FAK competes for Src to promote migration against invasion in melanoma cells.

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK(Y397F) reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.

Src activation and translocation from focal adhesions to membrane ruffles contribute to formation of new adhesion sites.

Cell migration requires the coordinated turnover of focal adhesions, a process that involves FAK phosphorylation. Since Src is the major kinase implicated in FAK phosphorylation, we focus here on the role of Src activation on adhesion remodelling. In astrocytoma cells, constitutively activated Src induces both FAK phosphorylation and adhesion rearrangement. To evaluate how Src controls these processes, we used a recently described Src reporter to monitor the dynamics of Src phosphorylation. Upon Src activation, focal adhesions started to disassemble while Src appeared highly expressed at newly formed membrane ruffles. Kinetic analysis of time-lapse movies showed that loss of phospho-Src at focal adhesions was time-correlated with the appearance of membrane ruffles containing phospho-Src. Moreover, FLIP analysis revealed a dynamic equilibrium of Src between focal adhesions and membrane ruffles. We conclude that upon phosphorylation, Src is directly translocated from focal adhesions to membrane ruffles, thereby promoting formation of new adhesion complexes.

The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression.

Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.

Postsynaptic target regulates functional responses induced by 5-HT3 serotonin receptors on axonal varicosities of NG108-15 hybrid neuroblastoma cells.

Rat brain presynaptic 5-HT3 serotonin receptors, members of the ligand-gated ion channel superfamily, induce changes in nerve terminal [Ca2+]i in a manner distinct from that found for somatic 5-HT3 receptors. Here, we assessed the role of postsynaptic target in regulating the nature of presynaptic receptor-induced responses, using the hybrid neuroblastoma cell line NG108-15 as a model neuronal system that expresses 5-HT3 receptors. Using immunocytochemistry, 5-HT3 receptors were found to be present on the presynaptic-like varicosities of differentiated NG108-15 cells, indicating that these receptors possess an inherent ability to localize to potential presynaptic sites. In the absence of postsynaptic target, 5-HT3 receptors localized to the varicosities induce rapid but transient changes in [Ca2+]i that were initiated by voltage-gated Ca2+ channels, as assessed using Ca2+ channel blockers, these properties being typical of those found for somatic 5-HT3 receptors. In co-cultures containing rat myotubes, with which NG108-15 cells form functional cholinergic synapses, the 5-HT3 receptor-induced changes in [Ca2+]i in the axonal varicosities shifted over time (three to 10 days) to that found for brain nerve endings: sustained responses that were insensitive to blockade by antagonists of voltage-gated Ca2+ channels. The effect of co-culturing myotubes with the NG108-15 cells was mimicked by conditioned media from myotube cultures. These results indicate that regulatory molecules from the target postsynaptic cell dictate the functional responses elicited by presynaptic 5-HT3 receptors. Because the target-induced changes required several days before they were evident, we hypothesize that changes in protein expression, perhaps the consequence of altered gene regulation, underlie the changes in the responses to 5-HT3 receptor activation in the axonal varicosities of this neuronal cell line.

Functional IP3- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108-15 (rodent neuroblastoma x glioma hybrid) cells.

Presynaptic varicosities of the model neuronal cell line NG108-15, a cholinergic neuroblastoma cell x glioma cell hybrid capable of innervating striated myotubes, were examined for the presence of inositol 1,4,5-trisphosphate (IP3)-sensitive and Ca2+-activated (ryanodine-sensitive) Ca2+ stores using confocal microscopic imaging of Ca2+-sensitive fluorescent dye loaded into the cells. Initial demonstration of the presence of IP3 receptors and ryanodine receptors in the NG108-15 varicosities was obtained using immunocytochemistry. Treatment of NG108-15 cells with bradykinin (0.1 microM), whose receptor is linked to IP3 generation, and separately, caffeine (10 mM), an activator of endoplasmic reticulum ryanodine receptors, resulted in substantial increases in [Ca2+]i in the varicosities. K+-evoked changes in [Ca2+]i in the varicosities were reduced (52 %) after emptying the ryanodine-sensitive Ca2+ store using caffeine (10 mM), but were not affected by prior depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 microM). Bradykinin-induced changes in [Ca2+]i were abolished following depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 microM) and were reduced (72 %) by prior emptying of the ryanodine-sensitive Ca2+ store with caffeine (10 mM). The same results were obtained when the varicosities of the NG108-15 cells had formed synaptic junctions with co-cultured rat hindlimb myotubes. Taken together, the results suggest that, in the varicosities, activation of the IP3 pathway evoked the release of Ca2+ from the IP3-sensitive store, which, in turn, secondarily induced the release of Ca2+ from the ryanodine-sensitive store via Ca2+-induced Ca2+ release, and that depolarization-induced Ca2+ entry evoked Ca2+-induced Ca2+ release only from the ryanodine-sensitive store. Thus, functional internal Ca2+ stores are inherent components of presynaptic varicosities in this neural cell line.

Mechanism of calcium oscillations in migrating human astrocytoma cells.

Numerous studies show that intracellular calcium controls the migration rate of different mobile cell types. We studied migrating astrocytoma cells from two human cell lines, U-87MG and A172, in order to clarify the mechanisms by which calcium potentially influences cell migration. Using the wound-healing model to assay migration, we showed that four distinct components of migration could be distinguished: (i) a Ca(2+)/serum-dependent process; (ii) a Ca(2+)-dependent/serum-independent process; (iii) a Ca(2+)/serum-independent process; (iv) a Ca(2+)-independent/serum-dependent process. In U-87MG cells which lack a Ca(2+)-dependent/serum-independent component, we found that intracellular Ca(2+) oscillations are involved in Ca(2+)-dependent migration. Removing extracellular Ca(2+) greatly decreased the frequency of migration-associated Ca(2+) oscillations. Furthermore, non-selective inhibition of Ca(2+) channels by heavy metals such as Cd(2+) or La(3+) almost completely abolished changes in intracellular Ca(2+) observed during migration, indicating an essential role for Ca(2+) channels in the generation of these Ca(2+) oscillations. However, specific blockers of voltage-gated Ca(2+) channels, including nitrendipine, omega-conotoxin GVIA, omega-conotoxin MVIIC or low concentrations of Ni(2+) were without effect on Ca(2+) oscillations. We examined the role of internal Ca(2+) stores, showing that thapsigargin-sensitive Ca(2+) stores and InsP(3) receptors are involved in Ca(2+) oscillations, unlike ryanodine-sensitive Ca(2+) stores. Detailed analysis of the spatio-temporal aspect of the Ca(2+) oscillations revealed the existence of Ca(2+) waves initiated at the leading cell edge which propagate throughout the cell. Previously, we have shown that the frequency of Ca(2+) oscillations was reduced in the presence of inhibitory antibodies directed against beta3 integrin subunits. A simple model of a Ca(2+) oscillator is proposed, which may explain how the generation of Ca(2+) oscillations is linked to cell migration.

Nicotinic receptors co-localize with 5-HT(3) serotonin receptors on striatal nerve terminals.

Nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors are present on presynaptic nerve terminals in the striatum, where they have been shown to be involved in the regulation of dopamine release. Here, we explored the possibility that both receptor systems function on the same individual nerve terminals in the striatum, as assessed by confocal imaging of synaptosomes. On performing sequential stimulation, nicotine (500 nM) induced changes in [Ca(2+)](i) in most of the synaptosomes ( approximately 80%) that had previously responded to stimulation with the 5-HT(3) receptor agonist m-chlorophenylbiguanide (mCPBG; 100 nM), whereas mCPBG induced [Ca(2+)](i) responses in approximately half of the synaptosomes that showed responses on nicotinic stimulation. The 5-HT(3) receptor-specific antagonist tropisetron blocked only the mCPBG-induced responses, but not the nicotinic responses on the same synaptosomes. Immunocytochemical staining revealed extensive co-localization of the 5-HT(3) receptor with the alpha4 nicotinic receptor subunit on the same synaptosomes, but not with the alpha3 and/or alpha5 subunits. Immunoprecipitation studies indicate that the 5-HT(3) receptor and the alpha4 nicotinic receptor subunit do not interact on the nerve terminals. The presence of nicotinic and 5-HT(3) receptors on the same presynaptic striatal nerve terminal indicates a convergence of cholinergic and serotonergic systems in the striatum.

Coupling of I(1) imidazoline receptors to the cAMP pathway: studies with a highly selective ligand, benazoline.

Clonidine and benazoline are two structurally related imidazolines. Whereas clonidine binds both to alpha(2)-adrenoceptors (alpha(2)R) and to I(1) imidazoline receptors (I(1)R), benazoline showed a high selectivity for imidazoline receptors. Although the alpha(2)R are negatively coupled to adenylate cyclase, no effect on cAMP level by activation of I(1)R has been reported so far. We therefore aimed to compare the effects of clonidine and benazoline on forskolin-stimulated cAMP levels in cell lines expressing either I(1)R only (PC12 cells), alpha(2)R only (HT29 cells), or I(1)R and alpha(2)R together (NG10815 cells). Clonidine proved able to decrease the forskolin-stimulated cAMP level in the cells expressing alpha(2)R and this effect could be blocked by rauwolscine. In contrast, in cells lacking these adrenoceptors, clonidine had no effect. On the other hand, benazoline and other I(1) receptor-selective imidazolines decreased forskolin-stimulated cAMP level in the cells expressing I(1)R, in a rauwolscine- and pertussis toxin-insensitive manner. These effects were antagonized by clonidine. According to these results, we demonstrated that 1) alpha(2)R and I(1)R are definitely different entities because they are expressed independently in different cell lines; 2) alpha(2)R and I(1)R are both implicated in the cAMP pathway in cells (one is sensitive to pertussis toxin and the other is not); and 3) I(1)R might be coupled to more then one transduction pathway. These new data will be essential to further understand the physiological implications of the I(1)R and the functional interactions between I(1) receptors and alpha(2)-adrenoceptors.

Migration of human vascular smooth muscle cells involves serum-dependent repeated cytosolic calcium transients.

Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca(2+)](i)were observed in single human VSMCs migrating on type I collagen. Such [Ca(2+)](i)transients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca(2+)](i) with BAPTA led to accumulation of (beta)1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca(2+)](i) transients in the rapid, serum-dependent migration of VSMCs. These [Ca(2+)](i)transients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB.

Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain.

The serotonin 5-hydroxytryptamine-3 receptor is a ligand-gated ion channel that is distributed widely in the nervous system. Within the CNS, a significant portion of the 5-hydroxytryptamine-3 receptors appears to be present on presynaptic nerve terminals and, using an imaging approach, it was shown previously that presynaptic 5-hydroxytryptamine-3 receptors on individual isolated nerve terminals (synaptosomes) from rat corpus striatum display a distinctive set of properties-slow onset, little desensitization and high apparent permeability for Ca2+-when compared to those observed for 5-hydroxytryptamine-3 receptors localized at postsynaptic sites on neuronal cell bodies. To consider whether their characteristic nature is a common feature of presynaptic 5-hydroxytryptamine-3 receptors across the brain, we used confocal microscopy to measure changes in intracellular Ca2+ concentration resulting from 5-hydroxytryptamine-3 agonist-induced responses in synaptosomes from representative rat brain regions, ranging in expression of overall levels of 5-hydroxytryptamine-3 receptors from relatively low (cerebellum) to intermediate (corpus striatum and hippocampus) to high (amygdala). Application of 100 nM m-chlorophenyl biguanide, a specific 5-hydroxytryptamine-3 receptor agonist, induced changes in relative intracellular Ca2+ concentration in subsets of synaptosomes from the corpus striatum (approximately 6% of total), hippocampus (approximately 3% of total), amygdala (approximately 30% of total) and cerebellum (approximately 32% of total). In order to assure the viability of the synaptosomes that did not respond to 5-hydroxytryptamine-3 agonist stimulation, KCl (45 mM) was subsequently added to depolarize the same population of synaptosomes, and increases in intracellular Ca2+ concentration were then seen in 80-90% of the synaptosomes from all four regions. The kinetics of the intra synaptosomal Ca2+ changes produced by K+-evoked depolarization were similar in all regions, showing a rapid rise to a peak followed by an apparent plateau phase. In contrast, the changes in intracellular Ca2+ concentration evoked by m-chlorophenyl biguanide displayed substantially slower kinetics, similar to previous findings, but which varied among responding synaptosomes from one region to another. In particular, m-chlorophenyl biguanide-induced changes were notably slower in synaptosomes from the amygdala (rise time constant, tau = 25 s), when compared to responses in synaptosomes from other regions (striatum, tau = 12 s; hippocampus, tau= 9.6 s; cerebellum, tau = 7 s). To independently demonstrate the presence of 5-hydroxytryptamine-3 receptors on nerve terminals in the various regions using a molecular approach, we double-immunostained the synaptosomes for the 5-hydroxytryptamine-3 receptor and the synaptic vesicle protein synaptophysin, using, respectively, a polyclonal antibody raised against an N-terminal peptide of the 5-hydroxytryptamine-3 receptor and a monoclonal anti-synaptophysin antibody, and observed 5-hydroxytryptamine-3 receptors in varying subsets of the synaptosomes from each region, providing direct support for the results obtained in our functional experiments. These results suggest that the distinctive properties of presynaptic 5-hydroxytryptamine-3 receptors are found throughout the brain, with evident differences in the kinetics of the responses to agonist stimulation observed across the brain regions studied. As expected, the proportion of the synaptosomal population that responded on application of 5-hydroxytryptamine-3 agonist varied in preparations from one region to another; however, the presence of a relatively high proportion of presynaptic 5-hydroxytryptamine-3 receptors in the cerebellum contrasts with previous binding studies demonstrating a relatively low overall density of 5-hydroxytryptamine-3 receptors in this region. We hypothesize that presynaptic 5-hydroxytryptamine-3 receptors present on nerve terminals regulate the

Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells.

The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation.

High calcium permeability of serotonin 5-HT3 receptors on presynaptic nerve terminals from rat striatum.

The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-D-glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 microM Co2+/Cd2+ or by 1 microM omega-conotoxin MVIIC/1 microM oemga-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 microM mCPBG was substantially reduced by 10 microM Co2+/Cd2+ and was completely blocked by 1 microM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant.

5-HT3 receptors induce rises in cytosolic and nuclear calcium in NG108-15 cells via calcium-induced calcium release.

A complete understanding of how excitatory ligand-gated ion channels regulate intracellular Ca2+ in nerve cells remains to be elucidated. Laser-scanning confocal microscopy was used here to measure Ca2+ changes in the neuroblastoma x glioma hybrid cell line NG108-15, employed as a model nerve cell line, upon activation by the 5-HT3 receptor, a serotonin-activated ligand-gated ion channel. Addition of the 5-HT3 agonist 1-m-(chlorophenyl)-biguanide (mCPBG) induced increases in [Ca2+]i in both the cytoplasm and the nuclei of the NG108-15 cells. Using high-time resolution line scanning, no delay was evident between the mCPBG-induced rise in cytosolic [Ca2+]i and the rise in nuclear [Ca2+]i. The agonist-induced responses were completely blocked by addition of EGTA to chelate external Ca2+ and by addition of the 5-HT3 receptor antagonist tropisetron or the L-type Ca2+ channel blocker nitrendipine. Caffeine, but not thapsigargin, treatment significantly reduced the mCPBG-induced responses in the nucleus and the cytoplasm, both to the same extent. We conclude that, upon 5-HT3 receptor activation, Ca2+ enters the cells through voltage-gated Ca2+ channels and then triggers the release of Ca2+ from ryanodine-sensitive intracellular stores, greatly amplifying the increases in Ca2+ in the cytoplasm and the nucleus.

Homologous desensitization of 5-hydroxytryptamine4 receptors in rat esophagus: functional and second messenger studies.

We have studied agonist-induced desensitization of 5-hydroxytryptamine (5-HT4) receptor-mediated relaxation and 5-HT4 receptor-mediated increases in cAMP in rat esophageal tunica muscularis mucosae. In both cases, the desensitization time course was biphasic. The first phase was very rapid because more than 50% of desensitization was obtained after a 5-min incubation period with 10 microM of 5-HT. The second phase was slower and led to a complete suppression of the response after 2 h. Desensitization progressively reduced the maximal relaxation of esophagus induced by 5-HT without significantly affecting the EC50. Desensitization was a receptor-mediated event because cross-desensitization was observed between two chemically unrelated 5-HT4 receptor agonists, 5-HT itself and (S)-zacopride. Inasmuch as the kinetics of desensitization were the same when second messenger production or final responses were measured, this suggests that the limiting step in the desensitization process is at the level of the receptor itself or in its coupling to adenylyl cyclase. The desensitization was of the homologous type because exogenously applied cAMP, 8-Bromo-cAMP, or compounds increasing cAMP in the esophageal tunica muscularis mucosae such as isoproterenol and forskolin, were unable to induce any desensitization of the 5-HT4 receptor-induced relaxation response. Homologous desensitization was not followed by a rapid down-regulation of 5-HT4 receptors because no decrease in the Bmax of [3H]-GR113808 binding was observed after 30-min incubation with 10 microM 5-HT.