Cell Death by Apoptosis in Epidermal Biology

Homeostasis in continually renewing tissues is maintained by a tightly regulated balance between cell proliferation, cell differentiation, and cell death. Until recently, proliferation was thought to be the primary point of control in the regulation of normal tissue kinetic homeostasis and as such has been the major focus of both understanding the etiology of disease and developing therapeutic strategies. Now, physiologic cell death, known as apoptosis (â-pôp-tō'sîs, â-pōp-tō'sîs [Thomas CL (ed.): Taber's Cyclopedic Medical Dictionary. F.A. Davis, Co., Philadelphia, 1989)] has gained scientific recognition as an active regulatory mechanism, complementary, but functionally opposite, to proliferation with important roles in shaping and maintaining tissue size and prevention of disease. In this review we will describe the concept of apoptosis and discuss possible molecular mechanisms of its regulation that may have implications for skin biology

Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma.

International audience: BackgroundIt is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse.ResultsUsing an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal.ConclusionsWe conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor

Interleukin-7 Regulates Adipose Tissue Mass and Insulin Sensitivity in High-Fat Diet-Fed Mice through Lymphocyte-Dependent and Independent Mechanisms

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions

Interactions fonctionnelles des voies de contrôle de l'apoptose et de la différenciation cellulaire lors du renouvellement physiologique de l'épiderme humain

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Caractérisation des cellules souches cancéreuses de la peau humaine (implication de la voie de signalisation de l'Epidermal Growth Factor Receptor dans le contrôle de la différenciation des cellules souches de l'épiderme)

L'épiderme humain est un tissu en renouvellement constant où l'équilibre entre les cellules nouvellement formées et les cellules perdues par desquamation est maintenu par la division asymétrique des cellules souches produisant deux cellules filles différentes : l'une possédant la capacité de s'auto-renouveler et l'autre engagée dans la différenciation en plusieurs lignées cellulaires reconstituant l'épiderme et ses appendices. De récentes découvertes suggèrent que les cellules souches altérées, appelées cellules initiatrices de tumeur ou cellules souches cancéreuses, seraient à l'origine du développement tumoral. Par conséquent, l'éradication ciblée de ces cellules pourrait traiter les cancers. Nos études avaient pour but d'identifier et de caractériser les cellules souches cancéreuses dans des tumeurs de peau et d'évaluer l'implication de la voie de signalisation de l'Epidermal Growth Factor Receptor (EGFR) dans le contrôle de leur auto-renouvellement et leur potentiel de différenciation. Nous avons trouvé de rare kératinocytes mitotiques avec une distribution asymétrique de l'EGFR et nous avons déterminé que sa présence à la surface était liée au destin normal ou cancéreux des cellules. Bien qu'essentiel pour la prolifération, la différenciation et la survie des cellules épithéliales, EGFR n'était pas présent à la surface des cellules aux propriétés de cellules souches comme la quiescence, leur compétence à produire des cellules filles fonctionnellement différentes, leurs potentiels de prolifération et de clonogénicité élevés, leur capacité à former des sphères et l'expression de marqueurs de cellules souches. Au contraire, les kératinocytes exposant l'EGFR ont acquis un phénotype plus différencié, démontrant que l'EGFR contrôle un basculement du compartiment de cellules souches à celui de cellules d'amplification transitoire. Ce basculement était associé avec des changements dans le profile d'expression de protéines du cycle cellulaire ou contrôlant la survie et la mitochondrie qui variaient entre les cellules normales et cancéreuses. De plus, nous avons déterminé que la croissance et la différenciation des kératinocytes étaient associées au statut phosphorylé de p66Shc, une cible en aval de l'EGFR et essentielle au métabolisme des espèces réactives de l'oxygène (ROS) et à l'activation de la voie de mort cellulaire dépendante de la mitochondrie. Donc, alors que p66Shc non phosphorylée était principalement présent dans le noyau des cellules normales et cancéreuses proliférant rapidement, sa forme fonctionnelle phosphorylée sur la sérine 36 était détectée dans les kératinocytes normaux quiescents et différenciés uniquement. A partir de nos résultats, nous proposons un modèle dans lequel EGFR fonctionne comme un déterminant du destin des kératinocytes qui équilibre entre la quiescence et la prolifération/différenciation des cellules souches épidermiques durant la mitose. Cet équilibre semble clairement mal fonctionner dans le cancer. Ainsi comme nous avons précédemment montré que la différenciation des kératinocytes partage certains aspects de la signalisation apoptotique et est stimulée par les ROS, nous proposons donc que l'acquisition de l'EGFR à la surface cellulaire active la prolifération cellulaire dépendante de p66Shc et par conséquent, la production des ROS et la phosphorylation de p66Shc. Le manque de p66Shc phosphorylé dans les tumeurs de peau montre son rôle essentiel dans la différenciation des kératinocytes se terminant par la mort cellulaire par desquamation. De façon importante, notre étude suggère fortement que les cellules EGFR négatives peuvent constituer le compartiment reproductif de la tumeur responsable du maintien et du développement tumoral, donc fournir un aperçu mécanistique de l'inefficacité des thérapies actuelles anti-EGFRLILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Etude des effets du NGF et du proNGF sur les cellules souches du cancer du sein

Malgré d importants progrès en recherche fondamentale et clinique, le cancer du sein reste à ce jour le premier cancer féminin en termes d incidence et de mortalité. Parmi les facteurs contribuant à son développement, notre laboratoire a mis en évidence le rôle des neurotrophines, en particulier le NGF (Nerve Growth Factor) et son précurseur (proNGF). D autre part, les études récentes ont permis de démontrer l importance des cellules souches cancéreuses (CSC) dans la tumorigenèse et dans les phénomènes de résistance thérapeutique et de métastase. Mon projet de thèse avait pour but d étudier les effets du NGF et du proNGF sur la régulation des CSC de sein et d évaluer la conséquence sur le développement tumoral. Nous avons pu démontrer que le NGF et le proNGF sont capables d enrichir la population de CSC dans plusieurs lignées de cellules épithéliales mammaires cancéreuses. Cet enrichissement est lié à la surexpression de plusieurs facteurs de transcription essentiels dans le maintien de la pluripotence des cellules souches. Les expériences de tumorigenèse chez les souris montrent que les cellules cancéreuses de sein MCF-7 cultivées en présence de NGF et de proNGF présentent une tumorigenèse accrue comparée aux cellules contrôles. De façon intéressante, seul le traitement au NGF induit la formation de métastases dans cerveau, poumons et foie, ainsi que une transition épithélio-mésenchymateuse. Ces travaux ont permis de monter que le NGF et le proNGF agissent sur le compartiment des CSC et contribuent à la régulation de la plasticité des cellules cancéreuses in vitro et in vivo.Despite significant advances in cancer research and treatment, breast cancer remains one of the leading causes of cancer-related death in women. NGF (Nerve Growth Factor) and its precursor (proNGF) are overexpressed in breast cancer cells, where they exert a mitotic, anti-apoptotic and pro-invasive effects through an autocrine loop. In the last few years, a raising number of experimental data indicate the importance of cancer stem cells (CSC) in tumour development, metastasis and resistance to therapies.The objective of my thesis was to evaluate the influence of NGF and proNGF on breast CSC biology and to determine their consequences on tumour development. We demonstrated that NGF and proNGF are able to enrich for CSC in several breast cancer cell lines. This enrichment is connected to the overexpression of several transcription factors essential for stem cells self-renewal and pluripotency. Tumorigenicity assays in SCID mice proved that cells from MCF-7 cell line cultured with NGF or proNGF are more tumorigenic than the control condition. Nevertheless, only cells pre-treated with NGF were able to develop metastases in brain, lung and liver and to go through an epithelial-to-mesenchymal transition, demonstrating that NGF and proNGF induce their effects through different signaling pathways. Taken together, our results have shown that NGF and proNGF contribute to the regulation of stem cells plasticity in vitro and in vivo. Future perspectives concern the comprehension of involved molecular mechanisms in order to better understand their implication in breast cancer development.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Variable Bax antigenicity is linked to keratinocyte position within epidermal strata and UV-induced apoptosis.

International audiencePro- and anti-apoptotic members of the Bcl-2 family are fundamental in the control of apoptosis. Among them, Bax plays a key role in apoptosis induction by mediating the release of apoptogenic factors from mitochondria to the cytosol. In this report, we investigated, by immunohistofluorescence, the in vivo distribution of Bax in normal human epidermis before and 24 h after exposure to solar-simulated radiation. Bax expression was evaluated with three different, Western blot pretested, anti-Bax antibodies (Ab) and correlated with markers of keratinocyte differentiation and apoptosis using anti-beta(1) integrin and anti-active caspase-3 Abs respectively. Using anti-Bax N20 and A-3533 polyclonal Ab, we found that, whereas undifferentiated keratinocytes of the basal proliferative compartment contained Bax in the cytosol, the differentiated suprabasal cells had Bax mainly in the nucleus. This immunoreactivity pattern was not modified by skin irradiation. Interestingly, the well known apoptosis-related Bax redistribution to mitochondria in response to a cell death signal, could be detected only with yet another, the 2D2 monoclonal Ab. This relocalization occurred specifically in apoptotic, active caspase-3 positive cells of irradiated epidermis. Our data highlight the differentiation- and apoptosis-associated changes in the pattern of Bax subcellular and cellular distribution as uncovered by different anti-Bax Abs and suggest that Bax undergoes successive activation that progresses in parallel with keratinocyte differentiation and apoptosis

TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma.

Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes. The role of calcium in keratinocyte differentiation is uncontested but the mechanisms controlling calcium-induced differentiation have yet to be completely elucidated. This study was designed to investigate the role of calcium-permeable TRPC channels in human keratinocyte differentiation and BCC, using a combination of molecular and cell biology approaches, involving electrophysiology and Ca(2+)-imaging, on the HaCaT cell line, primary cultures of normal human keratinocytes, and BCC cells. We demonstrated that TRPC1/TRPC4 channel expression was important for keratinocyte differentiation, as knocking out these channels (by siRNA strategy) prevented the induction of Ca(2+)-induced differentiation. TRPC1/TRPC4-mediated calcium entry and endoplasmic reticulum Ca(2+) content increased significantly in differentiated keratinocytes. However, the failure of BCC cells to differentiate was related to a lack of TRPC channel expression and calcium entry. In summary, our data demonstrate that TRPC1 and TRPC4 channels are key elements in keratinocyte Ca(2+) homeostasis and differentiation and may therefore be responsible for skin pathologies.Journal Articleinfo:eu-repo/semantics/publishe