Regulation Of Cellular Adhesion Research Articles

Perphenazine and prochlorperazine decrease glioblastoma U‑87 MG cell migration and invasion: Analysis of the ABCB1 and ABCG2 transporters, E‑cadherin, α‑tubulin and integrins (α3, α5, and β1) levels

Glioblastoma multiforme is the most frequent type of malignant brain tumor, and is one of the most lethal and untreatable human tumors with a very poor survival rate. Therefore, novel and effective strategies of treatment are required. Integrins play a crucial role in the regulation of cellular adhesion and invasion. Integrins and α-tubulin are very important in cell migration, whereas E-cadherin plays a main role in tumor metastasis. Notably, drugs serve a crucial role in glioblastoma treatment; however, they have to penetrate the blood-brain barrier (BBB) to be effective. ABC transporters, including ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily G member 2 (ABCG2), are localized in the brain endothelial capillaries of the BBB, have a crucial role in the development of multidrug resistance and are modulated by phenothiazine derivatives. The impact of perphenazine and prochlorperazine on the motility of human Uppsala 87 malignant glioma (U87-MG) cells was evaluated using a wound-healing assay, cellular migration and invasion were assessed by Transwell assay, and the protein expression levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins were determined by western blotting. The present study explored the effects of perphenazine and prochlorperazine on the levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins (α3, α5, and β1), as well as on the migratory and invasive ability of U87-MG cells. The results suggested that perphenazine and prochlorperazine may modulate the expression levels of multidrug resistance proteins (they decreased ABCB1 and increased ABCG2 expression), E-cadherin, α-tubulin and integrins, and could impair the migration and invasion of U-87 MG cells. In conclusion, the decrease in migratory and invasive ability following treatment with phenothiazine derivatives due to the increase in ABCG2 and E-cadherin expression, and decrease in α-tubulin and integrins expression, may suggest that research on perphenazine and prochlorperazine in the treatment of glioblastoma is worth continuing.

Calpain-calpastatin system and cancer progression.

The calpain system is required by many important physiological processes, including the cell cycle, cytoskeleton remodelling, cellular proliferation, migration, cancer cell invasion, metastasis, survival, autophagy, apoptosis and signalling, as well as the pathogenesis of a wide range of disorders, in which it may function to promote tumorigenesis. Calpains are intracellular conserved calcium-activated neutral cysteine proteinases that are involved in mediating cancer progression via catalysing and regulating the proteolysis of their specific substrates, which are important signalling molecules during cancer progression. μ-calpain, m-calpain, and their specific inhibitor calpastatin are the three molecules originally identified as comprising the calpain system and they contain several crucial domains, specific motifs, and functional sites. A large amount of data supports the roles of the calpain-calpastatin system in cancer progression via regulation of cellular adhesion, proliferation, invasion, metastasis, and cellular survival and death, as well as inflammation and angiogenesis during tumorigenesis, implying that the inhibition of calpain activity may be a potential anti-cancer intervention strategy targeting cancer cell survival, invasion and chemotherapy resistance.

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development

BackgroundCellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor.ResultsWe show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth.ConclusionsWe have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.

Abstract 4185: The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer

Abstract Breast cancer is the first cause of death in women globally. Metastatic breast cancer, which occurs when cancer cells migrate from the primary tumor and establish in distant organs, is estimated to affect more than a quarter of a million of women in the US. Regrettably, metastatic breast cancer still lacks effective treatment and remains the principal cause of breast cancer mortality. Therefore, it is imperative to develop new and more effective strategies to inhibit metastasis. A promising target for anti-metastatic cancer therapy is the Rho GTPase Rac because it plays a key role in the regulation of cellular adhesion, migration, proliferation and survival; processes which promote tumorigenesis and metastasis. Recently, our group developed Ehop-016, a small molecule that inhibits Rac in metastatic breast cancer cells with an IC50 of 1µM and reduces the activity of its downstream effector p21-activated kinase (PAK). Ehop-016 also inhibits tumor growth, angiogenesis, and metastasis in a mouse model of metastatic breast cancer. However, its relative bioavailability is moderate and should be further improved. Therefore, in an effort to find a compound with greater inhibitory capacity and bioavailability, we tested several Ehop-016 derivatives for their activity against Rac and their toxicity towards metastatic breast cancer cells. Using Rac pulldown assays we show that HV-107 at 250nM inhibits Rac activation by 55% in MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells. The effects of HV-107 on cell viability were tested by MTT assays and our results indicate HV-107 at ≥500nM significantly inhibits metastatic breast cancer cell viability, while showing minimal toxicity towards non-cancerous mammary epithelial cells. We also measured caspase 3 activity, in order to evaluate apoptosis as a potential mechanism of HV-107-induced cell death. Our results show HV-107 at 1000nM causes a significant 1.5 fold increase in caspase 3 activity in MDA-MB-231 cells, indicating HV-107 affects cell viability by inducing apoptosis. Finally, we tested HV-107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5mg/kg BW HV-107. Taken together, our results suggest HV-107 is approximately four times more efficient than the parent compound Ehop-016 and has potential as anti-breast cancer metastasis therapeutic. This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475-13) Sub-Award to LCP. Citation Format: Grace E. Velez Crespo, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane, Linette Castillo-Pichardo. The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4185.

Abstract 5044: CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome

Abstract Metabolic syndrome (MeS) is a cluster of pathophysiological disorders that comprise at least three of the following factors: abdominal obesity, elevated triglycerides, dyslipidemia, high blood pressure and impaired glucose tolerance. Several studies associated MeS with increased risk for several cancer types, including prostate cancer (PCa). C-terminal binding protein 1 (CtBP1) is a potent transcriptional co-repressor of tumor suppressor genes. This protein is activated by low NAD+/NADH ratio produced by highly energetic environment such as high fat diet (HFD) intake. Previously, we identified CtBP1 as a novel molecular link between MeS and prostate tumor growth. The aim of this work was to assess the CtBP1 related pathways in PCa and MeS. We developed a MeS in vivo model by chronically feeding male nude mice with HFD. Control diet (CD) fed animals were maintained at the same conditions. These mice were inoculated with PC3 stable CtBP1 depleted or control cells. RNA obtained from xenografts was used for Genome-wide expression profiles (Affymetrix) and Gene Set Enrichment Analysis (GSEA). These analyses yielded several important pathways regulated by CtBP1, such as “Cell Adhesion” (COL17A1, PRRS2, CDH3, ITGB4, LCN2, CDH1, GJB5, TGM2 and SPARC) and “Olfactory” (OR4C45, OR5P2, GUCA1C and CLCA2). CtBP1 significantly diminished the capability of PCa cell lines to adhere to a collagen matrix, repressing the epithelial marker CDH1 and inducing the mesenchymal marker VIM expressions. Interestingly, CtBP1 associated to ITGB4 promoter gene, strongly repressing its expression. CtBP1 depletion increased the plasma membrane of the cell attached to the substrate and the number of filopodia. From olfactory pathway, we particularly focus on Chloride Channel Accessory 2 (CLCA2), a reported breast cancer tumor suppressor gene that function inhibiting epithelial to mesenchymal transition (EMT) and invasion processes. We found that CtBP1 regulates the transcription of CLCA2 in xenografts generated on HFD-fed mice. Using a panel of luciferase reporter plasmids with variable length of the CLCA2 promoter region, we determined that CtBP1 represses CLCA2 promoter activity in PC3 cells. Moreover, we established that CtBP1 associates to the CLCA2 proximal promoter region by chromatin immunoprecipitation (ChIP). Finally, several proteins regulate CLCA2 promoter activity independently (p53, ET2 and BRCA1) or dependently of CtBP1 (p300 and HDAC2). Altogether, these results demonstrated a new role for CtBP1 in the regulation of cellular adhesion, EMT and invasion reinforcing a potential function for this protein in cancer progression. Hence, CtBP1 pathway might help to identify new molecular candidates for better prediction of PCa progression in a subset of patients with MeS Citation Format: Guillermo N. Dalton, Juliana Porretti, Cintia Massillo, Georgina Scalise, Paula Lucía Farré, Cristian P. Moiola, Alejandra Paez, Geraldine Gueron, Paola De Luca, Adriana De Siervi. CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5044.

148 FOLLICULAR FLUID microRNA SEQUENCES AS BIOMARKERS OF COMPETENT OOCYTES IN CATTLE

Oocyte developmental competence is correlated with antral follicle count through ill-defined mechanisms. Oocytes from ovaries with fewer than 10 mid-antral follicles of 2 to 6 mm in diameter (low group) show reduced competence compared with those from ovaries with more than 10 follicles (high group). To unravel mechanisms underlying this phenomenon, this work explored the role of follicular fluid microRNAs (miRNAs, short non-coding RNAs regulating gene expression at the post-transcriptional level). A total of 3 pools of 300 µL of follicular fluid (FF) were collected from mid-antral follicles of low (L) and high (H) groups, respectively. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisEqn 2000 (Illumina Inc., San Diego, CA, USA). Differentially expressed miRNAs were identified with R package edgeR (http://bioconductor.org/packages/release/bioc/html/edgeR.html). Target genes of differentially expressed miRNAs were predicted with DIANA miRPath using homologous human miRNA and gene union options (P < 0.01). Gene ontology (GO) analysis was carried out by Cytoscape (http://www.cytoscape.org/) using a bovine database. In total, 1279 miRNAs were identified in FF: 805 ± 139 in L and 862 ± 36 in H (P > 0.05). We found that 27 miRNAs were differentially expressed (false discovery rate ≤0.001): 17 were up-regulated in L and 10 in H. Up-regulated miRNAs in L group were predicted to target 121 genes, 39 of which are specific for ovarian function (e.g. BCL2, FOXO3, KIT, TP53, and PTK2). The GO analysis indicated that these genes were kinases, anti-apoptotic and oncogenic factors, and enriched stress-activated MAPK cascade mediated by oxygen reactive species, G1/S transition checkpoint, cellular response to interleukin-1, and negative regulation of cellular adhesion. Overexpressed miRNAs in H group were predicted to target 92 genes, 22 of which (e.g. MAPK, APC, JNK, PKA) are involved in folliculogenesis. These genes were represented by kinases, apoptotic and cytoskeleton remodelling factors, and enriched very important ovarian processes such as cell cycle, ephrin receptor, smoothened and phosphatidylinositol 3-kinase activity GO processes. Only 7 target genes were common between the 2 groups, 2 of which were important in ovarian functionality (CDKN1A and ITGA5). Interestingly, overexpressed miRNAs in both groups regulate several genes involved in processes apparently not related to folliculogenesis. Finally, regulation can be exerted also by low levels of specific miRNAs such as miR-320, which was reduced in L group and is known to be associated with premature ovarian senescence in women and decreased developmental potential of mouse oocytes. Our results indicate that the different oocyte quality is associated with a different miRNA blueprint, which may alter the expression of several genes relevant for intra- and extra-ovarian processes. Further studies will be necessary to determine if FF miRNAs can act outside the ovary and if their levels can be detected in the bloodstream thereby becoming possible noninvasive, real-time markers to determine oocyte quality in living animals. This study was supported by FP7-KBBE-2012-FECUND-312097.

Roles of galactose 3'-O- sulfation in signaling.

Galactose-3'-O- sulfation is specific and exists in many important molecules from various human tissues, and the sulfation modification results in alteration of host molecule recognition and interaction with partner molecules which lead to signaling. The modification is thus associated with the regulation of cellular adhesion and interaction, and involved in cell recognition and even in tumor metastasis process since the binding affinity to their extracellular ligands has changed. Sulfated glycoproteins or glycolipids may also trigger signaling in the cells, which is important in regulating cell-cell and cell-extracellular matrix interaction, and in adhesion molecule transcription activation.

Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

Mechanisms and consequences of oxidative damage to extracellular matrix

Considerable evidence exists for oxidative damage to extracellular materials during multiple human pathologies. Unlike cells, the extracellular compartment of most biological tissues is less well protected against oxidation than intracellular sites in terms of the presence of both antioxidants (low molecular mass and enzymatic) and repair enzymes. The extracellular compartment may therefore be subject to greater oxidative stress, marked alterations in redox balance and an accumulation of damage due to slow turnover and/or poor repair. The nature and consequences of damage to ECM (extracellular matrix) are poorly understood, despite the growing realization that changes in matrix structure not only have structural consequences, but also play a key role in the regulation of cellular adhesion, proliferation, migration and cell signalling. The ECM also plays a key role in cytokine and growth factor binding, and matrix modifications would therefore be expected to alter these parameters. In the present study, we review mechanisms of oxidative damage to ECM, resulting changes in matrix structure and how this affects cellular behaviour. The role of such damage in the development and progression of inflammatory diseases is also discussed with particular reference to cardiovascular disease.

Phosphorylated β-catenin localizes to centrosomes of neuronal progenitors and is required for cell polarity and neurogenesis in developing midbrain

β-catenin has well-established functions in cell growth and differentiation as part of the Wnt signaling pathway and in regulation of cellular adhesion with E-cadherin. Here we studied its significance in midbrain development by temporally controlled deletion of β-catenin allowing simultaneous analysis of complete (β-cat-null) and partial (β-cat-low) loss-of-function phenotypes in progenitor cells. β-cat-null cells did not contain centrosomes or a microtubule network and were unpolarized forming delaminated bulges. β-cat-low cells displayed defects in the orientation of the mitotic spindle, increased asymmetric cell divisions and premature differentiation in absence of alterations in polarity or adhesion. The spindle defect was associated with decreased centrosomal S33/S34/T41 phosphorylated β-catenin (p-β-cat) and centrosomal and microtubule defects. Interestingly, neural progenitor cells in mice expressing only unphosphorylatable β-catenin share several phenotypes with β-catenin loss-of-function mice with defects in microtubules and polarity. The results demonstrate a novel function for p-β-cat in maintaining neuroepithelial integrity and suggest that centrosomal p-ß-cat is required to maintain symmetric cleavages and polarity in neural progenitors.

Phosphoinositides in Cell Architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.

Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue

Syk, a non-receptor tyrosine kinase, is an essential signaling molecule in B cells and other hematopoietic cells. Recently, its unexpected diverse functions were recognized in the regulation of cellular adhesion, innate immune recognition, vascular development, and carcinogenesis. Despite its pleiotropic role, only a few substrate proteins have been identified. To find new substrate proteins for Syk, we performed a systemic in vitro kinase assay using GST fusion peptides to determine the substrate specificity surrounding the tyrosine residue to be phosphorylated. Substitution of amino acid residues surrounding tyrosine 178 of BLNK, a principal Syk substrate in B cell receptor-mediated signaling, revealed that acidic residues at sites −5 to −1 were necessary for phosphorylation by Syk. Valine at site +1 was also influential in phosphorylation and a substitution of Pro on site +3 to a basic amino acid residue, Lys, resulted in attenuated phosphorylation. On the basis of these results, a general consensus phosphorylation motif for Syk was determined and several new candidate target proteins were identified in protein database searches. Of the candidate proteins, BCAR-1 (breast cancer anti-estrogen resistance 1) was confirmed to be phosphorylated by Syk in an in vitro kinase assay using a full-length protein of BCAR-1. Furthermore, BCAR-1 was tyrosine phosphorylated upon the overexpression of Syk in HEK-293T cells. These results suggest that more Syk substrates can be found using an in vitro kinase approach and show for the first time that BCAR-1 is a physiological substrate of Syk.

Identification of Phosphorylation-Dependent Interaction Partners of the Adapter Protein ADAP using Quantitative Mass Spectrometry: SILAC vs 18O-Labeling

The immune adapter protein ADAP (adhesion and degranulation promoting adapter protein) plays an important role in integrin-dependent migration and adhesion processes as a consequence of T cell stimulation. ADAP undergoes multiple phosphorylation events during T cell receptor (TCR) or chemokine receptor stimulation. The role of individual phosphotyrosines for protein complex formation and the regulation of cellular adhesion are still under debate. Here, we use peptide pull-down assays and quantitative mass spectrometry to identify interaction partners of site-specifically phosphorylated ADAP sequences. Phosphotyrosine peptide motifs covering Y595, Y625, and Y771 and the corresponding nonphosphorylated sequences were covalently coupled to agarose beads and incubated with Jurkat T cell lysates. For unambiguous differentiation between phosphorylation-specific and nonspecific protein interaction, we employed two different isotope labeling techniques: stable isotope labeling of amino acids in cell culture (SILAC) and enzymatic (18)O-labeling, both in combination with high-resolution mass spectrometry. In addition to previously known SH2 domain-based interactions of ADAP with SLP76, we identified novel ADAP interaction partners - such as the Ras GTPase activating protein - which belong to the larger TCR proximal signaling complex. The results show that both isotope labeling techniques are well suited for distinguishing phosphorylation-specific peptide-protein interactions from the background.

Involvement of Sib Proteins in the Regulation of Cellular Adhesion in Dictyostelium discoideum

Molecular mechanisms ensuring cellular adhesion have been studied in detail in Dictyostelium amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in Dictyostelium, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a Dictyostelium population. In order to understand the mechanism underlying this regulation, we analyzed the expression of recently identified Dictyostelium adhesion molecules (Sib proteins) that present features also found in mammalian integrins. sibA and sibC are both expressed in vegetative Dictyostelium cells, but the expression of sibC is repressed strongly in conditions where cellular adhesion decreases. Analysis of sibA and sibC mutant cells further suggests that variations in the expression levels of sibC account largely for changes in cellular adhesion in response to environmental cues.

The Protective Effect of Ischemic Preconditioning Associated With Altered Gene Expression Profiles in Intestinal Grafts After Reperfusion

Ischemia-reperfusion injury after small bowel transplantation (SBT) has been considered as a trigger for early graft rejection. Ischemic preconditioning (IPC) may improve the tolerance of the small intestine to ischemic insults; however, the mechanism of IPC is still not completely elucidated. Accordingly, the aim of the present research is to investigate the molecular mechanism of IPC through a large-scale expression measurement of intragraft gene profiles by complementary DNA microarrays in SBT. Three groups of rats were studied: S group (sham group), SBT group, and ISBT group (IPC plus SBT), in which IPC was carried out before the graft was harvested. Samples were collected from the different groups 1 h after reperfusion. Gene expression profiles of intestinal graft from the SBT group or ISBT group relative to gene expression profiles of normal intestine from the S group were analyzed respectively using 4096 gene chips. Microarray analysis showed that 14 genes were up-regulated more than 2-fold after reperfusion compared with normal intestine, and 17 genes were down-regulated in the SBT group. In the ISBT group, 84 genes with differential expression were detected after reperfusion compared with normal intestine. It was found that 16 genes had differential expression in both the SBT and ISBT groups. These differentially expressed genes were primarily associated with cellular adhesion, energetic and substrate metabolism, and intracellular signal transduction. In addition, a number of novel genes were found. The protective effect of IPC was correlated with regulation of cellular adhesion, improvement on energetic and substrate metabolism, and activation of intracellular signal transduction.

Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell–cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.

Collagen Type I Induces Disruption of E-Cadherin–Mediated Cell-Cell Contacts and Promotes Proliferation of Pancreatic Carcinoma Cells

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein beta-catenin, which correlates with collagen type I-dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with beta1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of beta-catenin, which leads to an increasing expression of the beta-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin-mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells.

Src Phosphorylates Ezrin at Tyrosine 477 and Induces a Phosphospecific Association between Ezrin and a Kelch-Repeat Protein Family Member

Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.

Ezrin gene, coding for a membrane-cytoskeleton linker protein, is regionally expressed in the developing mouse neuroepithelium

Ezrin is a member of the Ezrin, Radixin, Moesin (ERM) proteins family that are proposed to act as linkers between the cytoskeleton and plasma membrane. Ezrin regulates cell–cell and cell–matrix interactions playing a role in the regulation of cellular adhesion, movement and morphology in epithelia. Alterations in the expression of Ezrin and other members of ERM family have also been observed in brain tumours. Here we report the expression pattern of Ezrin during mouse neural development, from early stages to postnatal stages. In young and middle gestation embryos, Ezrin is expressed in the roof plate of the neural tube, in the presumptive domain of the choroidal plexus, and in some precise domains of ventricular epithelium. These domains are distributed in basal and alar neuroepithelial regions, some of them in relation to the expression of cadherins. At later gestation and postnatal stages, Ezrin expression is maintained on the mature choroidal plexus and is weakly detected in the proliferative regions of the mature brain.

Positive and negative adaptors in T-cell signalling.

Positive and negative adaptors in T-cell signalling.