Tumor-derived fibulin-3 activates pro-invasive NF-κB signaling in glioblastoma cells and their microenvironment.

Molecular profiling of glioblastomas has revealed the presence of key signaling hubs that contribute to tumor progression and acquisition of resistance. One of these main signaling mechanisms is the nuclear factor-kappa B (NF-κB) pathway, which integrates multiple extracellular signals into transcriptional programs for tumor growth, invasion and maintenance of the tumor-initiating population. We show here that an extracellular protein released by glioblastoma cells, fibulin-3, drives oncogenic NF-κB in the tumor and increases NF-κB activation in peritumoral astrocytes. Fibulin-3 expression correlates with a NF-κB-regulated 'invasive signature' linked to poorer survival, being a possible tissue marker for regions of active tumor progression. Accordingly, fibulin-3 promotes glioblastoma invasion in a manner that requires NF-κB activation both in the tumor cells and their microenvironment. Mechanistically, we found that fibulin-3 activates the metalloprotease ADAM17 by competing with its endogenous inhibitor, TIMP3. This results in sustained release of soluble tumor necrosis factor alpha (TNFα) by ADAM17, which in turn activates TNF receptors and canonical NF-κB signaling. Taken together, our results underscore fibulin-3 as a novel extracellular signal with strong activating effect on NF-κB in malignant gliomas. Because fibulin-3 is produced de novo in these tumors and is absent from the normal brain, we propose that targeting the fibulin-3/NF-κB axis may provide a novel avenue to disrupt oncogenic NF-κB signaling in combination therapies for malignant brain tumors.

Prolonged exposure to hypobaric hypoxia transiently reduces GABA(A) receptor number in mice cerebral cortex.

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.

Acute hypoxic hypoxia transiently reduces GABA(A) binding site number in developing chick optic lobe.

The Central Nervous System is known to be critically affected in the prenatal-perinatal period by hypoxic-ischemic insults, which produce several disorders such as loss of neural projections, increased susceptibility to seizures, apoptosis and an imbalance in normal activity of glutamatergic and GABAergic neurones, resulting in acute cell excitotoxicity. The aim of the present work was to establish a chick embryo model of normobaric acute hypoxic hypoxia as well as to evaluate modifications in GABA(A) receptor complex from chick optic lobe, that may result from this injury. Fertile chicken (Gallus gallus domesticus) eggs from White Leghorn were incubated and at embryonic days (ED) 12 to 18, subjected to a stream of 8%O(2)/92%N(2) during1 h, and then were either returned to their shelves in the incubator for recovery, or immediately processed for biochemical studies. Hypoxic treatment produced a significant age dependent reduction in GABA binding sites showing the greatest decrease at the earliest stages studied (ED12-ED16). Saturation curves of GABA binding performed at ED12 showed a decrease in B(max), (control, 5.48+/-0.20, hypoxic, 3.90+/-0.39 pmol/mg protein), but no significant change in K(d). Following 48 h in normoxic atmosphere post-hypoxia reduction in [3H]GABA binding was reversed. Pharmacological properties of GABA(A) receptor at ED12 showed that positive allosteric modulation effects of the steroid 3alpha-hydroxy-5alpha-pregnan-20-one and the barbiturate pentobarbital sodium were enhanced by the treatment. This model of acute prenatal hypoxic hypoxia produced marked alterations in inhibitory CNS neurotransmission that proved reversible and age dependent.

Synaptic membrane freezing affects modulatory sites in avian central nervous system GABA(A) receptor.

Studies were carried out to determine whether barbiturates and neurosteroids share common recognition sites at the GABA(A) receptor complex in avian CNS. To achieve this, differentially prepared fresh and frozen synaptic membranes were used. Both the barbiturate, pentobarbital, and the neurosteroid, 3alpha-hydroxy-5alpha-pregnan-20-one, were able to stimulate GABA binding in both types of membranes. Stimulation differed markedly when both drugs were added jointly to different treated tissue. In frozen membranes drugs acted synergistically and were differentially displaced by picrotoxinin, while in fresh ones, where both compounds were inhibited by the convulsant, this additivity was absent. Post-freezing wash supernatants were collected and used as a source of putative endogenous factors involved in the above mentioned membrane differences. Addition of a high molecular weight fraction from supernatants to frozen synaptic membranes led to an inhibition of barbiturate and neurosteroid potentiation, as well as a loss of their additive effect. Our results indicate that GABA(A) receptor modulation by barbiturates and neurosteroids is affected by synaptic membrane treatment, with a common modulatory site in fresh membranes and separate recognition sites after a freeze-thawing procedure. There may also be endogenous factors involved in overlapping of modulatory sites, which would thus regulate GABA(A) receptor functionality by direct interaction with the complex.

Neurosteroid modulation of GABA binding sites in developing avian central nervous system.

Our aim was to examine the effect of the potent neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) on [3H]-GABA binding to its receptor sites in the chick optic lobe. Binding was performed on synaptic membranes isolated at different stages of development and two different membrane preparation procedures were applied to expose high and low affinity GABA binding sites. The addition of 3 alpha, 5 alpha-P was shown to increase [3H]-GABA binding in an age- and concentration-dependent manner. Maximal stimulation for low affinity GABA binding sites was observed at hatching (130% enhancement), in fresh-washed as well as in frozen membranes. Saturation analysis performed on both membrane types disclosed that 3 alpha, 5 alpha-P increases the affinity of low affinity GABA binding sites without altering their maximal binding capacity. On the other hand, the augmenting effect at high affinity sites, displayed only in frozen membranes, was roughly 50% for all developmental stages. However, their saturation binding parameters remained unaltered in the presence of the steroid, suggesting that stimulation of such sites seems due to interference exerted by the low affinity site population. Findings indicate that 3 alpha, 5 alpha-P acts as an allosteric modulator only for low affinity GABA binding sites, displaying an age-dependent profile probably related to plastic events during visual pathway development.

Comparative modulation by 3 alpha,5 alpha and 3 beta,5 beta neurosteroids of GABA binding sites during avian central nervous system development.

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABAA receptor complex. The presence of a 3 alpha-hydroxyl group and a 5 alpha-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [3H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3 alpha-hydroxy,5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 beta-hydroxy,5 beta-pregnan-20-one (3 beta,5 beta-P). Both steroids were found able to enhance [3H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3 alpha,5 alpha-P had greater potency (EC50 0.22 microM) and enhancing effect (Emax: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABAA receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3 alpha,5 alpha-P by its 3 beta,5 beta isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5 alpha-reduced progesterone metabolites present since early development, while 3 alpha,5 alpha-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5 beta-steroids in early avian development, we propose that 3 beta,5 beta-P, instead of the classical potent 3 alpha,5 alpha-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.

3 beta-OH-5 beta-pregnan-20-one enhances [3H]GABA binding in developing chick optic lobe.

The neurosteroids are synthesized in the CNS and act mainly through allosteric modulation of the GABAA receptor. Structure-activity relationship studies in mammalian CNS have shown that a 3 alpha-hydroxyl group and a 5 alpha-reduced A-ring are striking features for their biological activity, while the 3 beta,5 beta structures as in 3 beta,5 beta-P are completely inactive. In this work we report the enhancing activity of epipregnanolone on [3H]GABA binding to its receptor sites in the chick optic lobe. Concentration-effect curves for this neurosteroid showed a concentration-dependent activity with different potencies at the three developmental stages studied, the hatching stage being the most sensitive to the steroid stimulatory effect. The displacement of a potent 3 alpha,5 alpha steroid by epipregnanolone indicated that this steroid behaved as a partial agonist of the steroid recognition site. Considering the developmental profile for steroidogenesis in avian tissues and the biological relevance of 5 beta-reduced steroids in early development, we propose that 3 or its 3 alpha-epimer, pregnanolone, instead of the potent 3 alpha,5 alpha neurosteroids, modulates GABAA receptors in the chick optic lobe during development.

Pentobarbital modulatory effect on GABA binding sites in developing chick optic lobe.

Barbiturates are allosteric modulators of the CNS GABAA receptor, increasing [3H]-GABA binding to its receptor sites. In the present work we have studied the modulatory effect of the barbiturate pentobarbital on low-affinity GABA binding sites during ontogenetic development of the chick optic lobe. Our results indicate that [3H]-GABA binding enhancement by pentobarbital shows a differential profile during development, following a two-component enhancement model at early stages of development and a single-component enhancement model in the adult stage. Kinetic analysis performed at different stages of development showed that barbiturate enhancement was invariably due to an increase in [3H]-GABA binding affinity, while maximal binding capacity remained unchanged. Using GABA antagonists, picrotoxinin and bicuculline, convulsant sensitivity of high-affinity barbiturate modulatory sites was found at early stages. These data suggest that barbiturate action displays receptor heterogeneity during development, with high- and low-affinity modulatory sites only at early stages, while the high-affinity sites disappear between hatching and adulthood. Kinetic data indicate that both barbiturate modulatory sites are coupled to the GABAA receptor at early stages. The presence of high-affinity modulatory sites at early stages and at hatching suggests a major role during visual pathway maturation.