Involvement of the RasGAP derived fragment N in the resistance of pancreatic beta cells towards apoptosis

RESUME DESTINE AUX NON SCIENTIFIQUESLe diabète est une maladie associée à un excès de glucose (sucre) dans le sang. Le taux de glucose sanguin augmente lorsque l'action d'une hormone, l'insuline, responsable du transport du glucose du sang vers les tissus de l'organisme diminue, ou lorsque les quantités d'insuline à disposition sont inadéquates.L'une des causes communes entre les deux grands types de diabète connus, le type 1 et le type 2, est la disparition des cellules beta du pancréas, spécialisées dans la sécrétion d'insuline, par mort cellulaire programmée aussi appelée apoptose. Alors que dans le diabète de type 1, la destruction des cellules beta est causée par notre propre système immunitaire, dans le diabète de type 2, la mort de ces cellules, est principalement causée par des concentrations élevées de graisses saturés ou de molécules impliquées dans l'inflammation que l'on rencontre en quantités augmentées chez les personnes obèses. Etant donné l'augmentation épidémique du nombre de personnes obèses de par le monde, on estime que le nombre de personnes diabétiques (dont une majorité sont des diabétiques de type 2), va passer de 171 million en l'an 2000, à 366 million en l'an 2030, expliquant la nécessité absolue de mettre au point de nouvelles stratégies thérapeutique pour combattre cette maladie.L'apoptose est un processus complexe dont la dérégulation induit de nombreuses affections allant du cancer jusqu'au diabète. L'activation de caspase 3, une protéine clé contrôlant la mort cellulaire, était connue pour systématiquement mener à la mort cellulaire programmée. Ces dernières années, notre laboratoire a décrit des mécanismes de survie qui sont activés par caspase 3 et qui expliquent sans doute pourquoi son activation ne mène pas systématiquement à la mort cellulaire. Lorsqu'elle est faiblement activée, caspase 3 clive une autre protéine appelée RasGAP en deux protéines plus courtes dont l'une, appelée le fragment Ν a la particularité de protéger les cellules contre l'apoptose.Durant ma thèse, j'ai été impliqué dans divers projets destinés à mieux comprendre comment le fragment Ν protégeait les cellules contre l'apoptose et à savoir s'il pouvait être utilisé comme outil thérapeutique dans les conditions de survenue d'un diabète expérimental. C'est dans ce but que nous avons créé une souris transgénique, appelée RIP-N, exprimant le fragment Ν spécifiquement dans les cellules beta. Comme attendu, les cellules beta de ces souris étaient plus résistantes à la mort induite par des composés connus pour induire le diabète, comme certaines molécules induisant l'inflammation ou les graisses saturées. Nous avons ensuite pu montrer que les souris RIP-N étaient plus résistantes à la survenue d'un diabète expérimental que ce soit par l'injection d'une drogue induisant l'apoptose des cellules beta, que ce soit dans un fond génétique caractérisé par une attaque spontanée des cellules beta par le système immunitaire ou dans le contexte d'un diabète de type 2 induit par l'obésité. Dans plusieurs des modèles animaux étudiés, nous avons pu montrer que le fragment Ν protégeait les cellules en activant une voie protectrice bien connue impliquant successivement les protéines Ras, PI3K et Akt ainsi qu'en bloquant la capacité d'Akt d'activer le facteur NFKB, connu pour être délétère pour la survie de la cellule beta. La capacité qu'a le fragment Ν d'activer Akt tout en prévenant l'activation de NFKB par Akt est par conséquent particulièrement intéressante dans l'intégration des signaux régulant la mort cellulaire dans le contexte de la survenue d'un diabète.La perspective d'utiliser le fragment Ν comme outil thérapeutique dépendra de notre capacité à activer les signaux protecteurs induits par le fragment Ν depuis l'extérieur de la cellule ou de dériver des peptides perméables aux cellules possédant les propriétés du fragment N.2 SUMMARYDiabetes mellitus is an illness associated with excess blood glucose. Blood glucose levels raise when the action of insulin decreases or when insulin is provided in inappropriate amounts. In type 1 diabetes (T1D) as well as in type 2 diabetes (T2D), the insulin secreting beta cells in the pancreas undergo controlled cell death also called apoptosis. Whereas in T1D, beta cells are killed by the immune system, in T2D, they are killed by several factors, among which are increased blood glucose levels, increased levels of harmful lipids or pro-inflammatory cytokines that are released by the dysfunctional fat tissue of obese people. Given the epidemic increase in the number of obese people throughout the world, the number of diabetic people (a majority of which are type 2 diabetes) is estimated to rise from 171 million affected people in the year 2000 to 366 million in 2030 explaining the absolute requirement for new therapies to fight the disease.Apoptosis is a very complex process whose deregulation leads to a wide range of diseases going from cancer to diabetes. Caspase 3 although known as a key molecule controlling apoptosis, has been shown to have various other functions. In the past few years, our laboratory has described a survival mechanism, that takes place at low caspase activity and that might explain how cells that activate their caspases for reasons other than apoptosis survive. In such conditions, caspase 3 cleaves another protein called RasGAP into two shorter proteins, one of which, called fragment N, protects cells from apoptosis.We decided to check whether fragment Ν could be used as a therapeutical tool in the context of diabetes inducing conditions. We thus derived a transgenic mouse line, called RIP-N, in which the expression of fragment Ν is restricted to beta cells. As expected, the beta cells of these mice were more resistant ex-vivo to cell death induced by diabetes inducing factors. We then showed that the RIP-N transgenic mice were resistant to streptozotocin induced diabetes, a mouse model mimicking type 1 diabetes, which correlated to fewer number of apoptotic beta cells in the pancreas of the transgenic mice compared to their controls. The RIP-N transgene also delayed overt diabetes development in the NOD background, a mouse model of autoimmune type 1 diabetes, and delayed the occurrence of obesity induced hyperglycemia in a mouse model of type 2-like diabetes. Interestingly, fragment Ν was mediating its protection by activating the protective Akt kinase, and by blocking the detrimental NFKB factor. Our future ability to activate the protective signals elicited by fragment Ν from the outside of cells or to derive cell permeable peptides bearing the protective properties of fragment Ν might condition our ability to use this protein as a therapeutic tool.3 RESUMELe diabète est une maladie associée à un excès de glucose plasmatique. La glycémie augmente lorsque l'action de l'insuline diminue ou lorsque les quantités d'insuline à disposition sont inadéquates. Dans le diabète de type 1 (D1) comme dans le diabète de type 2 (D2), les cellules beta du pancréas subissent la mort cellulaire programmée aussi appelée apoptose. Alors que dans le D1 les cellules beta sont tuées par le système immunitaire, dans le D2 elles sont tuées par divers facteurs parmi lesquels on trouve des concentrations élevées de glucose, d'acides gras saturés ou de cytokines pro-inflammatoires qui sont sécrétées en concentrations augmentées par le tissu adipeux dysfonctionnel des personnes obèses. Etant donné l'augmentation épidémique du nombre de personnes obèses de par le monde, on estime que le nombre de personnes diabétiques (dont une majorité sont des diabétiques de type 2), va passer de 171 million en l'an 2000, à 366 million en l'an 2030, justifiant la nécessité absolue de mettre au point de nouvelles stratégies thérapeutique pour combattre cette maladie.L'apoptose est un processus complexe dont la dérégulation induit de nombreuses affections allant du cancer jusqu'au diabète. Caspase 3, bien que connue comme étant une protéine clé contrôlant l'apoptose a bien d'autres fonctions démontrées. Ces dernières années, notre laboratoire a décrit un mécanisme de survie qui est activé lorsque caspase 3 est faiblement activée et qui explique probablement comment des cellules qui ont activé leurs caspases pour une autre raison que l'apoptose peuvent survivre. Dans ces conditions, caspase 3 clive une autre protéine appelée RasGAP en deux protéines plus courtes dont l'une, appelée le fragment Ν a la particularité de protéger les cellules contre l'apoptose.Nous avons donc décidé de vérifier si le fragment Ν pouvait être utilisé comme outil thérapeutique dans les conditions de survenue d'un diabète expérimental. Pour se faire, nous avons créé une souris transgénique, appelée RIP-N, exprimant le fragment Ν spécifiquement dans les cellules beta. Comme attendu, les cellules beta de ces souris étaient plus résistantes ex-vivo à la mort induite par des facteurs pro-diabétogènes. Nous avons ensuite pu montrer que les souris RIP-N étaient plus résistantes à la survenue d'un diabète induit par la streptozotocine, un drogue mimant la survenue d'un D1 et que ceci était corrélée à une diminution du nombre de cellules en apoptose dans le pancréas des souris transgéniques comparé à leurs contrôles. L'expression du transgène a aussi eu pour effet de retarder la survenue d'un diabète franc dans le fond génétique NOD, un modèle génétique de diabète de type 1 auto-immun, ainsi que de retarder la survenue d'une hyperglycémie dans un modèle murin de diabète de type 2 induit par l'obésité. Dans plusieurs des modèles animaux étudiés, nous avons pu montrer que le fragment Ν protégeait les cellules en activant la kinase protectrice Akt ainsi qu'en bloquant le facteur délétère NFKB. La perspective d'utiliser le fragment Ν comme outil thérapeutique dépendra de notre capacité à activer les signaux protecteurs induits par le fragment Ν depuis l'extérieur de la cellule ou de dériver des peptides perméables aux cellules possédant les propriétés du fragment

Female thyroid cancer : the role of reproductive and hormonal factors in Switzerland

We conducted a study on 91 women with thyroid cancer and 306 controls in hospital for acute nonneoplastic, non-hormone-related disorders in order to investigate the role of reproductive and hormonal factors in the etiology of epithelial thyroid cancer in the Canton of Vaud, Switzerland. Non-significant increases in cancer risk with an increasing number of full-term pregnancies (odds ratio, OR, after allowance for age and previous benign thyroid disease = 1.6, for > or = 3 vs. 0 full-term pregnancies, 95% confidence interval, CI: 0.7-3.6) and spontaneous abortions (OR = 2.0 for > or = 2 vs. 0 spontaneous abortions, 95% CI: 0.7-5.2) were seen. A significantly elevated OR (2.8, 95% CI: 1.1-7.2) was found in those women whose first pregnancy ended with an abortion. Whereas most other reproductive, menstrual and hormonal factors examined did not seem to affect the risk of thyroid cancer significantly, a clue emerged of an association between thyroid cancer and artificial menopause (OR = 6.3, for women who underwent artificial menopause vs. premenopausal women, 95% CI: 1.7-23.2). Although not necessarily causal, the relationship between the risk of epithelial thyroid cancer and the occurrence of spontaneous abortions and artificial menopause deserves attention in future studies, in the light of the high incidence of thyroid cancer in young and middle-aged women.

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA.

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3' tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA.

Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures.

INTRODUCTION: Breast cancer subtyping and prognosis have been studied extensively by gene expression profiling, resulting in disparate signatures with little overlap in their constituent genes. Although a previous study demonstrated a prognostic concordance among gene expression signatures, it was limited to only one dataset and did not fully elucidate how the different genes were related to one another nor did it examine the contribution of well-known biological processes of breast cancer tumorigenesis to their prognostic performance. METHOD: To address the above issues and to further validate these initial findings, we performed the largest meta-analysis of publicly available breast cancer gene expression and clinical data, which are comprised of 2,833 breast tumors. Gene coexpression modules of three key biological processes in breast cancer (namely, proliferation, estrogen receptor [ER], and HER2 signaling) were used to dissect the role of constituent genes of nine prognostic signatures. RESULTS: Using a meta-analytical approach, we consolidated the signatures associated with ER signaling, ERBB2 amplification, and proliferation. Previously published expression-based nomenclature of breast cancer 'intrinsic' subtypes can be mapped to the three modules, namely, the ER-/HER2- (basal-like), the HER2+ (HER2-like), and the low- and high-proliferation ER+/HER2- subtypes (luminal A and B). We showed that all nine prognostic signatures exhibited a similar prognostic performance in the entire dataset. Their prognostic abilities are due mostly to the detection of proliferation activity. Although ER- status (basal-like) and ERBB2+ expression status correspond to bad outcome, they seem to act through elevated expression of proliferation genes and thus contain only indirect information about prognosis. Clinical variables measuring the extent of tumor progression, such as tumor size and nodal status, still add independent prognostic information to proliferation genes. CONCLUSION: This meta-analysis unifies various results of previous gene expression studies in breast cancer. It reveals connections between traditional prognostic factors, expression-based subtyping, and prognostic signatures, highlighting the important role of proliferation in breast cancer prognosis.

The anti-apoptotic role of PPARbeta contributes to efficient skin wound healing.

PPARalpha and PPARbeta are expressed in the mouse epidermis during fetal development, but their expression progressively disappears after birth. However, the expression of PPARbeta is reactivated in adult mice upon proliferative stimuli, such as cutaneous injury. We show here that PPARbeta protects keratinocytes from growth factor deprivation, anoikis and TNF-alpha-induced apoptosis, by modulating both early and late apoptotic events via the Akt1 signaling pathway and DNA fragmentation, respectively. The control mechanisms involve direct transcriptional upregulation of ILK, PDK1, and ICAD-L. In accordance with the anti-apoptotic role of PPARbeta observed in vitro, the balance between proliferation and apoptosis is altered in the epidermis of wounded PPARbeta mutant mice, with increased keratinocyte proliferation and apoptosis. In addition, primary keratinocytes deleted for PPARbeta show defects in both cell-matrix and cell-cell contacts, and impaired cell migration. Together, these results suggest that the delayed wound closure observed in PPARbeta mutant mice involves the alteration of several key processes. Finally, comparison of PPARbeta and Akt1 knock-out mice reveals many similarities, and suggests that the ability of PPARbeta to modulate the Akt1 pathway has significant impact during skin wound healing.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Cigarette smoking and the risk of endometrial cancer.

The risk of endometrial cancer in relation to cigarette consumption was evaluated in a hospital-based case-control study of breast and genital neoplasms conducted in Milan, northern Italy. For the present analysis, 357 women (cases) with histologically confirmed endometrial cancer were compared to a group of 1122 women (controls) admitted for a large spectrum of acute conditions unrelated to smoking or to any of the known or potential risk factors for endometrial cancer. Compared with never-smokers, the multivariate relative risk estimates were for current 0.45 [95% confidence interval (CI) = 0.30-0.70] and 0.86 (95% CI = 0.50-1.46) for ex-smokers. The negative association of endometrial cancer with current smoking was not influenced by menopausal status as well as by other major identified potential confounding factors, i.e. menstrual and reproductive history, body mass index, oral contraceptive or estrogen replacement therapy use and family gynecologic cancer history. However, there was no evidence of a dose-risk effect, since the relative risks were similar in moderate and heavy smokers. The present study confirms that smoking is less frequent in cases hospitalized for endometrial cancer than in a comparison group of patients with non-smoking-related acute conditions. This negative association is perhaps explained in terms of reduced estrogen levels in smokers, though the influence and the importance of some uncontrolled selection bias (due, for instance, to longer hospital stay of smokers even when admission diagnosis was for non-smoking-related conditions) cannot be ruled out.

Solar Powered Highway Delineator System, HR-367, 1993

Currently, many drivers experience some difficulty in viewing the road ahead of them during times of reduced visibility, such as rain, snow, fog, or the darkness of night- Recent studies done by the National Safety Council provide a detailed contrast between fatal accidents occurring during the day and night. Revealed was that the motor vehicle night death rate (4.41 deaths per 100 million miles driven) was sharply higher than the corresponding death rate during daylight hours (1.21). By providing a delineating system powered by the natural resource of solar power, a constant source of visibility may be maintained throughout the evening. Along with providing enough light to trace the outline of the road, other major goals defined in producing this delineator system are as follows: 1. A strong and durable design that would protect the internal components and survive extreme weather conditions. 2. A low maintenance system where components need few repairs or replacements. 3. A design which makes all components accessible in the event that maintenance is needed, but also prevents vandalism. 4. A design that provides greater visibility to drivers and will not harm a vehicle or its passengers in the event of a collision. This solar powered highway delineator consists of an adjustable solar array, a light fixture, and a standard delineator pole. The solar array houses and protects the solar panels, and can be easily adjusted to obtain a maximum amount of sunlight. The light fixture primarily houses the battery, the circuit and the light assembly. Both components allow for easy accessibility and reduce vandalism using internal connections for bolts and wires. The delineator mounting pole is designed to extensively deform in the event of a collision, therefore reducing any harm caused to the vehicle and/or the passengers. The cost of a single prototype to be produced is approximately $70.00 excluding labor costs. However, these material and labor costs will be greatly reduced if a large number of delineators are produced. It is recommended that the Iowa Department of Transportation take full advantage of the research and development put into this delineator design. The principles used in creating this delineator can be used to provide an outline for drivers to follow, or on a larger scale, provide actual roadway lighting in areas where it was never before possible or economically feasible. In either event, the number of fatal accidents will be decreased due to the improved driver visibility in the evening.

Design, conduct, and analyses of Breast International Group (BIG) 1-98: a randomized, double-blind, phase-III study comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with receptor-positive, early breast cancer.

BACKGROUND: Aromatase inhibitors provide superior disease control when compared with tamoxifen as adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer. PURPOSE: To present the design, history, and analytic challenges of the Breast International Group (BIG) 1-98 trial: an international, multicenter, randomized, double-blind, phase-III study comparing the aromatase inhibitor letrozole with tamoxifen in this clinical setting. METHODS: From 1998-2003, BIG 1-98 enrolled 8028 women to receive monotherapy with either tamoxifen or letrozole for 5 years, or sequential therapy of 2 years of one agent followed by 3 years of the other. Randomization to one of four treatment groups permitted two complementary analyses to be conducted several years apart. The first, reported in 2005, provided a head-to-head comparison of letrozole versus tamoxifen. Statistical power was increased by an enriched design, which included patients who were assigned sequential treatments until the time of the treatment switch. The second, reported in late 2008, used a conditional landmark approach to test the hypothesis that switching endocrine agents at approximately 2 years from randomization for patients who are disease-free is superior to continuing with the original agent. RESULTS: The 2005 analysis showed the superiority of letrozole compared with tamoxifen. The patients who were assigned tamoxifen alone were unblinded and offered the opportunity to switch to letrozole. Results from other trials increased the clinical relevance about whether or not to start treatment with letrozole or tamoxifen, and analysis plans were expanded to evaluate sequential versus single-agent strategies from randomization. LIMITATIONS: Due to the unblinding of patients assigned tamoxifen alone, analysis of updated data will require ascertainment of the influence of selective crossover from tamoxifen to letrozole. CONCLUSIONS: BIG 1-98 is an example of an enriched design, involving complementary analyses addressing different questions several years apart, and subject to evolving analytic plans influenced by new data that emerge over time.

cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein.

IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1beta and TNF-alpha. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic beta-cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1beta-induced pancreatic beta-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic beta-cells and indicate that IB2 represents a novel candidate gene for diabetes.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

PGC-1α induces mitochondrial and myokine transcriptional programs and lipid droplet and glycogen accumulation in cultured human skeletal muscle cells

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a chief activator of mitochondrial and metabolic programs and protects against atrophy in skeletal muscle (skm). Here we tested whether PGC-1α overexpression could restructure the transcriptome and metabolism of primary cultured human skm cells, which display a phenotype that resembles the atrophic phenotype. An oligonucleotide microarray analysis was used to reveal the effects of PGC-1α on the whole transcriptome. Fifty-three different genes showed altered expression in response to PGC-1α: 42 upregulated and 11 downregulated. The main gene ontologies (GO) associated with the upregulated genes were mitochondrial components and processes and this was linked with an increase in COX activity, an indicator of mitochondrial content. Furthermore, PGC-1α enhanced mitochondrial oxidation of palmitate and lactate to CO2, but not glucose oxidation. The other most significantly associated GOs for the upregulated genes were chemotaxis and cytokine activity, and several cytokines, including IL-8/CXCL8, CXCL6, CCL5 and CCL8, were within the most highly induced genes. Indeed, PGC-1α highly increased IL-8 cell protein content. The most upregulated gene was PVALB, which is related to calcium signaling. Potential metabolic regulators of fatty acid and glucose storage were among mainly regulated genes. The mRNA and protein level of FITM1/FIT1, which enhances the formation of lipid droplets, was raised by PGC-1α, while in oleate-incubated cells PGC-1α increased the number of smaller lipid droplets and modestly triglyceride levels, compared to controls. CALM1, the calcium-modulated δ subunit of phosphorylase kinase, was downregulated by PGC-1α, while glycogen phosphorylase was inactivated and glycogen storage was increased by PGC-1α. In conclusion, of the metabolic transcriptome deficiencies of cultured skm cells, PGC-1α rescued the expression of genes encoding mitochondrial proteins and FITM1. Several myokine genes, including IL-8 and CCL5, which are known to be constitutively expressed in human skm cells, were induced by PGC-1α.

The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the 'fused genes' family.

  The skin is essential for survival and protects our body against biological attacks, physical stress, chemical injury, water loss, ultraviolet radiation and immunological impairment. The epidermal barrier constitutes the primordial frontline of this defense established during terminal differentiation. During this complex process proliferating basal keratinocytes become suprabasally mitotically inactive and move through four epidermal layers (basal, spinous, granular and layer, stratum corneum) constantly adapting to the needs of the respective cell layer. As a result, squamous keratinocytes contain polymerized keratin intermediate filament bundles and a water-retaining matrix surrounded by the cross-linked cornified cell envelope (CE) with ceramide lipids attached on the outer surface. These cells are concomitantly insulated by intercellular lipid lamellae and hold together by corneodesmosmes. Many proteins essential for epidermal differentiation are encoded by genes clustered on chromosomal human region 1q21. These genes constitute the 'epidermal differentiation complex' (EDC), which is divided on the basis of common gene and protein structures, in three gene families: (i) CE precursors, (ii) S100A and (iii) S100 fused genes. EDC protein expression is regulated in a gene and tissue-specific manner by a pool of transcription factors. Among them, Klf4, Grhl3 and Arnt are essential, and their deletion in mice is lethal. The importance of the EDC is further reflected by human diseases: FLG mutations are the strongest risk factor for atopic dermatitis (AD) and for AD-associated asthma, and faulty CE formation caused by TG1 deficiency causes life-threatening lamellar ichthyosis. Here, we review the EDC genes and the progress in this field.

Role of the serine protease CAP1/Prss8 and its inhibitor in the skin

The skin is the largest organ of the human body and protects it from water loss and mechanical damage. This barrier function is mainly provided by the epidermis, the outermost layer of the skin. This balance is regulated by several factors, including serine proteases, serine protease inhibitors and protease target substrates, such as receptors. Any mutations or alterations in the expression of these factors can lead to skin diseases. One of the players in this skin balance is the serine protease CAP1/Prss8, whose over-expression causes ichthyosis, hyperplasia and inflammation. This phenotype can be completely restored in the absence of PAR2 (protease-activated receptor 2) (Frateschi et al., 2011). During my thesis, I demonstrated that CAP1/Prss8 induces skin disease even if its catalytic triad is mutated. Additionally, I demonstrated an inhibitory effect of the serine protease-inhibitor nexin-1 (also called serpinE2, PN-1) on CAP1/Prss8, since nexin-1 negated the effects of both catalytically active and inactive CAP1/Prss8 over-expression. Indeed, CAP1/Prss8 and nexin-1 interact in vitro, but independent of the catalytic triad of CAP1/Prss8. These results demonstrate a novel mechanism of interaction between CAP1/Prss8 and nexin-1, and indicate that the catalytic triad of CAP1/Prss8 is dispensable for nexin-1 inhibition and PAR2 activation. These observations in vivo and in vitro could be helpful to specifically target drugs to treat ichthyoses-like skin diseases, like e.g. atopic dermatitis. - La peau est l'un des organes les plus importants du corps humain au regard de sa surface et de sa masse. Ses principales fonctions sont de nous protéger contre l'entrée de pathogènes et de former une barrière imperméable qui empêche la déshydratation. Ces fonctions sont principalement assurées par l'épiderme, la couche la plus superficielle de la peau, et garanties par plusieurs "acteurs", comme par exemple les sérine-protéases, les inhibiteurs de sérine- protéases ou les protéases cibles comme les récepteurs. Toute mutation ou altération de l'un de ces "acteurs" peut aboutir au déclanchement de maladies de la peau. Pour mieux comprendre les conséquences biologiques résultant d'une altération d'expression de CAP1/Prss8, une serine-protéase normalement exprimée au niveau de l'épiderme, nous avons généré des souris transgéniques surexprimant CAP1/Prss8 au niveau de la peau. Ces dernières présentent une peau squameuse, un épiderme hypertrophique, des processus inflammatoires et des prurits conséquents. Ces symptômes disparaissent si le gène du récepteur PAR2, qui régule l'activité des cellules de l'épiderme, est inactivé. Dans le but de vérifier si le phénotype observé chez les souris CAP1/Prss8 résulte de l'action du site catalytique de CAP1/Prss8, nous avons généré des souris CAP1/Prss8 chez lesquelles nous avons muté les trois acides aminés du site catalytique en alanine. Etonnement ces souris ont développé les mêmes problèmes de peau que les souris CAP1/Prss8, démontrant que l'effet de CAP1/Prss8, dans ce modèle animal, n'est pas lié à son site catalytique. Nous avons également montré in vivo, que la sérine-protéase nexin-1 (aussi appelée SERPINE2, PN-1) est capable d'exercer un effet inhibiteur sur CAP1/Prss8 indépendamment de l'activité du site catalytique de CAP1/Prss8. De plus, nous avons remarqué in vitro que CAP1/Prss8 et nexin-1 interagissent bien que la triade catalytique de CAP1/Prss8 soit enzymatiquement inactivée. Ces observations, in vivo et in vitro, pourraient être utilisées dans l'élaboration de médicaments contenant nexin-1, pour le traitement de pathologies de la peau telles l'ichthyose et la dermatite atopique.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.