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Chronological aging is defined as a time-dependent decline of tissue homeostasis which severely impacts skin. Understanding the mechanisms of skin aging is an active research area limited by the lack of relevant in vitro models. Being a component of aging, replicative or stress-induced senescence is repeatedly used to mimic skin aging in vitro, thus presenting only a partial view of the complexity of aging. Herein, we aimed to clarify whether primary normal human dermal fibroblasts retained age-related characteristics when cultured in 2D monolayer, and could be used as a relevant model for aging research. We compared three groups of fibroblasts isolated from different aged donors. We observed strongly decreased population doubling capacities, a reduced clonogenic ability, an impairment in extracellular matrix production together with modifications of respiratory metabolism with an increase in age. These disruptions were particularly marked when comparing fibroblasts isolated from old individuals (over 70 years old) to those isolated from young individuals (18–37 years old), while cells from middle-aged donors exhibited an intermediate profile. These alterations of cell features can be related to the signs of dermis aging, thus showing that cultured primary cells indeed retain some characteristics of the original tissue from which they were extracted.

International audience; Sarcoidosis is a multisystem disease characterized by the development and accumulation of granulomas, the hallmark of an inflammatory process induced by environmental and/or infectious and or genetic factors. This auto-inflammatory disease mainly affects the lungs, the gateway to environmental aggressions and viral infections. We have shown previously that genetic predisposition to sarcoidosis occurring in familial cases is related to a large spectrum of pathogenic variants with, however, a clustering around mTOR (mammalian Target Of Rapamycin)-related pathways and autophagy regulation. The context of the COVID-19 pandemic led us to evaluate whether such genetic defects may increase the risk of a severe course of SARS-CoV2 infection in patients with sarcoidosis. We extended a whole exome screening to 13 families predisposed to sarcoidosis and crossed the genes sharing mutations with the list of genes involved in the SARS-CoV2 host-pathogen protein-protein interactome. A similar analysis protocol was applied to a series of 100 healthy individuals. Using ENRICH.R, a comprehensive gene set enrichment web server, we identified the functional pathways represented in the set of genes carrying deleterious mutations and confirmed the overrepresentation of autophagy- and mitophagy-related functions in familial cases of sarcoidosis. The same protocol was applied to the set of genes common to sarcoidosis and the SARS-CoV2-host interactome and found a significant enrichment of genes related to mitochondrial factors involved in autophagy, mitophagy, and RIG-I-like (Retinoic Acid Inducible Gene 1) Receptor antiviral response signaling. From these results, we discuss the hypothesis according to which sarcoidosis is a model for studying genetic abnormalities associated with host response to viral infections as a consequence of defects in autophagy and mitophagy processes.

Background Visual and molecular changes occurring upon aging are rather well characterized. Still, aging signs show great significant inter-individual variations, and little is known concerning the link between perceived age and cutaneous microcirculation. Materials and methods To investigate this point, we recruited Caucasian women in their mid-50's to mid-70's and subsampled women looking older or younger than their age. We studied their facial skin color, as well as their microvascular reactivity to local heating assessed in the forearm skin. We also used skin biopsies from some of these women for gene expression or immunohistochemical analysis. Results Clinical and instrumental analysis of skin color revealed that subjects who look 5 years younger differ only by a higher glowing complexion. Our most striking result is that subjects looking 5 years younger than their age present a higher microcirculation reactivity in forearm skin. Transcriptome comparison of skin samples from women looking older or younger than their age revealed 123 annotated transcripts differentially expressed, among which MYL9 relates to microcirculation. MYL9 is downregulated in the group of women looking younger than their real age. Microscopy shows that the labeling of MYL9 and CD31 are altered and heterogeneous with age, as is the morphology of microvessels. Conclusion Therefore, assessing generalized vascular reactivity in non-photo-exposed skin to focus on the intrinsic aging allows subtle discrimination of perceived age within elderly healthy subjects.

The skin is a sentinel organ making easily visible the passing of time. Chronological and environmental aging weakens skin structure and functions. The skin barrier, the elastic and mechanical properties of the cutaneous tissue as well as its vascular reactivity are impacted by aging. The barrier dysfunction in aged skin is caused by defects in epidermal keratinocytes renewal and differentiation notably linked to abnormal expression of microRNAs regulating cell death and autophagy. An abnormal balance between synthesis and degradation of matrix proteins modifies the mechanical properties of the dermis in aged skin. Finally, a reduction of the vascular reactivity linked to endothelial dysfunctions is observed in elderly people. These biological processes can be targeted by therapeutic approaches either topical or systemic, especially using anti-oxydants or senolytics. These anti-aging strategies might contribute to restore, at least in part, the functional integrity of aged skin.Vieillissement et intégrité de la peau - De la biologie cutanée aux stratégies anti-âge.La peau est un organe sentinelle, soumis au vieillissement chronologique et environnemental qui fragilise sa structure et ses fonctions. La fonction barrière de la peau, ses propriétés élastiques et de résistance, ainsi que sa réactivité vasculaire sont atteintes par le vieillissement dans les compartiments épidermiques, dermiques et vasculaires. Les progrès de la recherche ont permis de révéler des processus biologiques sous-jacents, qui peuvent être ciblés par des approches médicamenteuses topiques ou globales à base notamment d’anti-oxydants ou de sénolytiques. Ces stratégies anti-âge pourront contribuer à restaurer, au moins en partie, l’intégrité fonctionnelle de la peau âgée.

Chronological aging is characterized by an alteration of the genes regulatory network. In human skin, epidermal keratinocytes fail to differentiate properly with aging, leading to the weakening of the epidermal function. MiR-30a is particularly overexpressed with epidermal aging, but the downstream molecular mechanisms are still uncovered. The aim of this study was to decipher the effects of miR-30a overexpression in the human epidermis, with a focus on keratinocyte differentiation. We formally identified the mitophagy receptor BNIP3L as a direct target of miR-30a. Using a 3D organotypic model of reconstructed human epidermis overexpressing miR-30a, we observed a strong reduction of BNIP3L expression in the granular layer. In human epidermal sections of skin biopsies from donors of different ages, we observed a similar pattern of BNIP3L decrease with aging. Moreover, human primary keratinocytes undergoing differentiation in vitro also showed a decreased expression of BNIP3L with age, together with a retention of mitochondria. Moreover, aging is associated with altered mitochondrial metabolism in primary keratinocytes, including decreased ATP-linked respiration. Thus, miR-30a is a negative regulator of programmed mitophagy during keratinocytes terminal differentiation, impairing epidermal homeostasis with aging.

MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.

Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKβ) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21 WAF1 and p27 Kip1 , decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. IMPORTANCE The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

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.

Background: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined. Results and methods: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal–epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT–PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival. Conclusion: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.

Connexins (Cx) are the protein subunits of gap junctions, which play an important role in cell-to-cell communication. We characterized the genomic structure of the human GJB6 gene, encoding connexin 30 (C x 30), and showed that it differs from most connexin-encoding genes. GJB6 presents six different exons, some of which can be alternatively spliced. We also mapped a basal promoter sequence active in a human keratinocyte cell line which responds to the activation of the EGF receptor. One of the non-encoding exons of GJB6, which has been described in brain C x 30 cDNA, was not found in cDNA obtained from human keratinocytes, suggesting tissue-specific splicing.

Clouston syndrome or hidrotic ectodermal dysplasia (HED) is a rare dominant genodermatosis characterized by palmoplantar hyperkeratosis, generalized alopecia and nail defects. The disease is caused by mutations in the human GJB6 gene which encodes the gap junction protein connexin30 (Cx30). To gain insight into the molecular mechanisms underlying HED, we have analyzed the consequences of two of these mutations (G11R Cx30 and A88V Cx30) on the functional properties of the connexons they form. Here, we show that the distribution of Cx30 is similar in affected palmoplantar skin and in normal epidermis. We further demonstrate that the presence of the wild-type protein (wt Cx30) improves the trafficking of mutated Cx30 to the plasma membrane where both G11R and A88V Cx30 co-localize with wt Cx30 and form functional intercellular channels. The electrophysiological properties of channels made of G11R and A88V Cx30 differ slightly from those of wt Cx30 but allow for dye transfer between transfected HeLa cells. Finally, we document a gain of function of G11R and A88V Cx30, which form functional hemichannels at the cell surface and, when expressed in HeLa cells, generate a leakage of ATP into the extracellular medium. Such increased ATP levels might act as a paracrine messenger that, by altering the epidermal factors which control the proliferation and differentiation of keratinocytes, may play an important role in the pathophysiological processes leading to the HED phenotype.

Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3 beta, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.

GATA3 belongs to the GATA transcription factor family and is a crucial regulator of lymphocyte differentiation. More recently, GATA3 was shown to be involved in skin cell lineage determination, in morphogenesis and maintenance of hair follicle keratinocytes as well as in epidermal barrier formation in mouse. In human, the potential role of GATA3 in the regulation of interfollicular epidermal homeostasis was still poorly explored. We thus investigated whether GATA3 could play a role in the regulation of proliferation and/or differentiation processes in human primary keratinocytes. We silenced the expression of GATA3 by small interfering RNA in either proliferating or differentiated human primary keratinocytes and analyzed the effect on cell proliferation and differentiation. We showed that GATA3 inhibition increased cell number, BrdU incorporation and expression of the proliferation markers PCNA and Ki67, demonstrating that GATA3 can inhibit keratinocyte proliferation. Moreover, GATA3 seems to be able to induce keratinocyte differentiation since its silencing leads to a decrease of both early and late differentiation markers such as Keratins 1 and 10, Involucrin and Loricrin. Our results demonstrate that GATA3 transcription factor inhibits proliferation and induces differentiation of primary keratinocytes, which suggest that it may regulate human interfollicular epidermal renewal.

Background MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. Results We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. Conclusions Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.

Background The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown. Results The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knock-down was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide microarrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGFβ signalling and stress response. Conclusion Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation.

International audience; p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The DeltaNp63alpha isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DeltaNp63alpha; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

Skin is the organ most exposed to various environmental aggressors, including ionizing radiation. Low-dose and low-dose-rate exposures to gamma rays account for most occupational, medical or environmental irradiations. To examine whether this type of exposure triggers specific molecular responses, cultured primary keratinocytes isolated from adult normal skin were irradiated with single acute doses of 1 cGy or 2 Gy. DNA microarrays containing 10,500 probes were used to assess transcriptional changes over a time course between 3 and 72 h postirradiation. Keratinocytes were studied at a differentiated stage to mimic the response of cells from the suprabasal layers of the epidermis. A major finding of this study was the identification of an important number of low-dose-specific genes (140), most of which were modulated at 48 h. Clustering analysis also revealed low-dose-specific profiles. One of these clusters (17 known genes) was further analyzed using Gibbs sampling algorithm, which led to the identification of 7 putative promoter sequences. These results show for the first time that low-dose ionizing radiation is able to induce specific transcriptional responses in human keratinocytes. Our findings support the potential usefulness of microarrays in biological dosimetry studies after low-dose exposures.

We performed a microarray study on human differentiated HaCaT keratinocytes exposed to ionizing radiation (2or 10 Gy).At 3 h after exposure, more than 150 known and unknown genes were found regulated in irradiated HaCaT keratinocytes. Among the genes regulated at 3 h,those involved in cell energy metabolism appeared to be the most abundant and the most responsive. Two mitochondrial ATP-synthases and several other genes involved in energy producing pathways, such as glucose metabolism, were induced, whereas many genes from energy requiring pathways were shutdown. These changes in energy metabolism were confirmed both in normal primary keratinocytes and in HaCaT keratinocytes by RT-PCR and proteins studies. Moreover, measures of intracellular ATP revealed a 50% increase in keratinocytes immediately after irradiation, supporting an energy procurement response. The overall results indicate that irradiation induces an immediate burst of ATP that seems to be a general response of human differentiated keratinocytes to the radiation stress. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/ suppi-nat/0730-231 2/suppmat/v95.html. J. Cell. Biochem. (c) 2005 Wiley-Liss, Inc.

Id2 plays a key role in epithelial cells, regulating differentiation, the cell cycle, and proliferation. Because human skin constantly renews itself and is the first target of irradiation, it is of primary interest to evaluate whether such a gene may be regulated in keratinocytes exposed to ionizing radiation. We show here that Id2 is induced in response to gamma-irradiation and have investigated the consequence of this regulation on cell fate. Using RNA interference, we observed that Id2 extinction significantly reduces cell growth in human keratinocytes through the control of the G(1)-S transition of the cell cycle. We have investigated whether the impact of Id2 on the cell cycle may have a physiological role on the cell's ability to cope with radiative stress. Indeed, when Id2 is down-regulated through interfering RNA, cells are more sensitive to irradiation. Conversely, when Id2 is overexpressed, this somehow protects the cell. We propose that Id2 favors reentering the cell cycle after radiation-induced cell cycle arrest to permit the recovery of keratinocytes exposed to ionizing radiation.

The knowledge of the mechanism of keratinocyte differentiation in culture is still uncompleted. The emergence of new technologies, such as cDNA microarrays or 2D electrophoresis followed by mass spectrometry analysis, has allowed the identification of genes and proteins expressed in biological processes in keratinocytes. Here, we report a genome wide analysis of proliferating versus differentiated human HaCaT keratinocytes. We found that genes and proteins which take part in the cell cycle control, carbohydrate metabolism, cell auto-immunity, adhesion and cytokine signal transduction pathways were regulated in differentiated HaCaT keratinocytes. In addition, we identified seven proteins and 33 transcripts that had not been previously described as differentially expressed in proliferating versus differentiated HaCaT cells. Furthermore, some of these transcripts or proteins were similarly regulated in human primary keratinocytes and in human epidermis. The present study opens new areas of investigation in the comprehension of keratinocyte differentiation.

The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.

The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.