p75 Neurotrophin Receptor-Mediated Signaling Promotes Human Hair Follicle Regression (Catagen)
2006; Elsevier BV; Volume: 168; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050163
ISSN1525-2191
AutoresEva M.J. Peters, Marit G. Stieglitz, Christiane Liezman, Rupert W. Overall, Motonobu Nakamura, E. Odette Hagen, Burghard F. Klapp, Petra Arck, Ralf Paus,
Tópico(s)Dermatologic Treatments and Research
ResumoNerve growth factor (NGF) and its apoptosis-promoting low-affinity receptor (p75NTR) regulate murine hair cycling. However, it is unknown whether human hair growth is also controlled through p75NTR, its high-affinity ligand pro-NGF, and/or the growth-promoting high-affinity NGF receptor tyrosine kinase A (TrkA). In microdissected human scalp anagen hair bulbs, mRNA for NGF, pro-NGF, p75NTR, and TrkA was transcribed. Immunohistomorphometry and in situ hybridization detected strong NGF and pro-NGF expression in terminally differentiating inner root sheath keratinocytes, whereas TrkA was co-expressed with p75NTR in basal and suprabasal outer root sheath keratinocytes. During spontaneous catagen development of organ-cultured human anagen hair follicles, p75NTR mRNA levels rose, and p75NTR and pro-NGF immunoreactivity increased dramatically in involuting compartments primarily devoid of TrkA expression. Here, TUNEL+ apoptotic cells showed prominent p75NTR expression. Joint pro-NGF/NGF administration inhibited hair shaft elongation and accelerated catagen development in culture, which was antagonized by co-administration of p75NTR-blocking antibodies. In addition, mRNA and protein expression of transforming growth factor-β2 increased early during spontaneous catagen development, and its neutralization blocked pro-NGF/NGF-dependent hair growth inhibition. Our findings suggest that pro-NGF/NGF interacts with transforming growth factor-β2 and p75NTR to terminate anagen in human hair follicles, implying that p75NTR blockade may alleviate hair growth disorders characterized by excessive catagen development. Nerve growth factor (NGF) and its apoptosis-promoting low-affinity receptor (p75NTR) regulate murine hair cycling. However, it is unknown whether human hair growth is also controlled through p75NTR, its high-affinity ligand pro-NGF, and/or the growth-promoting high-affinity NGF receptor tyrosine kinase A (TrkA). In microdissected human scalp anagen hair bulbs, mRNA for NGF, pro-NGF, p75NTR, and TrkA was transcribed. Immunohistomorphometry and in situ hybridization detected strong NGF and pro-NGF expression in terminally differentiating inner root sheath keratinocytes, whereas TrkA was co-expressed with p75NTR in basal and suprabasal outer root sheath keratinocytes. During spontaneous catagen development of organ-cultured human anagen hair follicles, p75NTR mRNA levels rose, and p75NTR and pro-NGF immunoreactivity increased dramatically in involuting compartments primarily devoid of TrkA expression. Here, TUNEL+ apoptotic cells showed prominent p75NTR expression. Joint pro-NGF/NGF administration inhibited hair shaft elongation and accelerated catagen development in culture, which was antagonized by co-administration of p75NTR-blocking antibodies. In addition, mRNA and protein expression of transforming growth factor-β2 increased early during spontaneous catagen development, and its neutralization blocked pro-NGF/NGF-dependent hair growth inhibition. Our findings suggest that pro-NGF/NGF interacts with transforming growth factor-β2 and p75NTR to terminate anagen in human hair follicles, implying that p75NTR blockade may alleviate hair growth disorders characterized by excessive catagen development. Half a century ago, the prototypic nerve growth factor (NGF) was discovered.1Levi-Montalcini R Meyer H Hamburger V In vitro experiments on the effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick embryo.Cancer Res. 1954; 14: 49-57PubMed Google Scholar, 2Levi-Montalcini R Aloe L Alleva A A role for nerve growth factor in nervous, endocrine and immune systems.Prog Neuroendocr Immunol. 1990; 3: 1-10Google Scholar Since then, almost every discipline that took up neurotrophin research unraveled new biological functions of NGF beyond the control of nerve growth.2Levi-Montalcini R Aloe L Alleva A A role for nerve growth factor in nervous, endocrine and immune systems.Prog Neuroendocr Immunol. 1990; 3: 1-10Google Scholar NGF released systemically or locally by glands, keratinocytes, mast cells, and lymphocytes3Di Marco E Marchisio PC Bondanza S Franzi AT Cancedda R De Luca M Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.J Biol Chem. 1991; 266: 21718-21722Abstract Full Text PDF PubMed Google Scholar, 4Reynolds M Alvares D Middleton J Fitzgerald M Neonatally wounded skin induces NGF-independent sensory neurite outgrowth in vitro.Brain Res Dev Brain Res. 1997; 102: 275-283Crossref PubMed Scopus (32) Google Scholar, 5Artuc M Hermes B Steckelings UM Grutzkau A Henz BM Mast cells and their mediators in cutaneous wound healing—active participants or innocent bystanders?.Exp Dermatol. 1999; 8: 1-16Crossref PubMed Scopus (197) Google Scholar is now established to operate as a growth factor for epithelial and mesenchymal tissues,6Botchkarev VA Metz M Botchkareva NV Welker P Lommatzsch M Renz H Paus R Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as “epitheliotrophins” in murine skin.Lab Invest. 1999; 79: 557-572PubMed Google Scholar, 7Pincelli C Marconi A Autocrine nerve growth factor in human keratinocytes.J Dermatol Sci. 2000; 22: 71-79Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Marconi A Terracina M Fila C Franchi J Bonte F Romagnoli G Maurelli R Failla CM Dumas M Pincelli C Expression and function of neurotrophins and their receptors in cultured human keratinocytes.J Invest Dermatol. 2003; 121: 1515-1521Crossref PubMed Scopus (73) Google Scholar, 9Micera A Puxeddu I Aloe L Levi-Schaffer F New insights on the involvement of nerve growth factor in allergic inflammation and fibrosis.Cytokine Growth Factor Rev. 2003; 14: 369-374Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10Kawamoto K Matsuda H Nerve growth factor and wound healing.Prog Brain Res. 2004; 146: 369-384Crossref PubMed Scopus (79) Google Scholar as an immunomodulator,11Bonini S Rasi G Bracci-Laudiero ML Procoli A Aloe L Nerve growth factor: neurotrophin or cytokine?.Int Arch Allergy Immunol. 2003; 131: 80-84Crossref PubMed Scopus (99) Google Scholar, 12Vega JA Garcia-Suarez O Hannestad J Perez-Perez M Germana A Neurotrophins and the immune system.J Anat. 2003; 203: 1-19Crossref PubMed Scopus (202) Google Scholar and as a key mediator of stress responses.13Alleva E Santucci D Psychosocial versus “physical” stress situations in rodents and humans: role of neurotrophins.Physiol Behav. 2001; 73: 313-320Crossref PubMed Scopus (99) Google Scholar, 14Peters EM Handjiski B Kuhlmei A Hagen E Bielas H Braun A Klapp BF Paus R Arck PC Neurogenic inflammation in stress-induced termination of murine hair growth is promoted by nerve growth factor.Am J Pathol. 2004; 165: 259-271Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar The growth-promoting effect of NGF is mediated by its high-affinity tyrosine kinase receptor TrkA.7Pincelli C Marconi A Autocrine nerve growth factor in human keratinocytes.J Dermatol Sci. 2000; 22: 71-79Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Barker PA p75NTR is positively promiscuous: novel partners and new insights.Neuron. 2004; 42: 529-533Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 16Botchkarev VA Botchkareva NV Peters EM Paus R Epithelial growth control by neurotrophins: leads and lessons from the hair follicle.Prog Brain Res. 2004; 146: 493-513Crossref PubMed Scopus (85) Google Scholar However, NGF signaling through TrkA can be enhanced by coupling to the low-affinity pan-neurotrophin receptor p75NTR,17Barker PA p75NTR: a study in contrasts.Cell Death Differ. 1998; 5: 346-356Crossref PubMed Scopus (123) Google Scholar a member of the tumor necrosis factor receptor family. In contrast, stimulation of p75NTR alone has been shown to induce apoptosis in cells lacking Trk expression,15Barker PA p75NTR is positively promiscuous: novel partners and new insights.Neuron. 2004; 42: 529-533Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 17Barker PA p75NTR: a study in contrasts.Cell Death Differ. 1998; 5: 346-356Crossref PubMed Scopus (123) Google Scholar, 18Botchkarev VA Botchkareva NV Albers KM Chen LH Welker P Paus R A role for p75 neurotrophin receptor in the control of apo-ptosis-driven hair follicle regression.FASEB J. 2000; 14: 1931-1942Crossref PubMed Scopus (85) Google Scholar on neurotrophin withdrawal,17Barker PA p75NTR: a study in contrasts.Cell Death Differ. 1998; 5: 346-356Crossref PubMed Scopus (123) Google Scholar or, most recently, on coupling to its newly defined high-affinity ligand, pro-NGF.19Ibanez CF Jekyll-Hyde neurotrophins: the story of pro-NGF.Trends Neurosci. 2002; 25: 284-286Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar The seemingly contradictory functions of NGF in the control of murine hair follicle cycling, a prototypic model of continuous tissue remodeling,16Botchkarev VA Botchkareva NV Peters EM Paus R Epithelial growth control by neurotrophins: leads and lessons from the hair follicle.Prog Brain Res. 2004; 146: 493-513Crossref PubMed Scopus (85) Google Scholar, 20Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1166) Google Scholar, 21Paus R Foitzik K In search of the “hair cycle clock”: a guided tour.Differentiation. 2004; 72: 489-511Crossref PubMed Scopus (244) Google Scholar therefore do not surprise. In this model, p75NTR signaling has been shown to inhibit hair follicle morphogenesis and to stimulate hair follicle regression.18Botchkarev VA Botchkareva NV Albers KM Chen LH Welker P Paus R A role for p75 neurotrophin receptor in the control of apo-ptosis-driven hair follicle regression.FASEB J. 2000; 14: 1931-1942Crossref PubMed Scopus (85) Google Scholar, 22Botchkareva NV Botchkarev VA Chen LH Lindner G Paus R A role for p75 neurotrophin receptor in the control of hair follicle morphogenesis.Dev Biol. 1999; 216: 135-153Crossref PubMed Scopus (55) Google Scholar, 23Botchkarev VA Yaar M Gilchrest BA Paus R p75 neurotrophin receptor antagonist retards apoptosis-driven hair follicle involution (catagen).J Invest Dermatol. 2003; 120: 168-169Crossref PubMed Scopus (18) Google Scholar This model is characterized by the physiologically occurring cyclic remodeling of the back skin pelage hair follicles and their surrounding skin between phases of growth (anagen), regression (catagen), and relative quiescence (telogen).20Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1166) Google Scholar, 21Paus R Foitzik K In search of the “hair cycle clock”: a guided tour.Differentiation. 2004; 72: 489-511Crossref PubMed Scopus (244) Google Scholar, 24Müller-Röver S Handjiski B van der Veen C Eichmüller 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, 25Paus R Cotsarelis G The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (1014) Google Scholar In this highly instructive model, p75NTR signaling was shown to act inhibitory of hair follicle development22Botchkareva NV Botchkarev VA Chen LH Lindner G Paus R A role for p75 neurotrophin receptor in the control of hair follicle morphogenesis.Dev Biol. 1999; 216: 135-153Crossref PubMed Scopus (55) Google Scholar whereas NGF has been shown to promote growth of early anagen hair follicles.26Peters EMJ Botchkarev V Müller-Röver S Gölz G Klapp BF Arck PC Paus R Nerve growth factor regulates hair cycle progression in humans and mice.J Invest Dermatol. 2004; 123: A58Google Scholar In contrast, NGF and p75NTR signaling can induce catagen and catagen-associated apoptosis of hair follicle keratinocytes.18Botchkarev VA Botchkareva NV Albers KM Chen LH Welker P Paus R A role for p75 neurotrophin receptor in the control of apo-ptosis-driven hair follicle regression.FASEB J. 2000; 14: 1931-1942Crossref PubMed Scopus (85) Google Scholar, 23Botchkarev VA Yaar M Gilchrest BA Paus R p75 neurotrophin receptor antagonist retards apoptosis-driven hair follicle involution (catagen).J Invest Dermatol. 2003; 120: 168-169Crossref PubMed Scopus (18) Google Scholar, 27Lindner 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 Also, NGF mediates stress-induced perifollicular inflammation and premature catagen entry in mice.14Peters EM Handjiski B Kuhlmei A Hagen E Bielas H Braun A Klapp BF Paus R Arck PC Neurogenic inflammation in stress-induced termination of murine hair growth is promoted by nerve growth factor.Am J Pathol. 2004; 165: 259-271Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar These murine data raise the expectation that NGF, via TrkA and/or p75NTR signaling, also acts as a potent hair growth modulator in humans. We have, therefore, adapted an organ culture model introduced by Philpott and colleagues28Philpott MP Kealey T Effects of EGF on the morphology and patterns of DNA synthesis in isolated human hair follicles.J Invest Dermatol. 1994; 102: 186-191Abstract Full Text PDF PubMed Google Scholar, 29Philpott 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: 55-72Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 30Philpott M In vitro maintenance of isolated hair follicles: current status and future development.Exp Dermatol. 1999; 8: 317-319PubMed Google Scholar to produce and analyze catagen-like human hair follicle regression. In this model, we have studied the course of neurotrophin and neurotrophin-receptor expression through the human hair cycle with regard to the following questions: 1. Are pro-NGF and NGF able to signal within the human hair follicle? 2. What is the expression pattern of pro-NGF, NGF, TrkA, and p75NTR in human anagen VI scalp hair follicles, and how does this expression pattern change when anagen hair follicles spontaneously undergo catagen transformation in hair follicle organ culture? 3. How do these expression patterns correspond to the intrafollicular foci of keratinocyte apoptosis during catagen development in culture? 4. Can NGF promote catagen development in organ-cultured human scalp skin anagen hair follicles, while p75NTR-blocking antibodies inhibit it? 5. Do NGF effects on human anagen hair follicles involve up-regulation of the key catagen promoter, transforming growth factor-β2 (TGF-β2)? Here, we show that NGF, pro-NGF, p75NTR, and TrkA mRNA are expressed at the transcript and protein levels in human anagen scalp skin hair follicles. The expression patterns dissected here demonstrate NGF/TrkA interactions in proliferating outer root sheath keratinocytes and pro-NGF/p75NTR interactions in the regressing catagen hair bulb corresponding to tissue foci of epithelial cell apoptosis. Pro-NGF/NGF inhibits hair shaft elongation, increases TGF-β2 transcription, and accelerates catagen development of organ-cultured human hair follicles in a p75NTR-dependent manner, with TGF-β2 likely acting downstream of NGF-induced signaling. These findings reveal an important role of the pro-NGF/NGF signaling cascade in terminating the growth of human anagen hair follicles and introduce human scalp hair follicle organ culture as an ideal, easily accessible, and highly instructive model for dissecting the complex, and often seemingly contradictory, functional effects of neurotrophin-induced signaling events in peripheral tissues. Anagen VI hair follicles were obtained from frontotemporal scalp skin and were immediately snap-frozen in liquid nitrogen for immunohistochemistry or whole skin polymerase chain reaction (PCR) analysis or were processed for in situ hybridization or microdissection and hair follicle organ culture as previously described.24Müller-Röver S Handjiski B van der Veen C Eichmüller 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, 29Philpott 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: 55-72Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 30Philpott M In vitro maintenance of isolated hair follicles: current status and future development.Exp Dermatol. 1999; 8: 317-319PubMed Google Scholar, 31Peters EMJ Hansen M Overall RW Nakamura M Pertile P Klapp BF Arck PC Paus R Control of human hair growth by neurotrophins: brain derived neurotrophic factor inhibits hair shaft elongation, induces catagen and stimulates follicular TGFβ2 expression.J Invest Dermatol. 2005; 124: 675-685Crossref PubMed Scopus (57) Google Scholar All samples were collected after obtaining informed consent, following accepted ethical guidelines and according to Helsinki guidelines. Of each sample, some hair follicles were immediately snap-frozen in liquid nitrogen for PCR analysis or snap-frozen embedded in a drop of OCT for histomorphometry. These follicles will be termed native hair follicles or hair follicles on day 0 throughout the remainder of the article. All other hair follicles were processed for organ culture as described below. RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany). Total RNA (0.8 μg) was reverse-transcribed using the First Strand cDNA synthesis kit (Roche, Mannheim, Germany) in an oligo-d(T)15-primed 20-μl reaction. cDNA (1 μl) was amplified by PCR using the Core PCR kit (Qiagen) and the following primers: 5′-TGCATAGCGTAATGTCCATG-3′ and 5′-AAGTCCAGATCCTGAGTGTC-3′ to amplify a 325-bp fragment corresponding to nucleotides 127 to 451 of NGFB mRNA (GenBank: NM_002506), 5′-CCATCGTGAAGAGTGGTCTC-3′ and 5′-GGTGACATTGGCCAGGGTCA-3′ to amplify a 476-bp fragment corresponding to nucleotides 346 to 821 of the TrkA mRNA (GenBank: NM_001007204) as published,32Hibino T Nishiyama T Role of TGF-beta2 in the human hair cycle.J Dermatol Sci. 2004; 35: 9-18Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar and 5′-TGGGCAGGACCTCAGAGTCC-3′ and 5′-TTCCTCCCTCTGAGTCTCTG-3′ to amplify a 283-bp fragment corresponding to nucleotides 1517 to 1799 of p75NTR mRNA (GenBank: NM_002507). The reactions were cycled for 5 minutes at 95°C, then 30 seconds at 95°C, 30 seconds at 60°C, 45 seconds at 72°C for 35 cycles, and finally 5 minutes at 72°C. For analysis of TGF-β2 and p75NTR mRNA during catagen-like hair follicle regression in cultured human anagen hair follicles, hair follicles were snap-frozen immediately after isolation (day 0) or after 4, 8, or 12 days in culture with complete Williams E medium (Table 1). Hair follicles cultured throughout 48 hours in the presence of 50 ng/ml of 7S NGF from mouse submaxillary gland (Sigma, St. Louis, MO) (Table 1) were also snap-frozen and processed as described above.Table 1Hair Follicle Organ-Culture Treatment SchedulesPeptideSpecificationSourceTreatmentNo. of scalp skin specimens from different donorsNone29Philpott 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: 55-72Abstract Full Text PDF PubMed Scopus (119) Google ScholarComplete Williams E hair follicle growth medium as published by Philpott and colleagues29Philpott 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: 55-72Abstract Full Text PDF PubMed Scopus (119) Google ScholarSee Materials and MethodsMedium change every 2nd day. Harvested after 0, 4, 8, or 12 days for PCR and IHC12NGF 7S32Hibino T Nishiyama T Role of TGF-beta2 in the human hair cycle.J Dermatol Sci. 2004; 35: 9-18Abstract Full Text Full Text PDF PubMed Scopus (177) Google ScholarIsolated from mouse submaxillary gland, active in humanSigma, St. Louis, MO5 or 50 ng/ml every other day. Harvested day 2, 4, or 10 for PCR (only day 4) and IHC10 (day 2, n = 1; day 4, n = 2; day 10, n = 7)p75NTR-neutralizing antibody50Monoclonal mouse IgG from ME20.4 hybridoma against human melanoma cell line WM245. recognizes an epitope within the third or fourth cysteine-rich repeats of p75NTRSigma5 or 50 ng/ml every other day. Harvested day 10 for IHC8 (medium control growth = 100%, n = 4; medium control growth = 60%, n = 4)The table gives information on growth factors and neutralizing antibodies used in organ-cultured human scalp skin anagen hair follicles. It also informs on timing of medium change, termination of culture (harvesting), and number of samples derived from different donors. Each donor donated nine hair follicles per experiment. IHC, immunohistochemistry. Open table in a new tab The table gives information on growth factors and neutralizing antibodies used in organ-cultured human scalp skin anagen hair follicles. It also informs on timing of medium change, termination of culture (harvesting), and number of samples derived from different donors. Each donor donated nine hair follicles per experiment. IHC, immunohistochemistry. The assay for the amplification of tumor growth factor β2 (TGF-β2) and p75NTR exploits the 5′nuclease activity of AmpliTaq Platin (Invitrogen, Karlsruhe, Germany) DNA polymerase to cleave a fluorogenic probe designed for pro-NGF/NGF (TipMolBiol, Berlin, Germany) and, to normalize our samples, a fluorogenic probe for the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) was used in real-time PCR. The sets of primers and probes were designed as follows: TGF-β2 probe: sequence 5′-6FAM-ACACGAACCCAAAGGGTACAATGCCAXT–PH-3′, reverse primer 5′-ATTGATTTCAAGAGGGATCTAGGGTG-3′, forward primer 5′-CAGGACCCTGCTGTGCTG-3′; p75NTR probe: sequence 5′-6FAM-CTCGGGCCTCGTGTTCTCCTGCXT–PH-3′, reverse primer 5′-TGGCCTCGTCGGAATACG-3′, forward primer 5′-CTTACGGCTACTACCAGGATGAG-3′; HPRT probe: sequence 5′-6FAM-CAAGCTTGCTGGTGAAAAGGACCCCXT–PH-3′, reverse primer 5′-GTCTGGCTTATATCCAACACTTCGT-3′, forward primer 5′-GGCAGTATAATCCAAAGATGGTCAA-3′. The real-time PCR reactions were developed as previously published33Knackstedt MK Zenclussen AC Hertwig K Hagen E Dudenhausen JW Clark DA Arck PC Th1 cytokines and the prothrombinase fgl2 in stress-triggered and inflammatory abortion.Am J Reprod Immunol. 2003; 49: 210-220Crossref PubMed Scopus (48) Google Scholar except that we used 0.2-μl probe at 20 μmol/L. Each analysis was normalized to HPRT by calculating the difference between the CT for HPRT and the CT for p75NTR or TGF-β2 as ΔCT = CT HPRT − CT p75NTR or TGF-β2. Amount of mRNA is expressed as increase or decrease over native hair follicles = 1. For the amplification of TGF-β2 from pro-NGF/NGF-cultured hair follicles, reactions were performed as 50-ml triplicates in a 96-well microplate format containing 1× SYBR Green Master Mix (PE Biosystems, Foster City, CA), first strand cDNA, and each primer set. Primers were designed using the Perkin Express 1.0 Software (PE Biosystems) and each set of primers are situated in different exons as human TGF-β2 5′-AAAGTGGACGTAGGC AGCAATTA-3′ forward primer, human TGF-β2 5′-GACCAACCGGCGGAAGA-3′ reverse primer, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5′-TGGGTGTGAACCATGAGAAG-3′ forward primer, and human GAPDH 5′-GCTAAGCAGTTGGTGGTGC-3′ reverse primer, respectively. The reaction mixture was subjected to the following thermal cycle conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Analysis of the reactions was performed in an ABI Prism 7700 sequence detection system (PE Biosystems) monitoring after each cycle. The expression of TGF-β2 was normalized with that of GAPDH. Each TaqMan experiment was performed in triplicate. Oligonucleotide probes to p75NTR, TrkA, and pro-NGF/NGF, labeled with fluorescein at both the 3′ and 5′ ends, were designed by and obtained from Biognostik (Göttingen, Germany). Paraffin sections (4 μm thick) were pretreated with proteinase K (Roche) and postfixed with 1% paraformaldehyde in phosphate-buffered saline (pH 7.4). Sections were then prehybridized in hybridization buffer [2× standard saline citrate (0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH 7.0), 500 ng/ml yeast tRNA (Sigma, Taufkirchen, Germany), 150 mmol/L NaCl, 50% formamide]. Hybridization was performed with 1.5-pmol probe in the hybridization buffer. Probes were detected using anti-fluorescein AP FAB fragments (Roche) in a standard NBT/BCIP color reaction. Cryostat sections (8 μm) fixed in acetone (at −20°C, 10 minutes), were preincubated with 10% normal goat serum and then incubated with the primary antisera to pro-NGF, NGF, TrkA, and p75NTR (Table 2) as described.31Peters EMJ Hansen M Overall RW Nakamura M Pertile P Klapp BF Arck PC Paus R Control of human hair growth by neurotrophins: brain derived neurotrophic factor inhibits hair shaft elongation, induces catagen and stimulates follicular TGFβ2 expression.J Invest Dermatol. 2005; 124: 675-685Crossref PubMed Scopus (57) Google Scholar Tetramethyl-rhodamine-isothiocyanate-conjugated F(ab)2 fragments of goat anti-rabbit IgG (Dianova, Hamburg, Germany) served as secondary antibodies. Then sections were stained with 4,6-diamidino-2-phenylindole (DAPI; Boehringer Mannheim, Mannheim, Germany) for identification of cell nuclei.Table 2Antibodies for ImmunofluorescenceAntigenSpecificationSourceDilution/protocolNo. of examined individualsPro-NGFRabbit, polyclonal, raised against murine pro-NGFChemicon, Temecula, CA1:150 Immunofluorescence8NGFRabbit, polyclonal, raised against a peptide mapping to hNGF N- terminusSanta Cruz Biotechnology, Santa Cruz, CA1:50 Immunofluorescence12NGFGoat, polyclonal, raised against rhβ-NGFR&D Systems, Minneapolis, MN1:20 Immunofluorescence8NGFMouse, monoclonal, raised against rhβ-NGFSigma, St. Louis, MOIn our hands no satisfactory immunostaining results3NGFRat, polyclonal, raised against hNGFPromega, Madison, WIIn our hands no satisfactory immunostaining results4TrkARabbit, polyclonal, raised against AA 763-777 within the C-terminus of Trk gp 140Santa Cruz Biotechnology1:75 Immunofluorescence12p75NTRMouse, monoclonal, raised against human melanoma cell line WM245, reacts against the third or fourth cysteine-rich repeatsSigma1:400 Immunofluorescence4p75NTRMouse, monoclonal (production discontinued)Chemicon1:500 Immunofluorescence12p75NTRRabbit, polyclonalChemicon1:1000 TSA7This table lists all antibodies employed for immunohistochemical detection of NGF, pro-NGF, TrkA, and p75NTR. For NGF we found two working antibodies yielding comparable staining results that mapped different epitopes of NGF. For pro-NGF only one antibody has recently been brought on the commercial market. To our knowledge, this is the first report on pro-NGF in human skin with this antibody. For TrkA only antibodies mapping the same epitope were commercially available. Both antibodies were available from Santa Cruz Biotechnology, we therefore chose these for immunofluorescence screening of all available skin samples. Three different antibodies yielding highly comparable results confirmed p75NTR immunoreactivity patterns. Open table in a new tab This table lists all antibodies employed for immunohistochemical detection of NGF, pro-NGF, TrkA, and p75NTR. For NGF we found two working antibodies yielding comparable staining results that mapped different epitopes of NGF. For pro-NGF only one antibody has recently been brought on the commercial market. To our knowledge, this is the first report on pro-NGF in human skin with this antibody. For TrkA only antibodies mapping the same epitope were commercially available. Both antibodies were available from Santa Cruz Biotechnology, we therefore chose these for immunofluorescence screening of all available skin samples. Three different antibodies yielding highly comparable results confirmed p75NTR immunoreactivity patterns. Two types of negative controls were run. Slides were incubated with the secondary antibody alone or with a mixture of the primary antibody and the control peptide for the specific antisera [incubated overnight at 4°C in tubes covered with 1% milk powder in Tris-buffered saline (TBS)] (20 times more concentrated than primary antibody, overnight, room temperature; Santa Cruz Biotechnology, Santa Cruz, CA) after preincubation with 10% normal goat serum. Labeling of nerve fibers and nerve fiber bundles (pro-NGF, NGF, p75NTR) and basal epidermal keratinocytes (TrkA) in full-thickness skin biopsies served as intrinsic positive controls. Stained sections were examined at ×250 magnification under a Zeiss Axioscope 2 microscope with a fluorescence device (Zeiss, Göttingen, Germany). In situ labeling or immunoreactivity (IR) was quantified by histomorphometry in the following compartments: the hair follicle ostium (hair follicle epithelium between epidermis and entry of the sebaceous gland into the hair canal), inner root sheath, outer root sheath, cuticle, dermal papilla, matrix, proximal hair bulb epithelium (below Auber's line), sebaceous gland, epidermis (separated into stratum basale, spinale, and granulosum), arrector pili muscle, sweat gland, blood vessels, nerve fibers, and melanocytes. Staining intensities were documented in arbitrary units as follows: 0 = negative, + = barely visible staining, ++ = apparent staining, +++ = strong staining. For each IR pattern follicular compartments were evaluated in at least four different anagen hair follicles per sample and the extrafollicular compartments in a minimum of 10 microscopic fields. The photodocumentation was done with the help of a modular imaging program (Openl
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