Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells.

Developmental regulation of expression of schizophrenia susceptibility genes in the primate hippocampal formation

The hippocampal formation is essential for normal memory function and is implicated in many neurodevelopmental, neurodegenerative and neuropsychiatric disorders. In particular, abnormalities in hippocampal structure and function have been identified in schizophrenic subjects. Schizophrenia has a strong polygenic component, but the role of numerous susceptibility genes in normal brain development and function has yet to be investigated. Here we described the expression of schizophrenia susceptibility genes in distinct regions of the monkey hippocampal formation during early postnatal development. We found that, as compared with other genes, schizophrenia susceptibility genes exhibit a differential regulation of expression in the dentate gyrus, CA3 and CA1, over the course of postnatal development. A number of these genes involved in synaptic transmission and dendritic morphology exhibit a developmental decrease of expression in CA3. Abnormal CA3 synaptic organization observed in schizophrenics might be related to some specific symptoms, such as loosening of association. Interestingly, changes in gene expression in CA3 might occur at a time possibly corresponding to the late appearance of the first clinical symptoms. We also found earlier changes in expression of schizophrenia susceptibility genes in CA1, which might be linked to prodromal psychotic symptoms. A number of schizophrenia susceptibility genes including APOE, BDNF, MTHFR and SLC6A4 are involved in other disorders, and thus likely contribute to nonspecific changes in hippocampal structure and function that must be combined with the dysregulation of other genes in order to lead to schizophrenia pathogenesis.

Mineralocorticoid receptor is essential for corticosteroid-induced up-regulation of L-type calcium currents in cultured neonatal cardiomyocytes.

Despite the fact that mineralocorticoid receptor (MR) antagonist drugs such as spironolactone and eplerenone reduce the mortality in heart failure patients, there is, thus far, no unambiguous demonstration of a functional role of MR in cardiac cells. The aim of this work was to investigate the activation pathway(s) mediating corticosteroid-induced up-regulation of cardiac calcium current (ICa). In this study, using neonatal cardiomyocytes from MR or glucocorticoid receptor (GR) knockout (KO) mice, we show that MR is essential for corticosteroid-induced up-regulation of ICa. This study provides the first direct and unequivocal evidence for MR function in the heart.

In pancreatic carcinoma, dual EGFR/HER2 targeting with cetuximab/trastuzumab is more effective than treatment with trastuzumab/erlotinib or lapatinib alone: implication of receptors' down-regulation and dimers' disruption.

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2(low) human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab')(2) fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.

Long-term outcome of idiopathic steroid-resistant nephrotic syndrome: a multicenter study.

Long-term outcome of idiopathic steroid-resistant nephrotic syndrome was retrospectively studied in 78 children in eight centers for the past 20 years. Median age at onset was 4.4 years (1.1-15.0 years) and the gender ratio was 1.4. Median follow-up period was 7.7 years (1.0-19.7 years). The disease in 45 patients (58%) was initially not steroid-responsive and in 33 (42%) it was later non-responsive. The main therapeutic strategies included administration of ciclosporine (CsA) alone (n = 29; 37%) and CsA + mycophenolate mofetil (n = 18; 23%). Actuarial patient survival rate after 15 years was 97%. Renal survival rate after 5 years, 10 years and 15 years was 75%, 58% and 53%, respectively. An age at onset of nephrotic syndrome (NS) > 10 years was the only independent predictor of end-stage renal disease (ESRD) in a multivariate analysis using a Cox regression model (P < 0.001). Twenty patients (26%) received transplants; ten showed recurrence of the NS: seven within 2 days, one within 2 weeks, and two within 3-5 months. Seven patients lost their grafts, four from recurrence. Owing to better management, kidney survival in idiopathic steroid-resistant nephrotic syndrome (SRNS) has improved during the past 20 years. Further prospective controlled trials will delineate the potential benefit of new immunosuppressive treatment.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0.

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.

Regulation of the AKAP-LBC signaling complex : molecular characterization and functional implications

RÉSUMÉ Les protéines d'ancrage de la protéine kinase A (AKAPs) constituent une grande famille de protéines qui ciblent la protéine kinase A (PKA) à proximité de ses substrats physiologiques pour assurer leur régulation. Une nouvelle protéine de cette famille, appelée AKAP-Lbc, a été récemment caractérisée et fonctionne comme un facteur d'échange de nucléotides guanine (GEF) pour la petite GTPase Rho. AKAP-Lbc est régulée par différents signaux qui activent et désactivent son activité Rho-GEF. Son activation est assurée par la sous-unité alpha de la protéine G hétérotrimérique G12, tandis que son inhibition dépend de son interaction avec la PKA et 14-3-3. AKAP-Lbc est principalement exprimée dans le coeur et pourrait réguler des processus importants tels que l'hypertrophie et la différenciation des cardiomyocytes. Ainsi, il est crucial d'élucider les mécanismes moléculaires impliqués dans la régulation de son activité Rho-GEF. Le but général de ce travail de thèse est la caractérisation de deux nouveaux mécanismes impliqués dans la régulation de l'activité de AKAP-Lbc. Le premier mécanisme consiste en la régulation de l'activité de AKAP-Lbc par son homo-oligomérisation. Mes travaux montrent que l'homo-oligomérisation maintient AKAP-Lbc inactive, dans une conformation permettant à la PKA ancrée et à 14-3-3 d'exercer leur effet inhibiteur sur l'activité de AKAP-Lbc. Le second mécanisme concerne la régulation de l'activité de AKAP-Lbc via une nouvelle interaction entre AKAP-Lbc et la protéine LC3. LC3 joue un rôle crucial dans l'autophagie, un processus cellulaire qui adresse les protéines cytoplasmiques au lysosome pour leur dégradation. Ce mécanisme est particulièrement important pour le survie des cardiomyocytes durant les périodes d'absence de nutriments. Mes travaux mettent en évidence que LC3 inhibe l'activité Rho-GEF de AKAP-Lbc, ce qui suggère que, au-delà son rôle bien établi dans l'autophagie, LC3 participerait à la régulation de la signalisation de Rho. Prises ensembles, ces études contribuent à comprendre comment le complexe de signalisation formé par AKAP-Lbc régule la signalisation de Rho dans les cellules. Au-delà de leur intérêt au niveau biochimique, ces travaux pourraient aussi contribuer à élucider les réseaux de signalisation qui régulent des phénomènes physiologiques dans le coeur. ABSTRACT A-kinase anchoring proteins (AKAPs) are a group of functionally related proteins, which target the cAMP dependent protein kinase A (PKA) in close proximity to its physiological substrates for ensuring their regulation. A novel PKA anchoring protein, termed AKAP-Lbc, has been recently characterized, which also functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rho. AKAP-Lbc is regulated in a bi-directional manner by signals which activate or deactivate its Rho-GEF activity. Activation is mediated by the alpha subunit of the heterotrimeric G protein G12, whereas inhibition occurs following its interaction with PKA and 14-3-3. AKAP-Lbc is predominantly expressed in the heart and might regulate important processes such as hypertrophy and differentiation of cardiomyocytes. Therefore ít is crucial to elucidate the molecular mechanisms involved in the regulation of the Rho-GEF activity of AKAP-Lbc. The general aim of the present thesis work is the characterization of two novel molecular mechanisms involved in the regulation of the Rho-GEF activity of AKAP-Lbc. The first mechanism consists of the. regulation of AKAP-Lbc activity through its homooligomerization. I report here that homo-oligomerization maintains AKAP-Lbc inactive, under a conformation suitable for ensuring the inhibitory effect of anchored PKA and 14-33 on AKAP-Lbc activity. The second mechanism concerns the regulation of AKAP-Lbc activity through a novel interaction between AKAP-Lbc and ubiquitin-like protein LC3. LC3 is a key mediator of autophagy, which is a cellular process that targets cytosolic proteins to the lysosome for degradation. This process is particularly important for cardiomyocyte survival during conditions of nutrient starvation. Here, I show that LC3 is a negative regulator of the Rho-GEF activity of AKAP-Lbc, which suggests that, beyond its well established role in autophagy, LC3 can participate in the regulation of Rho signaling in cells. Overall, these findings contribute to understand how the AKAP-Lbc signaling complex can regulate the Rho signaling in cells. Beyond its interest at the biochemical level, this work might also contribute to elucidate the signaling network that regulate physiological events in the heart.

Hyperammonemia: regulation of argininosuccinate synthetase and argininosuccinate lyase genes in aggregating cell cultures of fetal rat brain.

Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis. Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). The regulation of AS and AL genes during hyperammonemia is unknown in the brain. We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia. Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl. Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

Neural substrates of social emotion regulation: a FMRI study on imitation and expressive suppression to dynamic facial signals.

Emotion regulation is crucial for successfully engaging in social interactions. Yet, little is known about the neural mechanisms controlling behavioral responses to emotional expressions perceived in the face of other people, which constitute a key element of interpersonal communication. Here, we investigated brain systems involved in social emotion perception and regulation, using functional magnetic resonance imaging (fMRI) in 20 healthy participants. The latter saw dynamic facial expressions of either happiness or sadness, and were asked to either imitate the expression or to suppress any expression on their own face (in addition to a gender judgment control task). fMRI results revealed higher activity in regions associated with emotion (e.g., the insula), motor function (e.g., motor cortex), and theory of mind (e.g., [pre]cuneus) during imitation. Activity in dorsal cingulate cortex was also increased during imitation, possibly reflecting greater action monitoring or conflict with own feeling states. In addition, premotor regions were more strongly activated during both imitation and suppression, suggesting a recruitment of motor control for both the production and inhibition of emotion expressions. Expressive suppression (eSUP) produced increases in dorsolateral and lateral prefrontal cortex typically related to cognitive control. These results suggest that voluntary imitation and eSUP modulate brain responses to emotional signals perceived from faces, by up- and down-regulating activity in distributed subcortical and cortical networks that are particularly involved in emotion, action monitoring, and cognitive control.

Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding.

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPAR beta (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPAR beta mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPAR alpha, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPAR gamma 2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.

IL-10 controls cystatin C synthesis and blood concentration in response to inflammation through regulation of IFN regulatory factor 8 expression.

Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid β associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-β antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.

Molecular and social regulation of worker division of labour in fire ants.

Reproductive and worker division of labour (DOL) is a hallmark of social insect societies. Despite a long-standing interest in worker DOL, the molecular mechanisms regulating this process have only been investigated in detail in honey bees, and little is known about the regulatory mechanisms operating in other social insects. In the fire ant Solenopsis invicta, one of the most studied ant species, workers are permanently sterile and the tasks performed are modulated by the worker's internal state (age and size) and the outside environment (social environment), which potentially includes the effect of the queen presence through chemical communication via pheromones. However, the molecular mechanisms underpinning these processes are unknown. Using a whole-genome microarray platform, we characterized the molecular basis for worker DOL and we explored how a drastic change in the social environment (i.e. the sudden loss of the queen) affects global gene expression patterns of worker ants. We identified numerous genes differentially expressed between foraging and nonforaging workers in queenright colonies. With a few exceptions, these genes appear to be distinct from those involved in DOL in bees and wasps. Interestingly, after the queen was removed, foraging workers were no longer distinct from nonforaging workers at the transcriptomic level. Furthermore, few expression differences were detected between queenright and queenless workers when we did not consider the task performed. Thus, the social condition of the colony (queenless vs. queenright) appears to impact the molecular pathways underlying worker task performance, providing strong evidence for social regulation of DOL in S. invicta.