Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis.

Fas ligand (FasL) causes apoptosis of epidermal keratinocytes and triggers the appearance of spongiosis in eczematous dermatitis. We demonstrate here that FasL also aggravates inflammation by triggering the expression of proinflammatory cytokines, chemokines, and adhesion molecules in keratinocytes. In HaCaT cells and in reconstructed human epidermis (RHE), FasL triggered a NF-kappaB-dependent mRNA accumulation of inflammatory cytokines (tumor necrosis factor-alpha, IL-6, and IL-1beta), chemokines (CCL2/MCP-1, CXCL1/GROalpha, CXCL3/GROgamma, and CXCL8/IL-8), and the adhesion molecule ICAM-1. Oligomerization of Fas was required both for apoptosis and for gene expression. Inhibition of caspase activity abolished FasL-dependent apoptosis; however, it failed to suppress the expression of FasL-induced genes. Additionally, in the presence of caspase inhibitors, but not in their absence, FasL triggered the accumulation of CCL5/RANTES (regulated on activation normal T cell expressed and secreted) mRNA. Our findings identify a novel proinflammatory role of FasL in keratinocytes that is independent of caspase activity and is separable from apoptosis. Thus, in addition to causing spongiosis, FasL may play a direct role in triggering and/or sustaining inflammation in eczemas.

Targeted Gene Deletion and In Vivo Analysis of Putative Virulence Gene Function in the Pathogenic Dermatophyte Arthroderma benhamiae.

Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ΔacuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ΔacuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.

PPAR expression and function during vertebrate development.

The peroxisome proliferator activated receptors (PPARs) are ligand activated receptors which belong to the nuclear hormone receptor family. As with other members of this superfamily, it is thought that the ability of PPAR to bind to a ligand was acquired during metazoan evolution. Three different PPAR isotypes (PPARalpha, PPARbeta, also called 6, and PPARgamma) have been identified in various species. Upon binding to an activator, these receptors stimulate the expression of target genes implicated in important metabolic pathways. The present article is a review of PPAR expression and involvement in some aspects of Xenopus laevis and rodent embryonic development. PPARalpha and beta are ubiquitously expressed in Xenopus early embryos but become more tissue restricted later in development. In rodents, PPARalpha, PPARbeta and PPARgamma show specific time- and tissue-dependent patterns of expression during fetal development and in the adult animals. PPARs are implicated in several aspects of tissue differentiation and rodent development, such as differentiation of the adipose tissue, brain, placenta and skin. Particular attention is given to studies undertaken by us and others on the implication of PPARalpha and beta in rodent epidermal differentiation.

Analysis of epidermis- and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves.

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25,000 fragments estimated to represent about 17,000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissue.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

Background Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. Methods 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. Findings 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12. weeks versus 24 weeks (hazard ratio [HR] 1 98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00,0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. Interpretation While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA,exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population.

Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis.

PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-beta-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. beta-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Presence of an oligodendroglioma-like component in newly diagnosed glioblastoma identifies a pathogenetically heterogeneous subgroup and lacks prognostic value: central pathology review of the EORTC_26981/NCIC_CE.3 trial.

Glioblastoma (GBM) is a morphologically heterogeneous tumor type with a median survival of only 15 months in clinical trial populations. However, survival varies greatly among patients. As part of a central pathology review, we addressed the question if patients with GBM displaying distinct morphologic features respond differently to combined chemo-radiotherapy with temozolomide. Morphologic features were systematically recorded for 360 cases with particular focus on the presence of an oligodendroglioma-like component and respective correlations with outcome and relevant molecular markers. GBM with an oligodendroglioma-like component (GBM-O) represented 15% of all confirmed GBM (52/339) and was not associated with a more favorable outcome. GBM-O encompassed a pathogenetically heterogeneous group, significantly enriched for IDH1 mutations (19 vs. 3%, p = 0.003) and EGFR amplifications (71 vs. 48%, p = 0.04) compared with other GBM, while co-deletion of 1p/19q was found in only one case and the MGMT methylation frequency was alike (47 vs. 46%). Expression profiles classified most of the GBM-O into two subtypes, 36% (5/14 evaluable) as proneural and 43% as classical GBM. The detection of pseudo-palisading necrosis (PPN) was associated with benefit from chemotherapy (p = 0.0002), while no such effect was present in the absence of PPN (p = 0.86). In the adjusted interaction model including clinical prognostic factors and MGMT status, PPN was borderline nonsignificant (p = 0.063). Taken together, recognition of an oligodendroglioma-like component in an otherwise classic GBM identifies a pathogenetically mixed group without prognostic significance. However, the presence of PPN may indicate biological features of clinical relevance for further improvement of therapy.

Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem.

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H(+)-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.

NF-kappaB transmits Eda A1/EdaR signalling to activate Shh and cyclin D1 expression, and controls post-initiation hair placode down growth.

A novel function of NF-kappaB in the development of most ectodermal appendages, including two types of murine pelage hair follicles, was detected in a mouse model with suppressed NF-kappaB activity (c(IkappaBalphaDeltaN)). However, the developmental processes regulated by NF-kappaB in hair follicles has remained unknown. Furthermore, the similarity between the phenotypes of c(IkappaBADeltaN) mice and mice deficient in Eda A1 (tabby) or its receptor EdaR (downless) raised the issue of whether in vivo NF-kappaB regulates or is regulated by these novel TNF family members. We now demonstrate that epidermal NF-kappaB activity is first observed in placodes of primary guard hair follicles at day E14.5, and that in vivo NF-kappaB signalling is activated downstream of Eda A1 and EdaR. Importantly, ectopic signals which activate NF-kappaB can also stimulate guard hair placode formation, suggesting a crucial role for NF-kappaB in placode development. In downless and c(IkappaBalphaDeltaN) mice, placodes start to develop, but rapidly abort in the absence of EdaR/NF-kappaB signalling. We show that NF-kappaB activation is essential for induction of Shh and cyclin D1 expression and subsequent placode down growth. However, cyclin D1 induction appears to be indirectly regulated by NF-kappaB, probably via Shh and Wnt. The strongly decreased number of hair follicles observed in c(IkappaBalphaDeltaN) mice compared with tabby mice, indicates that additional signals, such as TROY, must regulate NF-kappaB activity in specific hair follicle subtypes.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

Odontopus brevirostris (Hustache, 1936) (Coleoptera, Curculionidae) associated with new host plants belonging to Annona (Annonaceae)

Odontopus brevirostris (Hustache, 1936) feeding on Annona squamosa L., A. cherimola Mill., A. glabra L., and A. muricata L. was observed. The last three host plants are recorded for the first time. The endophitic oviposition occurs in the veins of the ventral surface of the young leaves. The larvae, leaf miners, eat the parenchyma and the adults make small holes in the leaves. The pupation occurs in spherical cocoons protected by a sort of nest (pupation chamber) between the two epidermal layers.

Molecular and cellular analysis of genodermatoses

Summary Skin is the essential interface between our body and its environment; not only does it prevent water loss and protect us from external insults it also plays an essential role in the central nervous system acting as a major sense organ primarily for touch and pain. The main cell type present in skin, keratinocyte, undergoes a differentiation process leading to the formation of this protecting barrier. This work is intended to contribute to the understanding of how keratinocyte differentiates and skin functions. To do this, we studied two genetic skin diseases: Erythrokeratodermia variabilis and Mal de Meleda. Our approach was to examine the expression and localization of proteins implicated in these two pathologies in normal and diseased tissues and to determine the influence of mutant proteins at the molecular and cellular levels. Connexins are major components of gap junctions, channels allowing direct communication between cells. Our laboratory has identified mutations in both connexin 30.3 (Cx30.3) and 31 (Cx31) to be causally involved in erythrokeratodermia variabilis (EKV), an autosomal dominant disorder of keratinization. In the first chapter, we show a new mutation of Cx31, L209P-Cx31, in 3 EKV patients, extending the field of EKV-causing mutations although the mechanism by which connexin mutations lead to the disease is unclear. In the second chapter, we studied the effect of F137L-Cx30.3 on expression, trafficking and localization of cotransfected Cx31 and Cx30.3 in connexin-deficient HeLa cells. The F137 amino acid, highly conserved in connexin family, is oriented towards the channel pore and F137L mutation in either Cx30.3 or Cx31 lead to EKV. As two genes can lead to EKV when mutated, our hypothesis was that Cx31 and Cx30.3 might cooperate at a molecular level. We were able to demonstrate a physical interaction between Cx31 and Cx30.3. The presence of F137L-Cx30.3 disturbed the trafficking of both connexins, less connexins were integrated into gap junctions and thus, the coupling between cell was diminished. Connexins formed in the presence of F137L-Cx30.3 are degraded at their exit from the endoplasmic reticulum. In conclusion, our results indicate that the genetic heterogeneity of EKV is due to mutations in two interacting proteins. F137L-Cx30.3 has a dominant negative effect and affects Cx31, disturbing cellular communication in epidermal cells. Mal de Meleda is an autosomal recessive inflammatory and a keratotic palmoplantar skin disorder due to mutations in SLURP1 (secreted LY6/PLAUR-related protein 1). SLURP1 belongs to the LY6/PLAUR family of proteins and has the particularity of being secreted instead of being GPI-anchored. The high degree of structural similarity between SLURP1 and the three fingers motif of snake neurotoxins and LYNX 1-C suggests that this protein could interact with the neuronal acetylcholine receptors. In the third chapter, we show that SLURP1 potentiates responses of the a7 nicotinic acetylcholine receptor (nAchR) to acetylcholine. These results identify SLURP1 as a secreted epidermal neuromodulator that is likely to be essential for palmoplantar skin. In the fourth chapter, we show that SLURP1 is expressed in the granular layer of the epidermis but is absent from skin biopsies of Mal de Meleda patients. SLURP1 is also present in secretions such as sweat, tears or saliva. An in vitro analysis on two mutant of SLURP-I demonstrates that W15R-SLURP1 is absent in cells while G86R-SLURP1 is expressed and secreted, suggesting that SLURP1 can lead to the disease by either an absent or an abnormal protein. Finally, in the fifth chapter, we analyse the expression and biological properties of other LY6/PLAUR members, clustered around SLURP] on chromosome 8. Their GPI-anchored or secreted status were analysed in vitro. SLURP1, LYNX1-A and -B are secreted while LYPDC2 and LYNX 1-C are GPI anchored. Three of these proteins are expressed in the epidermis and in cultured keratinocytes. These results suggest that these LY6/PLAUR members may have an important role in skin homeostasis. Résumé Résumé La peau est la barrière essentielle entre notre corps et l'environnement, nous protégeant des agressions extérieures, de la déshydratation et assurant aussi un rôle dans le système nerveux central en tant qu'organe du toucher et de la douleur. Le principal type de cellules présent dans la peau est le kératinocyte qui suit un processus de différenciation aboutissant à la formation de cette barrière protectrice. Ce travail est destiné à comprendre la différenciation des kératinocytes et le fonctionnement de la peau. Pour cela, nous avons étudié deux maladies génodermatoses : l'Erthrokeratodermia Variabilis (EKV) et le Mal de Meleda. Nous avons examiné l'expression et la localisation des protéines impliquées dans ces deux pathologies dans des tissus normaux et malades puis déterminé l'influence des protéines mutantes aux niveaux moléculaires et cellulaires. Les connexines (Cx) sont les composants majeurs des jonctions communicantes, canaux permettant la communication directe entre les cellules. Notre laboratoire a identifié des mutations dans les Cx30.3 et Cx31 comme responsables de l'EKV, génodermatose de transmission autosomique dominante. Dans le ler chapitre, nous décrivons une nouvelle mutation de Cx31, L209-Cx31, et contribuons à l'établissement du catalogue des mutations de Cx31 entraînant cette maladie. Cependant, le mécanisme par lequel les mutations de Cx31 et C3x0.3 provoquent l'EKV est inconnu. Dans le 2ème chapitre, nous étudions les effets de la mutation F137L-Cx30.3 sur l'expression, le trafic et la localisation des Cx31 et Cx30.3 transfectées dans des cellules HeLa, déficientes en connexines. Comme deux gènes peuvent causer une EKV quand ils sont mutés, notre hypothèse était que Cx31 et Cx30.3 pourraient coopérer au niveau moléculaire. Nous avons montré l'existence d'une interaction physique entre ces deux connexines. La présence de la mutation F137L-Cx30.3 perturbe le trafic des deux connexines, moins de connexines sont intégrées dans les jonctions communicantes et donc le couplage entre les cellules est diminué. Les connexons formés en présence de cette mutation sont dégradés à leur sortie du réticulum endoplasmique. En conclusion, nos résultats indiquent que l'hétérogénéité génétique de EKV est due à des mutations dans deux protéines qui interagissent. F137L-Cx30.3 a un effet dominant négatif et affecte Cx31, perturbant la communication entre les cellules épidermiques. Le Mal de Meleda est une maladie récessive de la peau palmoplantaire due à des mutations dans SLURP1. SLURP1 appartient à la famille des protéines contenant un domaine LY6/PLAUR et a la particularité d'être sécrétée. La grande homologie de structure existant entre SLURP1, les neurotoxines de serpent et LYNX1-C suggère que la protéine pourrait interagir avec des récepteurs à acétylcholine (Ach). Dans le 3ème chapitre, nous montrons que SLURP1 module la réponse à l'Ach du récepteur nicotinique α7. Ces résultats identifient SLURP1 comme un neuromodulateur épidermique sécrété, probablement essentiel pour la peau palmoplantaire. Dans le 4ème chapitre, nous montrons que SLURP1 est exprimé dans la couche granuleuse de l'épiderme et qu'il est absent des biopsies des patients. SLURP1 a aussi été détecté dans des sécrétions telles que la sueur, les lamies et la salive. Une analyse in vitro de deux mutants de SLURP1 a montré que W15R-SLURP1 est absent des cellules tandis que G86R-SLURP1 est exprimé et sécrété, suggérant qu'une absence ou une anomalie de SLURP1 peuvent causer la maladie. Finalement, dans le 5ème chapitre, nous analysons l'expression et les propriétés biologiques d'autres membres de la famille LY6/PLAUR localisés autour de SLURP1 sur le chromosome 8. Leur statut de protéines sécrétées ou liées à la membrane par une ancre GPI est analysé in vitro. SLURP1, LYNXI-A et -B sont sécrétées alors que LYPDC2 et LYNX1-C sont liés à la membrane. Trois de ces protéines sont exprimées dans l'épiderme et dans des kératinocytes cultivés. Ces résultats suggèrent que la famille LY6/PLAUR pourrait avoir un rôle important dans l'homéostasie de la peau.

The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.