Nerve growth factor and keratinocytes: a role in psoriasis

ARTICLE

Nerve growth factor (NGF) belongs to a family of neurotrophic proteins termed the neurotrophin family which also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4 and NT-5 [1]. NGF was discovered more than forty years ago by the Italian Nobel Prize Rita Levi-Montalcini [2] and isolated by Stanley Cohen in 1960 [3]. NGF is responsible for the maintenance, development and differentiation of several cell types of the nervous system [4].

Cellular responses to NGF are mediated by two classes of transmembrane receptors, a low affinity receptor of ~75 kD (p75) [5] and a high affinity tyrosine kinase receptor of ~ 140 kD (TrkA) [6]. The high-affinity binding site requires expression of the TrkA protooncogene [7]. While TrkA can mediate NGF-induced effects in the absence of p75 [8, 9], the functional significance of this low-affinity receptor in NGF signal transduction is still a matter of intensive investigation [10]. p75 may modulate the function of trk receptors in that it enhances the affinity of TrkA for NGF [11]. Furthermore, p75 forms a complex with TrkA [12] and modulates TrkA trophic signals [13]. Only recently, it has become clear that p75 can signal, at least in certain cell types, in the absence of Trk. Indeed, Schwann cells that express substantial amounts of p75, but do not express TrkA, respond to NGF [14]. In addition, the structure of p75 is quite similar to that of p55 TNF receptor and Fas [15], proteins known to share a conserved sequence in their cytoplasmic domain, the so-called "death domain" motif which can signal cell death upon activation [16]. NGF binding to p75 in cells that do not express TrkA results in increased intracellular ceramide [17], activation of transcription factor NFkB [14] and apoptosis [18].

NGF plays a crucial role as a neurotrophic molecule also at the skin level [19]. Indeed, during cutaneous development, NGF is expressed at highest levels in the epidermis [20] and is retrogradely transported to the innervating neurons [4]. In particular, NGF synthesis begins in the skin with the arrival of the first axons to the epidermis, and the level of NGF mRNA increases throughout the period of skin innervation [20]. NGF neurotrophic activity in the skin is definitely provided by studies in transgenic mice: overexpression of NGF in the epidermis is associated with hypertrophy of the peripheral nerves, increase in the size of neurons and modulation of the expression of its own receptor [21-23].

NGF exerts a number of effects also on non-neuronal cells [24-27]. In particular, autocrine NGF rescues memory B lymphocytes from cell death [28] and exogenous NGF protects neutrophils from apoptosis [29]. NGF, synthesized and released by human keratinocytes [30], acts in a paracrine fashion on human melanocytes. Melanocytes express the low and the high affinity NGF receptors and display increased dendricity upon NGF stimulation [31]. In addition, NGF rescues melanocytes from UV-induced apoptosis [32]. NGF, secreted by keratinocytes, is also the key player of an autocrine system that could be of great relevance in several pathophysiological skin conditions. Autocrine NGF in human keratinocytes and its possible implication in the pathogenesis of psoriasis will be the topic of the present review.

Autocrine NGF in human keratinocytes

Exponentially growing keratinocytes, but not confluent keratinocytes, nor stratified keratinocyte cultures are the pool of epidermal cells that synthesize NGF [33]. Consistent with the above findings, greater amounts of NGF are secreted by proliferating, pre-confluent keratinocytes than by more differentiated, stratified cells [30]. This indicates that the basal proliferative cell compartment is the source of NGF in the epidermis. NGF secreted by keratinocytes is biologically active, since medium derived from these cells promote neurite outgrowth of sensory neurons [33] and of the pheocromocytoma cell line PC12 [34].

Human keratinocytes synthesize p75 and TrkA, both proteins being expressed in the basal layer of the epidermis [30]. NGF stimulates TrkA phosphorylation in human keratinocytes [35], while the role of p75 is still unclear. Although in human keratinocytes p75 mRNA and protein are increased during their exponential growth phase [36], K252, a potent inhibitor of TrkA phosphorylation, but not anti-p75, abrogates NGF-induced keratinocyte proliferation [30]. This seems to suggest that TrkA is the functional NGF receptor in human keratinocytes. In addition, TrkA overexpressing keratinocytes, in the absence of exogenous NGF, proliferate better than mock transfected cells, most likely because of more molecules of the high-affinity receptor becoming available for NGF released from keratinocytes. Moreover, the addition of an NGF mimicking anti-trk antibody induces an increased keratinocyte proliferation in TrkA overexpressing cells as compared to mock transfected keratinocytes [34]. These experiments strongly support the functional role of TrkA in mediating NGF activity in human keratinocytes.

Because NGF is synthesized and released by basal keratinocytes, which cells in turn express TrkA, an autocrine activity of this growth factor in keratinocytes could be hypothesized. Indeed, exogenous NGF up-regulates the synthesis of NGF mRNA in keratinocytes [34]. In addition, autocrine NGF may constitutively phosphorylate TrkA, as shown by the anti-phosphotyrosine antibody which precipitates a band of 140 kD MW even in absence of exogenous NGF [35]. Furthermore, the addition of K252, in the absence of exogenous NGF, strongly inhibits DNA synthesis in these cells [36]. To further stress the role of NGF as a mitogen for human keratinocytes, we have recently transfected normal human keratinocytes with NGF cDNA. First, NGF transfected cells synthesize and secrete the highest amounts of NGF. In addition, there is a constant increase in thymidine incorporation in mock transfected cells up to 168 hrs, most likely due to the release of autocrine NGF. As expected, the proliferation rate in NGF transfected keratinocytes is significantly higher than in mock transfected cells. Interestingly, the DNA synthesis is increased 3-fold in NGF overexpressing cells as compared to controls at 168 hrs. This indicates that keratinocytes overexpressing NGF can release this growth factor to an extent that allows them to proliferate significantly better than controls even after seven days in culture [37]. Taken together the above findings demonstrate that an autocrine loop exists in human keratinocytes where NGF and TrkA are the key players.

It has been proposed that cells carry a genetic death program and are destined to die unless they are rescued by "survival factors" from other cells [38-40]. Because NGF acts as a survival factor for many cell types [28, 29, 32], we postulated that autocrine NGF could rescue keratinocytes from apoptosis. In order to verify this hypothesis, keratinocytes were cultured with or without the addition of anti-NGF antibodies or K252. While the vaste majority of untreated keratinocytes survived up to six days in culture, keratinocytes treated with K252 or anti-NGF underwent apoptosis. By contrast, anti-p75 antibody failed to induce apoptotic cell death [41].

In normal epidermis, apoptosis appears to initiate in the basal cell layer [42, 43]. Apoptotic susceptibility seems to be determined by the cell cycle and its checkpoints [44]. In addition, evidence has been reported that entry into apoptosis requires cell cycling [45]. It would appear that dividing cells of the basal cell compartment are most sensitive to apoptosis, because they are undergoing a particular phase of the cell cycle [43] and an abortive entry into the cycle could lead to apoptotic cell death [46, 47]. NGF is released in increasing amounts by proliferating keratinocytes and TrkA receptor is expressed only in basal keratinocytes. Therefore, it is conceivable that autocrine NGF operates through its high-affinity receptor in the basal cell compartment to counteract the apoptotic program.

Interestingly, NGF mRNA and protein are markedly down-regulated by ultraviolet (UV) radiation [37]. Because UV irradiation induces apoptosis in human keratinocytes [48], the possibility exists that NGF could be involved in this process. Indeed, UV fail to affect NGF synthesis and release in keratinocytes overexpressing NGF. Moreover, UV irradiation down-regulates TrkA mRNA, but not p75 (unpublished data) in normal keratinocytes. By contrast, UV does not decrease TrkA mRNA levels in NGF transfected cells. Most importantly, NGF overexpression prevents caspase activation in human keratinocytes and cells are thus protected from UV-induced apoptosis [37]. These data suggest that first, down-regulation of the survival factor NGF takes part in the mechanisms of UV-induced apoptosis; second, high levels of endogenous NGF and TrkA prevent UV-induced keratinocyte apoptosis. After UV-irradiation, apoptotic keratinocytes ("sunburn cells") are largely found in the proliferative basal cell layer [48, 49]. Particularly, S-phase keratinocytes of this compartment have been shown to contribute most to the formation of "sunburn cells" [50]. Therefore, as in spontaneous apoptosis, it would appear that endogenous NGF acts as a survival factor through its high-affinity receptor mostly for basal keratinocytes. This is also consistent with the recent observation that human keratinocytes maintain strong anti-apoptotic defenses that can be altered by growth factor withdrawal [51].

Bcl-2 is the founding member of the large Bcl-2 family and protects many cell types from apoptosis [52, 53]. Bcl-x gene displays a close homology to Bcl-2 and produces two proteins, Bcl-x_L and Bcl-x_S. While Bcl-x_L protects cells from apoptosis, Bcl-x_S inhibits Bcl-2 and Bcl-x_L function, thus promoting apoptosis [54, 55]. NGF protects different cell types from apoptosis through a Bcl-2 dependent pathway [28, 32, 56]. In human keratinocytes, the addition of NGF enhances the expression of Bcl-2 protein levels [35], while anti-NGF antibody and K252 strikingly down-regulate Bcl-2, in the absence of exogenous NGF. Furthermore, HaCat cells overexpressing Bcl-2 are protected from apoptosis induced by K252, indicating that autocrine NGF prevents keratinocyte apoptosis in a Bcl-2-dependent manner [41]. Bcl-22 family plays an important role in UV-induced apoptosis. Indeed, human Bcl-2 expression delays UV-induced apoptosis in marsupial cells [57] and the addition of purified Bcl-2 protein blocks apoptosis in cytoplasmic extracts of cells exposed to UV light [58]. Furthermore, a decline in Bcl-2 transcripts is observed after UV radiation in rat skin [59]. Following UV radiation, the number of apoptotic cells in transgenic mice overexpressing bcl-x_L is markedly lower than in the skin of wild-type mice [60]. Recently, it has been shown that UV light completely abolishes Bcl-2 expression in human skin [61] and Bcl-2 transgenic mice exhibit reduced sensitivity to UV-B [62]. Interestingly, UV-B down-regulates the expression of Bcl-2 and Bcl-x_L in human keratinocytes, but not in the same cells overexpressing NGF [37]. This indicates that NGF antagonizes UV-B-induced apoptosis in human keratinocytes by modulating two antiapoptotic members of the Bcl-2 family. Because NGF, Trk and Bcl-2 are exclusively expressed in basal keratinocytes, one could speculate that an autocrine survival system sustained by NGF exists in the proliferative cell compartment: under normal conditions, autocrine NGF maintains constant levels of Bcl-2 in basal keratinocytes, most likely through the phosphorylation of Trk [24, 63]. When the activity of endogenous NGF is inhibited either by UV or by K252, which both affect Trk phosphorylation, levels of Bcl-2 dramatically decrease and basal keratinocytes undergo apoptosis .

NGF in cutaneous pathophysiology

How can the autocrine system sustained by NGF be relevant to the pathophysiology of the skin? The sprouting of sensory nerve fibers is typical of wound healing and disappears when the process is completed [64, 65]. Because NGF supports the survival of sensory nerves [66] and regulates the expression of neuropeptides [67], it could be suggested that proliferating keratinocytes, by secreting increasing amounts of NGF, regulate skin innervation during wound healing. Indeed, it has been known for many years that NGF accelerates the rate of wound healing [68]. Moreover, a recent study by Matsuda and co-workers has shown that the levels of NGF mRNA and protein increase in wounded skin tissues and topical application of NGF into the wounds accelerate the rate of wound healing [69].

Cell death exists in all tumors and accounts for the elimination of most cancer cells [70]. NGF, by inhibiting keratinocyte apoptosis, could be involved in skin tumorigenesis. It is interesting to note that K252, that specifically blocks NGF-induced TrkA phosphorylation, inhibits the growth of human prostatic carcinoma cell lines [71]. Sun exposure is the major environmental agent implicated in the induction of non-melanoma skin cancer [72, 73]. Sunlight induces mutations in the p53 tumor suppressor gene in chronically sun-exposed skin [74], in actinic keratosis, and in squamous cell carcinoma [75], pointing to a critical role for UV in tumorigenesis [76]. However, the apoptotic effects of UV may also be important in inhibiting the development of skin cancer [77]. NGF, by preventing UV-induced keratinocyte apoptosis, might have a role in the development of skin neoplasia. NGF is exclusively secreted by proliferating basal keratinocytes [30, 33]. Interestingly, it has been shown recently that the proliferative basal cell compartment of the epidermis contains clones of cells with characteristic UV-type p53 mutations which are more frequent and larger in sun exposed than in sun protected areas [74]. Therefore, an autocrine survival system sustained by NGF could be operational in the basal layer of the epidermis leading to the expansion of mutated keratinocytes and to the initiation of skin cancer.

NGF and psoriasis

Psoriatic lesions are characterized by an accelerated turnover of neural elements and by a more dense innervation [78, 79]. In addition, an alteration in the expression of neuropeptidergic fibers has been observed in psoriasis [80-83]. In particular, an imbalance of the neuropeptide (NP) content is a constant finding in psoriatic skin [84]. Although the mechanisms leading to biochemical alterations in the peptidergic neurons during peripheral inflammation are not completely understood, there is evidence that NGF could play an important role. Indeed, not only does NGF regulate skin innervation, but it also exerts a continuous control over NP synthesis in primary sensory neurons [85]. In the skin, when NGF content is increased in the epidermis, the expression of neuropeptides is highest. If NGF is inactivated by anti-NGF antibodies, no increase in neuropeptides is observed [86]. Moreover, NGF induces a complete recovery of NP content in injured cutaneous sensory nerves [66], and stimulates NP synthesis in dorsal root ganglia [67]. This suggests that NGF plays a regulatory role in vivo in the stimulation of neuropeptide synthesis [87]. Therefore NGF, through the regulation of NP, could take part in neurogenic inflammation which is known to be involved in the pathomechanisms of several dermatoses, including psoriasis [88-90]. Furthermore, because certain NP stimulate keratinocyte proliferation [84-91], it is conceivable that NGF, by regulating NP synthesis, can magnify the proliferative response. Thus, NGF could participate both in the inflammatory and in the hyperproliferative mechanisms associated with the formation of psoriatic lesions.

Cytokine dysregulation is an attractive concept to explain many of the observed abnormalities in psoriasis [92]. In particular, the levels and the functional activity of interleukin-1 alpha (IL-1 alpha) are constantly reduced in psoriatic lesions relative to normal skin [93-95]. Although the significance of this finding remains unknown, it is interesting that NGF down-regulates the release of IL-1alpha in cultured human keratinocytes (Fig. 1) and that K252, in the absence of exogenous NGF, strikingly up-regulates the expression of this cytokine (Fig. 2).

Elegant studies by Budtz in toad and human skin have demonstrated that apoptosis is a key event in epidermal homeostasis. Indeed, apoptotic cell death removes the excess cells, thus establishing the epidermal architecture and maintaining the proper cell number [96]. This implies that any dysfunction of this process could lead to pathological conditions characterized by epidermal thickness. More than 10 years ago Goldsmith, while he was commenting Budtz's experiments, first proposed the involvement of apoptotic cell death in the pathomechanisms of psoriasis [97]. Goldsmith's idea was first supported by the observation that psoriatic keratinocytes have abundant amounts of the cell survival protein bcl-x_L [98] and express Fas ligand upon UV-B irradiation [99]. Recently, it was shown that keratinocytes derived from psoriatic plaques are resistant to apoptosis compared with normal skin [100]. Finally, the anti-psoriatic drugs methotrexate, camptothecin and vitamine D3 reduce epidermal hyperplasia via the induction of apoptosis [101-103]. Interestingly, NGF protein levels are increased in psoriatic as compared to non-lesional and normal skin [104], and psoriatic keratinocytes express higher amounts of NGF than normal keratinocytes [105]. Furthermore, in psoriatic lesions, TrkA shows a pattern of expression quite similar to that of EGF-R [106]. Indeed, TrkA, which is only expressed in basal keratinocytes in normal skin, is detected in all epidermal layers in psoriatic lesions (Fig. 3). This has also been confirmed recently by "in situ hybridization" studies: it appears that TrkA synthesis takes place in the basal layer of the epidermis in normal skin, whereas TrkA mRNA is expressed throughout all epidermal layers in psoriatic skin [107]. Taken together, the above findings suggest that NGF, by preventing keratinocyte apoptosis through its high affinity receptor, takes part in the pathogenesis of the psoriatic lesion. A better understanding of the mechanisms by which NGF prevents keratinocyte apoptosis and in particular the study of the genes involved in this process will allow the design of new therapeutic approaches in the treatment of psoriasis and other hyperproliferative skin conditions.

Article accepted on 13/12/99

CONCLUSION

Acknowledgements

These studies were supported in part by the "Angela Serra" Association for Cancer Research. I would like to deeply thank Dr. Alessandra Marconi for her skillful technical assistance.

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