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 ...

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

a b s t r a c tGold nanoparticles (AuNPs) represent an effective choice for topical drug delivery systems thanks to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio. Even if systemic, methotrexate still plays an important role in psoriasis treatment: its topical use shows insufficient percutaneus penetration owing to limited passive diffusion, high molecular weight and dissociation at physiological pH. The aim of our study was to design a new drug delivery nanocarrier for Methotrexate and to improve its solubility, stability and biodistribution. AuNPs were on purpose prepared with a hydrophilic stabilizing layer, in order to improve the colloidal stability in water. Watersoluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were prepared and loaded with methotrexate (MTX). The loading efficiency of MTX on Au-3MPS was assessed in the range 70-80%, with a fast release (80% in one hour). The release was studied up to 24 h reaching the value of 95%. The Au-3MPS@MTX conjugate was fully characterized by spectroscopic techniques (UV-vis, FTIR) and DLS. Preliminary toxicity tests in the presence of keratinocytes monolayers allowed to assess that the used Au-3MPS are not toxic. The conjugate was then topically used on C57BL/6 mouse normal skin in order to trace the absorption behavior. STEM images clearly revealed the distribution of gold nanoparticles inside the cells. In vitro studies showed that Methotrexate conjugated with Au-3MPS is much more efficient than Methotrexate alone. Moreover, DL50, based on MTT analysis, is 20 folds reduced at 48 h, by the presence of nanoparticles conjugation. UV-vis spectra for in vivo tracing of the conjugate on bare mouse skin after 24 h of application, show increased delivery of Methotrexate in the epidermis and dermis using Au-3MPS@MTX conjugate, compared to MTX alone. Moreover we observed absence of the Au-3MPS in the dermis and in the epidermis, suggesting that these layers of the skin do not retain the nanoparticles. Based on our data, we found that the novel Au-3MPS@MTX conjugate is an effective non-toxic carrier for the satisfactory percutaneous absorption of Methotrexate and could help in possible topical treatment of psoriasis.

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brainderived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas b-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-jB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or b-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or b-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF-or b-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis.

Ultraviolet radiation is a potent inducer of apoptosis, whereas autocrine nerve growth factor protects human keratinocytes from programmed cell death. To evaluate the role of nerve growth factor in the mechanisms of ultraviolet B-induced apoptosis, cultured human keratinocytes were ultraviolet B irradiated following pretreatment with K252, a specific inhibitor of the tyrosine kinase high-affinity nerve growth factor receptor. Here we report that the addition of K252 significantly enhanced keratinocyte apoptosis. We then transfected normal human keratinocytes with pNUT-hNGF. Nerve growth factor overexpressing keratinocytes secreted the highest amounts of nerve growth factor in culture supernatants, were more viable, and had a higher rate of proliferation than mock-transfected cells. Whereas ultraviolet B radiation downregulated nerve growth factor mRNA and protein as well as the tyrosine kinase high-affinity nerve growth factor receptor in normal keratinocytes, it failed to do so in nerve growth factor-transfected cells. Moreover, nerve growth factor overexpressing keratinocytes were partially resistant to apoptosis induced by increasing doses of ultraviolet B at 24 and 48 h. These results indicate that downregulation of nerve growth factor function plays an important part in the mechanisms of ultraviolet B-induced apoptosis in human keratinocytes. In addition, ultraviolet B caused a decrease in BCL-2 and BCL-xL expression in mock-transfected keratinocytes, but not in nerve growth factor overexpressing cells. Finally, nerve growth factor prevented the cleavage of the enzyme poly(ADP-ribose) polymerase induced in human keratinocytes by ultraviolet B. These results are consistent with a model whereby the autocrine nerve growth factor protects human keratinocytes from ultraviolet B-induced apoptosis by maintaining constant levels of BCL-2 and BCL-xL, which in turn might block caspase activation.