Study of UVB-stress-induced premature senescence in normal human epidermal keratinocytes

Promoter

Dr. Florence CHAINIAUX, UNamur, Laboratory of Cellular and Molecular Biology (URBC)

Jury

• Prof. Patricia RENARD (département de biologie, UNamur), présidente
• Dr Florence CHAINIAUX (département de biologie, UNamur), secrétaire
• Dr Olivier PLUQUET (Institut Pasteur, France)
• Prof. Isabelle PETROPOULOS (Sorbonne Université, Paris, France)
• Prof. Yves POUMAY (département de médecine, UNamur)

Summary

The topic of this PhD thesis concerns the characterization of human normal epidermal keratinocytes (NHEKs) undergoing premature senescence induced by UVB stress (UVB-SIPS). The first part of this work consisted in the development of an in vitro model of UVBSIPS in NHEKs. By repeatedly exposing these cells to a subcytotoxic dose of UVB we induced a senescent phenotype showing an irreversible cell cycle arrest, an increase in the proportion of SA-betagalactosidase positive cells, unrepaired DNA damage and a long-term activation of the DNA damage response (DDR). In parallel, we also highlighted these markers in a model of senescence induced by cumulative doubling passage in culture (replicative-like senescence) in NHEKs. Based on RNA-seq transcriptomic analysis, we demonstrated that senescent NHEKs show a decrease in the expression of several amino acid transporters, associated, in UVB-SIPS NHEKs, with reduced intracellular levels of glycine, alanine and leucine. This could be recognized as a new marker of senescence, especially since it is also observed in fibroblasts undergoing replicative senescence. Moreover, chemical inhibition of the glycine transporter SLC6A9/glyt1 is able to induce some senescence biomarkers in NHEKs. In a second step, we studied the secretory profile of NHEKs in UVB-SIPS, by an a priori study of gene expression of interleukins IL-6 and IL-8, some growth factors and the metalloproteinase MMP-9, based on the SASP composition of senescent fibroblasts. This first approach allowed us to highlight a modification in the expression of these factors over time.

Subsequently, we performed a proteomic analysis to determine the SASP of NHEKS in UVB-SIPS at different time points after induction. It seems that this SASP, although presenting common factors to the different timings, displays also specific factors to each timing. Moreover, these secreted factors are partly found in the SASP of other cell types. We also report that senescent NHEKs (UVB-SIPS and replicative-like) secrete protein factors that influence the migration behavior of cSCC A431 cells, an effect that has never been reported before for the SASP of senescent NHEKs.