CELLULAR AND MOLECULAR STUDY OF SUBCORTICAL BAND HETEROTOPIA IN THE MOUSE MUTANT HECO

The HeCo mouse model is characterized by a subcortical heterotopia formed by misplaced neurons normally migrating into the superficial cortical layers. The mutant mouse has a tendency to epileptic seizures. In my thesis project we discovered the mutated Eml1 gene, a member of the echinoderm microtubule-associated protein (EMAP) family, in HeCo as well as in a family of three children showing complex malformation of cortical development. This discovery formed an important step in exploring the pathogenic mechanisms underlying the HeCo phenotype. In vitro results showed that during cell division the EML1 protein is associated with the midbody and a mutated version of Eml1 highlighted an important role of the protein in the astral MT array during cell cycle. In vivo, we found that already at an early age of cortical development (E13), ectopic progenitors such as RGs (PAX6) and IPCs (TBR2) accumulate in the IZ along the entire neocortex. We demonstrated that in the VZ of the HeCo mouse, spindle orientation and cell cycle exit are perturbed. In later stages (E17), RG fibers are strongly disorganized with deep layer (TBR1) and upper layer (CUX1) neurons trapped within an ectopic mass. At P3, columns of upper layer neurons were present between the heterotopia and the developing cortex; these columns were also present at P7 but at lesser extent. Time lapse video recording (E15.5) revealed that the parameters characterizing the migration of individual neurons are not disturbed in HeCo; however, this analysis showed that the density of migrating neuron was smaller in HeCo. In conclusion, truncated EML1 is likely to play a prominent role during cell cycle but also acts on the cytoskeletal architecture altering the shape of RG fibers thus influencing the pattern of neuronal migration. The signal transduction between external cues and intracellular effector pathways through MTs may be secondary but sustains the heterotopia development and further studies are needed to clarify the impact of EML1 in progenitors versus post-mitotic cells.

T cell receptor delta gene rearrangement and T early alpha (TEA) expression in immature alpha beta lineage thymocytes: implications for alpha beta/gamma delta lineage commitment.

Mature T cells comprise two mutually exclusive lineages expressing heterodimeric alpha beta or gamma delta antigen receptors. During development, beta, gamma, and delta genes rearrange before alpha, and mature gamma delta cells arise in the thymus prior to alpha beta cells. The mechanism underlying commitment of immature T cells to the alpha beta or gamma delta lineage is controversial. Since the delta locus is located within the alpha locus, rearrangement of alpha genes leads to deletion of delta. We have examined the rearrangement status of the delta locus immediately prior to alpha rearrangement. We find that many thymic precursors of alpha beta cells undergo VDJ delta rearrangements. Furthermore, the same cells frequently coexpress sterile T early alpha (TEA) transcripts originating 3' of C delta and 5' of the most upstream J alpha, thus implying that individual alpha beta lineage cells undergo sequential VDJ delta and VJ alpha rearrangements. Finally, VDJ delta rearrangements in immature alpha beta cells appear to be random, supporting models in which alpha beta lineage commitment is determined independently of the rearrangement status at the TCR delta locus.

Characterization of OsMIK in a rice mutant with reduced phytate content reveals an insertion of a rearranged retrotransposon.

The rice low phytic acid (lpa) mutant Os-lpa-XS110-1(XS-lpa) has ~45 % reduction in seed phytic acid (PA) compared with the wild-type cultivar Xiushui 110. Previously, a single recessive gene mutation was shown to be responsible for the lpa phenotype and was mapped to a region of chromosome 3 near OsMIK (LOC_Os03g52760) and OsIPK1 (LOC_Os03g51610), two genes involved in PA biosynthesis. Here, we report the identification of a large insert in the intron of OsMIK in the XS-lpa mutant. Sequencing of fragments amplified through TAIL-PCRs revealed that the insert was a derivative of the LINE retrotransposon gene LOC_Os03g56910. Further analyses revealed the following characteristics of the insert and its impacts: (1) the inserted sequence of LOC_Os03g56910 was split at its third exon and rejoined inversely, with its 5' and 3' flanking sequences inward and the split third exon segments outward; (2) the LOC_Os03g56910 remained in its original locus in XS-lpa, and the insertion probably resulted from homologous recombination repair of a DNA double strand break; (3) while the OsMIK transcripts of XS-lpa and Xiushui 110 were identical, substantial reductions of the transcript abundance (~87 %) and the protein level (~60 %) were observed in XS-lpa, probably due to increased methylation in its promoter region. The above findings are discussed in the context of plant mutagenesis, epigenetics and lpa breeding.

MOLECULAR MECHANISMS OF A ROOT SYSTEM TO SOIL ADAPTATION

Contrairement aux animaux, les plantes sont des organismes sessiles qui ne possèdent pas de mécanismes de fuite quand les conditions environnementales ne sont plus optimales. Les plantes sont physiquement ancrées à l'endroit où elles ont germées et aux conditions environnementales qui parfois peuvent être extrêmes. Les possibilités d'acclimatation de différentes espèces, parfois même de groupes de plantes au sein d'une même espèce, peuvent varier mais repose sur une adaptation génétique de la plante. L'adaptation est un long processus qui repose sur l'apparition spontanée de mutations génétiques, leur mise à l'épreuve face aux conditions environnementales, et dans le cas où la mutation a un impact positif sur la survie dans cet habitat particulier, elle sera maintenue dans une population donnée de plantes. De telles populations, appelées écotypes, sont le matériel de départ pour la découverte de gènes qui induisent un bénéfice pour la plante dans un environnement donné. La plante la plus étudiée en biologie moléculaire est Arabidopsis thaliana, l'arabette des prés. Dans une étude précédente, les racines d'écotypes naturels d'Arabidopsis ont été comparées et un écotype, Uk-1, avait le système racinaire le plus particulier. Cet écotype possède des racines beaucoup plus courtes et plus ramifiées que tous les autres écotypes. Des analyses plus poussées ont montré qu'une seule mutation dans un gène était la cause de ce phénotype, le gène BREVIS RADIX (BRX), mot latin signifiant 'racine courte'. Bien que l'on connaisse le gène BRX, on connaît finalement peu de choses sur son importance adaptative. Dans cette étude, nous avons montré que la mutation dans le gène BRX rend la plante plus résistante aux sols acides. Dans l'optique de mieux comprendre cette valeur adaptative du mutant brx, nous avons analysé dans quels tissus le gène BRX jouait un rôle important. Nous avons pu mettre en évidence que BRX est important pour le développement du protophloème. Le protophloème est un élément du système vasculaire de la plante. En général, les plantes supérieures possèdent deux systèmes de transport à longue distance. L'un d'eux, appelé xylème, transporte l'eau et les nutriments absorbés du sol par les racines vers les feuilles. Les feuilles sont le siège du processus de photosynthèse au cours duquel sont produits des sucres qui devront être distribués partout dans les autres parties de la plante. Le tissu cellulaire chargé de livrer les produits de la photosynthèse, ainsi que les régulateurs de croissance, est le phloème. Ce dernier regroupe le métaphloème et le protophloème. Le protophloème est essentiel pour la livraison des sucres synthétisés ainsi que des signaux de croissance aux pointes des racines, centres organogéniques responsables de la production de nouvelles cellules durant la phase de croissance de la racine. La structure du protophloème peut être décrite comme des tubes continus, vides et résistants, faits de cellules spécialisées qui permettent un transport efficace et rapide. Nous avons montré que dans les mutants brx ces canaux de transports sont discontinus car certaines cellules n'ont pas terminé leur cycle de différenciation. Ces cellules obstruent le conduit ce qui fait que les sucres et les signaux de croissance, comme l'auxine, ne peuvent plus être transportés aux méristèmes. En conséquence, la prolifération de l'activité des méristèmes est compromise, ce qui explique les racines courtes. Au lieu d'être délivré aux méristèmes, l'auxine se concentre en amont des méristèmes où cela provoque l'apparition de nouvelles racines branchées et, très probablement, l'activation des pompes à protons. Sur des sols acides, la concentration en ion H+ est très élevée. Ces ions entrent dans les cellules de la racine par diffusion et perturbent notablement la croissance des racines et de la plante en général. Si les cellules de la racine possédaient des pompes à protons hyperactives, elles seraient capable d'évacuer le surplus d'ions H+ en dehors de la cellule, ce qui leur assurerait de meilleures chances de survie sur sols acides. De fait, le mutant brx est capable d'acidifier le milieu de culture dans lequel il est cultivé plus efficacement que la plante sauvage. Ce mutant est également capable de donner plus de progéniture sur ce type de milieu de croissance que les plantes sauvages. Finalement, nous avons trouvé d'autres mutants brx en milieu naturel poussant sur sols acides, ce qui suggère fortement que la mutation du gène BRX est une des causes de l'adaptation aux sols acides. -- Plants as sessile organisms have developed different mechanisms to cope with the complex environmental conditions in which they live. Adaptation is the process through which traits evolve by natural selection to functionally improve in a given environmental context. An adaptation to the environment is characterized by the genetic changes in the entire populations that have been fixed by natural selection over many generations. BREVIS RADIX (BRX) gene was found through natural Arabidopsis accessions screen and was characterized as a root growth regulator since loss-of-function mutants exhibit arrested post-embryonic primary root growth in addition to a more branched root system. Although brx loss-of-function causes a complete alteration in root architecture, BRX activity is only required in the root vasculature, in particular in protophloem cell file. Protophloem is a part of the phloem transport network and is responsible for delivery of photo-assimilates and growth regulators, coming from the shoot through mature phloem component - metaphloem, to the all plant primary meristems. In order to perform its function, protophloem is the first cell file to differentiate within the root meristem. During this process, protophloem cells undergo a partial programmed cell death, during which they build a thicker cell wall, degrade nucleus and tonoplast while plasma membrane stays functional. Interestingly, protophloem cells enter elongation process only after differentiation into sieve elements is completed. Here we show that brx mutants fail to differentiate protophloem cell file properly, a phenotype that can be distinguished by a presence of a "gap" cells, non-differentiated cells between two flanking differentiated cells. Discontinuity of protophloem differentiation in brx mutants is considered to be a consequence of local hyperactivity of CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) - BARELY ANY MERISTEM 3 (BAM3) signaling module. Interestingly, a CLE45 activity, most probably at the level of receptor binding, can be modulated by apoplastic pH. Altogether, our results imply that the activity of proton pumps, expressed in non-differentiated cells of protophloem, must be maintained under certain threshold, otherwise CLE45-BAM3 signaling pathway will be stimulated and in turn protophloem will not differentiate. Based on vacuolar morphology, a premature cell wall acidification in brx mutants stochastically prevents the protophloem differentiation. Only after protophloem differentiates, proton pumps can be activated in order to acidify apoplast and to support enucleated protophloem multifold elongation driven by surrounding cells growth. Finally, the protophloem differentiation failure would result in an auxin "traffic jam" in the upper parts of the root, created from the phloem-transported auxin that cannot be efficiently delivered to the meristem. Physiologically, auxin "leakage" from the plant vasculature network could have various consequences, since auxin is involved in the regulation of almost every aspect of plant growth and development. Thus, given that auxin stimulates lateral roots initiation and growth, this scenario explains more branched brx root system. Nevertheless, auxin is considered to activate plasma membrane proton pumps. Along with this, it has been shown that brx mutants acidify media much more than the wild type plants do, a trait that was proposed as an adaptive feature of naturally occurring brx null alleles in Arabidopsis populations found on acidic soils. Additionally, in our study we found that most of accessions originally collected from acidic sampling sites exhibit hypersensitivity to CLE45 treatment. This implies that adaptation of plants to acidic soil involves a positive selection pressure against upstream negative regulators of CLE45-BAM3 signaling, such as BRX. Perspective analysis of these accessions would provide more profound understanding of molecular mechanisms underlying plant adaptation to acidic soils. All these results are suggesting that targeting of the factors that affect protophloem differentiation is a good strategy of natural selection to change the root architecture and to develop an adaptation to a certain environment. -- Les plantes comme organismes sessiles ont développé différents mécanismes pour s'adapter aux conditions environnementales complexes dans lesquelles elles vivent. L'adaptation est le processus par lequel des traits vont évoluer via la sélection naturelle vers une amélioration fonctionnelle dans un contexte environnemental donné. Une adaptation à l'environnement est caractérisée par des changements génétiques dans des populations entières qui ont été fixés par la sélection naturelle sur plusieurs générations. Le gène BREVIS RADIX (BRX) a été identifié dans le crible d'une collection d'accessions naturelles d'Arabidopsis et a été caractérisé comme un régulateur de la croissance racinaire étant donné que le mutant perte-de-fonction montre une croissance racinaire primaire arrêtée au stade post-embryonnaire et présente de plus un système racinaire plus ramifié que la plante sauvage. Bien que le mutant perte-de-fonction brx cause une altération complète de l'architecture racinaire, l'activité de BRX n'est requise que dans la vascularisation racinaire, en particulier au niveau du protophloème. Le protophloème est un composant du réseau de transport du phloème et est responsable du transit des dérivés de la photosynthèse ainsi que des régulateurs de croissances, venant de la partie aérienne par le phloème mature (métaphloème) vers tous les méristèmes primaires de la plante. Pour pouvoir réaliser sa fonction, le protophloème est la première file de cellules à se différencier à l'intérieur du méristème de la racine. Pendant ce processus, les cellules du protophloème subissent une mort cellulaire programmée partielle durant laquelle elles épaississent leur paroi cellulaire, dégradent le noyau et le tonoplaste tandis que la membrane plasmique demeure fonctionnelle. De manière intéressante, les cellules du protophloème entament le processus d'allongement seulement après que la différenciation en tubes criblés soit complète. Ce travail montre que le mutant brx est incapable de mener à bien la différenciation de la file de cellules du protophloème, phénotype qui peut être visualisé par la présence de cellules 'trous', de cellules non différenciées entourées de deux cellules différenciées. La discontinuité de la différenciation du phloème dans le mutant brx est considérée comme la conséquence de l'hyperactivité localisée du module de signalisation CLA VA TA3/EMBRYO SURROUNDING REGION 45 (CLE45) - BARELY ANY MERISTEM 3 (BAM3). De manière intéressante, l'activité de CLE45, très probablement au niveau de la liaison avec le récepteur, peut être modulé par le pH apoplastique. Pris ensemble, nos résultats impliquent que l'activité des pompes à protons, actives dans les cellules non différenciées du protophloème, doit être maintenue en dessous d'un certain seuil autrement la cascade de signalisation CLE45-BAM3 serait stimulée, en conséquence de quoi le protophloème ne pourrait se différencier. D'après la morphologie vacuolaire, une acidification prématurée de la paroi cellulaire dans le mutant brx empêche la différenciation du protophloème de manière stochastique. Une fois que le protophloème se différencie, les pompes à protons peuvent alors être activées afin d'acidifier l'apoplaste et ainsi faciliter l'allongement des cellules énuclées du protophloème, entraînées par la croissance des cellules environnantes. Finalement, la différenciation défectueuse du protophloème produit une accumulation d'auxine dans la partie supérieure de la racine car le phloème ne peut plus acheminer efficacement l'auxine au méristème. Physiologiquement, la 'fuite' d'auxine à partir du réseau vasculaire de la plante peut avoir des conséquences variées puisque l'auxine est impliquée dans la régulation de la majorité des aspects de la croissance et développement de la plante. Etant donné que l'auxine stimule l'initiation et développement des racines latérales, ce scénario pourrait expliquer le système racinaire plus ramifié du mutant brx. En plus, l'auxine est considérée comme un activateur des pompes à protons. Par ailleurs, nous avons montré que les mutants brx ont la capacité d'acidifier le milieu plus efficacement que les plantes sauvages, une caractéristique des populations sauvages <¥Arabidopsis poussant sur des sols acides et contenant les allèles délétés brx. De plus, dans nos résultats nous avons mis en évidence que la plupart des accessions collectées originellement sur des sites acidophiles montre une hypersensibilité au traitement par CLE45. Ceci implique que l'adaptation des plantes aux sols acides repose sur la pression de sélection positive à rencontre des régulateurs négatifs de CLE45- BAM3, situés en amont de la cascade, tel le produit du gène BRX. Les analyses de ces accessions pourraient aboutir à une meilleure compréhension des mécanismes moléculaires responsables de l'adaptation des plantes aux sols acides. Tous nos résultats suggèrent que le ciblage des facteurs affectant la différenciation du protophloème serait une stratégie gagnante dans la sélection naturelle pour changer l'architecture de la racine et ainsi s'adapter efficacement à un nouvel environnement.

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD.

Transcriptional repression of peroxisome proliferator-activated receptor beta/delta in murine keratinocytes by CCAAT/enhancer-binding proteins.

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPARbeta promoter revealed an AP-1 site that is crucial for the regulation of PPARbeta expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPARbeta gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBPalpha and C/EBPbeta, but not C/EBPdelta, inhibit the expression of PPARbeta through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPARbeta promoter. Consistent with this repression, the expression patterns of PPARbeta and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPARbeta and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.

cGMP-dependent protein kinase type I is implicated in the regulation of the timing and quality of sleep and wakefulness.

Many effects of nitric oxide (NO) are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP). cGMP activates cGMP-dependent protein kinases (PRKGs), which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1) in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS) duration and in non-REM sleep (NREMS) consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG) power in the delta frequency range (1-4 Hz) under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep.

Les antagonistes du récepteur minératocorticoïde

Mineralocorticoid signaling pathway plays a pivotal role in cardiovascular physiopathology. Evidences from clinical and experimental studies have linked mineralocorticoid hormones with cardiovascular morbiditiy and mortality. Thus, antagonist of the mineralocorticoid receptor (AMR) has reappeared. In addition, a novel mineralocorticoid receptor antagonist has been developped, named eplerenone, which lack the side effect of former ARMs as gynecomastia. Based on two studies named RALES et EPHESUS, guidelines of the european and american societies of cardiology recommend the use of ARMs as a treatment for cardiac failure NYHA III and IV, and post-infarct cardiac failure (ejection fraction < 40%).

Epithelium-mesenchyme interactions control the activity of peroxisome proliferator-activated receptor beta/delta during hair follicle development.

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

Clinical correlates of frontal intermittent rhythmic delta activity (FIRDA).

Objectives: To investigate the clinical correlates of frontal intermittent rhythmic delta activity (FIRDA). Methods: we prospectively assessed all EEG studies recorded in our center over 3 months for the presence of frontal intermittent rhythmic delta activity (FIRDA). The FIRDA group was compared with a randomly selected control group from among EEGs recorded during the same period. Comparisons among FIRDA and non-FIRDA groups were performed using uni- and multi-variate analyses. Results: We found 36 patients with FIRDA among 559 EEG recordings (6%); the control group consisted of 80 subjects. While epilepsy was more frequent in the control group, structural brain lesions and encephalopathy were independently associated with the occurrence of FIRDA, but we could not identify any specific etiology. Asymmetric FIRDA was associated with an underlying brain lesion. Occasionally, FIRDA was recorded in otherwise healthy subjects during hyperventilation. Conclusion: FIRDA appears more common than previously reported, and is associated with a wide range of lesions and encephalopathic conditions. Significance: FIRDA occurrence should prompt investigations for toxic-metabolic disturbances and for structural lesions (particularly if asymmetric), but does not suggest an epileptic predilection.

Prostaglandin E(2) promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferator-activated receptor delta.

Cyclooxygenase-derived prostaglandin E(2) (PGE(2)) is the predominant prostanoid found in most colorectal cancers (CRC) and is known to promote colon carcinoma growth and invasion. However, the key downstream signaling pathways necessary for PGE(2)-induced intestinal carcinogenesis are unclear. Here we report that PGE(2) indirectly transactivates PPARdelta through PI3K/Akt signaling, which promotes cell survival and intestinal adenoma formation. We also found that PGE(2) treatment of Apc(min) mice dramatically increased intestinal adenoma burden, which was negated in Apc(min) mice lacking PPARdelta. We demonstrate that PPARdelta is a focal point of crosstalk between the prostaglandin and Wnt signaling pathways which results in a shift from cell death to cell survival, leading to increased tumor growth.

Induction of p38, tumour necrosis factor-α and RANTES by mechanical stretching of keratinocytes expressing mutant keratin 10R156H.

Background : Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma), characterized by ichthyotic, rippled hyperkeratosis, erythroderma and skin blistering, is a rare autosomal dominant disease caused by mutations in keratin 1 or keratin 10 (K10) genes. A severe phenotype is caused by a missense mutation in a highly conserved arginine residue at position 156 (R156) in K10. Objectives: To analyse molecular pathomechanisms of hyperproliferation and hyperkeratosis, we investigated the defects in mechanosensation and mechanotransduction in keratinocytes carrying the K10R156H mutation. Methods: Differentiated primary human keratinocytes infected with lentiviral vectors carrying wild-type K10 (K10wt) or mutated K10R156H were subjected to 20% isoaxial stretch. Cellular fragility and mechanosensation were studied by analysis of mitogen-activated protein kinase activation and cytokine release. Results: Cultured keratinocytes expressing K10R156H showed keratin aggregate formation at the cell periphery, whereas the filament network in K10wt cells was normal. Under stretching conditions K10R156H keratinocytes exhibited about a twofold higher level of filament collapse compared with steady state. In stretched K10R156H cells, higher p38 activation, higher release of tumour necrosis factor-alpha and RANTES but reduced interleukin-1 beta secretion compared with K10wt cells was observed. Conclusions: These results demonstrate that the R156H mutation in K10 destabilizes the keratin intermediate filament network and affects stress signalling and inflammatory responses to mechanical stretch in differentiated cultured keratinocytes.

Stage-specific integration of maternal and embryonic peroxisome proliferator-activated receptor delta signaling is critical to pregnancy success.

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P&lt;0.05) and pyruvatedehydrogenase kinase 4 (30%, P&lt;0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.