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Human Scalp Hair Follicles Are Both a Target and a Source of Prolactin, which Serves as an Autocrine and/or Paracrine Promoter of Apoptosis-Driven Hair Follicle Regression

2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2006.050468

1525-2191

Kerstin Foitzik, Karoline Krause, Franziska Conrad, Motonobu Nakamura, Wolfang Funk, Ralf Paus,

Skin and Cellular Biology Research

The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyper-prolactinemia. The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyper-prolactinemia. The polypeptide hormone prolactin (PRL) belongs to the PRL/growth hormone/placental lactogen gene family. The PRL gene is 10 kb in size, and transcription of the PRL gene is regulated by two different promoter regions. The proximal 5000-bp region directs pituitary-specific expression, whereas the more upstream promoter region is responsible for extrapituitary expression.1Freeman M Kanycisca B Lerant A Nagy G Prolactin: structure, function and regulation of secretion.Physiol Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1814) Google Scholar PRL has been shown to exert an exceptionally wide variety of bioactivities. Beyond lactation and reproduction, PRL is now recognized to modulate immune responses, osmoregulation, angiogenesis via induction of vascular endothelial growth factor, development, and hair growth.2Goldhar AS Vonderhaar BK Trott JF Hovey RC Prolactin-induced expression of vascular endothelial growth factor via Egr-1.Mol Cell Endocrinol. 2005; 232: 9-19Crossref PubMed Scopus (63) Google Scholar, 3Gootwine E Placental hormones and fetal-placental development.Anim Reprod Sci. 2004; 82–83: 551-566Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 4Soares MJ The prolactin and growth hormone families: pregnancy-specific hormones/cytokines at the maternal-fetal interface.Reprod Biol Endocrinol. 2004; 2: 51Crossref PubMed Scopus (225) Google Scholar, 5Larsen PR Kronenberg HM Melmed S Polonsky KS Williams Textbook of Endocrinology. ed 10. 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Academic Press, London1998Google Scholar, 10Paus R Does prolactin play a role in skin biology and pathology?.Med Hypoth. 1991; 36: 33-42Abstract Full Text PDF PubMed Scopus (39) Google Scholar It has been speculated that the mammotropic actions of PRL in humans may have evolved from more generalized actions of this versatile biomediator on integumental structures in other vertebrates, such as the epidermis of amphibians, the feathers of birds, or hair and sebaceous glands in mammals.11Hadley MC Endocrinology. ed 2. Prentice Hall, Englewood Cliffs1988Google Scholar Released most prominently by the pituitary gland and binding to specific receptors in the skin, PRL has been hypothesized to act as a neuroendocrine modulator of epithelial proliferation and of the skin immune system.9Malley BWO Hormones and Signaling. vol 1. Academic Press, London1998Google Scholar, 12Slominski A Wortsman J Neuroendocrinology of the skin.Endocr Rev. 2000; 5: 457-487Google Scholar The role of PRL in hair growth regulation has been intensely studied in mammals with seasonally dependent cycles of pelage replacements. PRL has been shown to stimulate hair growth, moulting, and shedding in sheep and mink, and contradictory data report of induction of both anagen (hair growth) and catagen (HF regression) in seasonal dependent HFs by PRL.13Martinet L Allain D Weiner C Role of prolactin in the photoperiodic control of moulting in the mink (Mustela vison).J Endocrinol. 1984; 103: 9-15Crossref PubMed Scopus (65) Google Scholar, 14Duncan MJ Goldman BD Hormonal regulation of the annual pelage colour cycle in the Djungarian hamster, Phodopus sungorus. II. Role of prolactin.J Exp Zool. 1984; 203: 97-103Crossref Scopus (116) Google Scholar, 15Rougeot J Allain D Martinet L Photoperiodic and hormonal control of seasonal coat changes in mammals with special reference to sheep and mink.Acta Zool Fennica. 1984; 171: 13-18Google Scholar, 16Ibraheem M Galbraith H Scaife J Ewen S Growth of secondary hair follicles of the cashmere goat in vitro and their response to prolactin and melatonin.J Anat. 1994; 185: 135-142PubMed Google Scholar, 17Alonso LC Rosenfield RL Molecular genetic and endocrine mechanisms of hair growth.Horm Res. 2003; 60: 1-13Crossref PubMed Scopus (46) Google Scholar, 18Puchala R Pierzynowski SG Wuliji T Goetsch AL Soto-Navarro SA Sahlu T Effects of prolactin administered to a perfused area of the skin of Angora goats.J Anim Sci. 2003; 81: 279-284PubMed Google Scholar Although PRL and melatonin stimulate hair shaft elongation in culture in cashmere goats,16Ibraheem M Galbraith H Scaife J Ewen S Growth of secondary hair follicles of the cashmere goat in vitro and their response to prolactin and melatonin.J Anat. 1994; 185: 135-142PubMed Google Scholar increased levels of PRL after experimentally increased photoperiods have been shown to decrease hair growth in vitro.7Nixon AJ Ford CA Wildermoth JE Craven AJ Pearson AJ Regulation of prolactin receptor expression in ovine skin in relation to circulating prolactin and wool follicle growth status.J Endocrinol. 2002; 172: 605-614Crossref PubMed Scopus (55) Google Scholar, 19Pearson AJ Parry AL Ashby MG Choy VJ Wildermoth JE Craven AJ Inhibitory effect of increased photoperiod on wool follicle growth.J Endocrinol. 1996; 148: 157-166Crossref PubMed Scopus (40) Google Scholar Increasing PRL levels in spring was even shown to reactivate telogen HFs and induce anagen in cashmere goats.20Dicks P The role of prolactin and melatonin in regulating the timing of the spring moult in the cashmere goat.Eur Fine Fibre Netw Occasional Publ. 1994; 2: 109-125Google Scholar PRL likely also plays a role in seasonally independent hair cycles, as they are characteristic for mice and man.21Stenn K Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1110) Google Scholar We have recently demonstrated that PRL and its receptor are expressed in a hair cycle-dependent manner in HF keratinocytes in mice in vivo. Treatment of murine anagen HFs with PRL leads to HF regression (catagen) accompanied by decreased proliferation in murine skin organ culture.22Foitzik K Krause K Nixon AJ Ford CA Ohnemus U Pearson AJ Paus R Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen.Am J Pathol. 2003; 162: 1611-1621Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Also, disruption of the PRL receptor (PRL-R) gene in mice results in hair cycle perturbations: PRL-R knockout mice show premature fur molting and premature entry of their HFs into the next hair cycle.23Craven A Ormandy C Robertson F Kelly P Nixon A Pearson A Prolactin signalling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice.Endocrinology. 2001; 142: 2533-2539Crossref PubMed Scopus (63) Google Scholar The role of PRL in human hair growth control is still unclear. Hyperprolactinemia is accompanied by an androgenetic alopecia-like hair loss pattern, amenorrhea, infertility, acne vulgaris, and hirsutism.24Moltz L Hormonal diagnosis in so-called androgenetic alopecia in the female.Geburtshilfe Frauenheilkd. 1988; 48: 203-214Crossref PubMed Scopus (13) Google Scholar, 25Schmidt JB Hormonal basis of male and female androgenic alopecia: clinical relevance.Skin Pharmacol. 1994; 7: 61-66Crossref PubMed Scopus (33) Google Scholar, 26Schmidt JB Lindmaier A Trenz A Schurz B Spona J Hormone studies in females with androgenic hair loss.Gynecol Obstet Invest. 1991; 31: 235-239Crossref PubMed Scopus (46) Google Scholar This may be related to the fact that PRL can increase adrenal androgen production, although it can attenuate 5-α-reductase activity both in vivo and in vitro.27Serafini P Lobo RA Prolactin modulates peripheral androgen metabolism.Fertil Steril. 1986; 45: 41-46PubMed Google Scholar However, in men presenting premature balding before the age of 30, a recent study has reported subnormal PRL serum levels.28Starka L Cermakova I Duskova M Hill M Dolezal M Polacek V Hormonal profile of men with premature balding.Exp Clin Endocrinol Diabetes. 2004; 112: 24-28Crossref PubMed Scopus (24) Google Scholar In women, hair loss (telogen effluvium) may also be seen as a side-effect of treatment with bromocriptine, a dopaminergic inhibitor of pituitary PRL secretion.29Fabre N Montastruc JL Rascol O Alopecia: an adverse effect of bromocriptine.Clin Neuropharmacol. 1993; 16: 266-268Crossref PubMed Scopus (17) Google Scholar, 30Blum I Leiba S Increased hair loss as the side-effect of bromocriptine treatment.N Engl J Med. 1980; 303: 1418PubMed Google Scholar, 31Sinclair RD Banfield CC Dawber RPR Handbook of Diseases of the Hair and Scalp. Blackwell Science, Oxford1999Google Scholar A particularly intriguing issue is from where the ligands arise that stimulate cutaneous PRL-Rs. It is now recognized that, besides the pituitary gland, a number of extrapituitary tissues (such as placenta, uterus, mammary gland, brain, and lymphocytes) can synthesize PRL.1Freeman M Kanycisca B Lerant A Nagy G Prolactin: structure, function and regulation of secretion.Physiol Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1814) Google Scholar, 32Harris J Stanford PM Oakes SR Ormandy CJ Prolactin and the prolactin receptor: new targets of an old hormone.Ann Med. 2004; 36: 414-425Crossref PubMed Scopus (64) Google Scholar The PRL receptor (PRL-R) is a single-pass membrane-bound protein that belongs to the cytokine receptor family and transduces its signal by binding Janus kinases (JAKs) and by activating signal transducers and activators of transcription (Stat) proteins. Crosslinking of PRL-Rs by PRL brings the JAK2 that is bound to the cytoplasmic tail of each PRL-R together, leading to PRL-R phosphorylation. Subsequently, this activation of the PRL-R by JAK2 turns the receptor into a receptor tyrosine kinase, which phosphorylates inactive STATs and causes them to dimerize and translocate as activated transcription factors into the nucleus, where they bind to specific DNA regions and stimulate transcription of PRL target genes.32Harris J Stanford PM Oakes SR Ormandy CJ Prolactin and the prolactin receptor: new targets of an old hormone.Ann Med. 2004; 36: 414-425Crossref PubMed Scopus (64) Google Scholar, 33Kelly PA Binart N Freemark M Lucas B Goffin V Bouchard B Prolactin receptor signal transduction pathways and actions determined in prolactin receptor knock-out mice.Biochem Soc Trans. 2001; 29: 48-52Crossref PubMed Google Scholar, 34Ormandy CJ Binart N Helloco C Kelly PA Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3′-exon splicing.DNA Cell Biol. 1998; 17: 761-770Crossref PubMed Scopus (50) Google Scholar In addition, the biological activity of PRL is triggered via a hormone-induced receptor homodimerization process that is regulated by tertiary features of the hormone. This feature plays an important role in the regulation of these systems by producing binding surfaces with dramatically different binding affinities to the receptor.35Kossiakoff AA The structural basis for biological signaling, regulation, and specificity in the growth hormone-prolactin system of hormones and receptors.Adv Protein Chem. 2004; 68: 147-169Crossref PubMed Scopus (36) Google Scholar Such PRL-R are expressed by human epidermal keratinocytes in vitro36Poumay Y Jolivet G Pittelkow MR Herphelin F De Potter IY Mitev V Houdebine LM Human epidermal keratinocytes upregulate expression of the prolactin receptor after the onset of terminal differentiation, but do not respond to prolactin.Arch Biochem Biophys. 1999; 364: 247-253Crossref PubMed Scopus (22) Google Scholar; in the dermal papilla, matrix, outer root sheath, lower regions of the inner root sheath, and connective tissue sheath in wool follicles of sheep37Choy VJ Nixon J Pearson AJ Distribution of PRL receptor immunoreactivity in ovine skin and changes during the wool follicle growth cycle.J Endocrinol. 1997; 155: 265-275Crossref PubMed Scopus (42) Google Scholar, 38Choy VJ, Wildermoth JE, Nixon AJ, Pearson AJ: Localisation of target tissues in ovine skin. Proc Endocr Soc Aust 1994, pp NZ12Google Scholar; and in the outer root sheath of anagen HFs of mice.22Foitzik K Krause K Nixon AJ Ford CA Ohnemus U Pearson AJ Paus R Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen.Am J Pathol. 2003; 162: 1611-1621Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 23Craven A Ormandy C Robertson F Kelly P Nixon A Pearson A Prolactin signalling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice.Endocrinology. 2001; 142: 2533-2539Crossref PubMed Scopus (63) Google Scholar This expression pattern suggests that PRL can directly alter skin and HF functions by targeting cognate, locally expressed receptors. In human skin, PRL mRNA has been reported to be expressed in and released by dermal fibroblasts and sweat glands in vitro.39Richards RG Hartman SM Human dermal fibroblast cells express prolactin.J Invest Dermatol. 1996; 106: 1250-1255Crossref PubMed Scopus (42) Google Scholar Another group found PRL-R to be expressed in differentiated human keratinocytes in vitro.36Poumay Y Jolivet G Pittelkow MR Herphelin F De Potter IY Mitev V Houdebine LM Human epidermal keratinocytes upregulate expression of the prolactin receptor after the onset of terminal differentiation, but do not respond to prolactin.Arch Biochem Biophys. 1999; 364: 247-253Crossref PubMed Scopus (22) Google Scholar However, it has been recently reported that PRL RNA cannot be detected in truncal skin by reverse transcriptase (RT)-polymerase chain reaction (PCR).40Slominski A Malarkey WB Wortsman J Asa SL Carlson A Human skin expresses growth hormone but not the prolactin gene.J Lab Clin Med. 2000; 6: 476-481Abstract Full Text Full Text PDF Scopus (38) Google Scholar In this study we, therefore, wished to clarify further the role of PRL in human skin, with emphasis on its role in human hair growth. We investigated by immunohistology where exactly PRL and its receptor are expressed in HFs and by RT-PCR whether PRL is even synthesized in HFs. In addition, we wanted to know whether PRL is able to modulate human hair growth in vitro and whether it shows any influence on follicular apoptosis and proliferation. We show for the first time that PRL mRNA and protein are expressed in human skin and isolated organ-cultured HFs. In addition, we show that PRL induces premature catagen in isolated anagen scalp HFs. These data support the hypothesis that PRL is locally produced in the skin and acts directly as a hormonal regulator of HF regression in human anagen scalp HFs, possibly as a cutaneous response to stress or as a part in the pathogenesis of androgenetic alopecia in females. Williams E medium (Life Technologies, Inc., Rockville, MD) was supplemented with l-glutamine, penicillin, and streptomycin. Human recombinant PRL was purchased from R&D Systems (Minneapolis, MN). Goat anti-human PRL antibody was obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA) and sheep anti-human PRL-R from DFC, Biermann GmbH (Bad Nauheim, Germany). Cryosections from isolated human HFs were fixed in acetone, washed in Tris-buffered saline, and incubated with 3% H2O2, followed by avidin and biotin application. Additionally, cryosections from full-thickness human scalp skin were treated the same way to look for PRL protein expression in the skin and without the wounding trauma of microdissected HFs. The samples were blocked with 10% donkey serum and 3% bovine serum albumin for 20 minutes and incubated with goat anti-human PRL antibody (1:100, Santa Cruz Biotechnology) overnight at 4°C (polyclonal goat antibody raised against a peptide mapping near the carboxy terminus of PRL of human origin). After further washing biotin-marked donkey anti-goat secondary antibody (1:200: Jackson ImmunoResearch, Hamburg, Germany) was applied for 45 minutes. Washes and incubation with ABC-Kit (Vector Laboratories, Burlingame, CA) for 30 minutes followed. AEC+ was used as substrate (DAKO, Hamburg, Germany), and sections were counterstained with hematoxylin and mounted using Kaiser's glycerol gelatin. Human pituitary gland sections were used as positive controls. Sections without primary antibody served as negative controls. For detecting PRL-R, cryosections were treated the same way as for the anti-PRL staining. Blocking solution of 10% rabbit serum and 3% bovine serum albumin were applied for 20 minutes, followed by incubation of the primary antibody sheep anti-human PRL-R (1:100; DFC, Biermann) overnight at 4°C. Biotin-marked rabbit anti-sheep IgG (1:200, Jackson ImmunoResearch) was used as secondary antibody and incubated for 45 minutes at room temperature. AEC+ (DAKO) was used as substrate. Human mammary gland sections served as positive control. Sections omitting the primary antibody served as negative controls. Excess anagen HFs from occipital human scalp skin, obtained with informed consent during routine hair transplant or face-lift surgery, were isolated and cultured within 24 hours after surgery as previously described by Philpott and colleagues.41Philpott MP Sanders D Westgate GE Kealey T Human hair growth in vitro: a model for the study of hair follicle biology.J Dermatol Sci. 1994; 7: S55-S72Abstract Full Text PDF PubMed Scopus (115) Google Scholar The total number of organ-cultured HFs in anagen VI stage was 180, derived from 12 different individuals 25 to 55 years of age. After separation of epidermis and dermis from subcutaneous fat under a binocular dissecting microscope, anagen HFs were isolated from the subcutis by using watchmaker's forceps. HFs were then cultured under serum-free conditions in a 24-well plate containing 500 μl of Williams E medium supplemented with insulin, l-glutamine, hydrocortisone, streptomycin, and penicillin. Three follicles per well were incubated for 8 days at 5% CO2 with addition of 400 ng/ml of human recombinant PRL (R&D Systems). Medium was changed every second day. Cultured HFs without PRL served as vehicle controls. After 4 days in culture, human HFs were washed in phosphate-buffered saline and embedded in OCT for cryosectioning. Normal PRL levels in humans vary between nonpregnant females (30 to 80 ng/ml), pregnant females (150 to 600 ng/ml), and males (5 to 20 ng/ml).5Larsen PR Kronenberg HM Melmed S Polonsky KS Williams Textbook of Endocrinology. ed 10. Saunders, Philadelphia2003Google Scholar, 8Felig P Frohmann L Endocrinology and Metabolism. ed 4. McGraw-Hill, Columbus2001Google Scholar, 42Hadley ME Endocrinology. ed 4. Simon and Schuster, Upper Saddle River1996Google Scholar Hair shaft length was measured every second day using a binocular dissecting microscope and hair-cycle stages were assessed according to defined morphological criteria and photodocumented by light microscopy. Longitudinally cut HFs (n = 60/group) were counted, and the hair-cycle stage of each HF was assessed according to defined morphological criteria, classified by morphological criteria, and assigned to their respective hair-cycle stages, following quantitative hair-cycle histomorphometry techniques described for murine catagen development.43Muller-Rover S Handjiski B van-der-Veen C Eichmuller S Foitzik K McKay IA Stenn KS Paus R A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Crossref PubMed Google Scholar, 44Kligman AM The human hair cycle.J Invest Dermatol. 1959; 5: 307-316Crossref Scopus (243) Google Scholar The hair-cycle score (HCS) was assessed and calculated as described.45Maurer M Handjiski B Paus R Hair growth modulation by topical immunophilin ligands: induction of anagen, inhibition of massive catagen development, and relative protection from chemotherapy-induced alopecia.Am J Pathol. 1997; 150: 1433-1441PubMed Google Scholar, 46Foitzik K Spexard T Nakamura M Halsner U Paus R Towards dissecting the pathogenesis of retinoid-induced hair loss: all-trans retinoic acid induces premature hair follicle regression (catagen) by upregulation of TGF-β2 in the dermal papilla.J Invest Dermatol. 2005; 124: 1119-1126Crossref PubMed Scopus (77) Google Scholar The data of all experiments were pooled and statistical analysis was calculated by Mann-Whitney U-test for unpaired samples. To demonstrate proliferating and apoptotic cells at the same time, we combined the established protocols for Ki-67 (Dianova, Hamburg, Germany) and TUNEL (Apop-tag; Oncor Appligene, Heidelberg, Germany) immunohistochemistry.46Foitzik K Spexard T Nakamura M Halsner U Paus R Towards dissecting the pathogenesis of retinoid-induced hair loss: all-trans retinoic acid induces premature hair follicle regression (catagen) by upregulation of TGF-β2 in the dermal papilla.J Invest Dermatol. 2005; 124: 1119-1126Crossref PubMed Scopus (77) Google Scholar, 47Lindner G Botchkarev VA Botchkareva NV Ling G van der Veen C Paus R Analysis of apoptosis during hair follicle regression.Am J Pathol. 1997; 1: 1601-1617Google Scholar, 48Foitzik K Lindner G Müller-Röver S Maurer M Botchkareva N Botchkarev V Handjiski B Metz M Hibino T Soma T Dotto G Paus R Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.FASEB J. 2000; 14: 752-760Crossref PubMed Scopus (270) Google Scholar Briefly, 5-μm cryosections of human HFs were air-dried, fixed in 1% paraformaldehyde, and postfixed in an ethanol:acetic acid mixture (2:1) at −20°C. After incubation with TdT enzyme for 1 hour at 37°C, TUNEL-positive cells were visualized by an anti-digoxigenin fluorescein antibody. Subsequently, tissue sections were preincubated with 10% goat serum, followed by an application of mouse anti-human Ki-67 antiserum (1:20, Dianova). To detect Ki-67 immunoreactivity, rhodamine-conjugated goat anti-mouse secondary antibody (1:200, Jackson ImmunoResearch) was used. Sections were then counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1:5000, Hoechst 33342). Negative controls for the TUNEL staining were made by omitting TdT enzyme, and murine spleen sections served as positive control. For Ki67, positive controls were run by comparison with tissue sections from the back skin of mice in anagen VI stage of the depilation-induced hair cycle. Sections were examined under a Zeiss Axioscope microscope, using the appropriate excitation-emission filter systems for studying the fluorescence induced by DAPI, fluorescein, and rhodamine. The number of cells positive for Ki-67 and TUNEL immunoreactivity was counted per hair bulb and statistical significance was calculated by Mann-Whitney U-test for unpaired samples. Total RNA was isolated from ∼1 g of each frozen scalp skin by grinding to powder under liquid nitrogen in a freezer mill (SPEX 7700; Glen Creston Ltd., Middlesex, UK) and extraction with TRIzol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. RNA concentration was measured by spectrophotometry at 260 nm, and RNA integrity was verified by Northern blotting. A surgically obtained full-thickness human scalp skin, 30 freshly isolated human HFs and human pituitary gland (positive control) were snap-frozen in liquid nitrogen and homogenized using an electronic homogenizer. Total RNA was isolated with an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cDNA was synthesized by reverse transcription of 1 μg of total RNA using First Strand cDNA synthesis kit for RT-PCR (AMV) (Roche, Mannheim, Germany). The following sets of oligonucleotide primers were used: hPRL forward, 5′-CCC TTG CCC ATC TGT CCC GGC G-3′; hPRL reverse, 5′-ATC GCA ATA TGC TGA CTA TCA G-3′; 5-GAPDH, 5′-TGGGTGTGAACCATGAGAAG-3′; 3-GAPDH: 5′-GCTAAGCAGTTGGTGGTGC-3′. Primers for PRL are located in different exons according to the reported sequences in GenBank (accession number, NM000948). Amplification was performed using PCR core kit (Qiagen) for more than 40 cycles using an automated thermal cycler (Biometra, Göttingen, Germany). Each cycle consisted of denaturing at 94°C (30 seconds), annealing at 63°C (1 minute), and extension at 72°C (1 minute). PCR conditions for GAPDH (GenBank accession number, AY340484.1), which gave a 168-bp PCR product, involved 94°C (5 minutes); 28 cycles of 94°C (1minute), 58°C (1 minute), and 72°C (1 minute); and 72°C (10 minutes). PCR products were analyzed by agarose gel electrophoresis. PCR fragment identity was verified with digestion with restriction enzyme EcoRI and NcoI (Roche), which gave expected sized digested fragments through gel electrophoresis (data not shown). In isolated human anagen VI HFs, PRL protein was expressed in a thin layer of keratinocytes between inner and outer root sheath. Matrix keratinocytes and dermal papilla were negative (Figure 1A). Prolactin receptor (PRL-R)-like immunoreactivity was detected in the outer root sheath and additionally in the proximal inner root sheath and matrix keratinocytes. No immunoreactivity could be observed in the dermal papilla (Figure 1B). The positive and negative controls confirmed the sensitivity and specificity of these immunostaining results. Sections from full-thickness human scalp skin showed a similar expression pattern for PRL and PRL-R as in microdissected, organ-cultured human HFs. Besides the HF immunoreactivity, PRL and PRL-R were seen in the epidermis and arrector pili muscle. The capsule of the sebaceous gland stained positive for PRL-R, while the immunoreactivity of the receptor could be detected in the whole sebaceous gland (data not shown). Interestingly, in catagen III HFs, the immunoreactivity for PRL and its receptor was more intense than in anagen VI HFs. PRL and PRL-R-like protein could be detected in keratinocytes of the outer root sheath and matrix, while the inner root sheath and dermal papilla were negative (Figure 1, C and D) in contrast to anagen VI HFs, where the proximal inner root sheath was positive (Figure 1, A and B). Although this could not be further quantitated, this suggests a slight up-regulation of intrafollicular PRL/PRL-R signaling during the anagen-catagen transformation of human scalp HFs. In addition, we investigated whether PRL directly exerts growth-modulating effects on human HFs. PRL was added to the microdissected hair bulbs of organ-cultured human anagen VI HFs. In the current study, we cultured human anagen VI HFs from male occipital scalp skin up to 6 days and observed a hair shaft elongation of ∼0.3 mm per day during this time (Figure 1, Figure 2). Human HFs treated with 400 ng/ml of PRL every other day showed a significantly reduced hair shaft