Integrin αVß3 Function Influences Citalopram Immobility Behavior in the Tail Suspension Test.

Human studies first identified genetic and expression interactions between integrin ß3 and serotonin (5-HT) transporter (SERT) genes. This association has been further strengthened by our discovery that integrin ß3-containing receptors (αvß3) physically interact with, and thereby define, a subpopulation of SERTs that may represent the main target of selective serotonin reuptake inhibitors (SSRIs). In this study, we examine how integrin αvß3 function influences the behavioral response to the highly SSRI citalopram in the tail suspension test. Mice bearing a conditional deletion of the integrin ß3 gene in neurons, or those expressing a constitutively active αvß3 receptor, have decreased sensitivity to citalopram, when compared to wild-type littermates. To identify potential signaling pathways downstream of integrin αvß3 that could be altered in these mouse lines, and consequently influence citalopram response in vivo, we performed antibody array analyses of midbrain synaptosomes isolated from mice bearing genetically altered integrin ß3. We then pharmacologically targeted focal adhesion (FAK) and extracellular-signal-regulated (ERK) kinases and determined that FAK and ERK activity are critical for the actions of citalopram. Taken together, our studies have revealed a complex relationship between integrin αvß3 function, SERT-dependent 5-HT uptake, and the effective dose of citalopram in the TST, thus implicating a role for integrin signaling pathways in the behavioral response to SSRIs.

Disabling the Gßγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes.

G protein-coupled receptors (GPCRs) that couple to Gi/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gßγ subunits from activated G proteins decreases the activity of voltage-gated Ca2+ channels (VGCCs), decreasing excitability. A less understood Gßγ-mediated mechanism downstream of Ca2+ entry is the binding of Gßγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABAB receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT1b receptors and adrenergic α2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by Gi/o-coupled GPCRs through the Gßγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

p120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia.

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

The Gain-of-Function Integrin ß3 Pro33 Variant Alters the Serotonin System in the Mouse Brain.

Engagement of integrins by the extracellular matrix initiates signaling cascades that drive a variety of cellular functions, including neuronal migration and axonal pathfinding in the brain. Multiple lines of evidence link the ITGB3 gene encoding the integrin ß3 subunit with the serotonin (5-HT) system, likely via its modulation of the 5-HT transporter (SERT). The ITGB3 coding polymorphism Leu33Pro (rs5918, PlA2) produces hyperactive αvß3 receptors that influence whole-blood 5-HT levels and may influence the risk for autism spectrum disorder (ASD). Using a phenome-wide scan of psychiatric diagnoses, we found significant, male-specific associations between the Pro33 allele and attention-deficit hyperactivity disorder and ASDs. Here, we used knock-in (KI) mice expressing an Itgb3 variant that phenocopies the human Pro33 variant to elucidate the consequences of constitutively enhanced αvß3 signaling to the 5-HT system in the brain. KI mice displayed deficits in multiple behaviors, including anxiety, repetitive, and social behaviors. Anatomical studies revealed a significant decrease in 5-HT synapses in the midbrain, accompanied by decreases in SERT activity and reduced localization of SERTs to integrin adhesion complexes in synapses of KI mice. Inhibition of focal adhesion kinase (FAK) rescued SERT function in synapses of KI mice, demonstrating that constitutive active FAK signaling downstream of the Pro32Pro33 integrin αvß3 suppresses SERT activity. Our studies identify a complex regulation of 5-HT homeostasis and behaviors by integrin αvß3, revealing an important role for integrins in modulating risk for neuropsychiatric disorders.SIGNIFICANCE STATEMENT The integrin ß3 Leu33Pro coding polymorphism has been associated with autism spectrum disorders (ASDs) within a subgroup of patients with elevated blood 5-HT levels, linking integrin ß3, 5-HT, and ASD risk. We capitalized on these interactions to demonstrate that the Pro33 coding variation in the murine integrin ß3 recapitulates the sex-dependent neurochemical and behavioral attributes of ASD. Using state-of-the-art techniques, we show that presynaptic 5-HT function is altered in these mice, and that the localization of 5-HT transporters to specific compartments within the synapse, disrupted by the integrin ß3 Pro33 mutation, is critical for appropriate reuptake of 5-HT. Our studies provide fundamental insight into the genetic network regulating 5-HT neurotransmission in the CNS that is also associated with ASD risk.

p120-catenin controls contractility along the vertical axis of epithelial lateral membranes.

In vertebrate epithelia, p120-catenin (hereafter referred to as p120; also known as CTNND1) mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120's activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120's cadherin-stabilizing and RhoA-suppressing activites. Unexpectedly, removing p120's Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120's distinct activities towards E-cadherin and Rho are molecularly and functionally coupled and this, in turn, enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development.

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements.

A membrane fusion protein αSNAP is a novel regulator of epithelial apical junctions.

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.

Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin.

The dynamic functional linkage of cadherins with the underlying actin cytoskeleton is tightly regulated to achieve proper cell-cell adhesion. p120-catenin (p120) regulates both cadherin stability and actin dynamics, but the relationship between these two functions remains unclear. Using a novel proteomic approach called reversible cross-link immunoprecipitation, or ReCLIP, we previously identified a physical interaction between p120 and Rho-associated protein kinase 1 (ROCK1), a major effector of RhoA. In this paper, we show that a discrete fraction of cellular ROCK1 coimmunoprecipitates with p120 and precisely colocalizes to adherens junctions (AJs). Manipulation of AJs using a calcium-switch assay and cadherin-blocking antibodies indicates direct recruitment of ROCK1 to newly forming junctions. Importantly, we find that p120 links ROCK1 to the cadherin complex, as ROCK1 coimmunoprecipitates with wild-type but not p120-uncoupled E-cadherin. Moreover, depletion of ROCK1 using short-hairpin RNA results in dramatic mislocalization of the cadherin complex and junctional actin. These data are consistent with a model in which p120 dynamically regulates Rho-GTPase activity at the cadherin complex through transient interaction with several of its up- and downstream effectors, including ROCK1.

An essential role for p120-catenin in Src- and Rac1-mediated anchorage-independent cell growth.

p120-catenin regulates epithelial cadherin stability and has been suggested to function as a tumor suppressor. In this study, we used anchorage-independent growth (AIG), a classical in vitro tumorigenicity assay, to examine the role of p120 in a different context, namely oncogene-mediated tumorigenesis. Surprisingly, p120 ablation by short hairpin RNA completely blocked AIG induced by both Rac1 and Src. This role for p120 was traced to its activity in suppression of the RhoA-ROCK pathway, which appears to be essential for AIG. Remarkably, the AIG block associated with p120 ablation was completely reversed by inhibition of the downstream RhoA effector ROCK. Harvey-Ras (H-Ras)-induced AIG was also dependent on suppression of the ROCK cascade but was p120 independent because its action on the pathway occurred downstream of p120. The data suggest that p120 modulates oncogenic signaling pathways important for AIG. Although H-Ras bypasses p120, a unifying theme for all three oncogenes is the requirement to suppress ROCK, which may act as a gatekeeper for the transition to anchorage independence.

p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho.

Integration of receptor tyrosine kinase, integrin, and cadherin activities is crucial for normal cell growth, motility, and adhesion. Here, we describe roles for p120-catenin (p120) and p190RhoGAP that coordinate crosstalk between these systems and regulate cadherin function. Surprisingly, PDGFR-induced actin remodeling in NIH3T3 cells is blocked in the absence of p120, and the cells are partially transformed via constitutive activation of Rho. We have traced the mechanism to unexpected codependent roles for p120 and p190RhoGAP in regulating Rac-dependent antagonism of Rho. Receptor-induced Rac activity causes translocation of p190RhoGAP to adherens junctions (AJs), where it couples to the cadherin complex via interaction with p120. AJ formation is dependent on this p120-p190RhoGAP interaction and fails altogether if either of these proteins are compromised. We propose that Rac activation links diverse signaling systems to AJ assembly by controlling transient p190RhoGAP interactions with p120 and localized inhibition of Rho.

Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes.

PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.