Control of Human Hair Growth by Neurotrophins: Brain-Derived Neurotrophic Factor Inhibits Hair Shaft Elongation, Induces Catagen, and Stimulates Follicular Transforming Growth Factor β2 Expression
2005; Elsevier BV; Volume: 124; Issue: 4 Linguagem: Inglês
10.1111/j.0022-202x.2005.23648.x
ISSN1523-1747
AutoresEva M.J. Peters, Marit G. Hansen, Rupert W. Overall, Motonobu Nakamura, Paolo Pertile, Burghard F. Klapp, Petra Arck, Ralf Paus,
Tópico(s)Skin and Cellular Biology Research
ResumoNeurotrophins are important modulators of epithelial–mesenchymal interactions. Previously, we had shown that brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tyrosine kinase B (TrkB) are prominently involved in the control of murine hair follicle cycling. We now show that BDNF and TrkB are also expressed in the human hair follicle in a manner that is both hair cycle dependent and suggestive of epithelial–mesenchymal cross-talk between BDNF-secreting dermal papilla fibroblasts of anagen hair follicles and subpopulations of TrkB+ hair follicle keratinocytes. As functional evidence for an involvement of BDNF/TrkB in human hair growth control, we show in organ-cultured human anagen hair follicles that 50 ng per mL BDNF significantly inhibit hair shaft elongation, induce premature catagen development, and inhibit keratinocyte proliferation. Quantitative real-time rtPCR analysis demonstrates upregulation of the potent catagen inducer, transforming growth factor β2 (TGFβ2) by BDNF, whereas catagen induction by BDNF was partially reversible through co-administration of TGFβ-neutralizing antibody. This suggests that TrkB-mediated signaling promotes the switch between anagen and catagen at least in part via upregulation of TGFβ2. Thus, human scalp hair follicles are both a source and target of bioregulation by BDNF, which invites to target TrkB-mediated signaling for therapeutic hair growth modulation. Neurotrophins are important modulators of epithelial–mesenchymal interactions. Previously, we had shown that brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tyrosine kinase B (TrkB) are prominently involved in the control of murine hair follicle cycling. We now show that BDNF and TrkB are also expressed in the human hair follicle in a manner that is both hair cycle dependent and suggestive of epithelial–mesenchymal cross-talk between BDNF-secreting dermal papilla fibroblasts of anagen hair follicles and subpopulations of TrkB+ hair follicle keratinocytes. As functional evidence for an involvement of BDNF/TrkB in human hair growth control, we show in organ-cultured human anagen hair follicles that 50 ng per mL BDNF significantly inhibit hair shaft elongation, induce premature catagen development, and inhibit keratinocyte proliferation. Quantitative real-time rtPCR analysis demonstrates upregulation of the potent catagen inducer, transforming growth factor β2 (TGFβ2) by BDNF, whereas catagen induction by BDNF was partially reversible through co-administration of TGFβ-neutralizing antibody. This suggests that TrkB-mediated signaling promotes the switch between anagen and catagen at least in part via upregulation of TGFβ2. Thus, human scalp hair follicles are both a source and target of bioregulation by BDNF, which invites to target TrkB-mediated signaling for therapeutic hair growth modulation. brain-derived neurotrophic factor immunoreactivity recombinant human BDNF transforming growth factor β2 tyrosine kinase B terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin Nick end labeling Since its discovery more than 20 y ago (Barde et al., 1982Barde Y.A. Edgar D. Thoenen H. Purification of a new neurotrophic factor from mammalian brain.EMBO J. 1982; 1: 549-553Crossref PubMed Scopus (1308) Google Scholar), the neurotrophin brain-derived neurotrophic factor (BDNF), the second member of the nerve growth factor family of growth factors, has become known to guide and sustain sensory and autonomic neuron development and their differentiation within peripheral neural networks (Barde et al., 1982Barde Y.A. Edgar D. Thoenen H. Purification of a new neurotrophic factor from mammalian brain.EMBO J. 1982; 1: 549-553Crossref PubMed Scopus (1308) Google Scholar; Botchkarev et al., 1998bBotchkarev V.A. Botchkareva N.V. Lommatzsch M. et al.BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin.Eur J Neurosci. 1998; 10: 3276-3283Crossref PubMed Scopus (24) Google Scholar; Ernfors, 2001Ernfors P. Local and target-derived actions of neurotrophins during peripheral nervous system development.Cell Mol Life Sci. 2001; 58: 1036-1044Crossref PubMed Scopus (66) Google Scholar; Metsis, 2001Metsis M. Genes for neurotrophic factors and their receptors: Structure and regulation.Cell Mol Life Sci. 2001; 58: 1014-1020Crossref PubMed Scopus (12) Google Scholar; Farinas et al., 2002Farinas I. Cano-Jaimez M. Bellmunt E. Soriano M. Regulation of neurogenesis by neurotrophins in developing spinal sensory ganglia.Brain Res Bull. 2002; 57: 809-816Crossref PubMed Scopus (46) Google Scholar). BDNF is produced by peripheral innervation targets such as visceral epithelia (Lommatzsch et al., 1999Lommatzsch M. Braun A. Mannsfeldt A. et al.Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived neurotrophic functions.Am J Pathol. 1999; 155: 1183-1193Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and skin including the murine and rat hair follicle (Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar,Botchkarev et al., 1999bBotchkarev V.A. Metz M. Botchkareva N.V. et al.Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as "eliotrophins" in murine skin.Lab Invest. 1999; 79: 557-572PubMed Google Scholar; Bergman et al., 2000Bergman E. Ulfhake B. Fundin B.T. Regulation of NGF-family ligands and receptors in adulthood and senescence: Correlation to degenerative and regenerative changes in cutaneous innervation.Eur J Neurosci. 2000; 12: 2694-2706Crossref PubMed Scopus (35) Google Scholar; Peters et al., 2003Peters E.M.J. Botchkarev V.A. Paus R. Neurotrophins act as regulators of hair follicle morphogenesis and cycling.in: Van N.D. Hair Science and Technology. Skinterface, Tournai, Belgium2003Google Scholar) and cutaneous nerve fibers express the high-affinity receptor for BDNF tyrosine kinase B (TrkB) in mice (Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar) and humans (Peters et al., 2003Peters E.M.J. Botchkarev V.A. Paus R. Neurotrophins act as regulators of hair follicle morphogenesis and cycling.in: Van N.D. Hair Science and Technology. Skinterface, Tournai, Belgium2003Google Scholar). As a result of more recent research efforts, neurotrophins such as BDNF have also been shown to hold growth- and immunmodulatory capacities far beyond their sole engagement as neurotrophins that was previously assigned to them. They evolve as potent neuro-immunmodulators, non-neuronal growth factors, and even as stress mediators in various tissues, transgressing their role in the regulation of nerve damage repair and peripheral neuronal plasticity (Alleva and Santucci, 2001Alleva E. Santucci D. Psychosocial versus "physical" stress situations in rodents and humans: Role of neurotrophins.Physiol Behav. 2001; 73: 313-320Crossref PubMed Scopus (91) Google Scholar; Renz, 2001Renz H. The role of neurotrophins in bronchial asthma.Eur J Pharmacol. 2001; 429: 231-237Crossref PubMed Scopus (29) Google Scholar; Marshall and Born, 2002Marshall L. Born J. Brain–immune interactions in sleep.Int Rev Neurobiol. 2002; 52: 93-131Crossref PubMed Scopus (79) Google Scholar; Bayas et al., 2003Bayas A. Kruse N. Moriabadi N.F. et al.Modulation of cytokine mRNA expression by brain-derived neurotrophic factor and nerve growth factor in human immune cells.Neurosci Lett. 2003; 335: 155-158Crossref PubMed Scopus (37) Google Scholar). TrkB for example is expressed by dendritic cells and macrophages in lymphoid organs (Hannestad et al., 1998Hannestad J. Germana A. Catania S. Laura R. Ciriaco E. Vega J.A. Neurotrophins and their receptors in the pigeon caecal tonsil. An immunohistochemical study.Vet Immunol Immunopathol. 1998; 61: 359-367Crossref PubMed Scopus (16) Google Scholar) and BDNF is expressed by T cells (Barouch et al., 2000Barouch R. Appel E. Kazimirsky G. Braun A. Renz H. Brodie C. Differential regulation of neurotrophin expression by mitogens and neurotransmitters in mouse lymphocytes.J Neuroimmunol. 2000; 103: 112-121Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar; Moalem et al., 2000Moalem G. Gdalyahu A. Shani Y. et al.Production of neurotrophins by activatedT cells: Implications for neuroprotective autoimmunity.J Autoimmun. 2000; 15: 331-345Crossref PubMed Scopus (292) Google Scholar). BDNF is also required for the sustenance of corneal epithelial cells (You et al., 2000You L. Kruse F.E. Volcker H.E. Neurotrophic factors in the human cornea.Invest Ophthalmol Vis Sci. 2000; 41: 692-702PubMed Google Scholar) and the survival and proliferation of pigment cells during neural crest development and in the retina (Sieber-Blum, 1998Sieber-Blum M. Growth factor synergism and antagonism in early neural crest development.Biochem Cell Biol. 1998; 76: 1039-1050Crossref PubMed Scopus (52) Google Scholar; dos Santos et al., 2003dos Santos A.A. Medina S.V. Sholl-Franco A. de Araujo E.G. PMA decreases the proliferation of retinal cells in vitro: The involvement of acetylcholine and BDNF.Neurochem Int. 2003; 42: 73-80Crossref PubMed Scopus (13) Google Scholar), and may also play a role in bone formation (Asaumi et al., 2000Asaumi K. Nakanishi T. Asahara H. Inoue H. Takigawa M. Expression of neurotrophins and their receptors (TRK) during fracture healing.Bone. 2000; 26: 625-633Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). As a very densely innervated mini-organ, the hair follicle is subject to life-long recurring tissue-remodeling events (hair cycle) with alternating phases of rapid growth and pigmentation (anagen), apoptosis-driven regression (catagen), and relative quiescence (telogen). These tissue remodeling events during hair follicle cycling involve both the organ itself (Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1033) Google Scholar), its innervation (Botchkarev et al., 1997Botchkarev V.A. Eichmüller S. Johansson O. Paus R. Hair cycle-dependent plasticity of skin and hair follicle innervation in normal murine skin.J Comp Neurol. 1997; 386: 379-395Crossref PubMed Scopus (108) Google Scholar,Botchkarev et al., 1998bBotchkarev V.A. Botchkareva N.V. Lommatzsch M. et al.BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin.Eur J Neurosci. 1998; 10: 3276-3283Crossref PubMed Scopus (24) Google Scholar,Botchkarev et al., 1999cBotchkarev V.A. Peters E.M. Botchkareva N.V. Maurer M. Paus R. Hair cycle-dependent changes in adrenergic skin innervation, and hair growth modulation by adrenergic drugs.J Invest Dermatol. 1999; 113: 878-887Crossref PubMed Scopus (69) Google Scholar; Peters et al., 2001Peters E.M. Botchkarev V.A. Botchkareva N.V. Tobin D.J. Paus R. Hair-cycle-associated remodeling of the peptidergic innervation of murine skin, and hair growth modulation by neuropeptides.J Invest Dermatol. 2001; 116: 236-245Crossref PubMed Scopus (93) Google Scholar), its vasculature (Mecklenburg et al., 2000Mecklenburg L. Tobin D.J. Müller-Röver S. et al.Active hair growth (anagen) is associated with angiogenesis.J Invest Dermatol. 2000; 114: 909-916Crossref PubMed Scopus (161) Google Scholar), and profound changes of the skin architecture and immune function (Paus et al., 1999aPaus R. Christoph T. Müller-Röver S. Immunology of the hair follicle: A short journey into terra incognita.J Investig Dermatol Symp Proc. 1999; 4: 226-234Abstract Full Text PDF PubMed Scopus (93) Google Scholar). BDNF expression in the murine hair follicle fluctuates in a strikingly hair cycle-dependent manner and BDNF overexpression causes premature catagen development (Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar) and alters cutaneous innervation density (Botchkarev et al., 1998bBotchkarev V.A. Botchkareva N.V. Lommatzsch M. et al.BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin.Eur J Neurosci. 1998; 10: 3276-3283Crossref PubMed Scopus (24) Google Scholar). Since nervous system and hair follicle epithelium share a common ectodermal origin, it is not surprising that TrkB is also expressed by cutaneous epithelial cells in mice and humans (Shibayama and Koizumi, 1996Shibayama E. Koizumi H. Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues.Am J Pathol. 1996; 148: 1807-1818PubMed Google Scholar; Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar,Botchkarev et al., 1999bBotchkarev V.A. Metz M. Botchkareva N.V. et al.Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as "eliotrophins" in murine skin.Lab Invest. 1999; 79: 557-572PubMed Google Scholar). In regressing hair follicles of C57BL/6 mice, TrkB is expressed on apoptotic keratinocytes during catagen and BDNF skin content is highest during anagen–catagen transformation (Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar). Simultaneous expression of BDNF by these cells suggests para- and autocrine growth-regulatory effects of BDNF on epithelial tissues (Shibayama and Koizumi, 1996Shibayama E. Koizumi H. Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues.Am J Pathol. 1996; 148: 1807-1818PubMed Google Scholar; Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar). Although epidermal keratinocyte proliferation is stimulated by BDNF (Botchkarev et al., 1999bBotchkarev V.A. Metz M. Botchkareva N.V. et al.Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as "eliotrophins" in murine skin.Lab Invest. 1999; 79: 557-572PubMed Google Scholar), a growth-inhibitory effect of BDNF on selected keratinocyte subpopulations was demonstrated with the report that BDNF induces premature catagen development in anagen hair follicles of C57BL/6 mice (Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar). Given the very substantial inter-species differences that a given bioregulatory compound can exert, e.g. on murine versus human hair follicles (cf.Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Eng J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar; Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1033) Google Scholar) and the contrasting effects of BDNF on epidermal (stimulation of proliferation) (Botchkarev et al., 1999bBotchkarev V.A. Metz M. Botchkareva N.V. et al.Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as "eliotrophins" in murine skin.Lab Invest. 1999; 79: 557-572PubMed Google Scholar) and hair follicle keratinocytes (promotion of apoptosis) (Botchkarev et al., 1999aBotchkarev V. Botchkareva N. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar) in mice, it remains totally unknown whether BDNF/TrkB signaling plays any role in human hair biology. With these limitations in mind, we have addressed the following specific questions: 1.Do human hair follicle keratinocytes and fibroblasts in normal scalp skin express BDNF and/or its high-affinity receptor TrkB mRNA and immunoreactivity (IR)?2.Does BDNF promote catagen development in organ-cultured human anagen hair follicles, as it does in mice?3.If so, does this include the recruitment of recently recognized common mechanisms of catagen induction in human hair follicles, such as downregulation of hair follicle keratinocyte proliferation, upregulation of keratinocyte apoptosis, and transforming growth factor β (TGFβ)-mediated catagen induction?4.Can negative BDNF effects on human hair growth in vitro be antagonized? These questions were addressed by analyzing the expression pattern of BDNF and TrkB in human scalp skin biopsies as well as in isolated anagen VI hair follicles by rtPCR analysis, in situ hybridization, and immunofluorescence. Analysis of human hair follicle cycling is difficult. Most available hair follicles for rtPCR, in situ analyses, or immunohistochemistry derive from scalp skin excised during elective plastic surgery. In such biopsies, 80%–90% of all hair follicles are in anagen VI, and the remainder are in telogen, with catagen or early anagen hair follicles being detectable only extremely rarely (Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Eng J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar). Therefore, in situ expression analyses of growth factors and receptors largely are limited to studying anagen VI and telogen hair follicles. In addition, human anagen VI scalp skin hair follicles were organ cultured (Philpott et al., 1994Philpott M.P. Sanders D. Westgate G.E. Kealey T. Human hair growth in vitro: A model for the study of hair follicle biology.J Dermatol Sci. 1994; 7: 55-S72Abstract Full Text PDF PubMed Scopus (110) Google Scholar; Philpott, 1999Philpott M. In vitro maintenance of isolated hair follicles: Current status and future development.Exp Dermatol. 1999; 8: 317-319PubMed Google Scholar) to investigate the impact of recombinant human BDNF (rhBDNF) or BDNF plus TGFβ-neutralizing antibodies on active hair shaft elongation and maintenance of the hair shaft-producing unit in the anagen hair bulb. rtPCR revealed that micro-dissected human scalp skin anagen hair bulbs, which include the hair follicle epithelium proximal to the isthmus and bulge region as well as the dermal papilla and the proximal connective tissue sheath of the hair follicle, contained transcripts of the BDNF and TrkB genes (Figure 1). This offered an indication, that, just like their murine counterparts, human anagen hair follicles are both sources and targets of BDNF/TrkB-mediated signalling. BDNF and TrkB transcription in human scalp anagen VI hair follicles was confirmed and localized by in situ hybridization using fluorescein-labeled oligonucleotide probe (Figure 2). All negative and positive controls (see Materials and Methods) confirmed the specificity and sensitivity of the used in situ stainings. Staining intensity for BDNF transcripts was strongest in the proximal inner root sheath and distal outer root sheath (Figure 2), and BDNF transcript-related signals were also detected in the dermal papilla. TrkB transcripts were present in the proximal inner and distal outer root sheath, but not in the dermal papilla (Figure 2). BDNF and TrkB translation in human scalp anagen VI hair follicles was confirmed by immunohistochemistry (Figure 2). All negative and positive controls confirmed the specificity and sensitivity of the used immunohistochemical stainings: specific staining patterns were absent in negative controls without primary antibody, or were strongly diminished after preincubation with specific blocking peptide. Positive controls showed the expected IR patterns in human pancreatic epithelia (Miknyoczki et al., 1999Miknyoczki S.J. Lang D. Huang L. Klein-Szanto A.J. Dionne C.A. Ruggeri B.A. Neurotrophins and Trk receptors in human pancreatic ductal adenocarcinoma: Expression patterns and effects on in vitro invasive behavior.Int J Cancer. 1999; 81: 417-427Crossref PubMed Scopus (140) Google Scholar). All extrafollicular IR patterns were not analyzed in detail, since they are the subject of a separate, ongoing study, and the schemes that summarize BDNF and TrkB IR have only been sketched in preliminary form (Figure 2). Semiquantitative analysis of BDNF IR demonstrated strongest protein expression in more differentiated keratinocyte cell populations such as the proximal inner root sheath and in the mesenchymal compartment, e.g. the dermal papilla. TrkB IR, in contrast, was strongest in more undifferentiated keratinocyte cell populations such as the basal layer of the hair follicle ostium. TrkB IR was also detected in the hair matrix and proximal outer root sheath but not in the dermal papilla (Table I).Table ISummary of BDNF and TrkB staining intensities in various compartments of anagen VI scalp skin hair folliclesBDNFTrkBmRNAProteinmRNAProteinHair follicle ostium++++++aBasal layer, rest is + like epidermis.Inner root sheath+++++++++Outer root sheath (including the isthmus and bulge region)++++++-++Proximal hair follicle bulb++++Hair matrix(+)(+)(+)0Cuticle of the hair shaft00++Dermal papilla++++00The table lists the mean staining intensities from nine (TrkB) or 13 (BDNF) different human scalp skin samples.BDNF, brain-derived neurotrophic factor; TrkB, tyrosine kinase B.a Basal layer, rest is + like epidermis. Open table in a new tab The table lists the mean staining intensities from nine (TrkB) or 13 (BDNF) different human scalp skin samples. BDNF, brain-derived neurotrophic factor; TrkB, tyrosine kinase B. Because of their low incidence in human scalp skin (10%–20% of all hair follicles), telogen hair follicles were detected only rarely in normal human scalp skin sections. In 13 (BDNF) and nine (TrkB) different donor samples (which contained more than 40 anagen hair follicles), only four telogen hair follicles, each of a different donor, were found per investigated antigen. Hair follicles in catagen or early anagen were not detected at all. Weakly positive BDNF staining (IR and in situ) was detected in the outer root sheath and secondary hair germ epithelium of all telogen hair follicles (Figure 2). BDNF staining was pronounced in the outer root sheath basal layer. TrkB staining was weak in the hair follicle epithelium (Figure 2). Interestingly and in contrast to our findings in anagen skin, in the telogen stage of the hair cycle the dermal papilla-stained negative for both antigens (Figure 2). In order to assess the functional relevance of BDNF/TrkB signaling in the anagen hair follicle, microdisected lower anagen hair follicles from normal human scalp skin were cultured in the presence of 1, 5, 25, 50, and 150 ng per mL rhBDNF. These experiments revealed a significant reduction of hair growth in hair follicles cultured with 50 and 150 ng per mL BDNF, whereby hair follicles treated with 150 ng per mL exhibited dystrophic features such as pigment incontinence as a sign of toxification. Therefore 50 ng per mL was chosen for the treatment of a larger number of samples (Figure 3). Hair cycle staging of hair follicles treated with 50 ng per mL rhBDNF after 48 h in culture did not show catage-like development. First catagen-like hair follicles could be detected after 96 h, whereas none were seen in control hair follicles (not shown). This difference became highly significant after 10 d in culture with 50 ng per mL rhBDNF (Figure 4). Quantitative histomorphometry of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin Nick end labeling (TUNEL) or Ki67 IR nuclei in organ-cultured human hair bulbs revealed significantly less Ki67+, proliferating keratinocytes in hair follicles treated for 10 d with 50 ng per mL rhBDNF when compared with controls (Figure 5). Complementary hair follicles showed significantly more TUNEL+ cells after 10 d of culture with 50 ng per mL rhBDNF (Figure 5). Quantitative TUNEL/Ki67 histomorphometry of hair follicles harvested after only 2 or 4 d of culture did not show significant differences (not shown). To investigate possible signaling loops recruited by BDNF in the control of catagen induction, we performed quantitative real-time rtPCR analysis of TGFβ2 steady-state mRNA levels in hair bulbs, since TGFβ2 is now appreciated as a key catagen-promoting growth factor in the human system (Soma et al., 2002Soma T. Tsuji Y. Hibino T. Involvement of transforming growth factor-beta2 in catagen induction during the human hair cycle.J Invest Dermatol. 2002; 118: 993-997Crossref PubMed Scopus (100) Google Scholar; Tsuji et al., 2003Tsuji Y. Denda S. Soma T. Raftery L. Momoi T. Hibino T. A potential suppressor of TGF-beta delays catagen progression in hair follicles.J Investig Dermatol Symp Proc. 2003; 8: 65-68Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar; Hibino and Nishiyama, 2004Hibino 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 (149) Google Scholar). This showed substantial upregulation of TGFβ2 mRNA in hair bulbs that were cultured in the presence of 50 ng per mL rhBDNF over 48 h (Figure 6). Simultaneous treatment of cultured human anagen hair follicles with 50 ng per mL BDNF and 10 (not shown) or 25 (Figure 7) μg per mL TGFβ-neutralizing antibodies completely abrogated the catagen-inductive and anti-proliferative effects of 50 ng per mL rhBDNF, e.g. hair shaft elongation rates did not differ from controls after 4 d in culture, when rhBDNF inhibition of hair shaft elongation became significantly different from control (Figure 7). Likewise, hair cycle staging, apoptosis (TUNEL), and proliferation analysis (Ki67) on hair follicles cultured over 4 or 10 d revealed anagen to early catagen hair bulbs with high numbers of Ki67+ proliferating and low numbers of TUNEL+ apoptotic keratinocytes in the control as well as the groups treated with BDNF together with TGFβ-neutralizing antibody (not shown). Hair follicles that were treated with 50 ng per mL rhBDNF showed striking upregulation of TGFβ2 expression in the hair bulb by immunohistochemistry especially in the hair follicle matrix, the inner root sheath, and the outer root sheath (Figure 7), whereas TGFβ2 expression was restricted to the most differentiated layer of the inner root sheath in control hair follicles and hair follicles treated with 50 ng per mL BDNF and 25 μg per mL TGFβ-neutralizing antibodies. Since it was first shown in mice that mammalian skin and especially keratinocytes may be a source for neurotrophins (Tron et al., 1990Tron V.A. Coughlin M.D. Jang D.E. Stanisz J. Sauder D.N. Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212).J Clin Invest. 1990; 85: 1085-1089Crossref PubMed Scopus (114) Google Scholar), a body of data has accumulated, in support of the concept that the skin epithelium is also an important target of neurotrophin signaling (Paus et al., 1994bPaus R. Luftl M. 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Nerve growth factor promotes corneal healing: Structural, biochemical, and molecular analyses of rat and human corneas.Invest Ophthalmol Vis Sci. 2000; 41: 1063-1069PubMed Google Scholar). More recently, our own studies have highlighted that neurotrophins are prominently involved in the control of murine hair follicle development and cycling and that the murine hair follicle is actually a prominent source of neurotrophins (Botchkarev et al., 1998aBotchkarev V.A. Botchkarev N.V. Albers K.M. van der Veen C. Lewin G.R. Paus R. Neurotrophin-3 involvement in the regulation of hair follicle morphogenesis.J Invest Dermatol. 1998; 111: 279-285Crossref PubMed Scopus (48) Google Scholar,Botchkarev et al., 1998cBotchkarev V.A. Welker P. Albers K.M. et al.A new role for neurotrophin-3: Involvement in the regulation of hair follicle regression (catagen).Am J Pathol. 1998; 153: 785-799Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar,Botchkarev et al., 1999aBotchkarev V. 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