François Brégégère

The ubiquitin-proteasome system at the crossroads of stress-response and ageing pathways : A handle for skin care?

The regulation of gene expression at the transcriptional level has been considered for long as the main mechanism of cellular adaptive responses. Since the turn of the century, however, it is becoming clear that higher organisms developed a complex, sensitive and maybe equally important network of regulatory pathways, relying largely on protein interactions, post-translational modifications and proteolysis. Here we review the involvement of the ubiquitin-proteasome pathway of protein degradation at different levels of cellular life in relation with ageing, and with a special focus on skin. It comes out that the ubiquitin system plays a major role in signal transduction associated with stress and ageing, in skin in particular through the control of retinoid and NF-kappaB pathways. The understanding of specific proteolytic targeting by E3 ubiquitin-ligases paves the way for a new generation of active molecules that may control particular steps of normal and pathological ageing.

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin

Background:  Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known.

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage.

Background:  Ultraviolet (UV) irradiation is a major cause of skin damage, of long‐term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV‐induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non‐invasive procedures.

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas‐mediated apoptosis. Here, we describe the induction of the Fas‐dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV‐IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV‐IgG‐mediated apoptotic death and culture lesions. Together with immunosenescence, this phenomenon may explain the late onset of pemphigus disease.

Aged keratinocyte phenotyping : Morphology, biochemical markers and effects of Dead Sea minerals

The aging process and its characterization in keratinocytes have not been studied in depth until now. We have assessed the cellular and molecular characteristics of aged epidermal keratinocytes in monolayer cultures and in skin by measuring their morphological, fluorometric and biochemical properties. Light and electron microscopy, as well as flow cytometry, revealed increase in cell size, changes in cell shape, alterations in mitochondrial structure and cytoplasmic content with aging. We showed that the expression of 16 biochemical markers was altered in aged cultured cells and in tissues, including caspases 1 and 3 and beta-galactosidase activities, immunoreactivities of p16, Ki67, 20S proteasome and effectors of the Fas-dependent apoptotic pathway. Aged cells diversity, and individual variability of aging markers, call for a multifunctional assessment of the aging phenomenon, and of its modulation by drugs. As a test case, we have measured the effects of Dead Sea minerals on keratinocyte cultures and human skin, and found that they stimulate proliferation and mitochondrial activity, decrease the expression of some aging markers, and limit apoptotic damage after UVB irradiation.

Evaluation of topically applied copper(II) oxide nanoparticle cytotoxicity in human skin organ culture.

The increasing use of nano-sized materials in our environment, and in many consumer products, dictates new safety concerns. In particular, adequate experimental models are needed to evaluate skin toxicity of metal oxide ions, commonly found in cosmetic and dermatologic preparations. We have addressed the biological effects of topically applied copper oxide (CuO) nanoparticles in human skin organ cultures, using light and electron microscopy, and biochemical tests. Nanoparticles were more toxic than micro-sized particles, and their effects were stronger when supplied in growth medium than in topical application. Still topically applied CuO nanoparticles induced inflammatory cytokine secretion and necrosis, especially in epidermis deprived of its protective cornea. Since nanoparticle penetration was not seen, we propose that they may adhere to skin surface, react with the local acidic environment, and generate soluble ions that make their way to inner sites. This work illustrates the abilities of skin organ culture to evaluate the biological effects of topically-applied materials on skin in vitro.

Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load

In order to assess the activity of cellular proteasome, we developed a method to permeabilize keratinocyte monolayers and measure proteasome activities intracellularly, using fluorogenic peptide substrates. The observed K(m) did not differ significantly in situ and in soluble extracts, and the K(i) of proteasome inhibitor MG132 was slightly higher in situ (34nM instead of 4nM). Inhibition studies in permeabilized cells showed that MG132 followed competitive inhibition patterns, and clasto-lactacystin beta-lactone non-competitive patterns, as expected. The observed velocities in situ (500pmoles/min/mg protein) were comparable to the best values of proteasome activity in crude cellular extracts. These features altogether allowed to identify the in situ activity as that of proteasome. To characterize proteasome complexes present in human keratinocytes, we analyzed cellular lysates by ultracentrifugation and gel filtration: most proteasome activity was associated with PA700-bound, presumably 26S, particles. PA28 activator was detected only when cells were treated by gamma interferon. Proteasome activities were determined using the in situ method in keratinocytes at different stages of replicative senescence. Only a slight decrease of proteasome activity per cell was seen at intermediate passages, followed by a slight increase in senescent cells. In the same time, the amount of total proteins increased notably with cellular ageing. Thus, proteasome activity decreased relatively to total proteins, but not relatively to cell numbers. Flow cytometry confirmed that the size of aged keratinocytes increased with the ageing marker beta-galactosidase.

Enhancement of Fas-mediated apoptosis in ageing human keratinocytes

Cellular senescence and apoptosis are two metabolically related and seemingly synergistic processes that are involved in tissue maintenance and homeostasis, anti-tumor protection, and age-related diseases. Despite this apparent co-operativity, senescence can inhibit apoptosis in certain conditions. Here, we describe senescence-apoptosis relationships in human epidermal cells by comparing apoptosis-related effector concentrations in keratinocyte cultures and epidermal skin cells at various stages of ageing. Using western blots, flow cytometry, enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence, we determined the amounts of apoptotic effectors in aged cells compared to young ones, in parallel with beta-galactosidase activity at neutral pH (senescence-associated beta-galactosidase, SA beta-gal), found to be a good indicator of cellular ageing. We observed increased levels of several Fas-mediated apoptosis effectors (Fas, Fas ligand, FADD, FLICE), both in cell cultures at advanced passages and in skin cells of aged donors (above 45 years). Furthermore, we found that while the pro-apoptotic p53 increased, the anti-apoptotic Bcl-2 declined. In spite of this, the extent of spontaneous apoptosis did not change in senescent keratinocyte cultures. The cells, however, became notably more susceptible to apoptosis when kept in exhausted growth medium, or upon Fas receptor activation by anti-Fas antibody binding. Our results are consistent with recent findings in senescent fibroblasts, showing that the death-signaling pathway is enhanced at senescence.

Penetration and biological effects of topically applied cyclosporin A nanoparticles in a human skin organ culture inflammatory model

Systemic antipsoriatic therapies have potentially life‐threatening, long‐term side effects. The efficacy of topical drugs is poor, but may be improved by the use of delivery systems based on drug nanoparticles. To produce nanoparticles (NP) composed of cyclosporin A, a classical antipsoriatic drug, and to investigate their penetration and biological effects in human skin affected by psoriatic symptoms, poly‐ε‐caprolactone (PCL) and cyclosporin A (CsA) NP were prepared by the solvent evaporation method. Skin penetration was followed using fluorescently labeled NP in human skin organ cultures (hSOC). Psoriatic symptoms were mimicked in hSOC by the treatment with epidermal growth factor (EGF) and bacterial lipopolysaccharide (LPS). Cell viability in hSOC was evaluated by the resazurin test, and cytokine secretion into the growth medium was measured by immunodetection. We showed that topically applied NP diffused throughout the epidermis within two hours and through the dermis within the following day. They significantly reduced the secretion of inflammatory cytokines IL–1β, IL–6, IL–8, IL–20 and IL–23. At active doses, no cytotoxicity was detected. This type of NP display relevant properties for the use as topical anti‐inflammatory agents and may help to resorb psoriatic lesions.