Growth promoting signaling by tenascin-C [corrected].

Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of beta-catenin and stimulated the expression of the beta-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Limited role of the c-Jun N-terminal kinase pathway in a neonatal rat model of cerebral hypoxia-ischemia

D-JNKI1, a cell-permeable peptide inhibitor of the c-Jun N-terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D-JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia-ischemia. Seven-day-old rats underwent a permanent ligation of the right common carotid artery followed by 2h of hypoxia (8% oxygen). Treatment with D-JNKI1 (0.3mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase-3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic and autophagic. However the size of the lesion was unchanged. Further analysis showed that neonatal hypoxia-ischemia induced an immediate decrease in JNK phosphorylation (reflecting mainly P-JNK1) followed by a slow progressive increase (including P-JNK3 54kDa), whereas c-jun and c-fos expression were both strongly activated immediately after hypoxia-ischemia. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral hypoxia-ischemia in neonatal rats and the observed positive effects of D-JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D-JNKI1 can only be considered in association with other therapies.

Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Expressão de fos após pulso de escuro no núcleo pré- geniculado do tálamo do sagui (Callithrix jacchus)

The pregeniculate nucleus (PGN) of the primate s thalamus is an agglomerate neuronal having a cap shaped located dorsomedially to the main relay visual information to the cerebral cortex, the dorsal lateral geniculate nucleus (GLD). Several cytoarchitectonic, neurochemical and retinal projections studies have pointed PGN as a structure homologous to intergeniculate leaflet (IGL) of rodents. The IGL receives retinal terminals and appears to be involved in the integration of photic and non-photic information relaying them, through geniculo-hypothalamic tract (TGH), to the main circadian oscillator in mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus. Thus, the IGL participates in the control of the biological rhythm by modulating the activity of the SCN. Pharmacological and IGL injury studies conclude that it is critical in the processing of non-photic information which is transmitted to the SCN. Other studies have found that especially neurons immunoreactive to neuropeptide Y (NPY) respond to this type of stimulation, determined by its colocation with the FOS protein. Has not been determined if the PGN responds, expressing the FOS protein, to the non-photic stimulus nor the neurochemical nature of these cells. Thus, we apply a dark pulse in the specifics circadian phases and analyze the pattern of expression of FOS protein in PGN of the marmoset (Callithrix jacchus). We found that in all animals analyzed the FOS expression was higher in the experimental than in the control group. There was a higher expression of FOS when the dark pulse was applied during the subjective day between the groups. Still, a subregion of the PGN, known by immunoreactive to NPY, had a greater number of FOS-positive cells in relation to his other just close dorsal region. Our data corroborate the theory that the PGN and IGL are homologous structures that were anatomically modified during the evolutionary process, but kept its main neurochemical and functional characteristics. However, injury and hodological studies are still needed for a more accurate conclusion

Neuronal NOS Inhibitor and Conventional Antidepressant Drugs Attenuate Stress-induced Fos Expression in Overlapping Brain Regions

Recent evidence indicates that the administration of inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant-like effects in animal models such as the forced swimming test (FST). However, the neural circuits involved in these effects are not yet known. Therefore, this study investigated the expression of Fos protein, a marker of neuronal activity, in the brain of rats submitted to FST and treated with the preferential nNOS inhibitor, 7-nitroindazole (7-NI), or with classical antidepressant drugs (Venlafaxine and Fluoxetine). Male Wistar rats were submitted to a forced swimming pretest (PT) and, immediately after, started receiving a sequence of three ip injections (0, 5, and 23 h after PT) of Fluoxetine (10 mg/kg), Venlafaxine (10 mg/kg), 7-NI (30 mg/kg) or respective vehicles. One hour after the last drug injection the animals were submitted to the test session, when immobility time was recorded. After the FST they were sacrificed and had their brains removed and processed for Fos immunohistochemistry. Independent group of non-stressed animals received the same drug treatments, or no treatment (naive). 7-NI, Venlafaxine or Fluoxetine reduced immobility time in the FST, an antidepressant-like effect. None of the treatments induce significant changes in Fos expression per se. However, swimming stress induced significant increases in Fos expression in the following brain regions: medial prefrontal cortex, nucleus accumbens, locus coeruleus, raphe nuclei, striatum, hypothalamic nucleus, periaqueductal grey, amygdala, habenula, paraventricular nucleus of hypothalamus, and bed nucleus of stria terminalis. This effect was attenuated by 7-NI, Venlafaxine or Fluoxetine. These results show that 7-NI produces similar behavioral and neuronal activation effects to those of typical antidepressants, suggesting that these drugs share common neurobiological substrates.

Chronic unpredictable mild stress alters an anxiety-related defensive response, Fos immunoreactivity and hippocampal adult neurogenesis

Previous results show that elevated T-maze (ETM) avoidance responses are facilitated by acute restraint. Escape, on the other hand, was unaltered. To examine if the magnitude of the stressor is an important factor influencing these results, we investigated the effects of unpredictable chronic mild stress (UCMS) on ETM avoidance and escape measurements. Analysis of Fos protein immunoreactivity (Fos-ir) was used to map areas activated by stress exposure in response to ETM avoidance and escape performance. Additionally, the effects of the UCMS protocol on the number of cells expressing the marker of migrating neuroblasts doublecortin (DCX) in the hippocampus were investigated. Corticosterone serum levels were also measured. Results showed that UCMS facilitates ETM avoidance, not altering escape. In unstressed animals, avoidance performance increases Fos-ir in the cingulate cortex, hippocampus (dentate gyrus) and basomedial amygdala, and escape increases Fos-ir in the dorsolateral periaqueductal gray and locus ceruleus. In stressed animals submitted to ETM avoidance, increases in Fos-ir were observed in the cingulate cortex, ventrolateral septum, hippocampus, hypothalamus, amygdala, dorsal and median raphe nuclei. In stressed animals submitted to ETM escape, increases in Fos-ir were observed in the cingulate cortex, periaqueductal gray and locus ceruleus. Also, UCMS exposure decreased the number of DCX-positive cells in the dorsal and ventral hippocampus and increased corticosterone serum levels. These data suggest that the anxiogenic effects of UCMS are related to the activation of specific neurobiological circuits that modulate anxiety and confirm that this stress protocol activates the hypothalamus-pituitary-adrenal axis and decreases hippocampal adult neurogenesis.

Molekulare Mechanismen Proteinkinase C-induzierter Nox4-Hochregulation in humanen Endothelzellen

Eine verstärkte Transkription von NADPH-Oxidasen (Nox) wird mit der Entstehung von atherosklerotischer Veränderungen in Verbindung gebracht. Die Arbeit unserer Gruppe zeigte, dass die Aktivität der Proteinkinase C (PKC) zu einer Nox4-Hochregulation führt, der dominanten NOX Isoform in endothelialen Zellen. Die vorliegende Arbeit zielte auf die Aufdeckung der dowm-stream gelegenen Mechanismen. Die Behandlung von humanen EA.hy 926-Zellen mit dem PKC Aktivator Phorbol-12-Myristat-13-Acetat (PMA) für 48 h führte in eine signifikante Nox4-mRNA-Hochregulation, welche mittels PKC-Inhibitoren oder PKC alpha siRNA abgewendet werden konnte. PMA führte zu einer andauernden Aktivierung der MAP-Kinase Erk1/2. Die PMA vermittelte Nox4-Expression konnte durch Erk1/2-Inhibitoren oder durch Erk1/2-Knock-down geblockt werden. Down-stream konnte die Involvierung der Erk1/2-Substarte Elk-1 und c-Fos mittels siRNA-Experimente gezeigt werden. Darüber hinaus blockte die Inhibierung der Histondeacetylasen (HDACs) mit Scriptaid oder durch HDAC3-Knock-down mittels siRNA die PMA-induzierte Nox4-Expression in EA.hy 926-Zellen, weswegen eine Rolle für HADC3 in der Regulation der Nox4-Expression angezeigt wurde. Abschließend reduzierte ein Knock-down von p53 (siRNA) deutlich die basale Expression von Nox4, hatte aber nur einen kleinen Effekt auf die PMA-induzierte Nox4-Expression. Zusammenfassend zeigen die Daten der vorliegenden Arbeit, dass in einer PKC alpha induzierten Nox4-mRNA-Hochregulation Erk1/2, Elk-1, cFos und HDAC3 involviert sind.

The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

Alpha C protein of group B Streptococcus as a potential vaccine candidate

Group B Streptococcus (GBS) is a leading cause of life-threatening infection in neonates and young infants, pregnant women, and non-pregnant adults with underlying medical conditions. Immunization has theoretical potential to prevent significant morbidity and mortality from GBS disease. Alpha C protein (α C), found in 70% of non-type III capsule polysaccharide group B Streptococcus, elicits antibodies protective against α C-expressing strains in experimental animals and is an appealing carrier for a GBS conjugate vaccine. We determined whether natural exposure to α C elicits antibodies in women and if high maternal α C-specific serum antibody at delivery is associated with protection against neonatal disease. An ELISA was designed to measure α C-specific IgM and IgG in human sera. A case-control design (1:3 ratio) was used to match α C-expressing GBS colonized and non-colonized women by age and compare quantified serum α C-specific IgM and IgG. Sera also were analyzed from bacteremic neonates and their mothers and from women with invasive GBS disease. Antibody concentrations were compared using t-tests on log-transformed data. Geometric mean concentrations of α C-specific IgM and IgG were similar in sera from 58 α C strain colonized and 174 age-matched non-colonized women (IgG 245 and 313 ng/ml; IgM 257 and 229 ng/ml, respectively). Delivery sera from mothers of 42 neonates with GBS α C sepsis had similar concentrations of α C-specific IgM (245 ng/ml) and IgG (371 ng/ml), but acute sera from 13 women with invasive α C-expressing GBS infection had significantly higher concentrations (IgM 383 and IgG 476 ng/ml [p=0.036 and 0.038, respectively]). Convalescent sera from 5 of these women 16-49 days later had high α C-specific IgM and IgG concentrations (1355 and 4173 ng/ml, respectively). In vitro killing of α C-expressing GBS correlated with total α C-specific antibody concentration. Invasive disease but not colonization elicits α C-specific IgM and IgG in adults. Whether α C-specific IgG induced by vaccine would protect against disease in neonates merits further investigation. ^

Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^

Estrogen induced desensitization of c -{\it fos\/} messenger RNA expression {\it in vivo\/}

In this thesis, we investigated the regulation of the nuclear proto-oncogene, c-fos by estrogen in vivo. In the uterus, estrogen causes a rapid, dramatic and transient induction of c-fos mRNA and this occurs by transcriptional activation. We have discovered a previously unrecognized regulatory mechanism by which fos becomes desensitized to estrogen following the transient induction. We investigated three aspects of this desensitization: (1) the kinetics and general characteristics of the phenomenon; (2) the molecular mechanism of the desensitization; and (3) the relationship of desensitization to estrogen stimulated DNA synthesis. The desensitization occurs between 3-24 hours after initial hormonal stimulation and is reversible within 72 hours. The desensitization is not species specific, in that it occurs in both the rat and mouse. The desensitization also occurs in at least two estrogen responsive tissues, the uterus and vagina. The desensitization is not unique to c-fos, since both c-myc and c-jun show similar patterns of desensitization. However, the desensitization is not observed with creatine kinase B (CKB), indicating that not all estrogen inducible genes become desensitized. In the second general area, we determined the desensitization is at the transcriptional level. The desensitization is homologous, but not heterologous, since estrogen induction does not desensitize c-fos to other agents. Other studies show that the desensitization is not due to the lack of functional estrogen receptors. Taken together, these findings suggest that the desensitization occurs at the level of the estrogen responsive element. In the third major area, we demonstrated that the desensitization appears to be related to estrogen induced DNA synthesis. Support for this suggestion comes from the observation that short acting estrogens which induce fos, but not DNA synthesis, do not produce desensitization. ^

GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for Gαi subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC–a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.

The small GTP-binding protein Rho links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gαs and Gαi2 families were found in human tumors; and members of the Gαq and Gα12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα12 family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ1γ2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Loss of haloperidol induced gene expression and catalepsy in protein kinase A-deficient mice

The antipsychotic drug, haloperidol, elicits the expression of neurotensin and c-fos mRNA in the dorsal lateral region of the striatum and produces an acute cataleptic response in rodents that correlates with the motor side effects of haloperidol in humans. Mice harboring a targeted disruption of the RIIβ subunit of protein kinase A have a profound deficit in cAMP-stimulated kinase activity in the striatum. When treated with haloperidol, RIIβ mutant mice fail to induce either c-fos or neurotensin mRNA and the acute cataleptic response is blocked. However, both wild-type and mutant mice become cataleptic when neurotensin peptide is directly injected into the lateral ventricle, demonstrating that the kinase deficiency does not interfere with the action of neurotensin but rather its synthesis and release. These results establish a direct role for protein kinase A as a mediator of haloperidol induced gene induction and cataleptic behavior.