Abstract. The molecular mechanism underlying the promotion of wound healing by TGF-/31 is incompletely understood. We report that TGF-/5'I regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-~I: (a) strongly upregulates the expression of the fibronectin receptor c~5~1, the vitronectin receptor t~v/~5, and the collagen receptor t~2/~l by differentially modulating the synthesis of their ot and/~ subunits; (b) downregulates the multifunctional oL3/~l heterodimer; (c) induces the de novo expression and surface exposure of the av#6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both #1 and ~4 dimers; and (f) causes a pericellular redistribution of ~v/~5. These data suggest that ot5~l, ave6, and av/~5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-~I reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-/5'l-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells. HUMAr~ epidermis, the outermost layer of skin, is a stratified squamous epithelium mainly composed of a single cell type, the keratinocyte. The epidermis survives through a self-renewal process (Green, 1980). Small progenitor keratinocytes (Barrandon and Green, 1987b), forming the innermost epidermal basal layer, regularly undergo mitosis, differentiation, and upward migration to replace terminally differentiated cornified cells that are continuously shed into the environment (Green, 1980;Watt, 1989;Fuchs, 1990). Basal epidermal keratinocytes rest on a basement membrane composed of a specific subset of extracellular matrix proteins such as laminin, type IV collagen, kalinin, nidogen, and heparan sulfate proteoglycan. The firm adhesion of basal keratinocytes, hence of the whole epidermis, to the basal lamina is mediated by hemidesmoAddress all correspondence to Dr. Michele De Luca, Unit of Epithelial Biology and Biotechnology, CBA, Centro di Biotecnologie Avanzate, Viale Benedetto XV no. 10, 16132, Genoa, Italy. Ph.: (39) (10) 5737423. Fax: (39) (10) 5737405.somes. These structures link the epithelial intermediate filament network to the dermal anchoring fibrils, which are mainly composed of type VII collagen and extend from the basement membrane to anchoring plaques in the papillary dermis (Jones et al., 1994). The keratinocyte behavior changes dramatically when ...

Neurotrophins (NTs) belong to a family of growth factors, which control the development, maintenance, and apoptotic death of neurons and also fulfill multiple regulatory functions outside the nervous system. Biological effects induced by NTs strongly depend on the pattern of NT receptor/co-receptors expression in target cells, as well as on the set of intracellular adaptor molecules that link NT signalling to distinct biochemical pathways. In this review, we summarize data on the molecular mechanisms underlying the involvement of NTs in the control of non-neuronal functions in normal skin (e.g. keratinocyte proliferation, melanocyte development and apoptosis, hair growth). We also review the data on the role for NTs and their receptors in a number of pathological skin conditions (stress-induced hair loss, psoriasis, atopic dermatitis). Although additional efforts are required to fully understand mechanisms underlying the involvement of NTs and their receptors in controlling functions of normal and pathologically altered skin cells, substantial evidence suggests that modulation of NT signalling by NTs receptor agonists/antagonists may be developed as intervention modalities in distinct skin and hair growth pathologies.

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Melanoma is a highly aggressive skin tumor that originates in the epidermis from melanocytes. As melanocytes share with the nervous system a common neuroectodermal origin and express all neurotrophins (NTs), we evaluated the expression and function of NTs and their receptors in melanoma. We report that primary and metastatic melanoma cell lines synthesize and secrete all NTs. Moreover, melanoma cells express the low-affinity (p75NTR) and the high-affinity tyrosine kinase NT receptors (Trk). The inhibition of Trk receptors by either K252a or Trk/Fc chimeras prevents proliferation, indicating that autocrine NTs are responsible for this effect. NT-3, NT-4, and nerve growth factor (NGF) induce cell migration, with a stronger effect on metastatic cell lines. Transfection with p75NTR small interfering RNA (p75NTRsiRNA) or treatment with K252a inhibits NT-induced melanoma cell migration, indicating that both the low- and high-affinity NT receptors mediate this effect. All melanoma cell lines express the p75NTR coreceptor sortilin by which proNGF stimulates migration in melanoma cells, but not in cells transfected with p75NTRsiRNA. These results indicate that NTs, through their receptors, play a critical role in the progression of melanoma.

The Fas/Fas ligand system triggers the extrinsic apoptotic pathway and is involved in several inflammatory conditions, also at the skin level. The Fas/Fas ligand cell death pathway plays a major role in anoikis, a type of apoptosis characterized by cell detachment. As pemphigus is characterized by loss of cell to cell adhesion, we evaluated the role of anoikis and Fas ligand in this bullous disease. We report that, in suprabasal epidermis from perilesional pemphigus skin, most keratinocytes are apoptotic. Moreover, Fas ligand levels are markedly increased in sera from pemphigus patients, whereas they are undetectable in sera from patients undergoing steroid treatment. Sera from untreated patients but not from patients under steroids induce keratinocyte apoptosis. Pemphigus-sera-induced cell death is partially inhibited by pretreatment with anti-Fas ligand antibodies and by incubation with caspase-8 inhibitor Z-IETD-FMK. Finally, caspase-8 is activated in keratinocytes provided with sera from pemphigus patients, whereas cleavage is partially blocked by pretreatment of sera with anti-Fas ligand antibody. These results suggest that increased Fas ligand in pemphigus sera is responsible for keratinocyte apoptosis, which occurs through the activation of a caspase-8-driven extrinsic apoptotic pathway.

Because inhibition of integrin signaling induces apoptosis, we investigated whether keratinocytes expressing L L1 and K K6L L4 integrins (enriched for stem cells) are protected from cell death. Keratinocytes rapidly adhering to type IV collagen expressed highest levels of L L1 and K K6L L4 and of the anti-apoptotic stem cell marker p63. Apoptotic cells were signi¢cantly higher in slowly adhering than in rapidly adhering keratinocytes. Anti-L L1 integrin caused a signi¢cant increase in apoptotic cells, while it decreased Bcl-2 levels in stem keratinocytes. Bax and Bad proteins were higher in slowly adhering than in rapidly adhering cells. By contrast, Bcl-2, Bcl-x and Mcl-1 proteins were highest in rapidly adhering keratinocytes and nearly absent in slowly adhering cells. After addition of anti-L L1 integrin, the apoptotic rate was signi¢cantly higher in HaCaT cells not expressing Bcl-2 than in controls. These results indicate that keratinocytes enriched for stem cells are protected from apoptosis via L L1 integrin, in a Bcl-2 dependent manner. ß

Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-⌬Ex3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2␣ tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking ␤1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of ␤1 integrin upregulates mRNA expression of survivin-2␣. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the "niche" protection by preventing anoikis in KSCs. STEM CELLS 2007;25:149 -155

Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by β-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-β1 (TGF-β1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-β1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with β-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.