05-417 Sigma-AldrichAnti-Paxillin Antibody, clone 5H11

Cell surface proteoglycans interact with numerous regulators of cell behavior through their glycosaminoglycan chains. The syndecan family of transmembrane proteoglycans are virtually ubiquitous cell surface receptors that are implicated in the progression of some tumors, including breast carcinoma. This may derive from their regulation of cell adhesion, but roles for specific syndecans are unresolved.The MDA-MB231 human breast carcinoma cell line was exposed to exogenous glycosaminoglycans and changes in cell behavior monitored by western blotting, immunocytochemistry, invasion and collagen degradation assays. Selected receptors including PAR-1 and syndecans were depleted by siRNA treatments to assess cell morphology and behavior. Immunohistochemistry for syndecan-2 and its interacting partner, caveolin-2 was performed on human breast tumor tissue arrays. Two-tailed paired t-test and one-way ANOVA with Tukey's post-hoc test were used in the analysis of data.MDA-MB231 cells were shown to be highly sensitive to exogenous heparan sulfate or heparin, promoting increased spreading, focal adhesion and adherens junction formation with concomitantly reduced invasion and matrix degradation. The molecular basis for this effect was revealed to have two components. First, thrombin inhibition contributed to enhanced cell adhesion and reduced invasion. Second, a specific loss of cell surface syndecan-2 was noted. The ensuing junction formation was dependent on syndecan-4, whose role in promoting actin cytoskeletal organization is known. Syndecan-2 interacts with, and may regulate, caveolin-2. Depletion of either molecule had the same adhesion-promoting influence, along with reduced invasion, confirming a role for this complex in maintaining the invasive phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover their expression levels were correlated in triple negative breast cancers.Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of β1-integrin by interference with recycling following normal endocytosis of inactive β1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces β1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin-actin interactions needed for recycling by G-clathrin during migration.

Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including ovarian cancer. Therefore, chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for ovarian cancer.In this study, we show that activation of Hh signaling promoted cellular migration and invasion, whereas blockade of Hh signaling with GANT61 suppressed cellular migration and invasion in ovarian cancer cells. After treatment with GANT61, cDNA microarray analyses revealed changes in many genes such as Integrin β4 subunit (ITGB4), focal adhesion kinase (FAK), etc. Furthermore, ITGB4 expression was up-regulated by Sonic Hedgehog (Shh) ligand and down-regulated by Hh signaling inhibitor. The Shh-mediated ovarian cell migration and invasion was blocked by neutralizing antibodies to ITGB4. In addition, phosphorylations of FAK were increased by Shh and decreased by Hh signaling inhibitor. Inhibition of Gli1 expression using siRNA mimicked the effects of GANT61 treatment, supporting the specificity of GANT61. Further investigations showed that activation of FAK was required for Shh-mediated cell migration and invasion. Finally, we found that down-regulation of Gli reduced the expression of ITGB4 and the phosphorylated FAK, resulting in the inhibition of tumor growth in vivo.The Hh signaling pathway induces cell migration and invasion through ITGB4-mediated activation of FAK in ovarian cancer. Our findings suggest that the diminishment of crosstalk between phosphorylated FAK and ITGB4 due to the down-regulation of Gli family transcription factors might play a pivotal role for inhibiting ovarian cancer progression.

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.

Cytotoxic lymphocytes kill target cells through polarized release of the content of lytic granules at the immunological synapse. In human NK cells, signals for granule polarization and for degranulation can be uncoupled: Binding of β(2) integrin LFA-1 to ICAM is sufficient to induce polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. In this study, we investigated the basis for this difference. IL-2-expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR ζ-chain, tyrosine kinase Syk, and phospholipase C-γ. Whereas CD16 activated Ca(2+) mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, phospholipase C-γ, and protein kinase C, as well as by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca(2+)-dependent degranulation and paxillin-dependent granule polarization.

The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in approximately 30% of human breast cancers. c-Jun, the first cellular proto-oncogene, is overexpressed in human breast cancer. However, the role of endogenous c-Jun in mammary tumor progression is unknown. Herein, transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-jun(f/f) transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration, invasion, and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal.

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton collapse. In contrast to Hsp27 overexpression, siRNA-mediated knockdown had the opposite effect on these parameters. Since ischemia activates c-Src, a tyrosine kinase that disrupts both cell-cell and cell-substrate interactions, the relationship between Hsp27 and c-Src was examined. Although Hsp27 and c-Src did not coimmunoprecipitate and Hsp27 overexpression failed to inhibit whole cell c-Src activation during injury, manipulation of Hsp27 altered active c-Src accumulation at cell contact sites. Specifically, Hsp27 overexpression reduced, whereas Hsp27 knockdown increased active p-(416)Src detected at contact sites in intact cells as well as in a purified cell membrane fraction. Together, this evidence shows that Hsp27 overexpression prevents sublethal REC injury at cell contact sites possibly by a c-Src-dependent mechanism. Further exploration of the biochemical link between Hsp27 and c-Src could yield therapeutic interventions for ameliorating ischemic renal cell injury and organ dysfunction.,