Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance

Authors: Emilie Bauwens, Tom Parée, Sébastien Meurant, Inès Bouriez, Clotilde Hannart, Anne-Catherine Wéra, Alexis Khelfi, Antoine Fattaccioli, Sophie Burteau, Catherine Demazy, Maude Fransolet, Clémentine De Schutter, Nathalie Martin, Julien Théry, Gauthier Decanter, Nicolas Penel, Marina Bury, Olivier Pluquet, Marjan Garmyn, Florence Debacq-Chainiaux.
17 January 2023


Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an in vitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated β-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.


Cellular senescence is characterized by a permanent cell cycle arrest associated with an active metabolism (Hayflick and Moorhead, 1961). Referred to as replicative senescence, it was later associated with critical attrition of the telomeres (Harley et al., 1990). Oncogene expression, mitochondrial dysfunction, or exposures to various oxidative and/or genotoxic stress can also induce senescence (Hernandez-Segura et al., 2018; Wiley et al., 2016). Senescent cells display a common phenotype mainly characterized by irreversible cell cycle arrest, enlarged and flattened cell morphology, senescence-associated β-galactosidase (SA-βgal) activity, persistent DNA damage, and resistance to apoptosis (González-Gualda et al., 2021). They also secrete a set of cytokines, chemokines, growth factors, proteases, extracellular vesicles, and bioactive lipids, referred to as the senescence-associated secretory phenotype (SASP) (Coppé et al., 2008; Narzt et al., 2021), whose composition can vary depending on the cell type, the senescence trigger, and the time (Basisty et al., 2020). Finally, senescent cells exhibit several alterations in their metabolic state, including altered lipid metabolism, dysregulated autophagy, and disruption of the homeostasis of transition metals (for a review, see the study by Wiley and Campisi [2021]). Senescent cells were shown to accumulate with age in tissues such as the skin (Dimri et al., 1995; Ressler et al., 2006). However, it is still unknown how they appear in vivo. It is unlikely that their presence is related to the exhaustion of their proliferative potential, but because they are notably detected at sites of chronic inflammation, they could therefore result from chronic stress and/or inflammation (Del Pinto and Ferri, 2018; Pilkington et al., 2021).

Some tissues, especially the skin, are largely exposed to such chronic stress. Skin aging is first impacted by age, that is, intrinsic or chronological aging, but in exposed areas, extrinsic aging superimposes, resulting from the impact of a series of environmental stresses referred to as the skin exposome (Krutmann et al., 2017; Wlaschek et al., 2001). Among them, UV rays are identified as the most damaging stressors, leading to tissue dysfunction, photoaging, and susceptibility to cancer (Mullenders, 2018). Senescent cells accumulate with age in the dermis but also in the basal layer of the epidermis (Ressler et al., 2006; Waaijer et al., 2012). Dermal fibroblasts exhibit the common biomarkers of senescence. Of note, epidermal keratinocytes (KCs) in culture reach a senescence plateau only after a few passages. This senescence is independent of telomeres shortening but is associated with oxidative stress and defects in DNA single-strand break repair mechanism (Nassour et al., 2016). Melanocytes show a gradual cell cycle arrest that is p16INK-4A and telomere-shortening dependent and accumulate with age (Bandyopadhyay et al., 2001; Victorelli et al., 2019). In addition, senescence can also be induced in these cell types after UVB exposures (Debacq-Chainiaux et al., 2005; Lewis et al., 2008; Victorelli et al., 2019). UVB (280‒320 nm) penetrates the epidermis, leading to oxidative stress and direct DNA damage through the formation of pyrimidine dimers (Ichihashi et al., 2003). KCs are the first cells to be impacted by UVB and potentially able to react with their neighboring cells after irradiation. UVB-induced senescence in KCs is characterized by growth arrest, increased SA-βgal activity, and lamin B1 (LMNB1) loss; is p53 dependent; and requires a functional IGF-1R (Bertrand-Vallery et al., 2010; Lewis et al., 2008; Wang et al., 2017).

In this study, we showed that the UVB-induced senescence of normal human epidermal KCs is associated with persistent DNA damage and a long-term DNA damage response (DDR) activation. We also reported that UVB-induced senescent KCs secreted SASP factors, which were able to influence the migration of cutaneous squamous cell carcinoma cells. RNA-sequencing analysis highlighted a downregulation of the expression of several amino acid transporters (AATs) in senescent KCs. This was corroborated by lower intracellular levels of glycine, leucine, and alanine. We showed that the inhibition of the glycine transporter SLC6A9/GlyT1 was able to prematurely induce senescence traits and that its overexpression protects from the induction of several biomarkers of senescence. These results revealed that SLC6A9/GlyT1 can be involved in the tuning of senescence in KCs.

Section snippets

Repeated UVB exposures induce premature senescence in KCs

We first developed a model consisting of three UVB exposures over a day at 675 mJ/cm2 (Supplementary Figure S1a), which is the maximal dose without induction of cytotoxicity, apoptosis, or differentiation (Supplementary Figure S1b‒e). Biomarkers of senescence were evaluated 3 and 7 days after the last UVB stress to focus on the long-term effects of UVB exposures and were compared with KCs reaching their senescence plateau after 10‒12 passages referred to in this study as replicative-like


UVB-induced senescent KCs display several common biomarkers of senescence with replicative-like senescence, except for the expression of p21 WAF-1A, as previously shown (Lewis et al., 2008; Nassour et al., 2016; Wang et al., 2017). Unlike replicative-like senescence in which oxidative stress leads to single-strand break DNA damage (Nassour et al., 2016), UVB-induced senescence is associated with persistent cyclobutane-pyrimidine dimers and 53BP1 foci and activation of the ATM/Chk2/p53/p21 axis

Cell culture, UVB-induced stress-induced premature senescence model, and bitopertin treatment

Normal human epidermal KCs were isolated from the foreskins of young donors (aged <6 years) (Clinique Saint Luc, Bouge, Belgium) as previously described (Gilchrest, 1983). Skin samples were obtained after written informed consent, in accordance with the Medical Ethical Committee of the Clinique Saint Luc in Bouge and according to the Declaration of Helsinki Principles. KCs and A431 cells were grown in specific media (Supplementary Materials and Methods).

For UVB-induced stress-induced premature


Emilie Bauwens:

Tom Parée:

Sébastien Meurant:

Inès Bouriez:

Clotilde Hannart:

Anne-Catherine Wéra:

Alexis Khelfi:

Antoine Fattaccioli:

Sophie Burteau:

Catherine Demazy: //

Conflict of Interest

The authors state no conflict of interest.


The authors thank Luc de Visscher for providing skin samples (Clinique Saint Luc, Bouge, Belgium). The authors also thank Emmanuel Di Valentin and the Viral Vectors Platform (GIGA, Liège, Belgium) for their technical help in viral vectors design and production as well as Wouter Coppieters and Arnaud Lavergne from the Genomics platform (GIGA) for their assistance in the analysis of the RNA-sequencing data. The authors especially thank Anabelle Decottignies for her support for the telomeric FISH