Expression pattern of p75 neurotrophin receptor protein in human scalp skin and hair follicles: Hair cycle-dependent expression Mohamed A. Adly, PhD,a,c Hanan A. Assaf, MD,b and Mahmoud Rezk A. Hussein, MD, PhD, FRCPath, EBPd Assuit and Sohag, Egypt; and Abha, Saudi Arabia Background: The p75 neurotrophin receptor (p75NTR) is a death factor (apoptosis-promoting protein) that belongs to the tumor necrosis factor receptor superfamily of membrane proteins. In the murine hair follicle (HF) model, p75NTR plays a critical role during HF morphogenesis, functioning as a receptor that negatively controls HF development. p75NTR signaling is involved in the control of keratinocyte apoptosis during catagen. To date, knowledge about the expression pattern of p75NTR protein in human scalp skin and HFs is limited. In this investigation we hypothesized that p75NTR protein is expressed in human scalp skin and its expression in HFs fluctuates with the transitions from anagen / catagen / telogen stages. Methods: To test this hypothesis, the immunoreactivity of p75NTR protein was examined in human scalp skin by immunofluorescent and immunoalkaline phosphatase methods. A total of 50 normal-appearing human scalp skin biopsy specimens were examined (healthy women age 53-57 years). In each case, 50 HFs were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). Results: We found variations in p75NTR protein expression with HF cycling. p75NTR expression was negligible in early, mid, and mature anagen and weak during late anagen. p75NTR expression was moderate during anagen-catagen transition. It was strong in both catagen and telogen HF. Also, p75NTR protein expression was strong in the stratum corneum (epidermis), dermal fibroblasts, blood vessels, nerve endings, adipocytes, and both sebaceous and sweat glands. Limitations: Our knowledge about other proteins (prosurvival and pro-apoptotic molecules) interacting with p75 is incomplete. Conclusions: Our investigation reports, for the first time, the expression patterns of p75NTR in human scalp skin and HFs. p75NTR protein expression exhibited significant hair cycle-dependent fluctuation, suggesting a possible role in human HF biology. ( J Am Acad Dermatol 2009;60:99-109.)
he p75 neurotrophin (NT) receptor (NTR) (p75NTR) is a death receptor (apoptosispromoting protein) that belongs to the tumor necrosis factor receptor superfamily of membrane
From the Departments of Zoologya and Dermatology and Venereology,b Faculty of Science, Sohag University; Department of Biology, Faculty of Science, King Khalid University, Abha, Saudi Arabiac; and Department of Pathology, Faculty of Medicine, Assir Central and Assiut University Hospitals, Assir and Abha.d Funding sources: None. Conflicts of interest: None declared. Reprint requests: Mahmoud R. Hussein, MD, PhD, FRCPath, EBP, Department of Pathology, Faculty of Medicine, Assir and Assuit University Hospitals. E-mail: [email protected]
0190-9622/$36.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.09.060
proteins.1 NTs have many growth-regulatory functions outside the nervous system.2 In murine hair follicle (HF), NTs show developmentally and spatiotemporally controlled expression, including nerve growth factor (NGF) and its receptor p75NTR protein.2,3 Follicular NTs and NTR expression exhibit remarkable, hair cycle-dependent fluctuations on the gene and protein level. These fluctuations are mirrored by changes in nerve fiber density and neurotransmitter/neuropeptide content in the perifollicular neural networks. In murine HF model, p75NTR stimulation inhibits HF development and stimulates catagen. As HFs represent both the target and key peripheral source of NTs, an understanding of the role of NTs in the control of HF morphogenesis and cycling provides an accessible, and easily manipulated, clinically relevant experimental model. 99
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Abbreviations used: DP: GDNF:
dermal papilla glial cell lineederived neurotrophic factor HF: hair follicle mRNA: messenger RNA NGF: nerve growth factor NT: neurotrophin NTN: neurturin NTR: neurotrophin receptor ORS: outer root sheath p75NTR: p75 neurotrophin receptor SG: sebaceous glands SwG: sweat glands TBS: Tris-buffered saline Trk: tyrosine kinase
Several studies indicate that p75NTR plays important roles during HF morphogenesis, functioning as a receptor that negatively controls HF development (ie, a hair growth terminator). During murine HF morphogenesis, p75NTR is the first growth factor receptor expressed by those fibroblasts that later develop into the dermal papilla (DP) of the HF. During HF development in fetal and neonatal C57BL/6 murine back skin, p75NTR is strongly expressed by fibroblasts of the DP and by skin nerves. In contrast, p75NTR immunoreactivity disappears from the DP in the fully developed HF and it is expressed only in the epithelial outer root sheath (ORS) of the HF. The p75NTR knockout (e/e) mice showed significant acceleration of HF morphogenesis. The DP fibroblasts of p75NTR knockout mice showed reduced proliferative activity in situ, indicating alterations in their transition from proliferation to differentiation.4 In humans, p75NTR protein is selectively expressed by basal epithelial cells in pterygia, conjunctiva, and limbus. Also, its expression is observed in selective regions of the human epidermis and HF bulge.5 In anagen hair bulbs of the microdissected human scalp HF, messenger RNA (mRNA) for p75NTR is transcribed. A strong p75NTR protein expression was seen in basal and suprabasal ORS keratinocytes. During spontaneous catagen development of organcultured human anagen HFs, p75NTR mRNA levels increased and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (1) apoptotic cells showed prominent p75NTR expression. These findings suggest that p75NTR plays a role in termination of HF cycle in human beings.6 To date, the expression pattern of p75NTR in the human scalp and HF is poorly understood. In this investigation we hypothesized that p75NTR protein is expressed in the human scalp skin and its expression in HF fluctuates with the transitions form anagen
/ catagen / telogen stages. To test this hypothesis and to fill this existing gap in literature, the immunoreactivity of p75NTR protein was examined in human scalp skin by immunofluorescent and immunoalkaline phosphatase staining methods. Our study revealed significant fluctuations of p75NTR protein expression with the transitions from anagen / catagen / telogen stages. A strong p75NTR protein expression was also seen in extrafollicular structures (sweat glands [SwG], sebaceous glands [SG], nerve endings, blood vessels, dermal blood vessels, and fibroblasts).
METHODS Skin samples A total of 50 normal-appearing human scalp skin specimens were obtained from 50 women (age: 53-57 years; after informed consent) undergoing elective cosmetic plastic surgery. None of these women had accompanying primary hair disorder. The specimens were obtained from both frontal and temporal regions of the scalp. After surgery, samples were maintained in Williams E medium (Biochrom KG Seromed, Berlin, Germany) for transportation at 48C for up to 24 hours. Skin specimens used for cryostat-cut sections were frozen abruptly in liquid nitrogen and stored at e808C until use. Before immunostaining, samples were embedded and processed for longitudinal cryosections (8 m). Sections were dried, fixed in cold acetone (e208C), and stored at e208C until used for immunohistochemistry. A total of 50 HF (35 anagen, 10 catagen, and 5 telogen) were examined in each case. There were no remarkable immunohistologic variations among these cases. Immunohistochemistry Cryosections of normal-appearing human scalp skin were immunostained using polyclonal rabbit antibody against human p75NTR (Santa Cruz Biotechnology, Santa Cruz, CA). Two labeling techniques were performed to visualize antigen-antibody complexes: avidin-biotin complex (Vector Laboratories Inc, Burlingame, CA) and the highly sensitive immunofluorescent tyramide signal amplification (PerkinElmer Life Science, Boston, MA). For the avidin-biotin complex labeling method, cryosections of normal-appearing human scalp skin were washed in Tris-buffered saline (TBS) (0.05 mol/L, pH 7.6) and preincubated with avidin-biotin blocking kit solution (Vector Laboratories Inc) followed by incubation with protein blocking agent (Immunotech, Krefeld, Germany) to prevent nonspecific binding. Sections were then incubated with the primary antibodies diluted in TBS (p75NTR 1:100) containing
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Table I. Expression values of p75 neurotrophin receptor protein in human scalp skin and hair follicles Structure
Early, mid, and mature anagen Late anagen Early, mid, mature, and late anagen Anagen-catagen transition Catagen Telogen Sebaceous glands and sweat glands
0.2 0.5 0.3 1.2 2.2 2.7 2.8
6 6 6 6 6 6 6
0.1 0.1 0.1 0.1 0.1 0.4 0.4
0.5 1.5 1.0 2.5 3.5 3.6 3.7
6 6 6 6 6 6 6
0.1 0.2 0.1 0.2 0.2 0.1 0.1
0.2 1.0 0.6 3.1 7.8 9.9 10.5
6 6 6 6 6 6 6
0.1 0.3 0.1 0.5 0.7 0.9 0.8
IR was evaluated by multiplying PP% and SI. First, PP% was scored as 0 for \5%, 1 for 5%-25%, 2 for 25%-50%, 3 for 50%-75%, and 4 for [75%. Second, SI was scored as 1 for weak, 2 for medium, and 3 for intense staining, following other groups. There were statistically significant differences (P \ .00) in expression values (SI, PP%, and IR) between anagen-catagen transition and between anagen (early, mid, mature, and late) and other stages of hair follicles (catagen and telogen). Differences in expression values (SI, PP%, and IR) among early, mid, mature, and late anagen were not significant (P \ .38). No statistically significant differences were observed between expression values in catagen/telogen and values in extrafollicular structures (sweat and sebaceous glands). IR, Immunoreactivity score; PP%, percentage of positive cells; SI, staining intensity.
2% goat serum for 1 hour at room temperature or overnight at 48C. Thereafter, sections were incubated with biotinylated secondary antibodies goat antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:200 in TBS containing 2% goat serum, for 30 minutes at room temperature. Next, sections were incubated with avidin-biotinalkaline phosphatase complex (Vecta-Stain kits, Vector Laboratories Inc) diluted in TBS (1:100) for 30 minutes at room temperature. The alkaline phosphatase color reaction was developed by applying staining protocols described before,3,7,8 using fast red tablets (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Finally, sections were counterstained with Meyer hematoxylene, covered with Kaiser Glycerol (Dako, Glostrup, Denmark) and stored at 48C for microscopic examination and analysis. For tyramide signal amplification labeling technique, cryosections were washed in Tris-acidTween buffer (pH 7.5), followed by washing in 3% hydrogen peroxide. Sections were then incubated with lower concentrations of primary antibodies diluted in Tris-acid-blocking buffer (pH 7.2, 1:1000) overnight at 48C. Next, sections were washed in Tris-acid-Tween buffer and incubated with tetramethylrhodamine-isothiocyanate-conjugated F (ab) 2 fragments of goat antirabbit IgG secondary antibodies (Jackson ImmunoResearch Laboratories) diluted in Tris-acid-blocking buffer (1:200) for 30 minutes at room temperature. Thereafter, sections were incubated with streptavidin horseradish peroxidase (1:50 in Tris-acid-blocking buffer) for 30 minutes at room temperature. Finally, tetramethylrhodamine-isothiocyanate-tyramide amplification reagent was administrated (1:50 in amplification diluent provided with the kit) for 30 minutes at room temperature, followed by counterstaining
with 4’,6’-diamidino-2-phenylindole and mounting in levamisole (Dako Corp, Carpenteria, CA). The tyramide amplification signals were visualized under a fluorescence microscope (Zeiss, Jena, Germany). Positive control The positive control specimens consisted of internal positive controls (melanocytes) and external positive controls (glial cells and neurons of the mouse cerebral cortex). Negative control Additional sections, running in parallel but with omission of the primary antibodies, served as the negative controls for p75NTR.8-10 Semiquantitation of p75NTR protein expression The immunoreactivity score was evaluated by multiplying the percentage of positive cells and the staining intensity. First, the percentage of positive cells was scored as 0 for less than 5%, 1 for 5% to 25%, 2 for 25% to 50%, 3 for 50% to 75%, and 4 for greater than 75%. Second, the staining intensity was scored as 1 for weak, 2 for medium, and 3 for intense staining, following other groups.3,7,8,11,12 Statistical analysis Statistical comparison of the protein expression values among anagen, catagen, and telogen was evaluated using analysis of variance. Calculations were done with a statistical software package (SPSS for Windows, Version 10.0, SPSS Inc, Chicago, IL). Statistical significance was defined as P less than .05.
RESULTS The positive (melanocytes and mouse brain tissue) and negative controls were positive and
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Fig 1. Expression pattern of p75 neurotrophin receptor (p75NTR) protein in human anagen hair follicle (HF ). Human scalp skin (anagen VI HF) was immunostained with both tyramide signal amplification (TSA) and avidin-biotin complex (ABC ) techniques. Distal (A), central (B), bulb (C), distal with sebaceous glands (SG) (D), and bulb and mid (E) regions. Negligible p75NTR protein expression is occasionally seen in some cells of the outer root sheath (ORS )
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negative, respectively, indicating the validity of our results. The p75NTR protein expression was seen in adult scalp skin both in HF and in the extrafollicular structures. In HF, p75NTR protein expression was negligible in early, mid, and mature anagen, and was weak in late anagen. The expression was moderate in anagen-catagen transition and strong in both catagen and telogen HFs (Table I and Figs 1 to 3). The expression values of p75NTR protein in the anagen-catagen transition HFs, catagen HFs, and telogen HFs were statistically significantly high compared with those of anagen (P\.00). The expression was also strong in the stratum corneum of the epidermis, SG, SwG, wall of blood vessels, nerve endings, dermal fibroblasts, and adipocytes (subcutis) (Table I and Figs 4 and 5). Negligible p75NTR protein expression in early, mid, and mature anagen HFs In early, mid, and mature anagen HF (anagen VI), p75NTR protein expression was negligible. A weak p75NTR protein expression was occasionally seen in the cells of the ORS and in some hair matrix cells. In the ORS, a weak expression was detected in sparse basal keratinocytes of the proximal bulb region (Fig 1, C and E ), mid-central region (Fig 1, B and E ), and distal region (Fig 1, A and D) of mature anagen HF. The number of p75NTR positive keratinocytes of ORS decreased toward the distal region of the HF. In the hair matrix, sparse hair matrix cells had a negligible p75NTR expression (Fig 1, C ). Weak p75NTR protein expression in late anagen and moderate p75NTR protein expression during anagen-catagen transition In the late anagen and in anagen-catagen transition HFs, p75NTR expression was weak and p75NTR immunostaining values were higher than those of mature anagen. Examination of the different regions of anagen HF (distal region vs upper central region vs lower central region vs bulb region) revealed higher expression values in the distal region. The p75NTR immunoreactivity was detected in the ORS, inner root sheath (IRS), DP, connective tissue sheath, and hair matrix cell (Fig 2). In the ORS, the immunostaining of p75NTR was moderate and slightly different in the different regions of the HF (Fig 2). In the proximal cycling portion, p75NTR
protein expression in ORS was moderate in the outer basal keratinocytes of the suprabulbar region (Fig 2, C ) and gradually increased toward the central and distal regions to include all layers of the ORS (Fig 2, A to C ). In the bulge and isthmus regions, p75NTR staining was confined to the basal and outer suprabasal layers of keratinocytes. In the infundibulum, p75NTR immunopositivity was similar to that of the epidermis, being strong in the innermost layer, which is continuous with the stratum corneum, and in the basal layer, which is continuous with the stratum basale (Fig 2, A and E ). In the IRS, p75NTR protein expression was moderate in the proximal bulbar region, and less intense in the mid-central region of the HF (Fig 2, B and C ). However, it decreased distally until it disappeared in the upper distal region (Fig 2, B and C ). In connective tissue sheath, p75NTR protein expression was moderate throughout all regions of the HF (Fig 2, A to C ). Strong p75NTR protein expression in catagen and telogen HFs During early catagen, the expression of p75NTR was strong, but slightly decreased toward the late catagen. The p75NTR protein immunopositivity decreased in the involuting keratinocytes of epithelial strand in the inner layers of the ORS and the club hair during catagen VI (Fig 3, A and B). In telogen HF, p75NTR protein expression was strong in all layers of the ORS (Fig 3, C ). p75NTR protein expression in human scalp skin epidermis and extrafollicular structures The p75NTR protein expression was detected in the epidermis, SG, SwG (Table I and Fig 4), some dermal fibroblasts (Fig 4, E ), and adipocytes of the subcutis (Fig 5, B). In the epidermis, p75NTR protein expression was strong in the stratum corneum (Fig 4, E ). In SG, p75NTR protein expression was strong, especially in the peripheral sebaceocytes (Fig 4, B and F ). In the SwG, p75NTR protein expression was also strong (Fig 4, C and G). A strong expression was seen in the blood vessels (Fig 5, A and B), nerve endings (Fig 5, C ), and adipocytes (Fig 5, B).
DISCUSSION This investigation reports for, to the best of our knowledge, the first time the differential expression
and hair matrix cells (HMC ). In the ORS, weak expression was detected in sparse basal keratinocytes of proximal bulb (C and E), mid-central (B and E), and distal (A and D) regions of mature anagen HFs. In the hair matrix, sparse HMCs had negligible p75NTR expression (C). (A to E, Original magnifications: A to D, 3200; E, 3160.) CTS, Connective tissue sheath; DP, dermal papilla; IRS, inner root sheath.
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Fig 2. Expression pattern of p75 neurotrophin receptor (p75NTR) protein human anagen hair follicle (HF) during anagencatagen transition: human scalp skin (anagen-catagen transition HF) was immunostained with both tyramide signal amplification (TSA) and avidin-biotin complex (ABC ) techniques. Distal (A and D), central (B and E), and bulb (C and F) regions. p75NTR immunoreactivity is detected in outer root sheath (ORS ), inner root sheath (IRS ), dermal papilla (DP), connective tissue sheath (CTS ), and hair matrix cells (HMCs ). In the ORS, immunostaining of p75NTR is moderate and slightly different in different regions of HF. In proximal cycling portion, p75NTR protein expression in the ORS is moderate in outer basal keratinocytes of suprabulbar region (C) and gradually increases toward central and distal regions to include all layers of the ORS (A to C). In the bulge and isthmus regions, p75NTR staining is confined to basal and 1 to 2 outer suprabasal layers of keratinocytes. In the infundibulum, p75NTR immunopositivity is strong in innermost layer (A and E). In the IRS, p75NTR protein expression is moderate in proximal bulbar region, and less intense in mid-central region of HF (B and C). It decreases distally until it disappears in upper distal region (B and C). In the CTS, p75NTR protein expression is moderate throughout all regions of the HF (A to C). (A to F, Original magnifications: 3200.) HS, Hair shaft.
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Fig 3. Expression pattern of p75 neurotrophin receptor (p75NTR) protein in the human catagen and telogen hair follicle (HF ). Human scalp skin (catagen VI [A and B] and telogen [C] HF) was immunostained with both tyramide signal amplification (B and C) and avidin-biotin complex (A) techniques. Strong p75NTR protein expression is seen in catagen and telogen HFs. (A to C, Original magnifications: 3200.) CTS, Connective tissue sheath; DP, dermal papilla; ES, epithelial strand; ORS, outer root sheath; SG, sebaceous glands.
of p75NTR protein in the human scalp skin and HFs. It also demonstrates that p75NTR protein expression fluctuates with HF cycle transitions (ie, weak expression in the late anagen, moderate expression in anagen-catagen transition, and strong expression in the catagen and telogen HF). Moreover, our study revealed a strong expression of p75NTR protein in the extrafollicular structures (SG, SwG, nerve endings, blood vessels, and adipocytes). Fluctuations of p75NTR protein expression with HF cycle transitions The p75NTR is a pan-NTR that functions together with pro-NGF as important hair growth terminators. We found significant hair cycle-dependent fluctuations of p75NTR protein expression during the transitions from anagen / catagen / telogen stages. The finding of weak expression of p75NTR during
late anagen, moderate expression in anagen-catagen transition, and strong expression in catagen and telogen HFs agrees with observations in the murine HF model and in organ-cultured human anagen HFs.2,13,14 Peters et al6 reported that mRNA for NGF, pro-NGF, p75NTR, and tyrosine kinase (Trk) A (p75NTR and Trk A are receptors for NGF) was transcribed in the microdissected human scalp anagen hair bulbs. In addition, immunohistomorphometry and in situ hybridization detected strong expression of p75NTR in basal and suprabasal ORS keratinocytes. During spontaneous catagen development of organ-cultured human anagen HFs, p75NTR mRNA levels increased, and p75NTR and pro-NGF immunoreactivity increased dramatically in involuting compartments. TUNEL (1) apoptotic cells showed prominent p75NTR expression.6 In murine skin high steady-state p75NTR mRNA skin levels
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were found during the anagen-catagen transition of HF. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL1/Bcl2e keratinocytes of the regressing ORS, but p75NTR was expressed by the nonregressing TUNELe/Bcl21 secondary hair germ keratinocytes. There was significant catagen retardation in p75NTR knockout mice as compared with wild-type controls. These findings suggest that p75NTR signaling is involved in the control of keratinocyte apoptosis during catagen.13 In this series, the variations in p75NTR protein expression with HF cycling concur with findings of our previous studies. We previously examined the status of several neuropeptides in the human scalp skin and HFs. The expression pattern of glial cell lineederived neurotrophic factor (GDNF) and a related family member, neurturin (NTN), and their cognate receptors (GDNF receptors, glial cell line derived neurotrophic factor receptor (GFR) alpha-1, and GFRalpha-2, respectively) were examined in human scalp skin using immunofluorescence and immunoalkaline phosphatase staining methods. The expression of GDNF protein was strong in the epidermis, SG, and SwG. The expression of NTN, GFRalpha-1, and GFRalpha-2 proteins was strong in the papillary dermis, SG, and SwG. In the epidermis, NTN protein expression was absent. The expression of GFRalpha-1 and GFRalpha-2 proteins was moderate in the epidermis. These proteins were strongly expressed in both epithelial and mesenchymal compartments of human anagen scalp HFs.7 GDNF, NTN, GFRalpha-1, and GFRalpha-2 proteins were weakly expressed in catagen and telogen HFs.10 A prominent expression of NGF and its high-affinity receptor, Trk A (members of the NT family) was strong in human scalp anagen HF, and weak or absent from catagen and telogen HF.3 NT-3 protein and its highaffinity receptor, Trk C (members of the NT family) were strongly expressed in human scalp anagen HF (anagen VI), whereas they were weakly expressed in catagen and reincreased in telogen HFs. Bone morphogenetic proteins, first isolated from bone extracts, are members of the transforming
growth factor-beta superfamily. They control cell proliferation, differentiation, and apoptosis in different body tissues, including the skin. A strong expression of bone morphogenetic proteins (bone morphogenetic protein-7) (a member of the transforming growth factor-beta superfamily) was found in anagen as compared with either catagen or telogen HF.9 The findings of this study suggest the involvement of p75NTR in HF cellular cycling events.6 The strong p75NTR protein expression in catagen and telogen HF suggests its role as HF terminator. Alternatively, the negligible p75NTR expression in early, mid, and mature anagen versus weak expression in late anagen indicates a role for p75NTR in anagen prolongation and/or catagen retardation in both human and murine HFs.2,13,14 Our data implicate p75NTR in human hair growth control. Accordingly, it is possible that selective p75NTR agonists and antagonists may become innovative therapeutic tools for the management of hair growth disorders (alopecia, effluvium, and hirsutism). p75NTR protein expression in the epidermis of human scalp skin and in the extrafollicular structures The expression of p75NTR protein in the extrafollicular structures (SwG, SG, fibroblasts, nerve endings, blood vessels, and adipocytes) concurs with previous studies.15-19 In our series, the strong p75NTR protein expression in the epidermis (stratum corneum) concurs with previous studies and suggests a possible role for p75NTR in the keratinocyte cell growth and renewal.18-20 The transplanted psoriatic plaques and normal-appearing skin demonstrated variable proliferation of p75NTR-positive nerve fibers.21 Because p75NTR protein is highly expressed in psoriasis, one could speculate that this protein may play a role in the mechanisms associated with this and other hyperproliferative skin conditions, including cancer.22 The strong p75NTR expression in the wall of the blood vessels concurs with finding in psoriatic skin and in neighboring blood vessels after peripheral transection of the
Fig 4. Expression pattern of p75 neurotrophin receptor (p75NTR) protein in human scalp skin epidermis and extrafollicular structures. Human scalp skin was immunostained with both tyramide signal amplification and avidin-biotin complex techniques. Epidermis (A and E), sebaceous glands (SG) (B and F), sweat glands (SwG) (C and G), negative control (D), and arrector pili muscle and nerve fibers (H). p75NTR protein expression was detected in the epidermis, SG, and SwG. In the epidermis, p75NTR protein expression was strong in the stratum corneum (E). In the SG, p75NTR protein expression was strong, especially in peripheral sebaceocytes (B and F). In the SwG, p75NTR protein expression was also strong (C and G). (A to C and E to G, Original magnifications: 3200.) APM, Arrector pili muscle; CTS, connective tissue sheath; DP, dermal papilla; HMC, hair matrix cell; HS, hair shaft; IRS, inner root sheath; ORS, outer root sheath.
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Fig 5. Expression pattern of p75 neurotrophin receptor protein in extrafollicular structures of human scalp skin. Human scalp skin was immunostained with both tyramide signal amplification (TSA) and avidin-biotin complex (ABC ) techniques. A and B, Blood vessels. C, Artery and encapsulated nerve ending (end bulb of Krause). Strong expression is seen in blood vessels (A and B), nerve endings (C), and adipocytes (B). (A to C, Original magnifications: 3200.) BV, Blood vessel; HF, hair follicle; ORS, outer root sheath.
facial nerve of adult mice.16,23 These observations suggest a role for p75NTR in the pathogenesis of inflammatory conditions by promoting cutaneous recruitment of inflammatory cells.16 The expression of p75NTR protein in dermal fibroblasts agrees with findings in murine HF. During HF morphogenesis, p75NTR reportedly is the first growth factor receptor found to be expressed by those fibroblasts that later develop into the DP of the HF.4 The p75NTR protein is a low-affinity NTR that binds all NTs with similar affinity. It facilitates development of specific populations of sympathetic neurons, for which it may support axon growth. The p75NTR is expressed on developing motor neurons and is re-expressed on adult motor neurons under pathological conditions such as nerve trauma or neurodegeneration.23-25 The strong expression of p75NTR protein in the nerve endings, in this investigation, concurs with previous studies26-28 and suggests an important role for p75NTR in the biology of the coetaneous nerves. Johansson et al18 examined
the localization and distribution of NGF and its low affinity receptor (p75NTR) in prurigo nodularis skin using NGF and p75NTR-NGF receptor double labeling. They found weak expression of these proteins in the epidermis and dermis of normal-appearing skin. However, in the dermis of prurigo nodularis, strong staining for both NGF and p75NTR-NGF receptor antibodies was seen. NGF receptor-immunoreactive nerves were denser in areas where there were more NGF-immunoreactive cells. These results suggest that NGF and p75NTR-NGF receptor may contribute to the neurohyperplasia in prurigo nodularis. Liang and Johansson26 examined the distribution of p75NTR in cutaneous nerve fibers (normal-appearing human skin) using both double immunofluorescence and immunoelectron microscopic studies. After immunofluorescence staining, the dermal nerves were strongly p75NTR immunoreactive with very few p75NTR nerve fibers seen in the epidermis. After ultrastructural immunostaining, the Schwann cell membrane was strongly p75NTR immunoreactive.
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These findingsethat in human cutaneous nerves it is mainly the Schwann cells that express p75NTR immunoreactivityefurther stress the critical role of the glial ensheathment in the control and maintenance of a normal-appearing skin innervation. Interestingly, mice bearing a null mutation in the p75 locus reportedly have pineal glands and SwG lacking innervation. The absence of adult innervation reflects the failure of axons to reach these targets during development rather than a target deficit.24 In conclusion, p75 was prominently expressed in human scalp skin (extrafollicular structures and stratum corneum) and HF (catagen and telogen) and showed hair cycle-dependent alterations, indicating its importance not only in murine, but in human skin and HF biology. The exact molecular interactions between p75 and other proapoptotic and prosurvival molecules during the HF cycling events are open for future studies. REFERENCES 1. Harry GJ, Lefebvre d’Hellencourt C, McPherson CA, Funk JA, Aoyama M, Wine RN. Tumor necrosis factor p55 and p75 receptors are involved in chemical-induced apoptosis of dentate granule neurons. J Neurochem 2008;106:281-98. 2. Botchkarev VA, Botchkareva NV, Peters EM, Paus R. Epithelial growth control by neurotrophins: leads and lessons from the hair follicle. Prog Brain Res 2004;146:493-513. 3. Adly MA, Assaf H, Hussein MR. Age-associated decrease of the nerve growth factor protein expression in the human skin: preliminary findings. J Dermatol Sci 2006;42:268-71. 4. Botchkareva 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;216:135-53. 5. Di Girolamo N, Sarris M, Chiu K, Cheema H, Coroneo MT, Wakefield D. Localization of the low affinity nerve growth factor receptor p75 in human limbal epithelial cells. J Cell Mol Med. Available at: http://www.blackwellpublishing.com/journal. asp?ref=1582-1838&site=1. Accessed February 24, 2008. 6. Peters EM, Stieglitz MG, Liezman C, Overall RW, Nakamura M, Hagen E, et al. p75 Neurotrophin receptor-mediated signaling promotes human hair follicle regression (catagen). Am J Pathol 2006;168:221-34. 7. Adly MA, Assaf HA, Hussein MR, Paus R. Analysis of the expression pattern of glial cell line-derived neurotrophic factor, neurturin, their cognate receptors GFRalpha-1 and GFRalpha-2, and a common signal transduction element cRet in the human scalp skin. J Cutan Pathol 2006;33:799-808. 8. Adly MA, Assaf HA, Hussein MR. Expression of the heat shock protein-27 in the adult human scalp skin and hair follicle: hair cycle-dependent changes. J Am Acad Dermatol 2006;54:811-7. 9. Adly MA, Assaf HA, Hussein MR. Expression of bone morphogenetic protein-7 in human scalp skin and hair follicles. Br J Dermatol 2006;154:551-4. 10. Adly MA, Assaf HA, Pertile P, Hussein MR, Paus R. Expression patterns of the glial cell line-derived neurotrophic factor, neurturin, their cognate receptors GFRalpha-1, GFRalpha-2, and a common signal transduction element c-Ret in the human skin hair follicles. J Am Acad Dermatol 2008;58:238-50.
11. Hussein MR, Haemel AK, Albert DM, Wood GS. Microsatellite instability and alterations of mismatch repair protein expression in choroidal melanomas. Arch Ophthalmol 2005;123:1705-11. 12. Adly MA, Assaf HA, Hussein MR, Neuber K. Age-associated decrease of CD1d protein production in normal human skin. Br J Dermatol 2006;155:186-91. 13. Botchkarev VA, Botchkareva NV, Albers KM, Chen LH, Welker P, Paus R. A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression. FASEB J 2000;14: 1931-42. 14. Peters EM, Hendrix S, Golz G, Klapp BF, Arck PC, Paus R. Nerve growth factor and its precursor differentially regulate hair cycle progression in mice. J Histochem Cytochem 2006;54:275-88. 15. Yaar M, Grossman K, Eller M, Gilchrest BA. Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J Cell Biol 1991;115:821-8. 16. Kristensen M, Chu CQ, Eedy DJ, Feldmann M, Brennan FM, Breathnach SM. Localization of tumor necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor. Clin Exp Immunol 1993;94:354-62. 17. Lopez SM, Perez-Perez M, Marquez JM, Naves FJ, Represa J, Vega JA. p75 and TrkA neurotrophin receptors in human skin after spinal cord and peripheral nerve injury, with special reference to sensory corpuscles. Anat Rec 1998;251:371-83. 18. Johansson O, Liang Y, Emtestam L. Increased nerve growth factor- and tyrosine kinase A-like immunoreactivities in prurigo nodularis skinean exploration of the cause of neurohyperplasia. Arch Dermatol Res 2002;293:614-9. 19. Dou YC, Hagstromer L, Emtestam L, Johansson O. Increased nerve growth factor and its receptors in atopic dermatitis: an immunohistochemical study. Arch Dermatol Res 2006;298: 31-7. 20. Liu PY, Bondesson L, Lontz W, Johansson O. The occurrence of cutaneous nerve endings and neuropeptides in vitiligo vulgaris: a case-control study. Arch Dermatol Res 1996;288: 670-5. 21. Raychaudhuri SP, Jiang WY, Raychaudhuri SK. Revisiting the Koebner phenomenon: role of NGF and its receptor system in the pathogenesis of psoriasis. Am J Pathol 2008;172:961-71. 22. Pincelli C, Marconi A. Autocrine nerve growth factor in human keratinocytes. J Dermatol Sci 2000;22:71-9. 23. Gschwendtner A, Liu Z, Hucho T, Bohatschek M, Kalla R, Dechant G, et al. Regulation, cellular localization, and function of the p75 neurotrophin receptor (p75NTR) during the regeneration of facial motoneurons. Mol Cell Neurosci 2003;24:307-22. 24. Lee KF, Bachman K, Landis S, Jaenisch R. Dependence on p75 for innervation of some sympathetic targets. Science 1994;263: 1447-9. 25. Jahed A, Kawaja MD. The influences of p75 neurotrophin receptor and brain-derived neurotrophic factor in the sympathetic innervation of target tissues during murine postnatal development. Auton Neurosci 2005;118:32-42. 26. Liang Y, Johansson O. Light and electron microscopic demonstration of the p75 nerve growth factor receptor in normal human cutaneous nerve fibers: new vistas. J Invest Dermatol 1998;111:114-8. 27. Bergman E, Ulfhake B, Fundin BT. Regulation of NGF-family ligands and receptors in adulthood and senescence: correlation to degenerative and regenerative changes in cutaneous innervation. Eur J Neurosci 2000;12:2694-706. 28. Sieber-Blum M, Szeder V, Grim M. The role of NT-3 signaling in Merkel cell development. Prog Brain Res 2004;146:63-72.