Prolactin and Its Receptor Are Expressed in Murine Hair Follicle Epithelium, Show Hair Cycle-Dependent Expression, and Induce Catagen
2003; Elsevier BV; Volume: 162; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64295-2
ISSN1525-2191
AutoresKerstin Foitzik, Karoline Krause, A. J. Nixon, Christine A. Ford, Ulrich Ohnemus, A.J. Pearson, Ralf Paus,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoHere, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle. Here, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle. Hair follicles are unusual in that they undergo lifelong cycles of growth and regression. Active hair growth (anagen) is accompanied by hair shaft elongation, melanogenesis, and by massive keratinocyte proliferation, whereas hair follicle regression (catagen) is characterized by terminal differentiation and apoptosis, resulting in the resting stage (telogen) and in hair shaft shedding (exogen). The molecular mechanisms that are responsible for this tightly controlled process are still not clear, but in the last decade a large, yet limited number of growth factors, cytokines, neuropeptides, neurotransmitters, and hormones have been shown to play important regulatory roles.1Paus R Cotsarelis G The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (939) Google Scholar, 2Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1091) Google Scholar, 3Cotsarelis G Millar SE Towards a molecular understanding of hair loss and its treatment.Trends Mol Med. 2001; 7: 293-301Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar A particularly intriguing issue in this context is the search for the set of locally generated hormones and neurotrophines that are involved in that growth control4Paus R Botchkarev VA Botchkareva NV Mecklenburg L Luger T Slominski A The skin POMC system (SPS). Leads and lessons from the hair follicle.Ann NY Acad Sci. 1999; 885: 350-363Crossref PubMed Scopus (57) Google Scholar, 5Paus R Muller-Rover S Botchkarev VA Chronobiology of the hair follicle: hunting the “hair cycle clock.”.J Invest Dermatol Symp Proc. 1999; 4: 338-345Abstract Full Text PDF PubMed Scopus (73) Google Scholar beyond the well-recognized effects of locally metabolized steroid hormones.6Hoffmann R Enzymology of the hair follicle.Eur J Dermatol. 2001; 11: 296-300PubMed Google Scholar It has recently been recognized that prolactin (PRL) is expressed in numerous extrapituitary sites (such as the placenta, mammary gland, brain, and lymphocytes7Freeman ME Kanyicska B Lerant A Nagy G Prolactin: structure, function, and regulation of secretion.Physiol Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1765) Google Scholar). Furthermore PRL has an amazingly versatile repertoire of bioregulatory functions beyond lactation, which includes immune response, osmoregulation, angiogenesis, development and hair growth modulation.7Freeman ME Kanyicska B Lerant A Nagy G Prolactin: structure, function, and regulation of secretion.Physiol Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1765) Google Scholar, 8Paus R Does prolactin play a role in skin biology and pathology?.Med Hypotheses. 1991; 36: 33-42Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 9Thomas DG Loudon ASI Jabbour HN Local infusion of prolactin stimulates early localised development of red deer (Cervus elaphus) summer coat.J Endocrinol. 1994; 143: P44Google Scholar, 10Pearson AJ Ashby MG Wildermoth JE Craven AJ Nixon AJ Effect of exogenous prolactin on the hair growth cycle.Exp Dermatol. 1999; 8: 358-360PubMed Google Scholar, 11Martinet 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 On the basis of earlier observations that implicated PRL in the hair growth regulation in diverse species,10Pearson AJ Ashby MG Wildermoth JE Craven AJ Nixon AJ Effect of exogenous prolactin on the hair growth cycle.Exp Dermatol. 1999; 8: 358-360PubMed Google Scholar, 11Martinet 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, 12Duncan MJ Goldman BD Hormonal regulation of the annual pelage color cycle in the Dungarian hamster, Phodopus sungorus II. Role of prolactin.J Exp Zool. 1984; 230: 97-103Crossref PubMed Scopus (116) Google Scholar, 13Rougeot J Allain D Martinet L Photoperiodic and hormonal control of seasonal coat changes in mammals with special reference to sheep and mink.Acta Zoologica Fennica. 1984; 171: 13-18Google Scholar, 14Rose J Oldfield J Stormshak F Apparent role of melatonin and prolactin in initiating winter fur growth in mink.Gen Comp Endocrinol. 1987; 65: 212-215Crossref PubMed Scopus (40) Google Scholar we had previously hypothesized that both intracutaneously generated and systemically delivered PRL might serve as a hair growth modulator.8Paus R Does prolactin play a role in skin biology and pathology?.Med Hypotheses. 1991; 36: 33-42Abstract Full Text PDF PubMed Scopus (39) Google Scholar Subsequently, prolactin receptor (PRLR) knockout mice were shown to have hair cycle abnormalities.15Craven AJ Ormandy CJ Robertson FG Wilkins RJ Kelly PA Nixon AJ Pearson AJ Prolactin signaling 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 In addition, in mammals with seasonally dependent cycles of pelage replacement, the increasing PRL during spring was shown to reactivate telogen follicles and induce anagen.16Dicks P The role of prolactin and melatonin in regulating the timing of spring moult in the cashmere goat.in: Laker JP Allain D Hormonal Control of Fibre Growth and Shedding. European Fine Fibre Network Occasional Publication No. 2. European Fine Fibre Network, Aberdeen1994: 109-127Google Scholar In cashmere goats, PRL and melatonin have been shown to stimulate hair shaft elongation in vitro.17Ibraheem 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 In contrast, Wiltshire sheep show increased PRL levels after experimentally increased photoperiods associated with a short-term inhibitory effect on growing anagen follicles.18Pearson 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, 19Nixon 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 This is consistent with previous observations that shortening of the photoperiod accompanied by reduced PRL plasma levels results in initiation of fiber growth of the winter fur.11Martinet 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, 13Rougeot J Allain D Martinet L Photoperiodic and hormonal control of seasonal coat changes in mammals with special reference to sheep and mink.Acta Zoologica Fennica. 1984; 171: 13-18Google Scholar, 20Allain D Thébault RG Rougeot J Martinet L Biology of fibre growth in mammals producing fine fibre and fur in relation to control by day length: relationship with other seasonal functions.in: Laker JP Allain D Hormonal Control of Fibre Growth and Shedding. European Fine Fibre Network Occasional Publication No. 2. European Fine Fibre Network, Aberdeen1994: 23-40Google Scholar Thus, systemic PRL levels seem to play a dual role during the seasonal dependent hair growth cycle by operating to induce both transitional phases: catagen and proanagen.10Pearson AJ Ashby MG Wildermoth JE Craven AJ Nixon AJ Effect of exogenous prolactin on the hair growth cycle.Exp Dermatol. 1999; 8: 358-360PubMed Google Scholar, 18Pearson 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, 19Nixon 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 In humans with their seasonally independent hair cycles,2Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1091) Google Scholar hyperprolactinemia is associated with androgenetic alopecia, amenorrhea, infertility, and hirsutism.21Moltz L Hormonal diagnosis in so-called androgenetic alopecia in the female.Geburtshilfe und Frauenheilkunde. 1988; 48: 203-214Crossref PubMed Scopus (13) Google Scholar, 22Schmidt JB Hormonal basis of male and female androgenic alopecia: clinical relevance.Skin Pharmacol. 1994; 7: 61-66Crossref PubMed Scopus (31) Google Scholar, 23Schmidt 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 PRL may increase adrenal androgen production, and can attenuate 5-α-reductase activity both in vivo and in vitro thus increasing dihydro testosterone (DTH) synthesis.24Serafini P Lobo RA Prolactin modulates peripheral androgen metabolism.Fertil Steril. 1986; 45: 41-46PubMed Google Scholar However, hair loss may also be a side-effect of treatment with the PRL inhibitor bromocriptine.25Fabre N Montastruc JL Rascol O Alopecia: an adverse effect of bromocriptine.Clin Neuropharmacol. 1993; 16: 266-268Crossref PubMed Scopus (17) Google Scholar, 26Blum I Leiba S Increased hair loss as the side-effect of bromocriptine treatment.N Engl J Med. 1980; 303: 1418PubMed Google Scholar, 27Sinclair RD Banfield CC Dawber RPR Handbook of Diseases of the Hair and Scalp. Blackwell Science, London1999Google Scholar The PRLR is a single-pass membrane-bound protein that belongs to the cytokine receptor family and transduces its signal by binding Janus kinases (JAKs) and activating signal transducers and activators of transcription (Stat) proteins.28Kelly PA Binart N Freemark M Lucas B Goffin V Bouchard B Prolactin receptor signal transduction pathways and actions determined in prolactin receptor knockout mice.Biochem Soc Trans. 2001; 29: 48-52Crossref PubMed Google Scholar, 29Ormandy 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 (49) Google Scholar Several isoforms of PRLR arise from alternative initiation sites of transcription and gene splicing. In mice, one long and three short forms of the PRLR have been described.29Ormandy 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 (49) Google Scholar All four receptors have been shown to bind the ligand, but only the long form of the PRLR is able to transduce a signal via the JAK/Stat pathway.28Kelly PA Binart N Freemark M Lucas B Goffin V Bouchard B Prolactin receptor signal transduction pathways and actions determined in prolactin receptor knockout mice.Biochem Soc Trans. 2001; 29: 48-52Crossref PubMed Google Scholar, 29Ormandy 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 (49) Google Scholar The PRLR is related structurally and functionally to the growth hormone (GH) receptor.30Goffin V Shiverick KT Kelly PA Martial JA Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen, and related proteins in mammals.Endocr Rev. 1996; 17: 385-410PubMed Google Scholar However, murine GH does not bind to lactogen receptors, in contrast to primate GHs.30Goffin V Shiverick KT Kelly PA Martial JA Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen, and related proteins in mammals.Endocr Rev. 1996; 17: 385-410PubMed Google Scholar On the other hand, murine placental lactogens are potent agonists of the PRLR.31MacLeod KR Smith WC Ogren L Talamantes F Recombinant mouse placental lactogen-I binds to lactogen receptors in mouse liver and ovary: partial characterization of the ovarian receptor.Endocrinology. 1989; 125: 2258-2266Crossref PubMed Scopus (34) Google Scholar And although PRL is incapable of binding to the receptors for GH, it has somatotrophic activity in rodents.32Byatt JC Staten NR Salsgiver WJ Kostelc JG Collier RJ Stimulation of food intake and weight gain in mature female rats by bovine prolactin and bovine growth hormone.Am J Physiol. 1993; 264: E986-E992PubMed Google Scholar PRLRs have been shown to be expressed in epidermal keratinocytes in humans,33Poumay 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 wool follicles of sheep,34Choy VJ Nixon AJ Pearson AJ Localisation of receptors for prolactin in ovine skin.J Endocrinol. 1995; 144: 143-151Crossref PubMed Scopus (30) Google Scholar, 35Choy VJ Nixon AJ Pearson AJ Distribution of prolactin receptor immunoreactivity in ovine skin and during the wool follicle growth cycle.J Endocrinol. 1997; 155: 265-275Crossref PubMed Scopus (40) Google Scholar and anagen hair follicles of mice15Craven AJ Ormandy CJ Robertson FG Wilkins RJ Kelly PA Nixon AJ Pearson AJ Prolactin signaling 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 suggesting that PRL operates directly on the skin. Recently, we showed that disruption of the PRLR gene in mice results in hair cycle perturbations and slightly longer and coarser hair.15Craven AJ Ormandy CJ Robertson FG Wilkins RJ Kelly PA Nixon AJ Pearson AJ Prolactin signaling 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 knockout mice exhibit advanced hair replacement cycles. However, PRLR deletion occurs throughout the animal and is accompanied by reduced estrogen and progesterone and elevated PRL blood levels.36Clement-Lacroix P Ormandy C Lepescheux L Ammann P Damotte D Goffin V Bouchard B Amling M Gaillard-Kelly M Binart N Baron R Kelly PA Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice.Endocrinology. 1999; 140: 96-105Crossref PubMed Scopus (139) Google Scholar Hence, it remained unclear whether these hair growth alterations reflect PRLR-mediated signaling in murine hair follicles or whether they are the indirect results of systemic changes in the level of other hormones and cytokines. To further clarify the influence of PRL on hair follicle growth independent of seasonal coat changes and systemic hormone interactions, we investigated the expression of PRL, PRLR, and the PRLR ligand placental lactogen 1 (PL1) during the depilation-induced murine hair cycle by immunohistochemistry and real-time polymerase chain reaction (PCR). We also adopted a functional approach to test the direct effect of PRL on anagen VI hair follicles in murine skin organ culture. Syngenic, female C57BL/6 mice (6 to 9 weeks of age) in the telogen stage of the hair cycle, or pregnant mothers, were purchased from Charles River (Sulzfeld, Germany). The mice were housed in community cages at the animal facilities of the Universitätsklinikum, Hamburg, under a 12-hour light:12 hour dark photoperiod and were fed mouse chow and water ad libitum. Anagen was induced in the back skin of mice in the telogen phase of the hair cycle (identified by their homogeneously pink back skin color) by applying a liquid 1:1 melted wax/rosin mixture under anesthesia as previously described.37Paus R Hofmann U Eichmueller S Czarnetzki BM Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.Br J Dermatol. 1994; 130: 281-289Crossref PubMed Scopus (81) Google Scholar After hardening, the wax/rosin mixture was peeled off the skin, plucking out all telogen hair shafts, which induces the homogeneous development of anagen follicles that are morphologically indistinguishable from spontaneous anagen follicles. At 0, 1, 3, 5, 8, 12, 17, 19, 20, and 25 days after depilation, mice were sacrificed and their back skin was harvested perpendicular to the paravertebral line to obtain longitudinal hair follicle sections. Skin samples were frozen in liquid nitrogen as previously described.38Paus R Muller-Rover S Van Der Veen C Maurer M Eichmuller S Ling G Hofmann U Foitzik K Mecklenburg L Handjiski B A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis.J Invest Dermatol. 1999; 113: 523-532Crossref PubMed Scopus (441) Google Scholar Cryosections from murine back skin (days 0 to 34 of the depilation-induced hair cycle) were fixed in acetone, washed in Tris-buffered saline, and incubated for 20 minutes at room temperature first with avidin, followed by biotin (ABC Kit; Vector Laboratories, Burlingame, CA). The samples were blocked with 10% goat serum and 3% bovine serum albumin for 20 minutes and incubated with rabbit anti-sheep PRL antiserum (AgResearch, Hamilton, New Zealand) 1:700 overnight at 4°C. After three washes in Tris-buffered saline, biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, Hamburg, Germany) 1:200 was applied for 45 minutes. Washes and incubation with Vectastain reagent (ABC kit, Vector Laboratories) 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. Sections from murine pituitary glands were taken as positive control. Incubation of cryosections with preimmune rabbit serum (AgResearch) served as negative control. Cryosections were treated the same way as for the anti-PRL staining. A blocking solution of 10% goat serum and 3% bovine serum albumin was applied overnight at 4°C followed by incubation with rabbit anti-sheep PRLR anti-serum (AgResearch), 1:200 for 1 hour. Biotin-labeled goat anti-rabbit IgG (Jackson ImmunoResearch) 1:200 was used as secondary antibody and AEC+ as substrate. Tissue sections from murine mammary gland and thymus served as a positive control and incubation of murine skin sections with the preimmune serum in place of the primary antibody served as negative control. Total RNA was isolated from 0.2 to 0.5 g of each frozen murine back skin sample by grinding to powder under liquid nitrogen in a freezer mill (SPEX 7700; Glen Creston Ltd., Middlesex, UK), and extracting with TRIzol reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer's instructions. RNA concentration was measured by spectrophotometry at 260 nm and RNA integrity was verified by agarose gel electrophoresis. Expression of PRL and PRLR mRNA in skin was detected by real-time PCR. First strand cDNA was generated from 0.25 μg of each RNA preparation by reverse transcription with the Superscript Preamplification System (Life Technologies, Inc.) using oligo-dT primers according to the manufacturer's instructions. Oligonucleotide primers were designed using Primer Express software (Applied Biosystems, Foster City, CA) for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PRL, PL1, and PRLR long form, and synthesized as custom primers (Life Technologies, Inc.). Sequences of these primer sets are shown in Table 1. PCR reactions in 20-μl volumes were assembled using the SYBR Green PCR Master Mix (Applied Biosystems), containing a passive reference dye to correct for well-to-well variation. Reactions were run on an Applied Biosystems 7700 thermocycler, as prescribed by the manufacturer. PCR consisted of an initial denaturing step at 94°C for 3 minutes, followed by 40 cycles of annealing at 55°C for 45 seconds, 72°C extension for 30 seconds, and 94°C denaturation for 30 seconds. The identities of PCR products were confirmed by DNA sequencing (DNA Sequencing Facility, University of Waikato, Hamilton, New Zealand). The relative concentration of mRNA of the target genes (PRL, PL1, PRLR-long form) was measured as the number of cycles of PCR required to exceed threshold fluorescence, normalized against that of an internal standard gene (GAPDH), according to the quantitation procedures recommended by Applied Biosystems.Table 1PCR PrimersTarget geneSequenceAmplicon size, bpPRL forward5′-CTCTCAGGCCATCTTGGAGAA-3′PRL reverse5′-GGCTGACCCCTGGCTGTT-3′68PL1 forward5′-CTTGAGGTGCCGAGTTGTCTT-3′PL1 reverse5′-GGAAAGCATTACAAGTCTGGTTCA-3′99PRLR long-form forward5′-ATAAAAGGATTTGATACTCATCTGCTAGAG-3′PRLR long-form reverse5′-TGTCATCCACTTCCAAGAACTCC-3′133GAPDH forward5′-TGCACCACCAACTGCTTAG-3′GAPDH reverse5′-GGATGCAGGGATGATGTTC-3′177 Open table in a new tab C57BL/6 mice (6 to 8 weeks old) were depilated as described above. At day 0 after depilation for the anagen development study and at day 16 after depilation for the catagen experiments, 4 -μm punch biopsies from dorsal back skin were prepared that contained only hair follicles in synchronized late anagen VI. Six skin punches per treatment from two different mice (each experiment) were placed on gelatin sponges in 6-well plates containing Dulbecco's modified Eagle medium supplemented with fetal calf serum, l-glutamine and antibiotic/anti-mycotic mixture. The skin samples were cultured for 72 hours at 5% CO2 with the addition of two concentrations (200 and 400 ng/ml) of ovine PRL (Sigma, Chemie, Deisenhofen, Germany) The medium was changed at 0, 24, and 48 hours. Normal PRL levels in mice vary between nonpregnant females (30 to 80 ng/ml), pregnant females (150 to 600 ng/ml), and males (5 to 20 ng/ml).39Sinha YN Molecular size variants of prolactin and growth hormone in mouse serum: strain differences and alterations of concentrations by physiological and pharmacological stimuli.Endocrinology. 1980; 107: 1959-1969Crossref PubMed Scopus (35) Google Scholar, 40Gee DM Flurkey K Mobbs CV Sinha YN Finch CE The regulation of luteinizing hormone and prolactin in C57BL/6J mice: effects of estradiol implant size, duration of ovariectomy, and aging.Endocrinology. 1984; 114: 685-693Crossref PubMed Scopus (37) Google Scholar After culturing, the tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for quantitative histomorphometry. H&E-stained paraffin sections were screened for longitudinal hair follicles. At least 20 follicles per biopsy punch (n = 6) were counted and the hair-cycle stage of each follicle was assessed and classified by morphological criteria and assigned to their respective hair-cycle stages, following our previously published guidelines.41Muller-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 The hair-cycle score was assessed for the catagen induction study and calculated as described.42Maurer 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 Statistical significance was calculated using the Mann-Whitney U-test. To evaluate proliferating cells we used our established protocol for Ki-67 immunohistochemistry.43Lindner G Botchkarev VA Botchkareva NV Ling G van der Veen C Paus R Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1617PubMed Google Scholar, 44Foitzik K Lindner G Mueller-Roever S Maurer M Botchkareva N Botchkarev V Handjiski B Metz M Hibino T Soma T Dotto GP Paus R Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.EMBO J. 2000; 14: 752-760Google Scholar Cryosections from murine skin organ culture were preincubated with 10% goat serum, followed by incubation with rabbit anti-mouse Ki-67 antiserum 1:100 (Dianova, Hamburg, Germany). To detect Ki-67 immunoreactivity rhodamine-conjugated goat anti-rabbit secondary antibody 1:200 (Jackson ImmunoResearch, Hamburg, Germany) was applied. Sections were then counterstained with 4,6-diamidino-2-phenylindole, 1:5000. Negative controls were made by omitting the primary antibody and 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. The number of positive cells for Ki-67 immunoreactivity was counted per hair bulb. At least 20 bulbi per biopsy punch (n = 6 per group) were counted and statistical significance was calculated by the Mann-Whitney U-test. To explore the distribution of PRL protein in the murine hair follicle in relation to the hair cycle, we looked first for PRL expression during the depilation-induced hair cycle. In telogen, PRL-like immunoreactivity was weakly present in ORS keratinocytes (Figure 1A). With the development of the inner root sheath (IRS) during early anagen (anagen III), PRL immunoreactivity could be detected in the inner layer of the proximal ORS and the IRS (Figure 1B). During later anagen stages (IV to VI), PRL staining extended with hair shaft elongation and could be seen in a restricted area that included the inner layer of the pr
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