Non-senescent keratinocytes organize in plasma membrane submicrometric lipid domains enriched in sphingomyelin and involved in re-epithelialization.

Membrane lipid raft model has long been debated, but recently the concept of lipid submicrometric domains has emerged to characterize larger (micrometric) and more stable lipid membrane domains. Such domains organize signaling platforms involved in normal or pathological conditions. In this study, adhering human keratinocytes were investigated for their ability to organize such specialized lipid domains. Successful fluorescent probing of lipid domains, by either inserting exogenous sphingomyelin (BODIPY-SM) or using detoxified fragments of lysenin and theta toxins fused to mCherry, allowed specific, sensitive and quantitative detection of sphingomyelin and cholesterol and demonstrated for the first time submicrometric organization of lipid domains in living keratinocytes. Potential functionality of such domains was additionally assessed during replicative senescence, notably through gradual disappearance of SM-rich domains in senescent keratinocytes. Indeed, SM-rich domains were found critical to preserve keratinocyte migration before senescence, because sphingomyelin or cholesterol depletion in keratinocytes significantly alters lipid domains and reduce migration ability.

HOXA5 localization in postnatal and adult mouse brain is suggestive of regulatory roles in postmitotic neurons.

Hoxa5 is a member of the Hox gene family, which plays critical roles in successive steps of the central nervous system formation during embryonic and fetal development. Hoxa5 expression in the adult mouse brain has been reported, suggesting that this gene may be functionally required in the brain after birth. To provide further insight into the Hoxa5 expression pattern and potential functions in the brain, we have characterized its neuroanatomical profile from embryonic stages to adulthood. While most Hox mapping studies have been based solely on transcript analysis, we extended our analysis to HOXA5 protein localization in adulthood using specific antibodies. Our results show that Hoxa5 expression appears in the most caudal part of the hindbrain at fetal stages, where it is maintained until adulthood. In the medulla oblongata and pons, we detected Hoxa5 expression in many precerebellar neurons and in several nuclei implicated in the control of autonomic functions. In these territories, the HOXA5 protein is present solely in neurons, specifically in γ-aminobutyric acid (GABA)ergic, glutamatergic, and catecholaminergic neurons. Finally, we also detected Hoxa5 transcripts, but not the HOXA5 protein, in the thalamus and the cortex, from postnatal stages to adult stages, and in the cerebellum at adulthood. We provide evidence that some larger variants of Hoxa5 transcripts are present in these territories. Our mapping analysis allowed us to build hypotheses regarding HOXA5 functions in the nervous system after birth, such as a potential role in the establishment and refinement/plasticity of precerebellar circuits during postnatal and adult life. J. Comp. Neurol. 525:1155-1175, 2017. © 2016 Wiley Periodicals, Inc.

Resveratrol induces DNA damage in colon cancer cells by poisoning topoisomerase II and activates the ATM kinase to trigger p53-dependent apoptosis.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural polyphenol synthesized by various plants such as grape vine. Resveratrol (RSV) is a widely studied molecule, largely for its chemopreventive effect in different mouse cancer models. We propose a mechanism underlying the cytotoxic activity of RSV on colon cancer cells. Our data show that resveratrol induces apoptosis, as observed by the cleavage of PARP-1 and chromatin condensation. We show that the tumor suppressor p53 is activated in response to RSV and participates to the apoptotic process. Additionally, we show that HCT-116 p53 wt colon carcinoma cells are significantly more sensitive than HCT-116 p53-/- cells to RSV. RSV induces DNA damage including double strand breaks, as evidenced by the presence of multiple γ-H2AX foci in 50% of cells after a 24 h treatment with 25 µM RSV. The formation of DNA damage does not appear to rely on a pro-oxidant effect of the molecule, inhibition of topoisomerase I, or DNA intercalation. Rather, we show that DNA damage is the consequence of type II topoisomerase poisoning. Exposure of HCT-116 cells to RSV leads to activation of the Ataxia Telangiectasia Mutated (ATM) kinase, and ATM is required to activate p53.

Meaning of relative gene expression in multilayered cultures of epidermal keratinocytes.

Reconstructed human epidermis (RHE) has become an in vitro model of choice for studying cell and tissue functions. Analysis of gene expression over the course of reconstruction must take into account the heterogeneous differentiation states of keratinocytes reconstituting the typical epidermal layers. In monolayer cultures, relative mRNA expression levels of differentiation markers are usually expressed as a ratio versus a classical reference gene (also named house-keeping gene) tested to be expressed equally in certain experimental conditions. Applied to complex tissues in which the cell number increases over time together with differentiation, calculation of relative gene expression does not take enough into account a crucial phenomenon: epidermal morphogenesis results in progressive restriction of differentiation markers, such as involucrin, to a specific layer, or in the delayed onset of mRNA expression of filaggrin or TMEM45A for instance following stratification. Our study illustrates that comparing the relative expression level of mRNAs to that of a basal layer-specific gene (e.g. ITGA6) better illustrates the contribution of specific differentiation markers to the process of epidermal morphogenesis.