The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular ...characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either
or
/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status.
In human skin, keratinopoiesis is based on a functional hierarchy among keratinocytes, with rare slow-cycling stem cells responsible for the long-term maintenance of the tissue through their ...self-renewal potential, and more differentiated daughter progenitor cells actively cycling to permit epidermal renewal and turn-over every month. Skin is a radio-responsive tissue, developing all types of radiation damage and pathologies, including early tissue reactions such as dysplasia and denudation in epidermis, and later fibrosis in the dermis and acanthosis in epidermis, with the TGF-beta 1 pathway as a known master switch. Also there is a risk of basal cell carcinoma, which arises from epidermal keratinocytes, notably after oncogenic events in PTCH1 or TP53 genes. This review will cover the mechanisms of adverse human skin reactions and carcinogenesis after various types of exposures to ionizing radiation, with comparison with animal data when necessary, and will discuss the possible role of stem cells and their progeny in the development of these disorders. The main endpoints presented are basal cell intrinsic radiosensitivity, genomic stability, individual factors of risk, dose specific responses, major molecular pathways involved and the cellular origin of skin reactions and cancer. Although major advances have been obtained in recent years, the precise implications of epidermal stem cells and their progeny in these processes are not yet fully characterized.
Stem cells have been described in most adult tissues, where they play a key role in maintaining tissue homeostasis. As they self-renew throughout life, accumulating genetic anomalies can compromise ...their genomic integrity and potentially give rise to cancer. Stem cells (SCs) may thus be a major target of radiation carcinogenesis. In addition, unrepaired genotoxic damage may cause cell death and stem cell pool depletion, impairing lineage functionality and accelerating aging. Developments in SC biology enabled the characterization of the responses of stem cells to genotoxic stress and their role in tissue damage. We here examine how these cells react to ionizing radiation (IR), and more specifically their radiosensitivity, stress signaling and DNA repair. We first review embryonic SCs, as a paradigm of primitive pluripotent cells, then three adult tissues, bone marrow, skin and intestine, capable of long-term regeneration and at high risk for acute radiation syndromes and long-term carcinogenesis. We discuss IR disruption of the fine balance between maintenance of tissue homeostasis and genomic stability. We show that stem cell radiosensitivity does not follow a unique model, but differs notably according to the turnover rates of the tissues.
MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate Coutier, Julien; Auvré, Frédéric; Lemaître, Gilles ...
Journal of investigative dermatology,
January 2023, 2023-01-00, 20230101, 2023, Letnik:
143, Številka:
1
Journal Article
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Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways ...regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor ex vivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for ex vivo expansion of human KC precursors.
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The functional definition of somatic adult stem cells is based on their regenerative capacity, which allows tissue regeneration throughout life. Thus, refining methodologies to characterize this ...capacity is of great importance for progress in the fundamental knowledge of specific keratinocyte subpopulations but also for preclinical and clinical research, considering the high potential of keratinocytes in cell therapy. We present here a methodology which we define as iterative xenografting, which originates in the classical model of human skin substitute xenografts onto immunodeficient recipient mice. The principle of this functional assay is first to perform primary xenografts to assess graft take and the quality of epidermal differentiation. Then, human keratinocytes are extracted from primary graft samples to perform secondary xenografts, to assess the presence and preservation of functional keratinocyte stem cells with long-term regenerative potential. In the example of experiments shown, iterative skin xenografting was used to document the high regenerative potential of epidermal holoclone keratinocytes.
The transcription factor “Kruppel-like factor 4” (KLF4) is a central player in the field of pluripotent stem cell biology. In particular, it was put under the spotlight as one of the four factors of ...the cocktail originally described for reprogramming into induced pluripotent stem cells (iPSCs). In contrast, its possible functions in native tissue stem cells remain largely unexplored. We recently published that KLF4 is a regulator of “stemness” in human keratinocytes. We show that reducing the level of expression of this transcription factor by RNA interference or pharmacological repression promotes the ex vivo amplification and regenerative capacity of two types of cells of interest for cutaneous cell therapy: native keratinocyte stem and progenitor cells from adult epidermis, which have been used for more than three decades in skin graft bioengineering, and keratinocytes generated by the lineage-oriented differentiation of embryonic stem cells (ESCs), which have potential for the development of skin bio-bandages. At the mechanistic level, KLF4 repression alters the expression of a large set of genes involved in TGF-β1 and WNT signaling pathways. Major regulators of TGF-β bioavailability and different TGF-β receptors were targeted, notably modulating the ALK1/Smad1/5/9 axis. At a functional level, KLF4 repression produced an antagonist effect on TGF-β1-induced keratinocyte differentiation.
For the general population, medical diagnosis is a major cause of exposure to low genotoxic stress, as various imaging techniques deliver low doses of ionizing radiation. Our study investigated the ...consequences of low genotoxic stress on a keratinocyte precursor fraction that includes stem and progenitor cells, which are at risk for carcinoma development. Human skin organoids were bioengineered according to a clinically-relevant model, exposed to a single 50 mGy dose of γ rays, and then xeno-transplanted in nude mice to follow full epidermis generation in an in vivo context. Twenty days post-xenografting, mature skin grafts were sampled and analyzed by semi-quantitative immuno-histochemical methods. Pre-transplantation exposure to 50 mGy of immature human skin organoids did not compromise engraftment, but half of xenografts generated from irradiated precursors exhibited areas displaying focal dysplasia, originating from the basal layer of the epidermis. Characteristics of epithelial-to-mesenchymal transition (EMT) were documented in these dysplastic areas, including loss of basal cell polarity and cohesiveness, epithelial marker decreases, ectopic expression of the mesenchymal marker α-SMA and expression of the EMT promoter ZEB1. Taken together, these data show that a very low level of radiative stress in regenerating keratinocyte stem and precursor cells can induce a micro-environment that may constitute a favorable context for long-term carcinogenesis.
Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is ...increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome.
The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5 Gy) and low (50-250 mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods.
Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity.
In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations.
Although it is well established that 5 to 15% of radiotherapy patients exhibit severe side-effects in non-cancerous tissues, the molecular mechanisms involved are still poorly known, and the links ...between cellular and tissue radiosensitivity are still debated. We here studied fibroblasts from non-irradiated skin of patients with severe sequelae of radiotherapy, to determine whether specific basal cell activities might be involved in susceptibility to side-effects in normal tissues. Compared to control cells, patient fibroblasts exhibited higher radiosensitivity together with defects in DNA repair. Transcriptome profiling of dermal fibroblasts from 16 radiotherapy patients with severe side-effects and 8 healthy individuals identified 540 genes specifically deregulated in the patients. Nuclear factor of activated T cells 2 (
) was the most differentially expressed gene, poorly expressed at both transcript and protein level, whereas the
gene region was hypermethylated. Furthermore,
expression correlated with cell survival after irradiation. Finally, silencing
in normal cells by RNA interference led to increased cellular radiosensitivity and defects in DNA repair. This study demonstrates that patients with clinical hypersensitivity also exhibit intrinsic cellular radiosensitivity in their normal skin cells. It further reveals a new role for NFATC2 as a potential regulator of cellular sensitivity to ionizing radiation.
Human skin protects the body against infection and injury. This protection involves immune and epithelial cells, but their interactions remain largely unknown. Here, we show that cultured epidermal ...keratinocytes inhibit allogenic CD4+ T-cell proliferation under both normal and inflammatory conditions. Inhibition occurs through the secretion of soluble factors, including TGFB1 and the cell-surface expression of HLA-G1 and PD-L1 immune checkpoints. For the first time, we here describe the expression of the HLA-G1 protein in healthy human skin and its role in keratinocyte-driven tissue immunomodulation. The overexpression of HLA-G1 with an inducible vector increased the immunosuppressive properties of keratinocytes, opening up perspectives for their use in allogeneic settings for cell therapy.
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