for publication April 26, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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