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Autocrine nerve growth factor in human keratinocytes
Abstract
Biologically active nerve growth factor (NGF) is synthesised and released by proliferating normal human keratinocytes. NGF up-regulates the expression of NGF mRNA in keratinocytes. Keratinocytes express both the low (p75)- and the high-affinity (TrkA) NGF-receptors, which are located in the basal layer of the epidermis. K252, a specific inhibitor of trk phosphorylation, blocks NGF-induced keratinocyte proliferation, in absence of exogenous NGF. Normal keratinocytes over-expressing TrkA proliferate better than control transfectants, while the NGF mimicking anti-Trk antibody induces an increased keratinocyte proliferation in Trk over-expressing cells as compared to mock transfected keratinocytes. In addition, NGF over-expressing keratinocytes proliferate better than mock transfected cells. K252, by blocking TrkA phosphorylation, induces apoptosis in normal keratinocytes, but not in keratinocytes over-expressing bcl-2. Furthermore, NGF transfected keratinocytes are protected from UV-B-induced keratinocyte apoptosis, by maintaining constant levels of Bcl-2 and Bcl-xL . Taken together these results support the concept of an autocrine survival system sustained by NGF and its high-affinity receptor in human keratinocytes. Because NGF and Trk levels are highly expressed in psoriasis. one could speculate that NGF autocrine system plays a role in the mechanisms associated with this and other hyperproliferative skin conditions, including cancer.
... As a member of the family of neurotrophins, NGF regulates the survival and differentiation of peripheral sensory and sympathetic neurons, and the synthesis of neuropeptides and neurotransmitters (Yaping et al., 2006). NGF acts on a low-affinity p75 receptor and a high-affinity tropomyosin-related kinase receptor A (TrkA) (Pincelli and Marconi, 2000). ...
... Keratinocytes (KCs) is a major source of NGF in the epidermis (Di Marco et al., 1991;Pincelli et al., 1994;Pincelli and Marconi, 2000). In addition to neuropeptides, TNFα and histamine could stimulate the production of NGF from KCs via the Raf/MEK/ERK pathway (Woolf et al., 1997;Takaoka et al., 2009;Wijaya et al., 2020a,b). ...
... In addition to neuropeptides, TNFα and histamine could stimulate the production of NGF from KCs via the Raf/MEK/ERK pathway (Woolf et al., 1997;Takaoka et al., 2009;Wijaya et al., 2020a,b). Human KCs expressed both p75 and TrkA receptors, suggesting the existence of an autocrine and/or paracrine system (Pincelli et al., 1994;Pincelli and Marconi, 2000). TrKA receptors were primarily located in stratum basale, where they stimulated proliferation and prevented apoptosis of KCs upon activation (Pincelli et al., 1994;Bronzetti et al., 1995;Marconi et al., 1999;Pincelli and Marconi, 2000). ...
Keratinocytes are the predominant block-building cells in the epidermis. Emerging evidence has elucidated the roles of keratinocytes in a wide range of pathophysiological processes including cutaneous nociception, pruritus, and inflammation. Intraepidermal free nerve endings are entirely enwrapped within the gutters of keratinocyte cytoplasm and form en passant synaptic-like contacts with keratinocytes. Keratinocytes can detect thermal, mechanical, and chemical stimuli through transient receptor potential ion channels and other sensory receptors. The activated keratinocytes elicit calcium influx and release ATP, which binds to P2 receptors on free nerve endings and excites sensory neurons. This process is modulated by the endogenous opioid system and endothelin. Keratinocytes also express neurotransmitter receptors of adrenaline, acetylcholine, glutamate, and γ-aminobutyric acid, which are involved in regulating the activation and migration, of keratinocytes. Furthermore, keratinocytes serve as both sources and targets of neurotrophic factors, pro-inflammatory cytokines, and neuropeptides. The autocrine and/or paracrine mechanisms of these mediators create a bidirectional feedback loop that amplifies neuroinflammation and contributes to peripheral sensitization.
... NGF exerts its effect by binding two classes of transmembrane receptors, a low affinity receptor of ∼ 75 kDa (p75) and a high affinity tyrosine kinase receptor of ∼ 140 kDa (TrkA). While TrkA can mediate NGF-induced effects in the absence of p75, the functional significance of p75 is still unclear (Pincelli & Marconi 2000). ...
... NGF acts as a survival factor for many cell types; for example, NGF rescues memory B lymphocytes from cell death and protects skin melanocytes from ultraviolet (UV)-induced apoptosis (Lotan & Lotan 1981, Cosgaya et al. 1996, Islam et al. 2000, Pincelli & Marconi 2000. Autocrine NGF can rescue keratinocytes from apoptosis (Pincelli & Marconi 2000). ...
... NGF acts as a survival factor for many cell types; for example, NGF rescues memory B lymphocytes from cell death and protects skin melanocytes from ultraviolet (UV)-induced apoptosis (Lotan & Lotan 1981, Cosgaya et al. 1996, Islam et al. 2000, Pincelli & Marconi 2000. Autocrine NGF can rescue keratinocytes from apoptosis (Pincelli & Marconi 2000). Thus, the paracrine NGF from keratinocytes might have a major role in regulating innervation, lymphocyte function, melanocytes growth, and differentiation in epidermal morphogenesis as well as in wound healing (Lotan & Lotan 1981, Cosgaya et al. 1996, Islam et al. 2000, Pincelli & Marconi 2000. ...
Retinoic acid (RA) inhibited proliferation of cultured human keratinocytes. Southern blot analysis at 24 h showed that RA inhibited nerve growth factor (NGF) mRNA synthesis in a dose-dependent manner. However, RA stimulated the production of NGF protein up to 187% of control after 96 h and the treatment of cells with RA did not enhance apoptosis, either in the presence of low or high concentration of Ca2+, when compared to the control with DMSO.
... Le NGF serait également le ligand préférentiel de TrkE et stimulerait la croissance kératinocytaire par un mécanisme autocrine (46). Les kératinocytes des couches basales épidermiques expriment les deux récepteurs du NGF et une hyperexpression de TrkA est observée dans certaines dermatoses comme le psoriasis (147). La stimulation, in vitro, des cellules basales (127) réarrangements somatiques du gène humain trk (7). ...
... Le récepteur TrkA est exprimé par les précurseurs des lignées hématopoïétiques (29, 34, 88, 99), dans les cellules folliculaires dendritiques de ganglions réactionnels (64) et dans les cellules réticulaires interdigitées ganglionnaires et thymiques (141). Il est également retrouvé dans l'épithélium amygdalien et dans les cellules épithéliales médullaires et sous-capsulaires thymiques (99), dans les cellules basales conjonctivales (17) et épidermiques(147). In vitro, il est exprimé dans les monocytes (57), les mastocytes(176), les lymphocytes B, les lymphocytes T activés (58), ainsi que dans les immunoblastes présents dans les adénites suppurées de patients infectés par le VIH (99). ...
Le Nerve Growth Factor (NGF), chef de file de la famille des neurotrophines, protéine essentielle à la survie, au développement et à la différenciation des neurones se lie à deux récepteurs p75[exposant NTR](Neurotrophin Receptor) et TrkA (Tropomyosin-related kinase). Ces molécules ont récemment été identifiées dans le tissu lymphoïde humain normal ou pathologique. Dans une étude anatomoclinique rétrospective de 25 lymphomes folliculaires (LF), nous avons recherché l'existence et l'intensité d'expression de ces molécules. Les techniques immunohistochimiques (HIC) ont été réalisées sur des tissus congelés ou fixés dans du formol. Elles ont fait appel à des montages en troiscouches utilisant la streptavidine-biotine, à des méthodes d'amplification par la tyramine biotinylée ainsi qu'à des systèmes de révélation de l'anticorps primaire par la peroxydase ou la phosphatase alcaline. Les transcrits ont été recherchés par RT-PCR. Avec ces techniques, nous n'avons pu mettre en évidence que les transcrits de GF. En IHC, le NGF et ses récepteurs étaient exprimésdans les cellules du micro-environnement. L'expression du NGF était otiservée dans les cellules réticulaires interdigitées ; celle de p75[exposant NTR] dans les filets nerveux périvasculaires et sous-capsulaires, les cellules endothéliales et les cellules folliculaires dendritiques ; et celle de TrkA dans les cellulesréticulaires interdigitées, les cellules endothéliales et les cellules folliculaires dendritiques. En revanche, l'expression de NGF, p75[exposant NTR] et TrkA a été observée pour la première fois, in vivo, dans les cellules tumorales. Aucune corrélation n'a été observée entre le niveau d'expression du NGFavec les critères péjoratifs habituels des LF (le stade d'Ann Arbor au moment du diagnostic, l'élévation sérique du taux de [bêta]2-microglobuline ou des LDH, l'existence de symptômes B, d'une altération de l'état général ou d'une «forte» masse tumorale) ou avec l'immunoréactivité P53, Bcl2, Bcl6 et Ki67. En revanche, l'hyperexpression de p75[exposant NTR] était corrélée avec le stade d'AnnArbor élevé au moment du diagnostic (r=0,45). Au vu de nos résultats acquis sur une petite série aux caractéristiques superposables à celles des grandes séries de la littérature, p75[exposant NTR] pourraitconstituer un facteur pronostic prédictif dans les LF de stade élevé. Toutefois, ces résultats devraient être vérifiés sur de grandes séries multicentriques.
... In the skin, NGF is expressed mostly by keratinocytes (Tron et al., 1990). NGF in cutaneous keratinocytes has been suggested to be a key player in an autocrine loop, acting as mitogen, and survival factor through its high-affinity receptor TrkA (Pincelli and Marconi, 2000). In keratinocytes, NGF production is influenced by neuropeptide release. ...
... We have also established that proNGF is able to induce apoptosis in cultured human keratinocytes, which is indirect evidence that the NGF/proNGF balance exists and may be important in skin homeostasis. This idea is supported by the fact that the production of mature NGF is increased in inflammatory skin conditions such as psoriasis accompanied by keratinocyte hyperproliferation (Pincelli and Marconi, 2000; Raychaudhuri and Raychaudhuri, 2004). Even less is known regarding the NGF receptor expression. ...
Sortilin, a member of the family of Vps10p domain receptors, has been shown to be able to bind the precursor peptide of nerve growth factor (proNGF). ProNGF interacts with sortilin and the p75(NTR) receptor on the cell surface to form a molecular complex capable of activating an apoptotic cascade. Keratinocytes can secrete proNGF and they have p75(NTR) on their surface. The expression of sortilin in normal human keratinocytes has not yet been clearly shown. In this study, we show that keratinocytes express sortilin mRNA, and the presence of sortilin protein is shown in cultured keratinocytes and in normal human skin. We have also shown that the cutaneous neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and galanin are able to reduce the expression of sortilin mRNA and sortilin protein in cultured human keratinocytes. In addition, each of the analyzed neuropeptides has the ability to arrest the proNGF-induced apoptosis of human keratinocytes. These results suggest that all the participants in the NGF/proNGF pathway are present in the keratinocytes, and cutaneous neuropeptides can modulate their expressions and actions. The NGF/proNGF balance and its regulation by neuropeptides may have an important role in skin homeostasis.
... 46 Keratinocytes release NT that, in turn, exert autocrine activities through the high-affinity receptors Trks and CD271. 47 We have previously shown that KSC express the highest levels of nerve growth factor 48 that acts as a mitogen and a survival factor for the neighbor cells. 49 We have also provided evidence that CD271 mediates apoptosis in human keratinocytes. ...
In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population “early” TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony forming efficiency in ETA cells are lower than in KSC, but higher than in “late” TA (LTA). Stemness, proliferation and differentiation markers confirmed that ETA cell display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67 and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low affinity neurotrophin receptor CD271, which regulates the KSC to TA cells transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of human epidermis.
... Исследования с использованием клеточных культур позволяют выявить источники нейромедиаторов в коже, возможное влияние нейромедиаторов на развитие и течение воспалительной реакции. Оказалось, что продукция нейромедиаторов может осуществляться не только компонентами нервной систе-мы, но и кератиноцитами, тучными клетками, лимфоцитами [13,[27][28][29][30][31][32]. ...
... Currently, NGF is known as a neurokine because it functions not only in the nervous system but also in other systems, including the immune system. NGF is produced by many cells of inflam-matory processes, including mast cells, keratinocytes, macrophages, T cells, and B cells [31][32][33][34][35][36]. Low-affinity p75NTR and high-affinity trkA NGF receptors are upregulated during the pathophysiological inflammatory process in skin, especially during chronic inflammation that develops into peripheral sensory neuropathy and thermal hyperalgesia with loss of neural fibres [37]. ...
Leprosy, also known as Hansen’s disease, continues to have a substantial impact on infectious diseases throughout the world. Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae and shows a wide clinical and immunopathological spectrum related to the immune response of the host. This disease affects the skin and other internal organs with a predilection to infect Schwann cells, which play an active role during axonal degeneration, affecting peripheral nerves and promoting neurological damage. This chronic inflammation influences immune function, leading to neuroimmune disorders. Leprosy is also associated with neuroimmune reactions, including type 1 (reverse) and type 2 (erythema nodosum leprosum) reactions, which are immune-mediated inflammatory complications that can occur during the disease and appear to worsen dramatically; these complications are the main concerns of patients. The reactions may induce neuritis and neuropathic pain that progressively worsen with irreversible deformity and disabilities responsible for the immunopathological damage and glial/neuronal death. However, the neuronal damage is not always associated with the reactional episode. Also, the efficacy in the treatment of reactions remains low because of the nonexistence of a specific treatment and missing informations about the immunopathogenesis of the reactional episode. There is increasing evidence that peripheral neuron dysfunction strongly depends on the activity of neurotrophins. The most important neurotrophin in leprosy is nerve growth factor (NGF), which is decreased in the course of leprosy, as well as the presence of autoantibodies against NGF in all clinical forms of leprosy and neuroimmune reactions. The levels of autoantibodies against NGF are decreased by the immunomodulatory activity of cyclosporin A, which mainly controls pain and improves motor function and sensitivity. Therefore, the suppression of anti-NGF and the regulation of NGF levels can be attractive targets for immunomodulatory treatment and for controlling the neuroimmune reactions of leprosy, although further studies are needed to clarify this point.
... Patient consent for experiments was not required because Italian laws consider human tissue left over from surgery as discarded material. Isolated cells were cultured as described by Pincelli and colleagues [28]. Cells were maintained in culture with KGM until pre-confluence conditions and utilized for Western blot analysis. ...
Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer.
... Epidermal cells produce and secrete NGF in the wound healing process. The secreted NGF causes nerve fibers to sprout and nerves to regenerate, and proliferation and migration of epidermal cells are stimulated in an autocrine fashion [2,3,8,9]. Many reports have investigated the involvement of NGF in the delay of wound healing of diabetic ulcers [1][2][3]10,11]. ...
... Indeed, NGF or NT-3 stimulates keratinocyte growth (25,61), and transfecting HaCat cells with TrkA enhance cell proliferation (62), indicating that NTs act as mitogens through their high-affinity receptor. Consistently, K252, a natural alkaloid that blocks Trk phosphorylation, thus inhibiting NT functions, prevents NGF-induced keratinocyte proliferation (23). ...
p75 neurotrophin receptor (p75NTR), also known as CD271, is the low-affinity receptor that, together with the tyrosine kinase receptor tropomyosin-receptor kinase (Trk), mediate neurotrophin (NT) functions. Beside their classic role in skin innervation, NT and their receptors constitute a complex cutaneous network associated with a number of autocrine and paracrine activities. In this context, the role of p75NTR is becoming more and more important. This review will focus on the intriguing functions of p75NTR in healthy and diseased skin. First, p75NTR counterbalances the proliferative and survival activities of its cognate receptor Trk by inducing keratinocyte apoptosis. In addition, p75NTR identifies an early transit-amplifying (TA) keratinocyte population and plays a critical role in keratinocyte stem cell transition to its progeny as well as in epidermal differentiation. p75NTR is absent in psoriatic TA cells, thus rendering these cells resistant to apoptosis. On the other hand, p75NTR infection restores NT-induced apoptosis in psoriatic keratinocytes. Taken together, these results provide evidence for a critical role of p75NTR in epidermal homeostasis, while its lack may account for the TA defect in psoriasis. While the issue of p75NTR as a marker of melanoma initiating cells is still to be solved, there is strong evidence that downregulation of this receptor is a precondition to melanoma invasion and metastasis in vitro and in vivo. All in all, this review points to p75NTR as a major actor in both physiologic and pathologic conditions at the skin level.
... In normal human skin, a NT network fulfils different autocrine and paracrine functions, as most cells produce and release NT and express their receptors (Botchkarev et al., 2006). In human keratinocytes, the NT nerve growth factor is mainly expressed in KSCs (Marconi et al., 2003) and stimulates cell proliferation and survival through TrkA receptor (Pincelli and Marconi, 2000), thus contributing to the maintenance of ''stemness''. On the other hand, the NT brain-derived neurotrophic factor and NT-4 induce keratinocyte apoptosis through CD271 (Truzzi et al., 2011). ...
CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the TNF-receptor superfamily. Because in human epidermis CD271 is predominantly expressed in transit amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSC) to progeny. Calcium induced an up-regulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 siRNA. Furthermore, CD271 overexpression provoked the switch of KSC to TA cells, while silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and K15 than CD271(-) cells, and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSC to TA cells.Journal of Investigative Dermatology accepted article preview online, 20 October 2014. doi:10.1038/jid.2014.454.
... DRG neurons [2,12,30]. The positive effect of NGF-b on epidermal cell proliferation observed in our organotypic human model is consistent with previous studies, which demonstrated that a topical application of NGF-b accelerated SWH by stimulating keratinocyte proliferation and by protecting cells from apoptosis via an autocrine loop [31][32][33]. Hence, a direct interaction between neurites and epidermal cells could be preceded by the effects of soluble factors. ...
Background
Close interactions exist between primary sensory neurons of the peripheral nervous system (PNS) and skin cells. The PNS may be implicated in the modulation of different skin functions as wound healing.
Objective
study the influence of sensory neurons in human cutaneous wound healing.
Methods
We incubated injured human skin explants either with rat primary sensory neurons from dorsal root ganglia (DRG) or different neuropeptides (vasoactive intestinal peptide or VIP, calcitonin gene-related peptide or CGRP, substance P or SP) at various concentrations. Then we evaluated their effects on the proliferative and extracellular matrix (ECM) remodeling phases, dermal fibroblasts adhesion and differentiation into myofibroblasts.
Results
Thus, DRG and all studied neuromediators increased fibroblasts and keratinocytes proliferation and act on the expression ratio between collagen type I and type III in favor of collagen I, particularly between the 3rd and 7th day of culture. Furthermore, the enzymatic activities of matrix metalloprotesases (MMP-2 and MMP-9) were increased in the first days of wound healing process. Finally, the adhesion of human dermal fibroblasts and their differentiation into myofibroblasts were promoted after incubation with neuromediators. Interestingly, the most potent concentrations for each tested molecules, were the lowest concentrations, corresponding to physiological concentrations.
Conclusion
Sensory neurons and their derived-neuropeptides are able to promote skin wound healing.
... 174 NGF promotes reepithelialization by stimulating keratinocytes proliferation and by protecting them to apoptosis via an autocrine loop. 175,176 In different mice or rat models, it could be observed that topical application of NGF accelerated wound healing. [177][178][179] NGF acts on the fibroblasts in wound healing by stimulating the differentiation of dermal fibroblasts into dermal myofibroblasts as well as their migration and contractile activity but not their proliferation. ...
Due to the close interactions between the skin and peripheral nervous system, there is increasing evidence that the cutaneous innervation is an important modulator of the normal wound healing process. The communication between sensory neurons and skin cells involves a variety of molecules (neuropeptides, neurohormones, and neurotrophins) and their specific receptors expressed by both neuronal and nonneuronal skin cells. It is well established that neurotransmitters and nerve growth factors released in skin have immunoregulatory roles and can exert mitogenic actions; they could also influence the functions of the different skin cell types during the wound healing process.
... To understand this paradigm, it is important to know that within the epidermis, NGF is secreted keratinocytes, making these cells primarily responsible for the innervation TrkA-expressing DRG nerve terminals (Albers et al., 1994;Bennett et al., 1998;Di Marco et al., 1993). These NGF-producing keratinocytes express low level TrkA receptor as an autocrine regulator of NGF secretion levels (Pincelli and Marconi, 2000). As our in vivo studies showed a decrease in axon innervation at the footpad, and Western blot analysis of cultured DRG neurons demonstrated a decrease in TrkA receptor expression following Vpr expression ( Figure 4) the increase in TrkA receptor levels at the epidermis ( Figure 1H) is not likely due to axonal TrkA expression. ...
Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1(-/-)) mice displayed allodynia (p<0.05), diminished epidermal innervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1(-/-)) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor antagonists indicated that NGF acted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr's effect on DRG neurons. Overall, NGF/TrkA signalling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.
... This further supports our hypothesis that ameloblastomas indicate the recapitulation of odontogenic epithelium to the original oral epithelium. Sensory nerve associated factors like Trk A and S100 are expressed in normal basal keratinocytes and also by the presence or absence of epithelial cell rests of Malassez as suggested by Yammamoto, et al., [22] Pincelli, et al., [23] Richard et al. [24] This augments our hypothesis that an immunohistochemistry (IHC) marker like S100 expressed in the epithelium can also be expressed in ameloblastomas supporting the histogenic concept of derivation from the oral epithelium. ...
Ameloblastoma is characterized as a slow growing, non-metastatic and a locally invasive tumor with a high risk of recurrence. Immunohistochemical evaluation of ameloblastomas using epithelial and connective tissue specific markers help in studying the histogenesis and assessing the biological behavior. The aim of the study was to study the expression patterns of cytokeratin, vimentin, smooth muscle actin (SMA), S100 and CD34 in ameloblastomas.
The material for the study consisted of 24 cases of ameloblastomas. The excised specimens were grossed and bits were taken from different areas of the specimen. Based on the histopathology, the cases were classified into different types and stained for immunohistochemistry.
The cases showed strong positivity to cytokeratin, vimentin, moderate positivity for SMA and S100. Five cases were also moderately positive for CD34 in blood vessels.
The results and hypothesis achieved from the study, proved to be consistent, not only augmenting the already existing hypothesis but also imparting new concepts of hypothesis.
... N erve growth factor (NGF) is a neurotrophin that plays a crucial role in promoting growth, differentiation, and function in sympathetic nerve cells (1). In recent years, NGF has been described as a ''pleiotropic'' molecule (2), involved in a variety of peripheral actions such as tissue inf lammation (3), neuropeptide expression regulation (4), skin physiology (5), and peripheral tissue regeneration (6,7). Furthermore, targetderived NGF, which binds to its low-and high-affinity receptors on nerve terminals and is retrogradely transported to neural sensory and sympathetic perikarya, is known to prevent natural cell death and to promote and modulate peripheral innervation (8 -10). ...
In postnatal tissues, angiogenesis occurs in nontumoral conditions
on appropriate stimuli. In the nervous tissue, hypoxia, neural graft,
increased neural function, and synaptic activity are associated with
neoangiogenesis. We have investigated the occurrence of neoangiogenesis
in the superior cervical ganglia (scg) of newborn rats treated for
8–21 days with 6-hydroxy-dopamine (6-OHDA), nerve growth factor (NGF),
or 6-OHDA + NGF. The two latter treatments induced a significant
increase in scg size. However, the increase after combined treatment
far exceeded that of NGF alone. Similarly, histological and
histochemical analysis revealed neuronal hypertrophy and endothelial
cell hyperplasia associated with stromal hypertrophy (as described by
laminin immunostaining) and increased vascular bed (as revealed by
platelet/endothelial cell adhesion molecule-1 immunostaining) in
6-OHDA + NGF-treated pups. NGF, either alone or associated with 6-OHDA,
also induced a significant up-regulation of NADPH diaphorase, neuronal
nitric oxide synthase, and vascular endothelial growth factor
expression in scg neurons. The present investigation suggests that the
increase of scg size induced by NGF and 6-OHDA + NGF is associated with
neoangiogenesis, and that the induction of vasoactive and angiogenic
factors in neurons represents a further and previously undisclosed
effect of NGF.
... The role of NGF on skin biology is particularly relevant from a clinical perspective. Production and utilization of NGF has been demonstrated in skin cells, as keratinocytes [132][133][134], and in immune cells that are resident or recruited in epidermal tissue following trauma or inflammation [135][136][137]. NGF deregulation has been described in diseased skin [137][138][139][140][141][142][143]. ...
The physiological role of the neurotrophin nerve growth factor (NGF) has been characterized, since its discovery in the 1950s, first in the sensory and autonomic nervous system, then in central nervous, endocrine and immune systems. NGF plays its trophic role both during development and in adulthood, ensuring the maintenance of phenotypic and functional characteristic of several populations of neurons as well as immune cells. From a translational standpoint, the action of NGF on cholinergic neurons of the basal forebrain and on sensory neurons in dorsal root ganglia first gained researcher's attention, in view of possible clinical use in Alzheimer's disease patients and in peripheral neuropathies respectively. The translational and clinical research on NGF have, since then, enlarged the spectrum of diseases that could benefit from NGF treatment, at the same time highlighting possible limitations in the use of the neurotrophin as a drug. In this review we give a comprehensive account for almost all of the clinical trials attempted until now by using NGF. A perspective on future development for translational research on NGF is also discussed, in view of recent proposals for innovative delivery strategies and/or for additional pathologies to be treated, such as ocular and skin diseases, gliomas, traumatic brain injuries, vascular and immune diseases.
... NGF was the first member of the neurotrophin family to be described [15]. This NT may be synthesized by neurons and non-neuronal cells, including cells from the salivary glands [16,17], epithelial [18][19][20][21] and mast cells [13]. NGF plays an essential role during the development of the peripheral nervous system, regulating the survival and function of postganglionic sympathetic neurons and small diameter primary afferents [6, 9, 22-24,]. ...
The lower urinary tract (LUT) comprises a storage unit, the urinary bladder, and an outlet, the urethra. The coordination between the two structures is tightly controlled by the nervous system and, therefore, LUT function is highly susceptible to injuries to the neuronal pathways involved in micturition control. These injuries may include lesions to the
spinal cord or to nerve fibres and result in micturition dysfunction. A common trait of micturition pathologies, irrespective of its origin, is an upregulation in synthesis and secretion of neurotrophins, most notably Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF). These neurotrophins are produced by neuronal and non-neuronal cells and exert their effects upon binding to their high-affinity receptors abundantly expressed in the neuronal circuits regulating
LUT function. In addition, NGF and BDNF are present in detectable amounts in the urine of patients suffering from various LUT pathologies, suggesting that analysis of urinary NGF and BDNF may serve as likely biomarkers to be studied in tandem with other factors when diagnosing patients. Studies with experimental models of bladder dysfunction
using antagonists of NGF and BDNF receptors as well as scavenging agents suggest that those NTs may be key elements in the pathophysiology of bladder dysfunctions. In addition, available data indicates that NGF and BDNF might constitute future targets for designing new drugs for better treatment of bladder dysfunction.
... Nerve growth factor (NGF), which belongs to the neurotrophin family, is mainly produced and released by the basal keratinocytes15-19 and has diverse activity in the skin. Immunohistochemistry of human skin shows that NGF is expressed in suprabasal keratinocytes and in basal keratinocytes.18 ...
Staphylococcus aureus (SA) is usually present not only in the skin lesions of atopic dermatitis (AD) but also in the atopic dry skin. SA discharges various toxins and enzymes that injure the skin, results in activation of epidermal keratinocytes, which produce and release IL-18. IL-18 that induces the super Th1 cells secreting IFN-γ and IL-13 is supposed to be involved in development of AD and its pathogenesis. Indeed, the number of SA colonies on the skin surface and the serum IL-18 levels in patients with AD significantly correlated with the skin scores of AD lesions. Also, there is strong positive correlation between the skin scores and serum IL-18 levels in DS-Nh mice (P<0.0001, r=0.64), which develop considerable AD-like legions when they are housed under conventional conditions, but develop skin legions with less severity and less frequency under specific pathogens free (SPF) conditions. Therefore, they are well-known as model mice of AD, in which SA is presumed to be critical factor for the development of AD lesions. Also, theses DS-Nh mice pretreated with Cy developed more remarkable AD-like lesions in comparison with non-treated ones. The levels of INF-r and IL-13 in the supernatants of the lymph node cell cultures stimulated with staphylococcal enterotoxin B (SEB) or ConA were increased in the Cy-treated mice, although the serum levels of total IgE were not. In this experiment, we revealed that Cy-treated mice, to which CD25 +CD4 + reguratory T cells taken from non-treated ones had been transferred, developed the AD-like legions with less severity and less number of SA colonies on the skin surface. Therefore, it is presumed that CD25 +CD4 + reguratory T cells might be involved in the suppression of super Th1 cells which are induced by IL-18 and are involved in the development of AD-like lesions rather than IgE production. The efficient induction of CD25 +CD4 + reguratory T cells is expected for the new type of treatment of AD. We also found that farnesol (F) and xylitol (X) synergistically inhibited biofilm formation by SA, and indeed the ratio of SA in total bacteria at sites to which the FX cream containing F and X had been applied was significantly decreased 1 week later, accompanied with improvement of AD, when compared with that before application and at placebo sites. Therefore, the FX cream is a useful skin-care agent for atopic dry skin colonized by SA. The nerve growth factor (NGF) in the horny layer (the horn NGF) of skin lesions on the cubital fossa was collected by tape stripping and measured using ELISA in AD patients before and after 2 and 4 weeks treatments. Simultaneously, the itch and eruptions on the whole body and on the lesions, in which the horn NGF was measured, were recorded, and also the peripheral blood eosinophil count, serum LDH level and serum total IgE level were examined. The level of NGF was significantly higher in AD patients than in healthy controls, correlated with the severity of itch, erythema, scale/xerosis, the eosinophil count and LDH level, and also significantly decreased after treatments with olopatadine and/or steroid ointment for 2 and 4 weeks. Therefore, the measurement of the NGF by this harmless method seems to be useful to assess the severity of AD and the therapeutic effects on AD. In AD patients, C-fiber in the epidermis increase and sprout, inducing hypersensitivity, which is considered to aggravate the disease. Semaphorin 3A (Sema3A), an axon guidance molecule, is a potent inhibitor of neurite outgrowth of sensory neurons. We administered recombinant Sema3A intracutaneously into the skin lesions of NC/Nga mice, an animal model of AD, and investigated the effect of Sema3A on the skin lesions and their itch. Sema3A dose-dependently improved skin lesions and attenuated the scratching behavior in NC/Nga mice. Histological examinations revealed a decrease in the epidermal thickness, the density of invasive nerve fibers in the epidermis, inflammatory infiltrate including mast cells and CD4 +T cells, and the production of IL-4 in the Sema3A-treated lesions. Because the interruption of the itch-scratch cycle likely contributes to the improvement of the AD-like lesions, Sema3A is expected to become a promising treatment of patients with refractory AD.
... Mitochondrienmembran lokalisiert (Krajewski et al., 1993) und wirkt inhibierend auf ein frühes Ereignis im Ablauf des programmierten Zelltods (Reed, 1994). Es wurde auch beschrieben, dass Überexpression von Bcl-2 in Keratinozyten diese gegenüber spontaner und UV-induzierter Apoptose resistent macht (Pincelli and Marconi, 2000;Qin et al., 2002). In der Epidermis lässt sich Bcl-2 nur in den basalen Keratinozyten nachweisen (Pincelli et al., 1997)Peus et al., 1998;Peus et al., 2000). ...
In Keratinozyten wird sowohl durch UVB als auch durch PUVA-Bestrahlung Apoptose induziert. Wir untersuchten die in Keratinozyten durch UVB, PUVA, UVA und Todesliganden wie TRAIL ausgelösten Apoptosewege näher. UVB und PUVA, nicht aber UVA-Bestrahlung lösen in vitro Keratinozytenapoptose aus. 2-4 h nach UVB beobachteten wir die Aktivierung von Caspasen. Nach PUVA setzt die Aktivierung von Caspasen wesentlich später ein, nämlich erst 12 h nach Bestrahlung. Passend dazu, ist ein Verlust des mitochondrialen Transmembranpotentials 6-8 h nach UVB und 12-14 h nach PUVA detektierbar. Die Überexpression des Proteins Bcl-2 verhindert den Verlust des mitochondrialen Transmembranpotentials und die Caspase-Aktivierung nach UVB, und vermittelt auch einen klonogen Schutz, unabhängig von der Bildung reaktiver Sauerstoffradikale. Im Gegensatz dazu verzögert es den Verlust des Transmembranpotentials und die Caspase-Aktivierung nach PUVA nur und verhindert sie nicht. PUVA-bestrahlte Zellen können sich nicht weiter teilen, sind also durch Bcl-2 nicht klonogen geschützt.
... In vitro studies indicate that proliferating keratinocytes are a major source of NGF, thus it is possible that keratinocyte production of NGF may decrease post-proliferation (i.e with keratinocyte maturation) thereby providing less support for intra-epidermal Schwann cells (Albers and Davis, 2007a;Marconi et al., 2003;Pincelli and Marconi, 2000;Pincelli et al., 1994). The Schwann cell ensheathment at the leading edge of the regenerating axons supports the finding that the Schwann cells are guiding the pathway for the axons to reach the target Guenard et al., 1992;Thompson and Buettner, 2006). ...
To characterize the regenerative pattern of cutaneous nerves in simian immunodeficiency virus (SIV)-infected and uninfected macaques, excisional axotomies were performed in nonglabrous skin at 14-day intervals. Samples were examined after immunostaining for the pan-axonal marker PGP 9.5 and the Schwann cell marker p75 nerve growth factor receptor. Collateral sprouting of axons from adjacent uninjured superficial dermal nerve bundles was the initial response to axotomy. Both horizontal collateral sprouts and dense vertical regeneration of axons from the deeper dermis led to complete, rapid reinnervation of the epidermis at the axotomy site. In contrast to the slower, incomplete reinnervation previously noted in humans after this technique, in both SIV-infected and uninfected macaques epidermal reinnervation was rapid and completed by 56 days postaxotomy. p75 was densely expressed on the Schwann cells of uninjured nerve bundles along the excision line and on epidermal Schwann cell processes. In both SIV-infected and uninfected macaques, Schwann cell process density was highest at the earliest timepoints postaxotomy and then declined at a similar rate. However, SIV-infection delayed epidermal nerve fiber regeneration and remodeling of new sprouts at every timepoint postaxotomy, and SIV-infected animals consistently had lower mean epidermal Schwann cell densities, suggesting that Schwann cell guidance and support of epidermal nerve fiber regeneration may account for altered nerve regeneration. The relatively rapid regeneration time and the completeness of epidermal reinnervation in this macaque model provides a useful platform for assessing the efficacy of neurotrophic or regenerative drugs for sensory neuropathies including those caused by HIV, diabetes mellitus, medications, and toxins.
... 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 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. ...
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.
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).
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.
Our knowledge about other proteins (prosurvival and pro-apoptotic molecules) interacting with p75 is incomplete.
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.
... Similarly, NGF (Geldof et al 1997) and FGF-7 could stimulate invasiveness of prostate cancer cells (Ropiquet et al. 1999). Autocrine release of growth factors is a common feature of some cells, such as keratinocytes involving NGF and TrkA receptors (Pincelli & Marconi 2000). In cancer, autocrine loops involving secretion of TGF-α and epidermal growth factor receptor (EGFr) activation have been found in prostate cancer (Sporn & Todaro 1980) and other adenocarcinomas (Hsieh et al. 2000). ...
Studies in varied settings have provided estimates of the prevalence of surrogate markers of benign prostatic hyperplasia (BPH). In population-based studies, the prevalence of moderate-to-severe lower urinary tract symptoms and depressed peak urinary flow rates increases across successively older age groups. Prostatic volume follows a similar pattern. Unlike clinic-based studies in which correlations are almost nonexistent, the population-based studies demonstrate a modest correlation among lower urinary tract symptoms, peak urinary flow rates, and prostatic volume. These cross-sectional observations extend to serum prostate-specific antigen levels and postvoid residual urine volumes. Data collected during the longitudinal follow-up study of men participating in the Olmsted County Study of Urinary Symptoms and Health Status Among Men provide a more detailed description of the natural history of changes in these surrogate markers of BPH. They also provide insights into their relation with each other and with long-term outcomes of BPH, such as acute urinary retention and treatment of BPH. These data demonstrate the progressive nature of BPH and are useful for the design and interpretation of clinical trials. Furthermore, they suggest that observational studies of etiology and prognosis should take advantage of the spectrum of disease reflected by the full range of values of these quantitative traits, rather than an arbitrary dichotomized outcome.
... It is believed that coexpression of p75 NTR and Trk receptors leads to signaling through Trk receptors and promotion of cell survival by stimulation of NTs, 20,21 whereas expression of p75 NTR alone without Trk receptors promotes apoptosis by increased intracellular ceramide, activation of the nuclear transcription factor B 22 , and modulation of growth factor production by target cells. 23,24 In epithelial tissues, synthesis of biologically active NGF has already been demonstrated in the human epidermis in vivo 25 and in vitro, 26,27 and NGF plays an important role in facilitating the degree of re-epithelialization, the thickness of the granulation tissue, and the density of extracellular matrix, when topically applied to the full-thickness skin wound in normal and healing-impaired diabetic mice. 28 -30 It is believed that NGF accelerates the rate of epidermis wound healing through its high-affinity receptor TrkA, and acts synergistically with the other cytokines or growth factors that are released in injured tissues. ...
Amniotic membrane (AM) transplantation facilitates rapid epithelialization in severe neurotrophic corneal ulcers. To elucidate its action mechanism, we investigated the expression of ligands and receptors of the neurotrophin family by human limbal epithelial (HLE) cells expanded on AM cultures.
Expression of nerve growth factor (NGF); neurotrophins (NT)3 and NT4; brain-derived neurotrophic factor (BDNF); tyrosine kinase-transducing receptors TrkA, TrkB, and TrkC; and a pan-NT low-affinity receptor (p75(NTR)) was examined by immunostaining in the normal human corneolimbus, HLE grown on intact epithelially denuded AM, and stratified HLE, after subcutaneous implantation in NIH-bg-nu-xid BR mice. NGF protein level was assayed by an ELISA in extracts of intact and epithelially denuded AM. K252a, a specific inhibitor of TrkA autophosphorylation, was added to test whether it would inhibit HLE expansion on AM culture.
Strong positive TrkA staining was confined to the basal epithelial cell layer of normal corneal and limbal epithelia, with the highest intensity noted in the limbus. TrkA staining was also strongly positive in the basal layer of HLE cells cultured on intact and epithelially denuded AM and in basal and some suprabasal layers of stratified HLE transplanted in nude mice. Positive staining of p75(NTR) was noted in the full-thickness of the corneal epithelium but was limited to the superficial layers of the limbus and in HLE cells cultured on intact and epithelially denuded AM, but was weak in HLE transplanted to nude mice. Weak staining of NT3 and TrkC was noted in the suprabasal layers of corneal and limbal epithelia but was negative in the stratified HLE in nude mice. Negative staining of NGF, NT4, BDNF, and TrkB was noted in all specimens tested. The NGF protein level was readily measured as 35.6 +/- 9.1 and 41 +/- 12.5 pg/mg protein in the homogenate of the intact and epithelially denuded AM, respectively (P = 0.0256). K252a significantly inhibited the HLE outgrowth on intact AM culture (P = 0.024).
The strong expression of TrkA but not p75(NTR) in the limbal basal epithelial cells in vivo suggests that NGF signaling favors limbal epithelial stem cell survival. Such a phenotype is preserved in HLE cells on AM. Blocking NGF signaling significantly retarded HLE expansion on AM, supporting the notion that NGF is important in expansion of limbal epithelial progenitor cells. Furthermore, a high and therapeutic level of NGF was present in AM. Collectively, these findings indicate that denervated neurotrophic ulcers are associated with poor epithelial stem cell function at the limbus. Future studies are needed to determine whether AM transplantation to heal such ulcers may include the promotion of nerve regeneration and survival of epithelial progenitor cells.
... Recently, Rende et al reported that exogenous NGF increased proliferation of normal myogenic cells but not cells lacking trkA (38). Moreover, NGF over-expressing keratinocytes proliferated more rapidly than mock-transfected cells (37). In both cases, K252a blocked NGF-induced cell proliferation. ...
Both nerve growth factor (NGF) and estrogen have been shown to stimulate proliferation of various cell types. Human urothelial cells (HUC) express the alpha- and beta-subtypes of the estrogen receptor (ER(alpha) and ER(beta)) as well as tyrosine kinase A (trkA), the high-affinity receptor for NGF. We investigated interactions between estrogen and NGF relative to cell proliferation using primary cultures of HUC. 17 beta-estradiol (E2) stimulated NGF synthesis by HUC, and E2 (50 nM), the ER(alpha) agonist 16 alpha-iodo-17 beta-estradiol (10 nM), or the ER(beta) agonist genistein (50 nM) each stimulated HUC proliferation, an effect that was abolished by the estrogen antagonist ICI-182,780 (100 nM). NGF (1-100 ng/ml) stimulated HUC proliferation, and this was abolished by NGF antiserum (0.1 microl/ml) or the trkA antagonist K252a (100 nM). HUC proliferation stimulated by E2 was also abolished by NGF antiserum or K252a. Finally, we observed that treatment of HUC with NGF (50 ng/ml) or E2 (50 nM) stimulated trkA phosphorylation, and this was abolished by K252a (100 nM) or NGF antiserum (0.1 microl/ml). These data indicate that the effects of ER activation on HUC proliferation at least partly involve activation of trkA by NGF.
... The epidermal sources of growth factors have not been finally resolved (Albers et al., 1994). In situ hybridization studies have suggested that the suprabasal keratinocytes are the predominant producers of NGF, but in vitro data indicate that proliferating (basal) keratinocytes are the major source (Albers et al., 1994; Pincelli and Marconi, 2000). The neurotrophins have also been implicated in controlling axon arborization (Kennedy and Tessier-Lavigne, 1995). ...
Two distinct patterns of reinnervation occur after injury to the cutaneous nerves: regenerative growth of the injured nerve and "collateral sprouting" of neighboring intact nerves. We describe two complementary models of regrowth of transected small sensory fibers in human skin. The "incision" model uses a circular incision that transects the subepidermal plexus, resulting in Wallerian degeneration of the nerve fibers that enter the incised cylinder, leaving a defined zone of denervated dermis and epidermis. The "excision" model utilizes an identical incision, followed by removal of the incised cylinder of skin, leaving a denervated area in which Schwann cells are absent. In the incision model, the earliest reinervation of denervated epidermis occurred by collateral sprouting from the terminals of epidermal axons from just outside the incision line. These axon terminals extended horizontally across the incision line and through the superficial layers of the epidermis, beneath the stratum corneum. By 13 days, numerous regenerating axons appeared in the deeper dermis derived from transected axons. These regenerating axons grew toward and ultimately into the epidermis, so that epidermal axonal density had normalized by 30-75 days. The invasion of these axons was associated with regression of the horizontally growing collateral sprouts. In the excision model, new fibers arose by terminal elongation of the epidermal axons outside the incision line, as in the incision model, and especially by collateral branching of epidermal fibers at the incision margins. These collaterals reached the epidermal surface of the basal lamina at the dermal-epidermal junction and then grew slowly toward the center of the denervated circle. In contrast to the incision model, however, complete reinnervation was not achieved even after 23 months. These models can be used to study reinnervation of denervated skin in man in different injury models and have relevance for exploring the stimuli for axonal growth and remodeling.
... Findings from several non-neuronal systems indicate that NGF also acts in a manner that departs from the classical neurotrophic relationship. NGF is synthesized and secreted, and activates autoreceptors in a number of non-neuronal cell types including macrophages (Garaci et al., 1999), keratinocytes (Pincelli and Marconi, 2000), mast cells (Nilsson et al., 1997), and various neoplastic cells (Pica et al., 2000;Satoh et al., 2001), including the adrenergic neuronlike PC12 cell (Gill et al., 1998), consistent with an autocrine role. NGF may also play an autocrine survival role in early embryonic development of rat cerebellar granular neuroblasts and avian retinal horizontal cells (Muller et al., 1997;Karlsson et al., 2001). ...
Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.
The skin, which is comprised of the epidermis, dermis, and subcutaneous tissue, is the largest organ in the human body and it plays a crucial role in the regulation of the body's homeostasis. These functions are regulated by local neuroendocrine and immune systems with a plethora of signaling molecules produced by resident and immune cells. In addition, neurotransmitters, endocrine factors, neuropeptides, and cytokines released from nerve endings play a central role in the skin responses to stress. These molecules act on the corresponding receptors in an intra-, juxta-, para- or autocrine fashion. The epidermis as the outer most component of skin forms a barrier directly protecting against environmental stressors. This protection is assured by an intrinsic keratinocyte differentiation program, pigmentary system and local nervous, immune, endocrine, and microbiome elements. These constituents communicate cross-functionally among themselves and with corresponding systems in the dermis and hypodermis to secure the basic epidermal functions to maintain local (skin) and global (systemic) homeostasis. The neurohormonal mediators and cytokines used in these communications regulate physiological skin functions separately or in concert. Disturbances in the functions in these systems lead to cutaneous pathology that includes inflammatory (psoriasis, allergic or atopic dermatitis) and keratinocytic hyperproliferative disorders (seborrheic and solar keratoses), dysfunction of adnexal structure (hair follicles, eccrine and sebaceous glands), hypersensitivity reactions, pigmentary disorders (vitiligo, melasma and hypo- or hyperpigmentary responses, premature aging, and malignancies (melanoma and non-melanoma skin cancers). These components preserve skin integrity and protect against skin pathologies.
Il existe des interactions étroites entre le système nerveux périphérique (SNP) et la peau. En effet, le SNP intervient dans le maintien de l’homéostasie de la peau mais également dans de nombreux processus physiologiques et physiopathologiques. La cicatrisation est un processus complexe qui regroupe différents mécanismes biologiques comme l’hémostase, l’inflammation, la ré-épithélialisation et la synthèse matricielle. Les mécanismes par lesquels le SNP agit sur les différentes étapes de la cicatrisation demeurent encore assez obscurs. Nous avons donc entrepris d’analyser plus précisément les effets du SNP et de certains neuromédiateurs dans les phases de prolifération et de remodelage du processus de cicatrisation. Pour cela, nous avons utilisé dans un premier temps, un modèle de co-culture entre un explant de peau humaine lésée et des neurones issus de ganglions rachidiens dorsaux (GRD) de rats nouveau-nés et analysé leurs effets sur la prolifération des cellules dermiques et épidermiques, sur l’activité des métalloprotéases matricielles 2 et 9 (MMP-2 et MMP-9), et sur la balance d’expression des collagènes de type I et III au cours du temps. Dans un second temps, nous avons analysé plus précisément l’effet de la Substance P (SP), du Calcitonin-Gene Related Peptide (CGRP) et du Vasoactive Intestinal Peptide (VIP) ainsi que celui du facteur de croissance neuronale, Nerve Growth Factor (NGF-β) sur les mêmes mécanismes et ajouté à cette étude, l’effet de ces neuromédiateurs sur la capacité d’adhérence et la différenciation des fibroblastes dermiques primaires humains en myofibroblastes. Ainsi, ce modèle nous a permis de mettre en évidence que les neurones sensoriels influençaient la prolifération des kératinocytes et des fibroblastes en favorisant celle-ci, et qu’ils agissaient aussi sur le remodelage tissulaire en augmentant l’activité des MMP-2 et -9 ainsi qu’en modulant le ratio d’expression entre les collagènes I et III en faveur du collagène de type I. De plus, nous montrons que les neuropeptides étudiés ainsi que le NGF- β favorisent également ces différents mécanismes de manière dose-dépendante. De plus, les neuromédiateurs testés favorisent la différenciation myofibroblastique des fibroblastes dermiques humains et augmentent leur capacité d’adhérence sur une matrice de fibronectine humaine. L’utilisation de notre modèle peut ouvrir de nouvelles études fondamentales mais également participer au développement de nouvelles molécules favorisant la cicatrisation. Nous avons également identifié pour la première fois la neuroserpine, un inhibiteur du système fibrinolytique, dans la peau.
The skin is a natural barrier between the interior milieu of the organism and its environment. This barrier has multiple physiological functions and may be affected by an array of pathologies including wounds and burns. The present study aims to determine the effect of the nervous system on wound healing. Specifically, this study tested the effect of denervation by chemical ablation on the burn wound healing process using guanethidine for denervation of the sympathetic postganglionic neurons and resiniferatoxin for denervation of the sensory capsaicin-sensitive fibres. Animals were divided into 8 different groups: (1) control group, (2) sensory denervated and burned, (3) sensory denervated non-burned, (4) sympathetic denervated and burned, (5) sympathetic denervated non-burned, (6) vehicle sensory burned, (7) vehicle sympathetic burned, (8) non-denervated burned. We measured different morphologic and biochemical parameters such as wound surface area, histological alterations and mast cells. In addition, NGF, IL-1β, IL-6 and IL-8 levels were determined using the ELISA technique. The gross observations, the histological data including mast cell modulation, as well as the molecular data, speak in favour of a significant delay in burn wound healing caused by sensory denervation. On the other hand, results support the positive role of sympathetic denervation in speeding up the healing process. The dual effect of the nervous system on burn wound healing is being documented in an animal model for the first time. © 2018, Mediterranean Club for Burns and Fire Disasters. All rights reserved.
The neurotrophin β-Nerve Growth Factor (β-NGF) is flourishing as a protein with important roles in the ovulation induction process in induced-ovulation species but data in rabbits are still inconclusive, probably due to the species-specificity effect of the neurotrophin to trigger the ovulation. Moreover, β-NGF seems to have a role in sperm function. To clarify these functionalities we aimed, in the present research: 1) to newly synthesize a functional recombinant β-NGF from rabbit (rrβ-NGF), 2) to reveal differences in the amino acid sequence of rabbit β-NGF compared to other sequences of induced and spontaneous ovulator species, and 3) to assess the effects of rrβ-NGF on sperm viability and motility. The nucleotide sequence of NGF from rabbit prostate was sequenced by Rapid Amplification of cDNA Ends (RACE) and annotated in GenBank ( KX528686 ). Then, rrβ-NGF was produced in CHO cells and purified by affinity chromatography. Western blot and MALDI-TOF analyses confirmed the correct identity of the recombinant protein. rrβ-NGF functionality was validated in PC12 cells through a successful dose-response effect along 8 days. The comparison of the amino acid sequences of NGF between rabbit and other species suggested some relevant substitutions at its binding site to both the high- (TrkA) and the low- (p75) affinity receptors. The addition of rrβ-NGF in rabbit sperm, in a time- and dose-response study, did not affect its viability but slightly changed some of its motility parameters at the highest concentration used (100 ng/ml). Thus, it can be considered that this new recombinant protein may be used for biotechnological and reproduction assisted techniques in ovulation-induced species.
Nerve growth factor (NGF), a neurotrophin that plays a critical role in developmental neurobiology, is released by proliferating keratinocytes and induces proliferation.
The aim of this study was to investigate the role of tyrosine kinase receptor A (TrkA), a high-affinity receptor of NGF, in human keratinocytes.
Expression of TrkA and NGF in skin diseases was investigated by immunohistochemistry. Expression of TrkA in cells was examined by Western blotting and RT-PCR. Cell proliferation was assessed by BrdU assay.
We first determined the expression of TrkA and NGF in skin samples from patients with atopic dermatitis, prurigo nodularis, psoriasis vulgaris, and seborrheic keratosis. TrkA was only expressed in proliferating basal cells, and its expression was enhanced in atopic dermatitis samples. NGF expression was enhanced in atopic dermatitis and prurigo nodularis samples and in some samples from seborrheic keratosis patients. Investigation of the role of TrkA in vitro using normal human epidermal keratinocytes (NHEK) revealed that TrkA was significantly enhanced by the T helper type 2 (Th2) cytokines interleukin (IL)-4 and IL-13 but not by other inflammatory cytokines, such as IL-1β, tumor necrosis factor α, interferon γ, or epidermal growth factor. On the other hand, expression of NGF was not altered by Th2 cytokines. Notably, inhibition of TrkA significantly reversed the effects of IL-4 on proliferation and differentiation. Furthermore, overexpression of TrkA enhanced proliferation of NHEK. These results indicate that IL-4-induced TrkA expression in keratinocytes modulates proliferation and differentiation of these cells.
Increased TrkA expression in keratinocytes in atopic dermatitis may contribute to the observed epidermal hyperproliferation in these patients.
Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
The skin lesions of inflammatory skin diseases (e.g., atopic dermatitis or psoriasis) accompany infiltration of inflammatory cells like macrophages, where abnormal sensory innervations and elevation of nerve growth factor (NGF) level are observed. It is thought that increased NGF mediates the abnormal innervations and this may cause the hypersensitivity of the skin. However, the mechanism of this increased NGF production in the skin is still unknown. Here, we show that tumor necrosis factor (TNF)-α, but not interferon-γ or interleukin-6, enhanced the NGF production in human keratinocytes. The enhanced NGF production was abolished by both Raf-1 kinase and MEK inhibitors, whereas specific inhibitors of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase did not. The extracellular signal-regulated kinase (ERK) phosphorylation and expression of NGF mRNA were accelerated by TNF-α treatment. Furthermore, serum was necessary for the NGF production and epidermal growth factor could substitute for serum in the effect on NGF secretion. These results indicate that TNF-α enhances NGF production via the Raf-1 / MEK / ERK pathway in human keratinocytes, suggesting that regulating TNF-α is a therapeutic target to control NGF production and subsequent sensory innervations.
Psoriasis is a common Th1-mediated skin disease whose etiology remains obscure. Loss of sweating caused by retention hyperkeratosis is seen in psoriasis plaques, which is likely to have resulted from autonomic dysfunction.
In the present study, we aimed to evaluate autonomic nervous system (ANS) functions with regard to psoriasis and to examine whether there is an underlying ANS dysfunction.
Fifty adult patients with plaque-type psoriasis and 20 healthy controls were enrolled in the present study. RESULTS In this study, R-R interval variation (RRIV) was used to evaluate the parasympathetic system. No statistically significant difference was detected when RRIV values of the patient and the control groups were evaluated during normal and deep breathing (P > 0.05). It was determined that parasympathetic activity in the patient group remained unaffected. The sympathetic skin response (SSR) method was used for the evaluation of the sympathetic system. It was found that SSR latency and amplitude values of upper and lower extremities of the patient group were prolonged in comparison to those of the control group. The change in SSR latency of the upper extremity was found to be statistically significant (P < 0.05), whereas the changes in hand amplitude, foot latency, and foot amplitude values were not statistically significant (P > 0.05). It was determined that sympathetic activity in the patient group was affected.
In conclusion, we detected that a dysfunction was present in sympathetic nervous systems of patients with psoriasis. The parasympathetic nervous system functions were normal in patients with psoriasis.
Nerve growth factor (NGF), a target-derived factor for survival and maintenance of peripheral and central neurons, has been implicated in inflammatory processes. Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions, and also play a role in diseases characterised by inflammation, including those of the nervous system like multiple sclerosis. Mast cells are capable of synthesising and responding to NGF, although the occurrence of other members of the NGF family of neurotrophins and their protein forms have not been described. Immunoblot analysis with highly selective neurotrophin antibodies has now been used to show that rat peritoneal mast cells express a higher molecular weight form (73 kDa) of NGF, but not the monomeric (13 kDa) NGF polypeptide. Mast cells also expressed 73 kDa forms of neurotrophin-4 and neurotrophin-3; brain-derived neurotrophic factor was not detected. Medium conditioned by degranulating peritoneal mast cells contained similar high molecular weight forms of NGF and neurotrophin-4 on Western blots, but no neurotrophin-3. Mast cell-derived neurotrophin immunoreactivities were inhibited by the respective peptide antigen, further demonstrating the specificity of the mast cell-derived neurotrophic protein. Mast cell-released proteins supported the survival of cultured chicken embryonic neural crest- and placode-derived sensory neurons; neurotrophic activities were inhibited by neutralising antibodies for NGF and neurotrophin-4, respectively. High molecular isoforms of neurotrophins have been reported to occur in experimental colitis and in the inflamed gut of patients with Crohn’s disease and ulcerative colitis, tissue sites rich in mast cells. The data suggest an important role for neurotrophins in the pathophysiology of inflammatory disease.
Skin cancer has reached epidemic proportions and is considered to be a direct consequence of ultraviolet (UV) radiation exposure. Excessive exposure of epidermal cells to UV results in apoptosis of irreparably damaged cells to avoid malignant transformation. The Bcl-2 family of proteins is emerging as a crucial regulator of epidermal homeostasis and cell's fate in the stressed skin. Not surprisingly, deregulation of Bcl-2 family members is also chiefly involved in skin carcinogenesis and response to cancer therapy. Here we discuss the physiopathological role of epidermal Bcl-2 family members, their implications in skin carcinogenesis and as potential targets in cancer therapy.
Synopsis
Neurosensory cutaneous discomfort in response to topical products is common, yet the relationship between symptoms such as stinging and visible irritation is currently unclear. The presence of a polymorphism at position ‐308 on the TNF‐α gene has been associated with skin irritation, i.e., erythema, dryness. Individuals with a G to A transition (AA/GA genotypes) have a lower threshold to experimentally induced irritation than those with the wild type (G allele, GG genotype). We investigated the effect of this polymorphism on neurosensory irritation (NSI). DNA genotyping was used to determine the allele type amongst a population of health care workers. The neurosensory response to lactic acid and water on the nasolabial folds and hands was assessed using a quantitative lactic acid sting test. Both genotypes had a more intense response to lactic acid compared with water on the face. The AA/GA genotypes had directionally higher scores from lactic acid ( P = 0.1) and significantly higher stinging intensities from water ( P = 0.001) on the face. For the hands, stinging intensities were higher for lactic acid and water amongst the AA/GA genotypes ( P = 0.03 and 0.006 respectively). NSI to lactic acid was significantly higher on the face than on the hands ( P < 0.05). Our findings indicate that subjects with the A transition at position ‐308 on the TNF‐α gene experience more intense NSI with common ingredients, i.e., lactic acid and water, than those with the wild type. TNF‐α polymorphism ‐308 may account for some of the inter‐individual variability in response to skin care practices.
Epidermal keratinocytes are the main component cells of the epidermis and their function is regulated by various kinds of growth factors, cytokines, and chemokines. Of these, members of the epidermal growth factor and fibroblast growth factor families, as wells as hepatocyte growth factor and insulin-like growth factor, play central roles in keratinocyte proliferation, while transforming growth factor-beta, vitamin D3, and interferon-gamma are important inhibitors of keratinocyte growth. Keratinocytes are known to produce many of the currently identified growth factors, cytokines and chemokines. Keratinocyte-derived growth factors and cytokines regulate immune and inflammatory responses, and play important roles in pathological skin conditions. This review focuses on the regulation of keratinocytes by growth factors, cytokines, and chemokines.
Der Haarfollikel ist zugleich Quelle und Wirkort verschiedener Zytokine und Neurotrophine. Zytokine können die Expression von Neurotrophinen und ihrer Rezeptoren in verschiedenen extrakutanen Geweben modifizieren. Daher wurde untersucht, ob Zytokine diese Wirkung auch am Haarfollikel entfalten können. Mittels Immunfluoreszenz wurden Kryostatschnitte von Rückenhaut der C57BL/6-Maus dahingehend untersucht, ob die intradermale Injektion von proinflammatorischen Zytokinen (IL-1 beta, TNF-alpha, IFN-gamma), topische Gabe von Dexamethason oder die intraperitoneale Behandlung mit Cyclosporin A (CsA) die intra- und extrafollikuläre Immunreaktivität von NGF, BDNF, NT-4, NT-3 oder ihrer hochaffinen Rezeptoren Trk-A (NGF), Trk-B (BDNF, NT-4), Trk-C (NT-3) oder des niedrigaffinen Rezeptors p75NTR verändert. Alle Haarfollikel befanden sich im Wachstumsstadium des Haarzyklus (Anagen VI). Alle drei untersuchten Zytokine sowie ein Cocktail von IL-1 beta, TNF-alpha und IFN-gamma regulierten die NGF-Immunreaktivität (IR) in der proximalen äußeren Wurzelscheide (ÄWS) und in der Haarmatrix (HM) von Anagen-VI-Haarfollikeln hoch. Der Zytokincocktail regulierte ebenfalls sehr deutlich die NT-3- und NT-4-IR in der murinen Epidermis hoch. Zusätzlich erhöhte dieser Cocktail die NT-4-IR in spezifischen Zellpopulationen von HM und proximaler ÄWS sowie die p75 NTR-Expression in der dermalen Papille. Interessanterweise erhöhte die Behandlung mit CsA in gleicher Weise wie die einzelnen Zytokine und der Zytokincocktail die NGF-IR in HM und proximaler ÄWS. Dexamethason bewirkte in dieser Arbeit keine Veränderungen der Expression von Neurotrophinen bzw. ihrer Rezeptoren. Somit konnte gezeigt werden, daß sowohl bestimmte proinflammatorische Zytokine als auch CsA die intra- und extrafollikuläre Expression von Neurotrophinen und ihrer Rezeptoren verändern.
We have previously shown that axonal growth from a subset of sensory neurons was promoted by keratinocytes when the two cell types were co-cultured in a low calcium medium. This phenomenon involves the production of one or several diffusible factors. Here we show that the neuritogenic effect of keratinocytes was significantly reduced in the case of rat primary sensory dorsal root ganglion (DRG) neurons, or completely suppressed in the case of the sensory neuron cell line ND7-23, when the activity of neurotrophin receptors (Trk receptors) was blocked with K252a. This trophic effect apparently involved the activation of tyrosine kinase receptors A and B (TrkA and TrkB) expressed by subpopulations of small- to medium-sized DRG neurons, or only of TrkA receptors in the case of ND7-23 neurons. A residual neurite growth promoting effect of keratinocytes persisted in a fraction of DRG neurons after Trk receptor blockade. This effect was mimicked by the steroid dehydroepiandrosterone (DHEA) but not by other steroids such as pregnenolone, progesterone or 17beta-estradiol. The use of pharmacological agents which inhibit different steps of steroidogenesis indicated that DHEA was probably synthesized from cholesterol in keratinocytes. Our results strongly suggest that DHEA might act as a neurotrophic signal derived from keratinocytes to promote axonal outgrowth from a subpopulation of sensory neurons.
Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR), we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target.
The expression of nerve growth factor (NGF) is known to increase in the skin of patients with atopic dermatitis (AD) and is related to disease aggravation. In the present study, we measured skin NGF levels in AD patients and determined whether they correlate to AD severity as well as treatment effects.
NGF in the horny layer (horn NGF) of skin lesions found on the cubital fossa of AD patients was collected via tape stripping and measured using ELISA before and after 2 and 4 weeks following initiation of treatments. Itching and eruptions on the lesions were also evaluated. Peripheral blood eosinophil count, serum LDH level and total serum IgE level were also examined.
The level of NGF was significantly higher in AD patients than in healthy controls, and correlated with the severity of itch, erythema, scale/xerosis, eosinophil count, and LDH level. The NGF level decreased significantly at 2 and 4 weeks of treatment with olopatadine, a histamine H(1) receptor antagonist, and/or topical steroid. The reduction in NGF correlated with the decrease in the severity of itching and erythema, papule, scale/xerosis and lichenification of the lesion, eosinophil count, and LDH level. In psoriatic lesional skin with itch, the horn NGF was significantly higher than in non-lesional skin of psoriasis, but the value was lower than NGF in atopic skin.
The level of horn NGF was found to reflect the severity of itching and eruptions in AD. Therefore, quantification of NGF in the samples collected directly from the horny layer appears to be useful in assessing severity and therapeutic effects in AD.
Nerve growth factor (NGF) has recently been presented as a possible effector of inflammation and bronchial hyperresponsiveness. However, the production of NGF in human airways as well as the regulation of its expression by inflammatory cytokines and glucocorticoids have received little attention. A549 epithelial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, and starved for 24 h. The effect of the pro-inflammatory cytokine interleukin-1beta (1-30 U/ml), and of the glucocorticoid dexamethasone (1 microM) on NGF secretion was studied and quantified by enzyme-linked immunosorbent assay (ELISA). In addition, NGF production within the cells was visualized by immunocytochemistry. Under basal conditions, A549 cells produced and secreted NGF (12.6+/-2.0 pg/ml). Stimulation by interleukin-1beta for 24 h induced a dose-dependent increase in NGF production (maximal at 10 U/ml with 59.6+/-3.5% increase, P<0.05). Dexamethasone (1 microM) markedly reduced the constitute NGF secretion by 44.9% (7.0+/-2.1 pg/ml, P<0.001). In addition, the interleukin-1beta-stimulated NGF secretion was inhibited to approximately the same low level (8.5+/-2.5 pg/ml, P<0.001). In conclusion, we here report that human airway A549 epithelial cells are capable of producing NGF. This production is positively regulated by the pro-inflammatory interleukin-1beta, and negatively regulated by dexamethasone.
Our understanding of wound-healing mechanisms has progressed over the past decade. Wound healing is traditionally divided into three phases--the inflammatory phase, the proliferation phase, and the remodeling phase--and involves a well-orchestrated interaction among blood vessels (platelets, macrophages, neutrophils, endothelial cells, and smooth muscle cells), epidermis (keratinocytes, melanocytes, and Langerhans cells), adnexal structures (outer root sheath cells and hair dermal papilla cells), dermis (fibroblasts and myofibroblasts), nervous system (neurons), and subcutaneous fatty layers (adipocytes). We review recent discoveries of basic and clinical aspects of wound healing including several revolutions that occurred in wound management: occlusive dressing therapy, use of living skin equivalents, and topical administration of growth factors. As we previously proposed, the use of tissue substitutes and autologous epidermal sheets led to a new concept of skin grafting through the keratinocyte activation phase in the graft healing mechanism. In this review, we also discuss a representative patient who presented with plantar wounds caused by calcaneal osteomyelitis and healed by the coverage of epidermal grafting.
Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, which may be regulated by several mechanisms including apoptosis. In this study, we detected DNA fragmentation by the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) method and immunohistochemically examined the expression of Bcl-x and Bax in psoriasis. We determined the expression of bcl-xL mRNA by RT-PCR, and also determined the effect of vitamin D(3) (VD3) on bcl-xL mRNA expression in cultured normal human keratinocytes by RT-PCR, and the expression of Bcl-xL in psoriatic lesions before and after topical application of VD3. A large number of TUNEL-positive cells as well as Bcl-xL - and Bax-positive cells were observed throughout the epidermis in psoriatic lesions. Whereas, in nonlesional and normal skin, only a few TUNEL-positive cells were observed and only the lower epidermis showed positive staining for Bcl-x and Bax. We also observed higher expression of bcl-xL mRNA in psoriatic lesions than in nonlesional and normal skin. The expression of bcl-xL mRNA in cultured normal human keratinocytes stimulated or not with IFN-gamma and PMA was suppressed by VD3 in a dose-dependent manner, and the expression of Bcl-xL, but not Bax, in psoriatic lesional skin decreased after topical application of VD3 for 4 weeks. In conclusion, it is suggested that the apoptotic process in psoriatic lesions is in part regulated by Bcl-xL, and decreasing the expression of Bcl-xL by treatment with VD3 might ameliorate psoriatic lesions by contributing to the completion of the apoptotic process.
Neuroimmunologic aspects of skin inflammation involve several interacting systems. The modulating influence of autonomic and sensory nerves has been known for a long time. Neurokinines derived from these nerves have recently been shown to interact with antigen presentation in dermal Langerhans cells and other key functions of allergic skin disease. While some mechanisms of afferent function and local reflex are known, the nature of efferent effects (from brain to periphery) remains to be elucidated. New aspects include involvement of the brain-derived neurotrophic factor in addition to the autonomic nervous system in mental stress response and insight in the immunomodulation by proopiomelanocortins. Novel strategies for therapy and prevention of chronicity may also result from understanding the role of increased secretion of nerve growth factor in atopic eczema and psoriasis.
We have previously demonstrated that profound and persistent neuropathic pain as displayed by mechanical and cold allodynia and thermal hyperalgesia can be produced by a lumbar 5 ventral root transection (L5 VRT) model in adult rats in which only the motor nerve fibers were injured without axotomy of sensory neurons. However, the underlying mechanisms remain to be determined. In this study, by examining its changes in expression and by inhibiting its functions using a neutralizing antibody, we have investigated whether nerve growth factor (NGF), a neurotrophic factor known to have a function in regulating nerve injury-induced pain, is involved in the development of neuropathic pain induced by L5 VRT. Motor nerve injury by L5 VRT resulted in a de novo expression of NGF mRNA in a subpopulation of small sensory neurons and pericellular satellite cells in ipsilateral L5 dorsal root ganglion. NGF protein expression was also increased by sensory neurons with various sizes and by keratinocytes in the target tissue ipsilateral skin. Systemic administration of NGF antiserum twice within 17 days markedly attenuated L5 VRT-induced mechanical allodynia but not the cold allodynia and thermal hyperalgesia. These findings suggest that NGF is an important pain mediator in the generation of mechanical sensitivity induced by L5 VRT.
We report here that human esophageal keratinocyte stem cells are characterized by the expression of the low-affinity neurotrophin receptor p75(NTR) and differentially expressed cell adhesion molecules, the beta1 and beta4 integrins. The candidate stem cells could be fractionated from keratinocytes as a minor cell subset by means of immunocytochemical cell sorting based on the different levels of expression of these cell surface molecules. Flow cytometric analysis revealed that this minor cell subset retained a relatively slow-cycling phenotype in vitro. These cells expressed low levels of involucrin and cytokeratin 13, indicating that the p75(NTR)-positive cell subset is immature relative to the other predominant subpopulations coexpressing beta1 integrin at higher levels. The p75(NTR)-positive cell subset was crucial for achieving longevity and the greatest output of keratinocytes comprising all distinguishable subpopulations in vitro. This process was associated with self-renewal and self-amplification of the p75(NTR)-positive cell subset. These findings strongly implicate p75(NTR) as a stem cell marker, which will be valuable for prospectively investigating stem cell regulation in association with different biological processes including neoplastic transformation of regenerative epithelia.
Ultraviolet (UV) irradiation to the skin causes apoptosis of keratinocytes. Melanocytes are more resistant to UV-induced apoptosis, due, in part, to high levels of antiapoptotic proteins such as Bcl-2. In vitro studies have shown that nerve growth factor (NGF), a neurotrophic polypeptide, is produced by keratinocytes and exerts a protective role for melanocytes by upregulating Bcl-2. The purpose of this study was to determine NGF and Bcl-2 modulations in UV-irradiated human skin.
Nine volunteers were irradiated with two minimal erythema doses using solar-simulated UV irradiation. Seventy-two hours post irradiation, skin biopsies were obtained from irradiated and sun-protected skin. The skin specimens were stained with anti-tyrosinase-related protein-1 monoclonal antibody IgG2a (Mel-5), anti-Bcl-2 (monoclonal antibody IgG-kappa), and with anti-NGF (polyclonal antibody IgG).
NGF staining was identified within the cytoplasm of epidermal melanocytes, similar to the staining observed for TRP-1 and Bcl-2. While no significant difference in the number of TRP-1- and Bcl-2-positive melanocytes was observed between irradiated and non-irradiated skin within 72 h, the number of NGF-positive melanocytes decreased significantly, 72 h after UV irradiation (p < 0.024). NGF was also identified within keratinocytes, and while non-irradiated skin exhibited cytoplasmic NGF staining throughout the epidermis, NGF staining was reduced in the lower epidermal layers after UV irradiation.
This is the first in vivo study showing NGF to be present in melanocytes, as well as showing modulations of NGF and Bcl-2 in melanocytes, following solar-simulated UV irradiation.
Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing.
Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.
The receptor tyrosine kinase Kit and its cognate ligand KL/steel factor are encoded at the white spotting (W) and Steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl loci affect hematopoiesis including the stem cell hierarchy, erythropoiesis, and mast cells, as well as gametogenesis and melanogenesis. In addition, mutant mice display an increased sensitivity to lethal doses of irradiation. The role of KL/c-kit in cell proliferation and survival under conditions of growth factor-deprivation and gamma-irradiation was studied by using bone marrow-derived mast cells (BMMC) as a model. Whereas apoptosis induced by growth factor deprivation in BMMC is a stochastic process and follows zero order kinetics, gamma-irradiation-induced apoptosis is an inductive process and follows higher order kinetics. In agreement with these results, gamma-irradiation-induced apoptosis in BMMC was shown to be dependent on p53 whereas apoptosis induced by deprivation is partly dependent on p53, implying that there are other mechanisms mediating apoptosis in KL-deprived BMMC. In the presence and in the absence of serum, KL stimulated proliferation by promoting cell cycle progression. The presence of KL was required only during the early part of the G1 phase for entry into the S phase. At concentrations lower than those required for proliferation, KL suppressed apoptosis induced by both growth factor-deprivation and gamma-irradiation, and internucleosomal DNA fragmentation characteristic of apoptosis. The ability of KL to suppress apoptosis was independent of the phase of the cell cycle in which the cells were irradiated and suppression of apoptosis was a prerequisite for subsequent cell cycle progression. Moreover, addition of KL to gamma-irradiated and growth factor-deprived cells could be delayed for up to 1 h after irradiation or removal of growth factors when cells became irreversibly committed to apoptosis. KL and IL-3 induce suppression of apoptosis in mast cells by different mechanisms based on the observations of induction of bcl-2 gene expression by IL-3 but not by KL. It is proposed that the increased sensitivity of W and Sl mutant mice to lethal irradiation results from paucity of the apoptosis suppressing and proliferative effects of KL.
Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.
Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing.
Expression of the cytokine interleukin 1 alpha (IL-1 alpha) was demonstrated in the rat PC12 pheochromocytoma cell line by (i) immunohistochemistry using rabbit polyclonal antisera raised against the recombinant murine IL-1 alpha, (ii) an ELISA, and (iii) a specific cell conversion bioassay based on the use of LBRM33-1A5 cells. IL-1 alpha mRNA was demonstrated in the PC12 cells, by PCR amplification. Constitutive expression of IL-1 alpha in PC12 cells was demonstrated in all experiments, although the cellular levels of IL-1 alpha-like immunoreactivity varied. The expression of IL-1 alpha, as studied at the mRNA level, was inducible by mouse nerve growth factor (7S NGF), and the gene product level was inducible in a dose- and time-dependent fashion by 7S NGF. The maximum induction corresponds to a 600% increase in IL-1 alpha-like immunoreactivity above the expression level found in noninduced cells and occurred after a 3-day incubation of the cells with NGF at 0.75 micrograms/ml of culture medium. The significance of the ability of NGF to induce IL-1 expression lies in the fact that IL-1 itself also acts as a growth factor that promotes glial proliferation and, even more importantly, IL-1 itself induces the expression of NGF at peripheral nerve injury [Lindholm, D., Heumann, R., Meyer, M. & Thoenen, H. (1987) Nature (London) 330, 658-659].
Nerve growth factor (NGF) is a neurotropic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. Much is now known of the structural and functional characteristics of NGF, whose gene has recently been cloned. Since it is synthesized in largest amounts by the male mouse submandibular gland, its role exclusively in nerve growth is questionable. NGF also causes histamine release from rat peritoneal mast cells in vitro, and we have shown elsewhere that it causes significant, dose-dependent, generalized mast cell proliferation in the rat in vivo when administered neonatally. Our experiments now indicate that NGF causes a significant stimulation of granulocyte colonies grown from human peripheral blood in standard hemopoietic methylcellulose assays. Further, NGF appears to act in a relatively selective fashion to induce the differentiation of eosinophils and basophils/mast cells. Depletion experiments show that the NGF effect may be T-cell dependent and that NGF augments the colony-stimulating effect of supernatants from the leukemic T-cell (Mo) line. The hemopoietic activity of NGF is blocked by polyclonal and monoclonal antibodies to NGF. We conclude that NGF may indirectly act as a local growth factor in tissues other than those of the nervous system by causing T cells to synthesize or secrete molecules with colony-stimulating activity. In view of the synthesis of NGF in tissue injury, the involvement of basophils/mast cells and eosinophils in allergic and other inflammatory processes, and the association of mast cells with fibrosis and tissue repair, we postulate that NGF plays an important biological role in a variety of repair processes.
Cell-free systems are valuable tools for the dissection of complex cellular processes. Here we show that cytoplasmic extracts from cells exposed to anti-Fas antibody or UV radiation contain an activity capable of reproducing morphological changes typical of apoptosis in nuclei added to these extracts, as well as internucleosomal cleavage of DNA and proteolysis of a protein known to be cleaved during the apoptosis of intact cells. Extracts from control cell populations were inactive in this respect. These effects were partly blocked by the addition of purified Bcl-2 protein or a competitive inhibitor peptide of interleukin-1 beta-converting enzyme to the extracts. Furthermore, apoptotic activity was induced in cytoplasmic extracts from untreated cells by the addition of ceramide, a lipid second messenger implicated recently in apoptosis signaling. These extracts should prove highly useful in the dissection of molecular events that occur during apoptosis.
Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.
Apoptosis is a required event in maintaining kinetic homeostasis within continually renewing tissues such as skin. However, no systematic study of the apoptotic process in epidermal keratinocytes of the skin has been performed. In this report, we examined the expression of proteins associated with promoting (Fas) or preventing (Bcl-2, Bcl-x, CD40) apoptosis in the normal, psoriatic, and malignant keratinocyte. Immunohistochemical staining and flow cytometry analysis revealed that normal cultured keratinocytes express low levels of Fas, CD40, and Bcl-x that was enhanced by cytokines including gamma-interferon (IFN-gamma) and a phorbol ester tumor promoter, TPA. Only faint Bcl-2 staining was detected in cultured keratinocytes exposed to IFN-gamma and TPA compared with the prominent expression of Bcl-x. Biopsies of normal skin, psoriatic plaques, and basal cell carcinomas were examined to extend the in vitro observations. Immunohistochemical staining revealed that while keratinocytes in normal epithelium express low to absent levels of Fas and Bcl-x, psoriatic keratinocytes expressed significantly higher levels of Fas and Bcl-x. In contrast, malignant keratinocytes in basal cell carcinomas expressed high levels of Bcl-2, but minimal Bcl-x, and no Fas. Immunoblot analysis revealed that the long form of Bcl-x (Bcl-xI), which prevents apoptosis in lymphocytes, is expressed by cultured keratinocytes and psoriatic plaque keratinocytes. We conclude that normal cytokine-activated keratinocytes can express an apoptotic (Fas) and an anti-apoptotic protein (Bcl-x). The overexpression of Bcl-x in psoriasis, or Bcl-2 in basal cell carcinomas, may contribute to the longevity of these cells by blocking the normal apoptotic process involved in the terminal differentiation program of epidermal keratinocytes.
The impact of increased levels of skin‐derived nerve growth factor (NGF) neurotrophin on sensory and sympathetic innervation to the mouse mystacial pad and postero‐orbital vibrissae was determined. Consistent with an approximate doubling of neuron number in trigeminal and superior cervical ganglia, many components of the sensory and sympathetic innervation were substantially enhanced. Although the increased number of neurons raised the possibility that all types of innervation were increased, immunohistochemical analysis indicated that enhanced NGF production had a differential effect upon sensory innervation, primarily increasing unmyelinated innervation. This increased innervation occurred in specific locations known to be innervated by small, unmyelinated fibers, suggesting that NGF modulated sensory innervation density, but not targeting. In contrast, sympathetic innervation was not only increased but also was distributed to some aberrant locations. In the intervibrissal fur of the mystacial pad, both the number of sensory axons and branches appeared increased, whereas in vibrissal follicle sinus complexes, only branching increased. In some areas, sensory ending density was lower than expected based upon the size of the source nerve bundles suggesting that many axons and branches were surviving but failing to form functional endings. Furthermore, the immunochemical profile of innervation was altered in some sensory populations as demonstrated by the coexistence of RT‐97 neurofilament labeling in calcitonin gene‐related peptide (CGRP) positive axons, by the loss of substance P colocalization in some CGRP axons, and by an absence of neuropeptide Y labeling in tyrosine hydroxylase positive sympathetic axons. Collectively, these results indicate that the NGF mediated increase in neuron number may be selective for particular sets of innervation and that increases among some populations may result from phenotypic switching. J. Comp. Neurol. 387:489–506, 1997. © 1997 Wiley‐Liss, Inc.
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The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.
Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.
We have introduced the human bcl-2 gene under the control of the human metallothionein MTIIA promoter into the rat kangaroo PtK2 cell line. Two independent clones were obtained in which the levels of Bcl-2 protein expression can be controlled by the addition of metals in the culture medium. These cell lines were employed to investigate the effects of this protein in UV-induced apoptosis. Overexpression of Bcl-2 in PtK2 cells resulted in a delay in the appearance of apoptosis markers, such as chromatin condensation and internucleosomal DNA fragmentation. However, colony survival after UV was not affected, suggesting that Bcl-2 did not impose a definitive block for cell death. The elimination of cyclobutane pyrimidine dimers through photoreactivation 24 h after irradiation in cells overexpressing Bcl-2 did not affect apoptosis. This indicates that irreversible events in the signaling pathway of apoptosis occur in the period between irradiation and photoreactivation even in the presence of high levels of Bcl-2 in the cell. Therefore, although the human Bcl-2 protein can delay the onset of UV-induced apoptosis in these marsupial cells, early events triggered by the pyrimidine dimers, upstream from the Bcl-2 action, lead the cell to a state committed to die.
Bcl-x, a member of the Bcl-2 family, has two alternatively spliced forms, Bcl-x and Bcl-x. Bcl-x, like Bcl-2, is able to protect cells from a wide variety of apoptotic stimuli. Bcl-x, as a result of alternative splicing, lacks 63 amino acids that comprise the region of greatest amino acid identity between
Bcl-x and Bcl-2. These amino acids contain the highly conserved BH1 and BH2 regions, which have been used to define the Bcl-2 family.
We show that both Bcl-x and Bcl-x are able to regulate cell survival in a dose-dependent fashion. Bcl-x is able to increase the cellular apoptotic threshold and is able to form stable complexes with Bax both in vitro and in vivo. In contrast, Bcl-x can effectively inhibit the protective effects of Bcl-x following growth factor withdrawal and chemotherapeutic drug treatment. However, compared with Bax, Bcl-x binds to Bcl-x weakly when assessed by in vitro binding assays. Coimmunoprecipitation from mammalian cells demonstrates that Bcl-x does not show an observable ability to form heterodimers with other Bcl-2 family members. In addition, overexpression of
Bcl-x does not alter the ability of Bax to heterodimerize with Bcl-xin vivo. Thus, Bcl-x does not appear to function by competitively disrupting the formation of dimers composed of other Bcl-2 family members. This
suggests that Bcl-x can enhance cellular sensitivity to apoptosis via a mechanism of action distinct from other Bcl-2 family members that promote
apoptosis.
The role of nerve growth factor (NGF), a potent mediator acting in the development and differentitation of both neuronal and immune cells, was examined in a mouse model of allergic asthma. NGF-positive cells were detected in the inflammatory infiltrate of the lung and enhanced levels of NGF were detected in serum and broncho-alveolar lavage fluids. Mononuclear cells in inflamed airway mucosa as well as broncho-alveolar macrophages were identified as one source of NGF production. Splenic mononuclear cells from allergen-sensitized mice produced NGF in response to allergen. They responded to exogenously added NGF with a dose-dependent increase in IL-4 and IL-5 production and augmented IgE and IgG1 synthesis. In contrast, IFN- and IgG2a levels remained unaffected. The effects were NGF specific, since they could be blocked by an anti-NGF-antibody. Nasal application of anti-NGF to allergen-sensitized mice significantly reduced IL-4 and prevented development of airway hyperreactivity. These results show that allergic airway inflammation is accompanied by enhanced local NGF production that acts as an amplifier for Th2 effector functions and plays an important role in the development of airway hyperreactivity. Therefore it is suggested that NGF may serve as a link between the immune and nerve system.
We investigated inhibitory activity of nerve growth factor (NGF) on apoptosis of murine peritoneal exudate neutrophils. During culture for 9h, apoptotic cells were identified by morphological changes under a light microscope: nuclear pyknosis and chromatin condensation with or without cytoplasmic vacuolation. The apoptotic state was confirmed by DNA fragmentation indicating the endogenous endonuclease activation. When neutrophils were incubated in the presence of NGF, the proportion of cells with the morphological changes was decreased in a dose-dependent manner, and the development of the characteristic DNA fragmentation was restricted. The apoptosis-suppressing activity of NGF was abolished by the addition of anti-NGF monoclonal antibody. These results suggest that NGF may suppress neutrophil apoptosis by preventing the endogenous endonuclease activation.
The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF.
Many investigators have reported proliferation of terminal cutaneous nerves and upregulation of various neuropeptides (substance P, vasoactive intestinal polypeptide, calcitonin gene-related peptide) in psoriatic lesions. Nerve growth factor promotes growth of nerves and causes upregulation of neuropeptides like substance P and calcitonin gene-related peptide. In this study we investigated the expression of nerve growth factor in psoriatic lesions, non-lesional psoriatic skin, lichen planus and normal control skin. Immunoperoxidase staining was applied on cryosections prepared from snap-frozen biopsy specimens. The primary antibody used was a polyclonal anti-NGF-beta antibody. Nerve growth factor was detected only in the keratinocytes. In psoriatic tissue the number of keratinocytes per square millimeter of epidermis positive for nerve growth factor was 84.7 +/- 46.3 compared to 44.8 +/- 29.9, 18.9 +/- 11.8 and 7.5 +/- 16.9, respectively, in non-lesional psoriatic skin, normal skin and lichen planus. Increased expression of nerve growth factor substantiates larger numbers of terminal cutaneous nerves and upregulations of substance P and calcitonin gene-related peptide in psoriatic lesions. In addition, nerve growth factor is mitogenic to keratinocytes, activates T-lymphocytes and can induce migration of inflammatory cellular infiltrates, histological features characteristic of psoriasis.
Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.
Programmed cell death occurs in most animal tissues at some stage of their development, but the molecular mechanism by which it is executed is unknown. For some mammalian cells, programmed death seems to occur by default unless suppressed by signals from other cells. Such dependence on specific survival signals provides a simple way to eliminate misplaced cells, for regulating cell numbers and, perhaps, for selecting the fittest cells. But how general is this dependence on survival signals?
Mark Bothwell Department of Physiology and Biophysics University of Washington Seattle, Washington 98195 In the manner of many other growth factors, the prototypi- cal neurotrophic factor, nerve growth factor (NGF), has recently been found to be a member of a small gene family encoding structurally and functionally related proteins, collectively referred to as neurotrophins. Individual neuro- trophins exert similar functional effects, but on different neuronal populations. Each of the neurotrophins de- scribed to date-NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin3 (NT-3)-interacts with cell- surface receptors that are heterogeneous with regard to binding affinity. For NGF and BDNF, analysis of equilib- rium binding data indicates the existence of two receptor populations with dissociation constants of about IO-” M (“high affinity”) and 10e9 M (“low affinity”) (Sutter et al., 1979; Rodriguez-TBbar and Barde, 1988). The low affinity receptor binds NGF, BDNF, and NT-3 with similar affinity, while high affinity receptors exist that bind either NGF or BDNF selectively (Rodriguez-TBbar et al., 1990; Squint0 et al., 1991). Functional response to aneurotrophin appar- ently is mediated specifically by the high affinity receptors. Rodriguez-TBbar et al. have proposed that there is a shared component of neurotrophin receptors, known as ~75, with the specificity of each receptor determined by a second component. A variety of studies have shown that low and high affinity NGF receptor forms are intercon- vertible. The interrelationshipsof thevarious neurotrophin recep- tors and of their high and low affinity forms have now been described. The emerging story has profound implications for neurobiology and quite probably for the biology of non- neural tissues also. The first neurotrophin receptor molecular clones iso- lated (Johnson et al., 1986; Radeke et al., 1987) encode a 75-80 kd intrinsic membrane protein (~75) that binds NGF, BDNF, and NT-3 with comparable low affinity (Kd of 10d9 M) when expressed in fibroblastic cells (Rodriguez- Tb Squintoet al., 1991). ~75 has a relatively small cytoplasmic domain containing none of the struc- tural motifs known to function in signal transduction in other receptors. Yet, transfection of ~75 cDNA clones into appropriate neuronal cell lines generates both high and low affinity NGF binding and renders these cells func- tionally responsive to NGF with regard to gene induction and enhanced neurite outgrowth (e.g., Hempstead et al., 1989). The response of such cell lines is weak in comparison with the response of primary neuronal cultures and the rat PC12 neuronal cell line, which expresses endogenous high affinity NGF receptors. The meager cellular response of transfected neuronal cell lines expressing ~7.5 may be
Two classes of receptors with distinct affinities for nerve growth factor (NGF) have been identified. The low affinity receptor (Kd approximately 10(-9) to 10(-8) M) is a cysteine-rich glycoprotein encoded by the previously characterized LNGFR gene. The structural nature of the high affinity receptor (Kd approximately 10(-11) to 10(-10) M) has yet to be established. In this study we show that the product of the human trk proto-oncogene (gp140trk) binds NGF with high affinity. Moreover, NGF could be chemically cross-linked to the endogenous gp140trk present in rat PC12 pheochromocytoma cells as well as to gp140trk ectopically expressed in mouse fibroblasts and in insect Sf9 cells. High affinity binding of NGF to gp140trk can occur in the absence of low affinity LNGFR receptors, at least in nonneural cells. Addition of NGF to PC12 cells elicits rapid phosphorylation of gp140trk on tyrosine residues and stimulates its tyrosine kinase activity. These results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.
Nerve growth factor (NGF) interacts with two different low-affinity receptors that can be distinguished by affinity crosslinking. Reconstitution experiments by membrane fusion and transient transfection into heterologous cells indicate that high-affinity NGF binding requires coexpression and binding to both the low-affinity NGF receptor and the tyrosine kinase trk gene product. These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation involving trk and the low-affinity NGF receptor.
The trk proto-oncogene encodes a 140-kilodalton, membrane-spanning protein tyrosine kinase (p140prototrk) that is expressed
only in neural tissues. Nerve growth factor (NGF) stimulates phosphorylation of p140prototrk in neural cell lines and in embryonic
dorsal root ganglia. Affinity cross-linking and equilibrium binding experiments with 125I-labeled NGF indicate that p140prototrk
binds NGF specifically in cultured cells with a dissociation constant of 10(-9) molar. The identification of p140prototrk
as an NGF receptor indicates that this protein participates in the primary signal transduction mechanism of NGF.
Cell deaths occur selectively in many types of tissues throughout development. These physiological deaths appear to follow an orderly process of internal cellular disintegration that is distinct from the process observed in cell death resulting from trauma. Studies of a variety of physiological cell deaths have revealed that this process appears generally to involve the active participation of the dying cell in its own death. In other words, physiological cell death seems to be a process of induced cellular self-destruction, or cell suicide. Whether a single, genetically determined mechanism is utilized in all cell suicides remains to be established. Nonetheless, while genome digestion and intracellular calcium rises are dissociable from, and thus neither necessary nor sufficient for, cell death, control of the cell cycle may be critical in all cases of induced cell suicide. It is proposed here that physiological cell death occurs through a process of abortive mitosis.
Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.
Nerve growth factor (NGF) is known to affect peripheral sympathetic and sensory neurons as well as defined populations of neurons in the central nervous system. This paper presents evidence that NGF is also active in modulation of B-cell-mediated immune responses. NGF receptors were immunoprecipitated from highly purified human B-cell populations, and to a lesser extent, from T-cell populations, by using a monoclonal antibody recognizing NGF receptors present on neural cells. NGF receptors were also detected in significant amounts in human spleen and lymph node tissue. In addition, NGF induced a dose-dependent increase in B-cell DNA synthesis as determined by incorporation of [3H]thymidine. This B-cell growth-promoting activity was inhibited by a neutralizing anti-NGF monoclonal antibody. Immunoglobulin secretion, principally affecting IgM synthesis, was also modulated by NGF. The concentrations that affected B-cell proliferation are consistent with the presence of functional high-affinity NGF receptors. The results suggest that NGF, in addition to its neurotrophic function, also acts as an immunoregulatory cytokine.
We show that nerve growth factor (NGF) synthesis in developing skin begins with sensory innervation and that sensory neurons do not express NGF receptors until their fibres reach their cutaneous targets. Both cutaneous epithelium and mesenchyme synthesize NGF, the concentration of messenger RNA for NGF being higher in the more densely innervated epithelium.
Dermatologists when telling young patients that toads may have caused their warts. The word "toad" itself is derived from the old English word tadige, which is of unknown origin, without cognates in other languages, and has obvious negative connotations when applied to people. The common European toad, Bufo bufo, is used for studying moulting and recently has had its epidermal kinetics and epidermal homeostasis investigated in detail. Dermatologists are interested in epidermal kinetics because of psoriasis, the ichthyoses, epidermal malignancies, and the response of the epidermis to chemotherapeutic drugs and the retinoids. When we read studies of the kinetics in those disorders, we may not be dispassionate students and often carry with us all of our experience and hang-ups concerning the disease in question. In thinking about the studies in the toad, hopefully there is less emotional investment.
Since man and toad first met each other, it has been known
The quantitative aspect of apoptosis in experimental tumours is often neglected. In this study, the apoptotic and mitotic indices for a range of tumours have been determined at light microscope level. It has been found that the apoptotic levels fall into a consistent range for all tumour types and agree well with those described by previous workers. It is suggested that these might be basic parameters of tumour expansion, as relevant to growth kinetics as mitotic levels.
The relationship of the rate of epidermal proliferation to ultraviolet (UV)-induced cell death, namely sunburn cell (SC) formation, was investigated in vivo using the guinea-pig model. The number of SCs was correlated with (1) cellophane tape stripping which stimulates the proliferation rate and (2) treatment with antimitotic and antimetabolic agents which suppress the proliferation rate. The tape stripping performed 14 h before UV irradiation significantly increased the number of SCs counted 24 h after UV irradiation from a mean of 17.4 to 62.6 per section, and the tritiated thymidine (TdR-3H)-labelling index (LI) increased from a mean of 8.1 to 27.4%. SC counts and LI were markedly decreased by the intradermal injection of colchicine, methotrexate, hydroxyurea or 5-fluorouracil given once immediately after the tape stripping. The autoradiographic study showed that 23.2% of SCs were labelled when TdR-3H was given intradermally 1 h before UV exposure, while no SCs were labelled when TdR-3H was given immediately after UV exposure. The labelled SC count (23.2%) which presumably represents S phase cells at the time of UV exposure, was higher than the LI (mean 8.1%). These results suggest that proliferating cells, possibly S phase cells, may be responsible for SC formation.
Members of the NTR/TNFR family mediate apoptosis in many tissues, yet sequence homology has not been detected in their intracellular domains except for a 'death domain' in TNFR-I and Fas. Here, a region of the 75 kDa neurotrophin receptor (NTR) has been aligned with this apoptosis-inducing motif. Peptides at the carboxyl terminus of each domain potentially form amphiphilic helices, one of which (in NTR) resembles mastoparan, a G-protein activating peptide. Molecular models of three death-region peptides suggest that observed sequence similarities reflect a common structure, perhaps capable of undergoing an induced coil to helix transition.
Programmed cell death, or apoptosis, is a highly regulated process used to eliminate unwanted or damaged cells from multicellular organisms. The morphology of cells undergoing apoptosis is similar to cells undergoing both normal mitosis and an aberrant form of mitosis called mitotic catastrophe. During each of these processes, cells release substrate attachments, lose cell volume, condense their chromatin, and disassemble the nuclear lamina. The morphological similarities among cells undergoing these processes suggest that the underlying biochemical changes also may be related. The susceptibility of cells to apoptosis frequently depends on the differentiation state of the cell. Additionally, cell cycle checkpoints appear to link the cell cycle to apoptosis. Deregulation of the cell cycle components has been shown to induce mitotic catastrophe and also may be involved in triggering apoptosis. Some apoptotic cells express abnormal levels of cell cycle proteins and often contain active Cdc2, the primary kinase active during mitosis. Although cell cycle components may not be involved in all forms of apoptosis, in many instances cell proliferation and cell death may share common pathways.
Density-arrested quiescent murine Balb/c-3T3 cells are dependent upon growth factors for their survival. Withdrawal of serum from their medium induces rapid cell death, the mechanism of which is not yet fully understood. We have studied the effect of serum deprivation on density-inhibited quiescent Swiss 3T3 cells and found that they undergo rapid cell death upon total withdrawal of serum. The nature of this cell death is similar to apoptosis, as shown by cellular and nuclear morphology and DNA fragmentation into oligonucleosomal fragments. Investigating the regulation of early cell-cycle genes during this process, we found that c-myc, c-jun, c-fos, and cdc2 protein presence is induced after serum deprivation, when the phosphorylated form of the RB protein also appears. The upregulation of these genes' protein products is coupled with the appearance of PCNA, a proliferation-specific nuclear antigen, as well as significant incorporation of BrdU, which may reflect DNA repair activity; in situ analysis shows that BrdU-positive cells are also positive for DNA fragmentation. These results suggest that en route to apoptosis, cells undergo events typical of early cell-cycle traverse by expressing early G1 genes and may even experience the late G1/S phase boundary, as shown by the presence of PCNA. However, the demonstrated ability of these cells to traverse the G1 phase of the cell cycle seems to be an abortive event, since they die shortly afterwards.
We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.
Single ultraviolet (u.v.) irradiation of mammalian cells in culture evokes the transcriptional activation of various proto-oncogenes, among them members of the fos/jun family which are known to play an important role in cell proliferation and differentiation. u.v. exposure of mammalian skin results in growth arrest and cell death followed by hyperproliferation of epidermal cells. To obtain information in vivo about a possible relationship between u.v.-induced proto-oncogene expression and cellular alterations, we have analysed the expression of c-fos, fosB, c-jun, junB, bcl-2 and bax in rat epidermis after single and chronic u.v. irradiation. We present data demonstrating that the transcripts of these genes are constitutively expressed in the epidermis and that expression is differentially modulated by u.v. exposure. Single u.v. irradiation causes a rapid and sustained increase in c-jun, junB and c-fos mRNA and a decline in bcl-2 transcripts, whereas expression of bax remained unchanged. c-Fos and c-Jun immunoreactivity was localized throughout the epidermal cell layers 1.5 h after single irradiation, but restricted to basal cells at 48 h suggesting an involvement in both u.v.-induced apoptosis and hyperproliferation. 48 h after chronic exposure a significantly higher induction and a totally different pattern of epidermal proto-oncogene expression was detectable which may be associated with malignancy. Superfusion of rat skin with c-fos antisense oligodeoxynucleotides inhibited the increase in c-Fos immunolabeled epidermal cells 1.5 h after single u.v. irradiation demonstrating that antisense oligodeoxynucleotides are capable of penetrating mammalian skin and modulating the u.v. response in vivo. However, suppression of the early c-Fos activation did not significantly affect the formation of sunburn cells in the u.v.-exposed epidermis. Thus, c-Fos does not seem to play a major role in u.v.-induced apoptosis or other members of the fos/jun family may compensate for a loss in c-Fos.
Squamous cell carcinoma of the skin (SCC) can progress by stages: sun-damaged epidermis, with individual disordered keratinocytes; actinic keratosis (AK), spontaneously regressing keratinized patches having aberrant cell differentiation and proliferation; carcinoma in situ; SCC and metastasis. To understand how sunlight acts as a carcinogen, we determined the stage at which sunlight mutates the p53 tumour-suppressor gene and identified a function for p53 in skin. The p53 mutations induced by ultraviolet radiation and found in > 90% of human SCCs were present in AKs. Inactivating p53 in mouse skin reduced the appearance of sunburn cells, apoptotic keratinocytes generated by overexposure to ultraviolet. Skin thus appears to possess a p53-dependent 'guardian-of-the-tissue' response to DNA damage which aborts precancerous cells. If this response is reduced in a single cell by a prior p53 mutation, sunburn can select for clonal expansion of the p53-mutated cell into the AK. Sunlight can act twice: as tumour initiator and tumour promoter.
Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti-low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.
The role of the low-affinity neurotrophin receptor (p75NTR) in signal transduction is undefined. Nerve growth factor can activate
the sphingomyelin cycle, generating the putative-lipid second messenger ceramide. In T9 glioma cells, addition of a cell-permeable
ceramide analog mimicked the effects of nerve growth factor on cell growth inhibition and process formation. This signaling
pathway appears to be mediated by p75NTR in T9 cells and NIH 3T3 cells overexpressing p75NTR. Expression of an epidermal growth
factor receptor-p75NTR chimera in T9 cells imparted to epidermal growth factor the ability to activate the sphingomyelin cycle.
These data demonstrate that p75NTR is capable of signaling independently of the trk neurotrophin receptor (p140trk) and that
ceramide may be a mediator in neurotrophin biology.