Peptide-based interference of the transmembrane domain of neuropilin-1 inhibits glioma growth in vivo.

Angiogenesis in glioblastoma is largely dependent on vascular endothelial growth factor (VEGF) signalling. Consistently, the VEGF coreceptor NRP1 promotes angiogenesis and tumour growth in gliomas. Here, we provide data showing that an innovative peptidic tool targeting the transmembrane domain of NRP1 efficiently blocks rat and human glioma growth in vivo. We show both in vivo and in vitro that the antitumour effect results from the anti-proliferative, anti-migratory and anti-angiogenic properties of the compound. The proposed NRP1 antagonizing peptide is therefore a promising novel class of anti-angiogenic drugs that might prolong glioma patient survival. Our results finally show for the first time that the transmembrane domain of important signalling receptors can be antagonized in vivo thereby providing a new avenue towards the development of atypical antagonists with strong therapeutic potential.

p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1.

Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21(WAF1) (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21(WAF1) gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.

The many faces of semaphorins: from development to pathology.

The semaphorin family is a large group of proteins controlling cell migration and axonal growth cone guidance. These proteins are bi-functional signals capable of growth promotion or growth inhibition. Initially described in the nervous system, the majority of studies related to semaphorins and semaphorin signalling are nowadays performed in model systems outside the nervous system. Here, we provide an exhaustive review of the many faces of semaphorins both during developmental, regulatory and pathological processes. Indeed, because of their crucial fundamental roles, the semaphorins and their receptors represent important targets for the development of drugs directed at a variety of diseases.

Implication of neuropilin 2/semaphorin 3F in retinocollicular map formation.

Neural representations of the environment within the brain take the form of topographic maps whose formation relies on graded expression of axon guidance molecules. Retinocollicular map formation, from retinal ganglion cells (RGCs) to the superior colliculus (SC) in the midbrain, is mainly driven by Eph receptors and their ligands ephrins. However, other guidance molecules participate in the formation of this map. Here we demonstrate that the receptor Neuropilin-2 is expressed in an increasing nasal-temporal gradient in RGCs, whereas one of its ligands, Semaphorin3F, but not other Sema3 molecules, presents a graded low-rostral to high-caudal expression in the SC when mapping is underway. Neuropilin-2 and its coreceptor Plexin A1 are present on RGC growth cones. Collapse assays demonstrate that Semaphorin3F induces significant growth cone collapse of temporal, but not nasal, RGCs expressing high levels of Neuropilin-2. Our results suggest that Neuropilin-2/Semaphorin3F are new candidates involved in retinotopy formation within the SC.

Functional interaction between matrix metalloproteinase-3 and semaphorin-3C during cortical axonal growth and guidance.

In the developing cortex, axons and dendrites extend progressively in response to environmental cues attracting or repelling growing processes. Recent evidence suggests the existence of a functional link between guidance molecules and metalloproteinases. Here, we analyzed the putative functional interaction of matrix metalloproteinases (MMPs) with guidance cues of the semaphorin family during growth and guidance of cortical axons. Our results demonstrate that the expression pattern and the proteolytic activity of MMP-3 are consistent with a role of this particular MMP during cortical axon outgrowth. We found that MMP-3 is required for an optimal axon extension and is involved in the Sema3C-dependent chemoattraction of cortical axons by modulating both the growth capacity and the orientation of growth. Interestingly, the inhibitory Sema3A decreased both the expression and activity of MMP-3. Taken together, our results reveal a molecular interaction between MMPs and semaphorins providing new insight into the molecular mechanism allowing axonal growth cone to respond to environmental guidance cues in the context of cortical development.

Inhibition by transmembrane peptides of chimeric insulin receptors.

Receptor tyrosine kinases play essential roles in cell proliferation and differentiation. We have recently shown that peptides corresponding to the transmembrane domains of the epidermal growth factor (EGF) and ErbB2 receptors inhibit their corresponding receptor activation in cancer cell lines. We extend this observation to cells transfected with chimeric insulin receptors where the transmembrane domain has been replaced by that of the EGF receptor or a mutated Erb2 domain. Peptides corresponding to the transmembrane domains of the EGF receptor and ErbB2 are able to inhibit specifically the autophosphorylation of insulin receptors with the corresponding domain. This inhibitory effect is correlated with the propensity of the different transmembrane domains to self-associate in a genetic reporter assay. Thus, our data strengthen the notion that transmembrane domains are involved in erbB receptor activation, and that these receptors can be modulated by inhibiting protein-protein interactions within the membrane.

New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A.

Catestatin (bCGA(344-364)), an endogenous peptide of bovine chromogranin A, was initially characterized for its effect on the inhibition of catecholamine release from chromaffin cells. Catestatin and its active domain (bCGA(344-358)) were identified in chromaffin cells and in secretion medium. The present study identified a potent antimicrobial activity of bCGA(344-358) in the lowmicromolar range against bacteria, fungi and yeasts, without showing any haemolytic activity. Confocal laser microscopy demonstrated penetration of the rhodaminated peptide into the cell membranes of fungi and yeasts and its intracellular accumulation. Time-lapse videomicroscopy showed arrest of fungal growth upon penetration of the labelled peptide into a fungal filament. We identified several catestatin-containing fragments in the stimulated secretion medium of human polymorphonuclear neutrophils, suggesting the N-terminal sequence of catestatin (bCGA(344-358)) (named cateslytin) as a novel component of innate immunity.

Differential MAP kinases activation during semaphorin3A-induced repulsion or apoptosis of neural progenitor cells.

Semaphorins are multifunctional factors implicated in various developmental processes. Little is known about the intracellular pathways ensuring appropriate signal transduction that encode the diverse functions observed. In this study, we investigated whether mitogen-activated protein kinases (MAPK), which are key elements of signal transduction in eukaryotic cells, were activated during semaphorin 3A (Sema3A)-induced repulsion or apoptosis of neural progenitor cells. We found that selective recruitment of the ERK1/2 pathway occurred during Sema3A-induced neural progenitor cell repulsion, whereas p38 MAPK activation was necessary for induction of apoptosis. Moreover, we provide evidence for the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) in the activation of ERK1/2. Additional experiments performed with native cerebellar progenitors confirmed such a selective recruitment of MAPK during Sema3A-dependent migration or apoptosis. Altogether, our results suggest a model to explain how a single factor can exert different functions for a given cell type by the selective recruitment of intracellular pathways.

Innate immunity: involvement of new neuropeptides.

Secretory granules of chromaffin cells from the adrenal medulla store catecholamines and a variety of peptides that are secreted in the extracellular medium during exocytosis. Among these fragments, several natural peptides displaying antimicrobial activities at the micromolar range have been isolated and characterized. We have shown that these peptides, derived from the natural processing of chromogranins (CGs), proenkephalin-A (PEA) and free ubiquitin (Ub), are released into the circulation and display antibacterial and antifungal activities. In this review we focus on three naturally secreted antimicrobial peptides corresponding to CGA1-76 (vasostatin-I), the bisphosphorylated form of PEA209-237 (enkelytin) and Ub. In addition, the antimicrobial properties of the synthetic active domains of vasostatin-I (CGA47-66 or chromofungin) and Ub (Ub65-76 or ubifungin) are reported.

Antimicrobial chromogranins and proenkephalin-A-derived peptides: Antibacterial and antifungal activities of chromogranins and proenkephalin-A-derived peptides.

The secretory granules from adrenal medullary chromaffin cells contain a complex mixture of low-molecular mass constituents such as catecholamines, ascorbate, nucleotides, calcium, peptides, and several high-molecular mass water-soluble proteins including chromogranins and proenkephalin-A. These proteins are sequestered into secretory granules in which processing yields a large variety of peptides. These fragments are released into the extracellular space upon cell stimulation and are recovered in blood, lymph, cerebrospinal fluid, and synovial fluid. Some of them have biological activity on cells in an autocrine, paracrine, or endocrine fashion. In addition, we have shown that peptides with antimicrobial activity are present with the secretory chromaffin granules and demonstrated that they are released from stimulated chromaffin cells. We have shown that posttranslational modifications modulate the antimicrobial activities. For some peptides, using confocal laser microscopy, we have examined the interaction of the rhodaminated peptides with biological membranes. In addition, we have shown that chromofungin, the antifungal peptide corresponding to chromogranin A(47-66), can bind calmodulin in the presence of calcium and induce inhibition of calcineurin, a calmodulin-dependent enzyme. Because these antibacterial peptides are colocalized with catecholamines, they may be activated during stress, playing a role as a first protective barrier against bacterial infection, and thus act as factors of the innate immunity shortly after infection and before the induction and mobilization of an adaptative immune system.

Gamma-hydroxybutyrate receptor function determined by stimulation of rubidium and calcium movements from NCB-20 neurons.

Gamma-Hydroxybutyrate is derived from GABA in brain and plays specific functional roles in the CNS. It is thought to exert a tonic inhibitory control on dopamine and GABA release in certain brain areas, through specific gamma-hydroxybutyrate receptors. Apart from modifying certain calcium currents, the specific transduction mechanism induced by stimulation of gamma-hydroxybutyrate receptors remains largely unknown. We investigated the possible contribution of K(+) channels to the hyperpolarization phenomena generally induced by gamma-hydroxybutyrate in brain, by monitoring (86)Rb(+) movements in a neuronal cell line (NCB-20 cells), which expresses gamma-hydroxybutyrate receptors. Physiological concentrations of gamma-hydroxybutyrate (5-25 microM) induce a slow efflux of (86)Rb(+), which peaks at 5-15 min and returns to baseline levels 20 min later after constant stimulation. This effect can be reproduced by the gamma-hydroxybutyrate receptor agonist NCS-356 and blocked by the gamma-hydroxybutyrate receptor antagonist 6,7,8,9-tetrahydro-5-[H]-benzocycloheptene-5-ol-4-ylidene. The GABA(B) receptor antagonist CGP 55845 has no effect on gamma-hydroxybutyrate-induced (86)Rb(+) efflux. The pharmacology of this gamma-hydroxybutyrate-dependent efflux of (86)Rb(+) is in favor of the involvement of tetraethylammonium and charybdotoxin insensitive, apamin sensitive Ca(2+) activated K(+) channels, identifying them as small conductance calcium activated channels. We demonstrated a gamma-hydroxybutyrate dose-dependent entry of calcium ions into NCB-20 neuroblastoma cells at resting potential. Electrophysiological data showed that this Ca(2+) entry corresponded mainly to a left-hand shift of the current/voltage relation of the T-type calcium channel. This process must at least partially trigger small conductance calcium activated channel activation leading to gamma-hydroxybutyrate-induced hyperpolarization.

Structural and biological characterization of chromofungin, the antifungal chromogranin A (47-66)-derived peptide.

The antifungal peptide named chromofungin is the most active vasostatin-I-derived peptide, corresponding to the sequence 47-66 of chromogranin A. (1)H-NMR analysis revealed that it adopts a helical structure. The mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied by penetration of monolayers and confocal laser microscopy. Chromofungin is able to interact with the cell wall, to cross the plasma membrane, to accumulate in the microorganism, and to inhibit calcineurin activity.

Effects of GABA on circular smooth muscle spontaneous activities of rat distal colon.

GABAergic regulation of intestinal motility through the modulation of non-adrenergic non-cholinergic (NANC) neurons remains poorly understood especially in rat colon where very few studies have been undertaken. Therefore, the effects of GABA on circular preparations of rat distal colon were investigated using classical organ bath chambers to record spontaneous mechanical activities (SMA). SMA was characterized by the occurrence of rhythmic phasic contractions (type-I) or by spontaneously occurring large contractions superimposed on small rhythmic contractions (type-II). In the presence of atropine and guanethidine (NANC conditions), these large contractions were inhibited by bicuculline, a GABA(A)-receptor antagonist as well as by TTX, L-NAME and apamin together, or L 732-138, a NK1-receptor antagonist. In NANC conditions, GABA induced a transient monophasic relaxation or a biphasic effect characterized by a relaxation followed by a tonic contraction in both type-I and -II preparations. Both the inhibitory and excitatory effects of GABA were blocked by TTX and L-NAME + apamin; the GABA-induced contraction was also sensitive to L 732-138. The responses to GABA were mimicked by the GABA(A)-receptor agonist, muscimol, whereas baclofen and CACA, respectively GABA(B) and GABA(C)-receptors agonists showed no effect. These results demonstrated that only GABA(A)-receptors seem to be involved in the regulation of SMA in rat distal colon in NANC conditions. Release of NANC inhibitory transmitter (NO and probably ATP) and NANC excitatory transmitter (maybe substance P) might be involved.

Structural and biological characterization of chromofungin, the antifungal chromogranin A-(47-66)-derived peptide.

Vasostatin-I, the natural fragment of chromogranin A-(1-76), is a neuropeptide able to kill a large variety of fungi and yeast cells in the micromolar range. We have examined the antifungal properties of synthetic vasostatin-I-related peptides. The most active shortest peptide, named chromofungin, corresponds to the sequence Arg(47)-Leu(66). Extensive (1)H NMR analysis revealed that it adopts a helical structure. The biophysical mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied, showing the penetration of this peptide with different lipid monolayers. In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.

Cyclic GMP-dependent protein kinase potentiates serotonin-induced Egr-1 binding activity in PC12 cells.

The NO/cyclic GMP (cGMP) signal transduction pathway, which involves the cGMP-dependent protein kinase (PKG), regulates transcription of several genes, including immediate early genes. Using transfection experiments with the PKG-Ialpha cDNA cloned from human aorta, we show here that addition of membrane-permeable cGMP analogues to PC12 cells slightly upregulated ERK MAP (mitogen-activated protein) kinase. Likewise, PKG-Ialpha was found to activate weakly DNA binding activity of the Egr-1 transcription factor. On the other hand, PKG-Ialpha overexpression was shown to tremendously amplify the Egr-1 binding activity induced by the neurotransmitter serotonin, which activates egr-1 gene expression also via the stimulation of the ERK MAP kinase pathway. Since this potentiation occurred neither at the level of ERK nor at the egr-1 transcriptional level, the mechanism of amplification probably results from the convergence of ERK and PKG pathways at the level of the transcription factor Egr-1.

Adrenal gland SNAP-25 expression is altered in thyroid hormone receptor knock-out mice.

SNAP-25 is a protein in neurons and neuroendocrine cells, which is involved, together with syntaxin and VAMP, in neurotransmitter release and neurite outgrowth. Since the thyroid hormone receptors TR alpha and TR beta are essential for nervous system development, their possible role in regulating the expression of these vesicle trafficking proteins was examined by analysing SNAP-25 levels in TR alpha and TR beta knock-out mice. Immunoblotting and RT-PCR showed that SNAP-25 levels are increased in the adrenal gland, but not in cerebellum, in knock-out mice, while syntaxin-1 and VAMP-2 are unaffected in either tissue. Treatment of the pheochromocytoma-derived cell line PC12 with the thyroid hormone L-3,5,3'-triiodothyronine (T3) decreased SNAP-25 expression. Together, these data suggest that thyroid hormones exert a negative regulatory effect on SNAP-25 in adrenal medullary neuroendocrine cells.

NGF enhances depolarization effects on SNAP-25 expression: induction of SNAP-25b isoform.

The 25 kDa synaptosomal associated protein (SNAP-25), which is implicated in neuronal plasticity and neurosecretion, exists as two isoforms generated by alternative splicing of exons 5a and 5b. The aim of the present study was to characterize factors influencing isoform expression. We report that chronic depolarization of PC12 cells alone or in the presence of NGF induces the expression of isoform-b, in addition to a 1.8- to 3-fold increase in SNAP-25 mRNA and protein as determined by immunoblotting and combined RT-PCR and Southern blot analysis. When cerebellar granule neurons were cultured in elevated K+, the predominant isoform switched from SNAP-25a to SNAP-25b. Taken together these results suggested that chronic depolarization regulates the transcription and processing of SNAP-25 mRNA.

Mechanisms underlying neuronal death induced by chromogranin A-activated microglia.

The neurotoxic effects of activated microglia in neurodegenerative diseases are well established. We recently provided evidence that chromogranin A (CGA), a multifunctional protein localized in dystrophic neurites and in senile plaques, induces an activated phenotype and secretion of neurotoxins by rat microglia in culture. In the present study, we focused on the mechanisms underlying neuronal degeneration triggered by CGA-activated microglia. We found that neuronal death exhibits apoptotic features, characterized by the externalization of phosphatidylserine and the fragmentation of DNA. Microglial neurotoxins markedly stimulate the phosphorylation and activity of neuronal p38 mitogen-activated protein kinase and provoke the release of mitochondrial cytochrome c, which precedes apoptosis. Inhibition of p38 kinase with SB 203580 partially protects neurons from death induced by CGA-activated microglia. Furthermore, neurons are also protected by Fas-Fc, which antagonizes the interactions between the death receptor Fas and its ligand FasL and by cell-permeable peptides that inhibit caspases 8 and 3. Thus, CGA triggers the release of microglial neurotoxins that mobilize several death-signaling pathways in neurons. Our results further support the idea that CGA, which is up-regulated in many neuropathologies, represents a potent endogeneous inflammatory factor possibly responsible for neuronal degeneration.

The hypophysis controls expression of SNAP-25 and other SNAREs in the adrenal gland.

SNAP-25 (Synaptosomal Associated Protein of 25 kDa), in association with two other SNARE (soluble NSF attachment protein receptor) proteins, syntaxin and Vesicle Associated Membrane Protein, VAMP, is implicated in regulated and constitutive exocytosis in neurones and neuroendocrine cells. Our previous studies have shown that it is expressed more by noradrenergic than adrenergic chromaffin cells in the rat adrenal gland. Since certain hormones under hypophyseal control play an essential role in determining chromaffin cell phenotype, the present study examined the effect of hypophysectomy on SNAP-25 expression. Hypophysectomy was found by immunoblotting and RT-PCR analysis to increase adrenal gland SNAP-25, syntaxin-1 and VAMP-2 levels, without modifying the relative expression of SNAP-25 isoforms: immunocytochemistry showed a dramatic increase in SNAP-25 expression in former adrenergic chromaffin cells. Since adrenal glucocorticoids are considerably reduced by hypophysectomy, the effect of corticosterone replacement therapy was investigated. This did not change levels of SNAP-25, syntaxin-1 or VAMP-2. SNARE expression was also unmodified in pheochromocytoma cells treated with a synthetic glucocorticoid. In contrast, subcutaneous injection of hypophysectomized rats with thyroid hormone decreased adrenal SNAP-25, demonstrating the potential importance of the pituitary-thyroid axis. The current data thus demonstrate that the hypophysis exerts an inhibitory control on adrenal gland SNARE proteins. They suggest that glucocorticoids are unlikely to be directly responsible for this but provide evidence that thyroid hormones are implicated in this phenomenon. The putative role of hormonal regulation on SNARE function is discussed.