Modulation of breast cancer progression and differentiation by the gp30/neregulin

In the last decade we have come to understand that the growth of cancer cells in general and of breast cancer in particular depends, in many cases, upon growth factors that will bind to and activate their receptors. One of these growth factor receptors is the erbB-2 protein which plays an important role in the prognosis of breast cancer and is overexpressed in nearly 30% of human breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. Our recent discovery of the gp30 has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. We have shown that gp30 induces a biphasic growth effect on cells with erbB-2 over-expression. We have recently determined the protein sequence of gp30 and cloned its full length cDNA sequence. We have also cloned two additional forms to the ligand, that are believed to be different isoforms. We are currently expressing the different forms in order to determine their biological effects. To elucidate the cellular mechanisms underlying cell growth inhibition by gp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells which overexpress erbB-2 and cells which express low levels of this protooncogene. High concentrations of ligand induced differentiation of cells overexpressing erbB-2, as measured by inhibition of cell growth, and increased synthesis of milk components, and modulation of E-cadherin and up- regulation of c-jun and c-fos. These findings indicate that ligand-induced growth inhibition in cells overexpressing erbB-2 is associated with an apparent induction of differentiation. The availability of gp30 derived synthetic peptides and its full cDNAs provides tools necessary to acquire a better understanding of the mechanism of action of the this ligands and the erbB-2 receptor in breast cancer.

Death pathways activated in the neurotrophic factor-deprived neurons

Programed cell death (PCD) is a fundamental biological process that is as essential for the development and tissue homeostasis as cell proliferation, differentiation and adaptation. The main mode of PCD - apoptosis - occurs via specifi c pathways, such as mitochondrial or death receptor pathway. In the developing nervous system, programed death broadly occurs, mainly triggered by the defi ciency of different survival-promoting neurotrophic factors, but the respective death pathways are poorly studied. In one of the best-characterized models, sympathetic neurons deprived of nerve growth factor (NGF) die via the classical mitochondrial apoptotic pathway. The main aim of this study was to describe the death programs activated in these and other neuronal populations by using neuronal cultures deprived of other neurotrophic factors. First, this study showed that the cultured sympathetic neurons deprived of glial cell line-derived neurotrophic factor (GDNF) die via a novel non-classical death pathway, in which mitochondria and death receptors are not involved. Indeed, cytochrome c was not released into the cytosol, Bax, caspase-9, and caspase-3 were not involved, and Bcl-xL overexpression did not prevent the death. This pathway involved activation of mixed lineage kinases and c-jun, and crucially requires caspase-2 and -7. Second, it was shown that deprivation of neurotrophin-3 (NT-3) from cultured sensory neurons of the dorsal root ganglia kills them via a dependence receptor pathway, including cleavage of the NT- 3 receptor TrkC and liberation of a pro-apoptotic dependence domain. Indeed, death of NT-3-deprived neurons was blocked by a dominant-negative construct interfering with TrkC cleavage. Also, the uncleavable mutant of TrkC, replacing the siRNA-silenced endogeneous TrkC, was not able to trigger death upon NT-3 removal. Such a pathway was not activated in another subpopulation of sensory neurons deprived of NGF. Third, it was shown that cultured midbrain dopaminergic neurons deprived of GDNF or brainderived neurotrophic factor (BDNF) kills them by still a different pathway, in which death receptors and caspases, but not mitochondria, are activated. Indeed, cytochrome c was not released into the cytosol, Bax was not activated, and Bcl-xL did not block the death, but caspases were necessary for the death of these neurons. Blocking the components of the death receptor pathway - caspase-8, FADD, or Fas - blocked the death, whereas activation of Fas accelerated it. The activity of Fas in the dopaminergic neurons could be controlled by the apoptosis inhibitory molecule FAIML. For these studies we developed a novel assay to study apoptosis in the transfected dopaminergic neurons. Thus, a novel death pathway, characteristic for the dopaminergic neurons was described. The study suggests death receptors as possible targets for the treatment of Parkinson s disease, which is caused by the degeneration of dopaminergic neurons.

Growth factor induction in intervertebral disc tissue: Observations in basic mechanisms of disc degeneration and rearrangement

The intervertebral disc is composed of concentrically arranged components: annulus fibrosus, the transition zone, and central nucleus pulposus. The major disc cell type differs in various parts of the intervertebral disc. In annulus fibrosus a spindle shaped fibroblast-like cell mainly dominates, whereas in central nucleus pulposus the more rounded chondrocyte-like disc cell is the major cell type. At birth the intervertebral disc is well vascularized, but during childhood and adolescence blood vessels become smaller and less numerous. The adult intervertebral disc is avascular and is nourished via the cartilage endplates. On the other hand, degenerated and prolapsed intervertebral discs are again vascularized, and show many changes compared to normal discs, including: nerve ingrowth, change in collagen turnover, and change in water content. Furthermore, the prolapsed intervertebral disc tissue has a tendency to decrease in size over time. Growth factors are polypeptides which regulate cell growth, extracellular matrix protease activity, and vascularization. Oncoproteins c-Fos and c-Jun heterodimerize, forming the AP-1 transcription factor which is expressed in activated cells. In this thesis the differences of growth factor expression in normal intervertebral disc, the degenerated intervertebral disc and herniated intervertebral disc were analyzed. Growth factors of particular interest were basic fibroblast growth factor (bFGF or FGF-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGFβ). Cell activation was visualized by the expression of the AP-1 transcription promoters c-Fos and c-Jun. The expression was shown with either mono- or polyclonal antibodies by indirect avidin-biotin-peroxidase immunohistochemical staining method. The normal control material was collected from a tissue bank of five organ donors. The degenerated disc material was from twelve patients operated on for painful degenerative disc disease, and herniated disc tissue material was obtained from 115 patients operated on for sciatica. Normal control discs showed only TGFβ immunopositivity. All other factors studied were immunonegative in the control material. Prolapsed disc material was immunopositive for all factors studied, and this positivity was located either in the disc cells or in blood vessels. Furthermore, neovascularization was noted. Disc cell immunoreaction was shown in chondrocyte-like disc cells or in fibroblast-like disc cells, the former being expressed especially in conglomerates (clusters of disc cells). TGFβ receptor induction was prominent in prolapsed intervertebral disc tissue. In degenerated disc material, the expression of growth factors was analyzed in greater detail in various parts of the disc: nucleus pulposus, anterior annulus fibrosus and posterior annulus fibrosus. PDGF did not show any immunoreactivity, whereas all other studied growth factors were localized either in chondrocyte-like disc cells, often forming clusters, in fibroblast-like disc cells, or in small capillaries. Many of the studied degenerated discs showed tears in the posterior region of annulus fibrosus, but expression of immunopositive growth factors was detected throughout the entire disc. Furthermore, there was a difference in immunopositive cell types for different growth factors. The main conclusion of the thesis, supported by all substudies, is the occurrence of growth factors in disc cells. They may be actively participating in a network regulating disc cell growth, proliferation, extracellular matrix turnover, and neovascularization. Chondrocyte-like disc cells, in particular, expressed growth factors and oncoproteins, highlighting the importance of this cell type in the basic pathophysiologic events involved in disc degeneration and disc rearrangement. The thesis proposes a hypothesis for cellular remodelling in intervertebral disc tissue. In summary, the model presents an activation pattern of different growth factors at different intervertebral disc stages, mechanisms leading to neovascularization of the intervertebral disc in pathological conditions, and alteration of disc cell shape, especially in annulus fibrosus. Chondrocyte-like disc cells become more numerous, and these cells are capable of forming clusters, which appear to be regionally active within the disc. The alteration of the phenotype of disc cells expressing growth factors from fibroblast-like disc cells to chondrocyte-like cells in annulus fibrosus, and the numerous expression of growth factor expressing disc cells in nucleus pulposus, may be a key element both during pathological degeneration of the intervertebral disc, and during the healing process after trauma.

Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPAR gamma)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within similar to 80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPAR gamma) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPAR gamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPAR gamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPAR gamma interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPAR gamma, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistingene transcription.Conclusion: Our findings suggest a complex interplay of Sp1 and PPAR gamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

Background: Bryophyllum pinnata (B. pinnata) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of B. pinnata we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle. Methods: A chloroform extract derived from a bulk of botanically well-characterized pulverized B. pinnata leaves was separated using column chromatography with step-gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells. Results: Results showed presence of growth inhibitory activity in the crude leaf extracts with IC50 at 552 mu g/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50: 50) and showed IC50 at 91 mu g/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A. Conclusions: Our study therefore demonstrates presence of anticancer and anti-HPV an activity in B. pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1

The molecular mechanism of antimony-resistant Leishmania donovani ((SbLD)-L-R)-driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (M phi s) has been investigated. This study showed that both promastigote and amastigote forms of (SbLD)-L-R, but not the antimony-sensitive form of LD, express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in (SbLD)-L-R (KD (SbLD)-L-R), compromises the ability to induce the above effects. Infection of M phi s with KD (SbLD)-L-R enhanced the sensitivity toward antimonials compared with infection with (SbLD)-L-R, and infection of BALB/c mice with KD (SbLD)-L-R caused significantly less organ parasite burden compared with infection induced by (SbLD)-L-R. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by (SbLD)-L-R to activate ERK and nuclear translocation of NF-kappa B involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway. Interestingly both recombinant IL-10 and (SbLD)-L-R up-regulate MDR1 in M. with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but M phi s derived from IL-10(-/-) mice are unable to show MDR1 up-regulation on infection with (SbLD)-L-R. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10-driven MDR1 up-regulation is c-Fos/c-Jun and not NF-kappa B, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs.

Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiency in Vitro and in Vivo

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T -> alanine A] or 9 K -> arginine R] single mutant or 2 double K -> R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T -> A and 7 K -> R vectors were significantly higher (similar to 63-90%) than the AAV2-WT vectors (similar to 30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated similar to 8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

Structural and functional studies of bacterial protein tyrosine kinases

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases.

Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system. Oncogene (2010) 29, 752-762; doi: 10.1038/onc.2009.379; published online 9 November 2009

Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling

Mammary epithelial cells cultured on a concentrated laminin-rich extracellular matrix formed 3D acinar structures that matured to polarized monolayers surrounding a lumen. In the absence of glucocorticoids mature acinus formation failed and the expression of an acinus-associated, activator protein 1 (AP1) and nuclear factor kappaB transcription factor DNA-binding profile was lost. Treatment with the JNK inhibitor, SP600125, caused similar effects, whereas normal organization of the mammary epithelial cells as acini caused JNK activation in a glucocorticoid-dependent manner. The forming acini expressed BRCA1, GADD45beta, MEKK4, and the JNK activating complex GADD 45beta-MEKK4 in a glucocorticoid-dependent fashion. JNK catalyzed phosphorylation of c-Jun was also detected in the acini. In addition, expression of beta4 integrin and in situ occupation of its promoter by AP1 components, c-Jun and Fos, was glucocorticoid dependent. These results suggest that glucocortocoid signaling regulates acinar integrity through a pathway involving JNK regulation of AP1 transcription factors and beta4 integrin expression.

Lacking cytokine production in ES cells and ES-cell-derived vascular cells stimulated by TNF-alpha is rescued by HDAC inhibitor trichostatin A

Inflammation and TNF-alpha signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-alpha receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-alpha stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c-fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c-jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c-fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-alpha stimulation due to low levels of c-fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Importance de la phosphorylation de la ligase Itch dans la reconnaissance et l'ubiquitylation des protéines à domaine SH3

Itch est une ligase de l’ubiquitine impliquée dans la reconnaissance et la dégradation des protéines par le protéasome. Itch contient trois sites phosphorylés par JNK et il a été démontré que la phosphorylation de ces résidus est nécessaire pour que Itch puisse reconnaître et ubiquityler les protéines c-Jun et JunB. Ces sites de phosphorylation se retrouvent dans le domaine PRD responsable des interactions de Itch avec les protéines à domaine SH3. Si la phosphorylation de Itch par JNK est importante pour réguler son activité avec c-Jun et JunB, on connaît peu de choses sur les interactions de Itch avec les protéines à domaine SH3 ainsi que l’implication de la phosphorylation dans leur régulation. Nous avons donc créé des mutants de Itch par mutagenèse dirigée où les sites de phosphorylation étaient remplacés par des alanines (mutant non phosphorylable) et où l’un des trois sites était remplacé par un acide aspartique (mutant constitutivement phosphorylé). Ces mutants sont utilisés dans des tests d’interaction et d’ubiquitylation, dans le but de déterminer l’impact de la phosphorylation de Itch dans la reconnaissance et l’ubiquitylation des protéines SH3. Nos résultats montrent que, contrairement au modèle proposé, la phosphorylation de Itch n’est pas essentielle à l’interaction de Itch avec l’endophiline, mais la phosphorylation de Itch module l’ubiquitylation ainsi que la dégradation de l’endophiline. La régulation de l’interaction de Itch avec ses substrats est donc différente selon le substrat.