Expression and Function of Neurotrophins and Their Receptors in Cultured Human Keratinocytes
2003; Elsevier BV; Volume: 121; Issue: 6 Linguagem: Inglês
10.1111/j.1523-1747.2003.12624.x
ISSN1523-1747
AutoresM. Terracina, J. Franchi, Frédéric Bonté, Graziela Gorete Romagnoli, Riccardo Maurelli, Cristina Maria Failla, Marc Dumas, Alessandra Marconi, C. Fila, Carlo Pincelli,
Tópico(s)Advancements in Transdermal Drug Delivery
ResumoWhereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription–polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis. Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription–polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis. nerve growth factor neurotrophin neurotrophin-3 brain-derived neurotrophic factor neurotrophin-4/5 tyrosine kinase keratinocyte stem cell The neurotrophins (NT) consist of a family of four related polypeptide growth factors: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) (Lewin and Barde, 1996Lewin G.R. Barde Y.A. Physiology of neurotrophins.Ann Rev Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1734) Google Scholar). These structurally and functionally related molecules exert their effects by binding two different classes of transmembrane receptors. The p75 NT receptor binds all NT with low affinity (Chao et al., 1986Chao M.V. Bothwell M.A. Ross A.H. Koprowski H. Lanahan A.A. Buck C.R. Sehgal A. Gene transfer and molecular cloning of the human NGF receptor.Science. 1986; 232: 518-521Crossref PubMed Scopus (342) Google Scholar) and modulates signaling initiated by the second class of NT receptors, the trk family of 140 kDa high-affinity tyrosine kinase receptors (Barbacid, 1994Barbacid M. The trk family of neurotrophin receptors.J Neuroimmunol. 1994; 25: 1386-1403Google Scholar). TrkA, trkB, and trkC selectively bind NGF, BDNF, and NT-3, respectively, and trkB can also be activated by NT-4/5 (Dechant, 2001Dechant G. Molecular interactions between neurotrophin receptors.Cell Tissue Res. 2001; 305: 229-238Crossref PubMed Scopus (91) Google Scholar). NT play a prominent role in the development of the vertebrate nervous system, by influencing differentiation, survival, and cell death of neurons (Snider, 1994Snider W.D. Functions of the neurotrophins during nervous system development: What the knockouts are teaching us.Cell. 1994; 77: 627-638Abstract Full Text PDF PubMed Scopus (1283) Google Scholar). NT can also act on non-neuronal tissues. It has been shown that NGF activates mast cells (Kawamoto et al., 2002Kawamoto K. Aoki J. Tanaka A. et al.Nerve growth factor activates mast cells through the collaborative interaction with lysophosphatidylserine expressed on the membrane surface of activated platelets.J Immunol. 2002; 168: 6412-6419Crossref PubMed Scopus (84) Google Scholar) and prevents their apoptosis (Kanbe et al., 2000Kanbe N. Kurosawa M. Miyachi Y. Kanbe M. Saitoh H. Matsuda H. Nerve growth factor prevents apoptosis of cord blood-derived human cultured mast cells synergistically with stem cell factor.Clin Exp Allergy. 2000; 30: 1113-1120Crossref PubMed Scopus (54) Google Scholar). Moreover, NGF stimulates the proliferation of human microvascular endothelial cells (Raychaudhuri et al., 2001Raychaudhuri S.K. Raychaudhuri S.P. Weltman H. Farber E.M. Effect of nerve growth factor on endothelial cell biology: Proliferation and adherence molecule expression on human dermal microvascular endothelial cells.Arch Dermatol Res. 2001; 293: 291-295Crossref PubMed Scopus (103) Google Scholar), whereas it is an autocrine survival factor for lymphocytes (Torcia et al., 1996Torcia M. Bracci-Laudiero L. Lucibello M. et al.Nerve growth factor is an autocrine survival factor for memory B lymphocytes.Cell. 1996; 85: 345-356Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Normal human keratinocytes synthesize and release high amounts of biologically active NGF. In addition, human keratinocytes express both the low-affinity (p75) and the high-affinity (trkA) NGF receptors (Pincelli et al., 1994Pincelli C. Sevignani C. Manfredini R. et al.Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.J Invest Dermatol. 1994; 103: 13-18Abstract Full Text PDF PubMed Google Scholar). NGF is the key player of an autocrine loop, acting as a mitogen and as a survival factor for human keratinocytes (Di Marco et al., 1993Di Marco E. Mathor M. Bondanza S. Cutuli N. Marchisio P.C. De Cancedda R. Luca M. Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.J Biol Chem. 1993; 268: 22838-22846Abstract Full Text PDF PubMed Google Scholar;Pincelli et al., 1997Pincelli C. Haake A.R. Benassi L. et al.Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (trk): A role for bcl-2.J Invest Dermatol. 1997; 109: 757-764Crossref PubMed Scopus (88) Google Scholar). In particular, autocrine NGF protects human keratinocytes from ultraviolet (UV)-induced apoptosis (Marconi et al., 1999Marconi A. Vaschieri C. Zanoli S. Giannetti A. Pincelli C. Nerve growth factor protects human keratinocytes from ultraviolet-B-induced apoptosis.J Invest Dermatol. 1999; 113: 920-927Crossref PubMed Scopus (39) Google Scholar). Moreover, NGF released from keratinocytes exerts paracrine functions on human melanocytes by stimulating their dendricity (Yaar et al., 1991Yaar M. Grossman K. Eller M. Gilchrest B.A. Evidence for nerve growth factor-mediated paracrine effects in human epidermis.J Cell Biol. 1991; 115: 821-828Crossref PubMed Scopus (165) Google Scholar) and protecting them from cell death (Pincelli and Yaar, 1997Pincelli C. Yaar M. Nerve growth factor: Its significance in cutaneous biology.J Investig Dermatol Symp Proc. 1997; 2: 31-36Abstract Full Text PDF PubMed Scopus (68) Google Scholar). Whereas NGF is a crucial neurotrophic molecule for skin innervation (Albers et al., 1994Albers K.M. Wright D.E. Davis B.M. Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system.J Neurosci. 1994; 14: 1422-1432PubMed Google Scholar), the above findings strongly indicate that NGF is to be regarded as an important growth factor in human epidermis as well (Pincelli and Marconi, 2000aPincelli C. Marconi A. Autocrine nerve growth factor in human keratinocytes.J Dermatol Sci. 2000; 22: 71-79Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In this respect, NGF could be supported by other members of the NT family, although little is known about these molecules in human skin (Yaar et al., 1994Yaar M. Eller M.S. DiBenedetto P. et al.The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.J Clin Invest. 1994; 94: 1550-1562Crossref PubMed Scopus (122) Google Scholar;Grewe et al., 2000Grewe M. Vogelsang K. Ruzicka T. Stege H. Krutmann J. Neurotrophin-4 production by human epidermal keratinocytes: Increased expression in atopic dermatitis.J Invest Dermatol. 2000; 114: 1108-1112Crossref PubMed Scopus (103) Google Scholar). This study reports a comprehensive analysis of the expression of all NT family members as well as of their receptors in normal human keratinocytes. In particular, we provide evidence of the mitogenic role for autocrine NT-3 in these cells and report the different sensitivity of NT to UV light. Finally, we describe an inter-regulation of NT among each other. Human (in vivo) studies and all animal studies were approved by the Institutional Review Board. Normal human keratinocytes were obtained from foreskin and cultured as described (Pincelli et al., 1997Pincelli C. Haake A.R. Benassi L. et al.Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (trk): A role for bcl-2.J Invest Dermatol. 1997; 109: 757-764Crossref PubMed Scopus (88) Google Scholar) after informed consent. Briefly, keratinocytes were plated on mytomycin C-treated 3T3 cells (2.4×104 per cm2, ATCC, Rockville, Maryland) and cultivated in Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12, 3:1 ratio) media (Seromed-Biochrom, Berlin, Germany). Subconfluent secondary cultures were trypsinized with 0.05% trypsin/0.02% ethylenediamine tetraacetic acid (Seromed) and replated for the experiments in DMEM/F12 on 3T3 cells or in defined serum-free medium (1×104 per cm2; keratinocyte growth medium (KGM), Clonetics Corp., San Diego, California) without hydrocortisone. In order to enrich cell population in keratinocyte stem cells (KSC), keratinocytes were divided into three populations and cultured in serum-free medium. They were first allowed to adhere to type IV collagen (Sigma, St Louis, Missouri) for 5 min (population 1, KSC), and the nonadherent cells were then transferred to fresh collagen-coated dishes and allowed to attach overnight (population 2, young transit amplifying cells, TA). Finally, keratinocytes not yet attached after one night were plated on to type IV collagen to obtain a third population (population 3, TA cells). Populations were cultured for 5 d up to subconfluency. The three subpopulations were characterized according to the β1 integrin expression and colony-forming efficiency as described (Tiberio et al., 2002Tiberio R. Marconi A. Fila C. et al.Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner.FEBS Lett. 2002; 524: 139-144Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). UV radiation was delivered with a battery of TL 20W/12 RS lamps for UVB and TL 20W/09N for UVA (Philips Medical Brakel, The Netherlands). Before irradiation, keratinocytes were washed once with phosphate-buffered saline and irradiated in the presence of phosphate-buffered saline. Controls were sham irradiated for identical periods. Doses were measured by Goldilux Smart Meter (Oriel Instruments, Stratford, Connecticut) right before irradiation. Total cellular RNA was extracted from cultures using TRI Reagent method performed as described by Sigma. One microgram of total cellular RNA extracted was reverse-transcribed and amplified as described (Pincelli et al., 1994Pincelli C. Sevignani C. Manfredini R. et al.Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.J Invest Dermatol. 1994; 103: 13-18Abstract Full Text PDF PubMed Google Scholar) with a DNA Thermal Cycler (Perkin-Elmer, Norwalk, Connecticut). In order to perform a semiquantitative evaluation of mRNA, β-actin was used as a house-keeping gene. The relative intensity of bands on autoradiograms was quantitated by scanning laser densitometry. The linearity of PCR reaction was obtained by plotting values from densitometric analysis in each band versus the cDNA concentration. Nucleotide sequences of the oligomers (MWG Biotech, Ebersberg, Germany) used were as follows: NGF: 5′-TCATCATCCCATCCCATCTT-3′ 5′-CTTGACAAAGGTGTGAGTCG-3′ (nucleotides: 533–547 and 777–796; fragment: 264 bp; 28 cycles); BDNF: 5′-AGCCTCCTCTGCTCTTTCTGCTGGA-3′, 5′-CTTTTGT-CTATGCCCCTGCAGCCTT-3′ (nucleotides: 870–894 and 1143–1167; fragment: 298 bp); NT-3: 5′-TTTCTCGCTTATCTCCGTGGCATCC-3′, 5′-GGCAGGGTGCTCTGGTAATTTTCCT-3′ (nucleotides 100–124 and 243–266; fragment: 167 bp); NT-4/5: 5′-ATGCTCCCTCTCCCC-TCAT-3′, 5′-GCATGGGTCTCAGGCCCG3′ (nucleotides 475–493 and 1099–1116; fragment: 642 bp); trkA: 5′-GGCTCCTCGGGACTGCGATG-3′, 5′-CAGGAGAGAGACTCCAGAGCG-3′ (nucleotides 214–233 and 459–479; fragment: 266 bp); trkB: 5′-CCGCTAGGATTTGGTGTACT-GAGCCTTCT-3′, 5′ CCACTGTCATCAGATGAAATGTTCGTTATC-CT-3′ (nucleotides 630–658 and 1262–1293; fragment: 664 bp), intra-cellular trkB: 5′-TCGCAGATGCTGCATATAGC-3′ 5′-ATCAGCTCATA-CACCTCCTC-3 (nucleotides 2481–2500 and 2868–2887; fragment: 407 bp); extracellular truncated trkB: 5′-GACACTCAGGATTTGTACTGCC-3′, 5′-TCCGTGTGATTGGTAACATGTATT-3′ (nucleotides 990–1011 and 1481–1504; fragment: 515 bp); trkC: 5′-GGAAAGGTCTTCCTGGCCG-AGTGC-3′, 5′-GCTTTCCATAGGTGAAGATCTCCC-3′ (nucleotides 1803–1826 and 2395–2418; fragment 616 bp); β-actin: 5′-TGGAT GATG-ATATCGCCGCGCTCG-3′, 5′-CACATAGGAATCCTTCTGACCCA-3′ (nucleotides 75–98 and 213–235; fragment 161 bp; 25 cycles). The PCR was carried out at least three times for each sample. No reverse-transcribed mRNA and buffer without template were used as controls. Keratinocytes were cultivated both in KGM and on a 3T3 feeder layer. In the latter case, the feeder layer was removed prior to keratinocyte lysis. NGF, BDNF, NT-3, and NT-4/5 quantitation was performed by a two-site enzyme immunoassay (Quantikine, Promega Madison WI) according to manufacturer's instructions. The samples concentration was determined by absorbance at 540 nm against recombinant human NT standard protein. Keratinocytes (6000 cells per well) were grown on 96-well plates in KGM. Forty-eight hours after seeding medium was changed and NT (Sigma) or anti-NT-3 neutralizing antibody (Bertollini et al., 1997Bertollini L. Ciotti M.T. Cherubini E. Cattaneo A. Neurotrophin-3 promotes the survival of oligodendrocyte precursors in embryonic hippocampal cultures under chemically defined conditions.Brain Res. 1997; 746: 19-24Crossref PubMed Scopus (23) Google Scholar) were added to the cultures. Forty-eight hours after addition of NT, 3H thymidine (1 mCi per well, Amersham-Pharmacia Biotech, Rainham, UK) incorporation was performed and cells collected 12 h later. The incorporated radioactivity was determined by β-counter. Keratinocytes were seeded at a density of 1×104 per cm2. Forty-eight hours later, cells were preincubated with NT (100 ng per mL), 48 h before UVB (50 mJ per cm2) or sham irradiation. UVB radiation was delivered with a battery of lamps (TL20W/12 RS UVB Philips Medical). Keratinocytes were directly stained on chamber slides by the "In situ cell death detection kit" (Roche Diagnostics, Mannheim, Germany), as described (Pincelli et al., 1997Pincelli C. Haake A.R. Benassi L. et al.Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (trk): A role for bcl-2.J Invest Dermatol. 1997; 109: 757-764Crossref PubMed Scopus (88) Google Scholar). Negative control was obtained by replacing the primary incubation with a nucleotide mixture without TdT. Fluorescent specimens were analyzed by a Confocal Scanning Laser Microscopy (Leica TCS4D) in conjunction with a conventional optical mxicroscope (Leica DM IRBE). As NGF levels are highest in proliferating keratinocytes and tend to disappear with keratinocyte differentiation (Di Marco et al., 1993Di Marco E. Mathor M. Bondanza S. Cutuli N. Marchisio P.C. De Cancedda R. Luca M. Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.J Biol Chem. 1993; 268: 22838-22846Abstract Full Text PDF PubMed Google Scholar;Pincelli et al., 1994Pincelli C. Sevignani C. Manfredini R. et al.Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.J Invest Dermatol. 1994; 103: 13-18Abstract Full Text PDF PubMed Google Scholar), we evaluated NT expression in preconfluent keratinocytes. Preconfluent keratinocytes express NT-3, BDNF, and NT-4/5 mRNA (Figure 1). We also evaluated trk expression in secondary cultures prior to divisions into subpopulations. Preconfluent keratinocytes express trkA and trkC, but do not express the full-length trkB receptor (Figure 2a). By contrast, keratinocytes express the truncated isoform of trkB, lacking the intracellular domain (Figure 2b). Keratinocytes release NGF, NT-3, NT-4/5, and BDNF, even though in low amounts. NGF levels are 5-fold more elevated than the other NT in supernatants and tend to be significantly higher in supernatants than in cell lysates (Figure 3). NGF mRNA is highest in KSC, whereas they tend to decrease in young TA cells and they almost disappear in TA cells (Figure 4a). ELISA assay confirms that NGF is released in higher amounts from KSC as compared with young TA and TA cells. On the other hand, whereas all NT are almost undetectable in TA cells, there is no difference in NT-3, NT-4/5, and BDNF between KSC and young TA cells (Figure 4b). We first tested the toxic effect of NT in human keratinocytes. Table I shows that NT do not exert toxic effects neither affect cell viability at 24 and 72 h. In order to analyze the functions of NT other than NGF in human keratinocytes, we first treated preconfluent keratinocytes with increasing concentrations of NT. Our results show that NT-3 stimulates keratinocyte proliferation. The effect is statistically significant only with 100 ng per mL. This effect is specifically blocked by addition of decreasing dilutions of anti-NT-3 neutralizing antibody, in the absence of exogenous NT-3. Indeed, anti-NT-3 treatment inhibits keratinocyte proliferation, indicating that autocrine NT-3 promotes keratinocyte proliferation. By contrast, BDNF and NT-4/5 do not exert any effect on keratinocyte proliferation (Figure 5).Table ITrypan blue 24h %+SDNeutral red 24h %+SDTrypan blue 72h %+SDNeutral red 72h %+SDcntrl95.4+4.50.41+0.0493.2+5.50.69+0.07NGF97.6+3.70.42+0.0694.6+3.20.71+0.06NT396.2+4.10.40+0.0996.0+2.60.77+0.08NT494.6+5.10.49+0.0490.5+4.10.75+0.03BDNF93.7+7.50.46+0.0391.0+4.50.68+0.05 Open table in a new tab Whereas NT-4/5 and BDNF do not affect other NT release, NGF strikingly upregulates NT-3 release. NT-3 in turn slightly increases NGF secretion (Figure 6). We have previously reported that UVB strikingly downregulates the expression of NGF mRNA and the secretion of NGF in keratinocytes. In order to evaluate the effect of UV light on NT secretion, keratinocytes were exposed to UVB and UVA irradiation and NT were measured. As expected, UVB diminishes NGF secretion in both cell lysates and cultured supernatants in a concentration-dependent manner. By contrast, UVB significantly augments NT-3 and NT-4/5 expression and release, whereas BDNF is not affected (Figure 7a,b). UVA irradiation increases NT-3 but not NGF release in both cell lysates and cultured supernatants. Whereas BDNF is slightly increased by UVA irradiation, NT-4/5 is minimally augmented with 3 J per cm2 only in cell lysates. (Figure 7c,d). NT exert different effects on neuronal cell survival (Casaccia-Bonefil et al., 1998Casaccia-Bonefil P. Kong H. Chao M.V. Neurotrophins: The biological paradox of survival factors eliciting apoptosis.Cell Death Differ. 1998; 5: 357-364Crossref PubMed Scopus (116) Google Scholar), whereas NGF over-expression protects human keratinocytes from UVB-induced apoptosis (Marconi et al., 1999Marconi A. Vaschieri C. Zanoli S. Giannetti A. Pincelli C. Nerve growth factor protects human keratinocytes from ultraviolet-B-induced apoptosis.J Invest Dermatol. 1999; 113: 920-927Crossref PubMed Scopus (39) Google Scholar). We first evaluated the effect of NT on keratinocyte apoptosis. NT do not seem to influence cell death in these cells (Figure 8a). We then analyzed the function of NT in relation to UVB-induced apoptosis. Keratinocytes were sham or UVB irradiated after the addition of NT. Whereas NGF exerts a protective effect against UVB-induced apoptosis, the other NT do not prevent cell death (Figure 8b). Keratinocytes are both the source and the target of a number of cytokines and growth factors that contribute to epidermal proliferation, differentiation and apoptosis (Nickoloff et al., 1995Nickoloff B.J. Turka L.A. Mitra R.S. Nestle F.O. Direct and indirect control of T-cell activation by keratinocytes.J Invest Dermatol. 1995; 105: 25S-29SCrossref PubMed Scopus (49) Google Scholar;Bernstein and Vaughan, 1999Bernstein I.A. Vaughan F.L. Cultured keratinocytes in in vitro dermatotoxicological investigation: A review.J Toxicol Environ Health B Crit Rev. 1999; 2: 1-30Crossref PubMed Scopus (25) Google Scholar). NGF is one of these growth factors: blockade of endogenous NGF inhibits keratinocyte proliferation and induces apoptosis, indicating that an important autocrine loop exists in the epidermis, where NGF plays a crucial part (Pincelli and Marconi, 2000bPincelli C. Marconi A. Keratinocyte nerve growth factor. more than just a neurotrophin.in: Suzuki H. Ono T. Merkel Cell, Merkel Cell Carcinoma and Neurobiology of the Skin. Elsevier, Elsevier, 2000: 181-189Google Scholar). This study demonstrates that NGF is synthesized and released by human keratinocytes in greater amounts than other NT. Moreover, NGF appears to be expressed and secreted in higher levels by KSC cells as compared with more differentiated cells. This is intriguing in view of the fact that KSC are protected from cell death (Tiberio et al., 2002Tiberio R. Marconi A. Fila C. et al.Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner.FEBS Lett. 2002; 524: 139-144Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and one could speculate that autocrine NGF exerts its anti-apoptotic activity mostly on this keratinocyte subpopulation. On the other hand, it is interesting to note that the other NT, which are not elevated in KSC, do not exert anti-apoptotic activity. We report that other NT family members are expressed in keratinocytes and could participate in a novel "neurotrophin network" within the epidermis. We present evidence that human keratinocytes synthesize and secrete all of the NT. Whereas others have reported that human keratinocytes fail to synthesize NT-3 (Grewe et al., 2000Grewe M. Vogelsang K. Ruzicka T. Stege H. Krutmann J. Neurotrophin-4 production by human epidermal keratinocytes: Increased expression in atopic dermatitis.J Invest Dermatol. 2000; 114: 1108-1112Crossref PubMed Scopus (103) Google Scholar), the presence of this NT is confirmed in the current study not only by detection of RNA but also by protein release. Whereas all of the NT are synthesized in human keratinocytes, only low levels of NT protein are detected, suggesting that release in the medium only takes place under certain circumstances and upon various stimuli. NT storage and release is a matter of debate also in the nervous system: it appears that NT undergo regulated secretion in response to depolarization by high potassium (Blochl and Thoenen, 1995Blochl A. Thoenen H. Characterization of nerve growth factor (NGF) release from hippocampal neurons: Evidence for a constitutive and an unconventional sodium-dependent regulated pathway.Eur J Neurosci. 1995; 7: 1220-1228Crossref PubMed Scopus (293) Google Scholar;Wang and Poo, 1997Wang X.H. Poo M.M. Potentiation of developing synapses by post-synapsis release of neurotrophin-4.Neuron. 1997; 19: 825-835Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar;Franke et al., 2000Franke B. Bayatti N. Engele J. Neurotrophins require distinct extracellular signals to promote the survival of CNS neurons in vitro.Exp Neurol. 2000; 165: 125-135Crossref PubMed Scopus (33) Google Scholar) or in response to calcium (Canossa et al., 1997Canossa M. Griesbeck O. Berninger B. Campana G. Kolbeck R. Thoenen H. Neurotrophin release by neurotrophins: Implications for activity-dependent neuronal plasticity.Proc Natl Acad Sci USA. 1997; 94: 13279-13286Crossref PubMed Scopus (228) Google Scholar;Kang and Schuman, 2000Kang H. Schuman E.M. Intracellular Ca(2+) signaling is required for neurotrophin-induced potentiation in the adult rat hippocampus.Neurosci Lett. 2000; 282: 141-144Crossref PubMed Scopus (45) Google Scholar). In preconfluent, still proliferating keratinocytes, used in this study, the K+ channel modulating Ca2+ influx is not yet active (Mauro et al., 1997Mauro T. Dixon D.B. Komuves L. Hanley K. Pappone P.A.J. Keratinocyte K+ channels mediate Ca2+-induced differentiation.J Invest Dermatol. 1997; 108: 864-870Crossref PubMed Scopus (49) Google Scholar). It is thus feasible that, under these conditions, NT release is not allowed. Moreover, it is possible that NT secretion in keratinocytes occurs only in denervated areas, such as in diabetic skin, as a compensatory mechanism (Kennedy et al., 1998Kennedy A.J. Wellmer A. Facer P. Saldanha G. Kopelman P. Lindsay R.M. Anand P. Neurotrophin-3 is increased in skin in human diabetic neuropathy.J Neurol Neurosurg Psychiatry. 1998; 65: 393-395Crossref PubMed Scopus (38) Google Scholar). Finally, NT, secreted either by keratinocytes themselves or possibly by other skin cells, may induce the release of other NT through the regulated secretory pathway, such as in the nervous system (Kruttgen et al., 1998Kruttgen A. Carsten Moller J. Heymach J.V. Shooter E.M. Neurotrophins induce release of neurotrophins by the regulated secretory pathway.Proc Natl Acad Sci USA. 1998; 95: 9614-9619Crossref PubMed Scopus (105) Google Scholar). Indeed, in this study, NGF and NT-3 stimulate each other release, possibly contributing to the amplification of the autocrine loop where both NT play a crucial part in keratinocyte proliferation. The expression of trk receptors in human keratinocytes has been reported previously. There is agreement on the localization of trkA that, with different techniques, has been shown to be expressed only in the basal epidermal layer (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;Terenghi et al., 1997Terenghi G. Mann D. Kopelman P.G. Anand P. Trk A and trk C expression is increased in human diabetic skin.Neurosci Lett. 1997; 228: 33-36Crossref PubMed Scopus (49) Google Scholar). This finding has been confirmed in a previous work by reverse transcription–PCR performed on proliferative basal keratinocytes and supports the role of this tyrosine kinase receptor in the autocrine survival loop sustained by NGF in human keratinocytes (Pincelli, 2000Pincelli C. Nerve growth factor and keratinocytes: A role in psoriasis.Eur J Dermatol. 2000; 10: 85-90PubMed Google Scholar). By contrast, whereas trkC mRNA was detected in basal keratinocytes (Terenghi et al., 1997Terenghi G. Mann D. Kopelman P.G. Anand P. Trk A and trk C expression is increased in human diabetic skin.Neurosci Lett. 1997; 228: 33-36Crossref PubMed Scopus (49) Google Scholar), immunohistochemical studies have not confirmed the protein expression in the same location (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). The present study provides evidence that proliferating human keratinocytes express trkC at the mRNA level, whereas NT-3 stimulation of keratinocyte proliferation supports the expression of the functional receptor protein. Failure of BDNF or NT-4/5 to exert any activity on keratinocytes confirms the absence of the full-length trkB in these cells. In fact, we show that human keratinocytes only express the truncated isoform of trkB, which has been postulated to act as a dominant inhibitory modulator of trkB signaling (Kryl and Barker, 2000Kryl D. Barker P.A. TTIP is a novel protein that interacts with the truncated T1 trk B neurotrophin receptor.Biochem Biophys Res Commun. 2000; 279: 925-930Crossref PubMed Scopus (23) Google Scholar). On the other hand, BDNF might induce cell cycle arrest or apoptosis in human keratinocytes expressing the low-affinity NT receptor p75, which mediates these effects in most cell systems (Bamji et al., 1998Bamji S.X. Majdan M. Pozniak C.D. et al.The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally-occurring sympathetic neuron death.J Cell Biol. 1998; 140: 911-923Crossref PubMed Scopus (432) Google Scholar). NT and NT receptors seem to be differently expressed in human and mouse skin. The work on NT carried out in mouse skin has shown that p75 is not expressed in basal epidermal keratinocytes (Botchkarev et al., 1998Botchkarev 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), at variance with human epidermis (Fantini and Johansson, 1992Fantini F. Johansson O. Expression of growth-associated protein 43 and nerve growth factor receptor in human skin: A comparative immunohistochemical investigation.J Invest Dermatol. 1992; 99: 734-742Abstract Full Text PDF PubMed Google Scholar). With respect to the trk receptors, murine epidermal keratinocytes express trkB but not trkC. In addition, no NT immunoreactivity is observed in nonhairy murine keratinocytes (Botchkarev et al., 1998Botchkarev 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). It would appear that, in the mouse system, NT are derived mostly from nerve fibers or dermal cells and are involved in the hair cycle control (Botchkarev et al., 1999bBotchkarev V.A. Botchkareva N.V. Welker P. et al.A new role for neurotrophins: Involvement of brain-derived neurotrophic factor and neurotrophin-4 in haircycle control.FASEB J. 1999; 13: 395-410PubMed Google Scholar,Botchkarev et al., 2000Botchkarev V.A. Botchkareva N.V. Albers L.M. Chen L.H. Welker P. Paus R. A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.FASEB J. 2000; 14: 1931-1942Crossref PubMed Scopus (79) Google Scholar). On the contrary, NT seem to play a more important part in the human system as growth factors. Autocrine NT-3, similarly to NGF, stimulates keratinocyte proliferation, most likely through trkC and trkA receptors, suggesting that these NT, also by upregulating each other release, can co-operate in the maintenance of epidermal homeostasis. As trkB is not functioning in human keratinocytes, it remains to be established whether NT-4/5 exerts paracrine activities on other cell types. With respect to apoptosis, it is interesting to note that only NGF is downregulated by UVB and protects keratinocytes from UVB-induced cell death, as previously reported (Marconi et al., 1999Marconi A. Vaschieri C. Zanoli S. Giannetti A. Pincelli C. Nerve growth factor protects human keratinocytes from ultraviolet-B-induced apoptosis.J Invest Dermatol. 1999; 113: 920-927Crossref PubMed Scopus (39) Google Scholar). On the other hand, other NT are not reduced by UVB. This confirms that, among NT, NGF only can be considered as "the survival factor" for human keratinocytes. More detailed studies utilizing keratinocytes from NT-3/trkC-knock-out mice are necessary, however, to rule out the possibility that this NT is actually not involved in the protection of keratinocytes from UVB-induced apoptosis. In conclusion, this study describes a network of NT that, together with their receptors, could act both in an autocrine and in a paracrine manner in human epidermis. Ongoing studies will clarify which cells other than keratinocytes are the target and/or the source of NT in human skin (Figure 9). If the observation of a relationship between NGF and keratinocyte-derived cytokines (Pincelli, 2000Pincelli C. Nerve growth factor and keratinocytes: A role in psoriasis.Eur J Dermatol. 2000; 10: 85-90PubMed Google Scholar) will be confirmed for other NT, one could envisage a complex system whereby epidermal NT and cytokines co-operate in the control of skin homeostasis and inflammation. We would like to thank MIUR (Ministero della Istruzione, della Università e della Ricerca) and the "Angela Serra" Association for Cancer Research for partially supporting this study.
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