Caractérisation de la différenciation alternative de kératinocytes humains induite par des expositions répétées aux UVB et étude fonctionnelle de TRIM29, une protéine impliquée dans la survie cellulaire

Abstract

Exposures to ultaviolet radiations (UV) is one of the most dangerous stresses to which human skin is exposed daily. Keratinocytes, the main cell type of the epidermis, constitue our first line of defense against UV. UVB are highly mutagenic and are the most deleterious component of sunlight reaching earth surface. We developed an original model of eight repeated sublethal exposures of human keratinocytes lacking p16INK-4a and expressing human telomerase (hTERT), while retaining other growth controls and the ability to fully differentiate in vitro in a reconstructed epidermis. While premature senescence can occur in primary keratinocytes after exposure to a single dose of UVB, we did not observe any significant increase of the proportion of senescence-associated beta-galactosidase activity positive cells following UVB exposure in this cell line in contrast to primary keratinocytes. This suggested a role for p16INK-4a in UVB-induced senescence. We detected severe oxidative stress, sustained DNA damage, prolonged cell cycle arrest, and induction of markers of epidermal differentiation suggesting an alternative differentiation. In vivo, keratinocyte differentiation is an important mechanism of defense against UVB. In order to characterize this phenotype, a proteomic profiling with fluorescent twodimensional difference in-gel electrophoresis (2D-DIGE) at 16, 40 and 64 h after repeated exposures to UVB allowed to identify 69 differentially abundant protein species. Among these protein species, Capping-protein Gelsolin-like protein (CapG), TRIparite Motif Protein 29 (TRIM29) and several phosphorylated cytokeratins had never been previously involved in the response of keratinocytes to UVB. Increased abundance of capping-protein Gelsolin-like protein was mostly localized on cell edges suggesting its participation in morphological changes through modulation of the actin cytoskeleton. Keratin 8 was phosphorylated on two sites as previously analysed in association with stress. Invalidation of TRIM29 in keratinocytes favored apoptosis after UVB exposure and also in reconstructed epidermis, suggesting a role in a cell survival pathway. Analysis with chemical inhibitors suggested that the increased abundance of TRIM29 was a PKC-dependent process. This work allows a better understanding of specific mechanisms of the defense of keratinocytes repeatedly exposed to UVB particularly through an alternative differentiation process. Furthermore, we revealed that TRIM29 is involved in the cell survival of keratinocytes exposed to UVB.

Date of Award	16 Dec 2009
Original language	French
Awarding Institution	University of Namur
Supervisor	Olivier TOUSSAINT (Supervisor), Patricia Renard (President), Yves Poumay (Supervisor), Isabelle Petropoulos (Jury) & Corinne Abbadie (Jury)