Decreased expression of GPC1 in human skin keratinocytes and epidermis during ageing.

BACKGROUND: Glypicans (GPCs) are heparan sulfate cell membrane proteoglycans containing glycosylphosphatidylinositol (GPI) anchor. They play important role in cell behavior by activating/presenting numerous growth factors and cytokines. OBJECTIVES: The expression of GPCs was investigated in primary culture of skin keratinocytes sampled from healthy donors of different age. MATERIALS AND METHODS: Primary keratinocytes from healthy female donors aged from 20 to 89 years old (n = 30) were either isolated from breast or abdominal skin samples (n = 27) or purchased (n = 3). GPCs expression was examined by qPCR, immunohistochemistry and western blot. Its role in proliferation induced by fibroblast growth factor 2 (FGF2) was also studied. RESULTS: Glypican 1 (GPC1) was the major expressed GPC in human keratinocytes. Its expression was up to two orders of magnitude higher than other GPCs and was significantly decreased with the age of the donors. It was localized at the cell surface and associated with intracellular granules. In skin sections, GPC1 was mainly localized in basal layer of epidermis. Shedding of GPCs decreased the proliferative effect of FGF2, confirming their role of modulator of growth factor effects on keratinocytes. These results established GPC1 as an important player in epidermis biology and skin ageing.

Dehydroepiandrosterone and neurotrophins favor axonal growth in a sensory neuron-keratinocyte coculture model.

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.

Glycation during human dermal intrinsic and actinic ageing: an in vivo and in vitro model study.

BACKGROUND: Non-enzymatic glycation occurring in normal human skin plays an important part in ageing. OBJECTIVES To visualize and quantify, in human subjects, the extent of glycation during human dermal intrinsic and actinic ageing, and to develop a reliable reproducible in vitro model for evaluating the efficacy of potential inhibitors of glycation. METHODS: By immunohistochemistry using a monoclonal antibody recognizing carboxymethyl lysine, an advanced glycation end-product (AGE) (first objective), and by incubating dead de-epidermized dermis (DED) with glucose to simulate ageing-induced glycation in a human dermal equivalent model (second objective). RESULTS: We found that glycation of the dermis generally arises after 35 years, then increases rapidly with intrinsic ageing. We also noticed an enhancement of glycation by solar irradiation that occurred via glycation of the elastic fibre network or solar elastosis tissue. In the model, production of AGEs appeared in a time-dependent way, mimicking glycation observed in vivo during chronological ageing. Irradiation of DED before incubation with glucose strongly enhanced induction of AGEs, corresponding to the effect of solar irradiation on AGEs observed in vivo. CONCLUSIONS: These results confirm a marked increase of AGEs during intrinsic ageing in normal human skin and also suggest that glycation is enhanced in photoaged skin.