Integrin Recycling Research Articles

Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

Pyk2 contributes to reepithelialization by promoting MMP expression. Focus on “Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice”

matrix metalloproteinases (MMPs) play an important role in wound healing (reviewed in Ref. [5][1]). In the skin, the hemidesmosome attachment between keratinocytes and basement membrane is disrupted by MMP activity, so that keratinocytes are released to migrate under the wound matrix and contact the

The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

On the Role of Rab5 in Cell Migration

Uncontrolled endosome trafficking is a common feature of certain cancer cells, which has been acknowledged during the last decade. Migration and invasiveness of metastatic tumor cells are both regulated by components of the endocytic machinery, including Rab proteins. Rab GTPases are essential in processes of endosome fusion, as well as targeting, tethering and transport along the cytoskeleton. In addition to this canonical role, some Rabs depict other functions, such as controlling cell proliferation, apoptosis, adhesion and motility. Here, we review our current knowledge on the role of Rab5, a key regulator of early endosome dynamics, in migration of normal and tumor cells. Rab5 promotes cell migration in vitro and in vivo by mechanisms described at different levels. One such mechanism is by controlling the rates of integrin internalization and recycling, thereby affecting its activation and availability at the cell surface. On the other hand, Rab5 promotes focal adhesion disassembly and modulates downstream pathways of integrin signaling, involving proteins such as Ras and Rho family GTPases. In this context, identification of upstream regulators and downstream effectors of Rab5, and their study represents a big challenge in order to understand how cancer cells depend on endosome control, in order to acquire more aggressive traits that lead to metastatic disease.

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

Arl13b and the non-muscle myosin heavy chain IIA are required for circular dorsal ruffle formation and cell migration.

The Arf-like protein Arl13b has been implicated in ciliogenesis and Sonic hedgehog signaling. Furthermore, we have previously shown that it regulates endocytic recycling traffic and interacts with actin. Herein, we report that the non-muscle myosin heavy chain IIA, also known as Myh9, is an Arl13b effector. Moreover, we found that both proteins localized to circular dorsal ruffles (CDRs) induced by platelet-derived growth factor stimulation and are required for their formation. CDRs are ring-shaped actin-dependent structures formed on the dorsal cell surface and are involved in diverse processes, such as macropinocytosis, integrin recycling, internalization of receptor tyrosine kinases and cell migration. We found that Arl13b or Myh9 silencing impaired cell migration, suggesting that Arl13b is required for this function through the interaction with Myh9. Moreover, Arl13b silencing impaired neural crest cell migration in zebrafish embryos. Furthermore, we showed that Arl13b is required for the formation of CDRs in migrating cells. Thus, our results indicate a new role for Arl13b in actin cytoskeleton remodeling through the interaction with Myh9, by driving the formation of CDRs necessary for cell migration.

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer.

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5β1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.

CLIC4 regulates cell adhesion and β1 integrin trafficking.

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking.

RhoC Interacts with Integrin α5β1 and Enhances Its Trafficking in Migrating Pancreatic Carcinoma Cells

Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin α5β1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin α5β1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin α5β1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin α5β1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin α5β1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.

Endocytosis and Cancer

Endocytosis entails selective packaging of cell-surface proteins, such as receptors for cytokines and adhesion components, in cytoplasmic vesicles (endosomes). The series of sorting events that determines the fate of internalized proteins, either degradation in lysosomes or recycling back to the plasma membrane, relies on intrinsic sequence motifs, posttranslational modifications (e.g., phosphorylation and ubiquitination), and transient assemblies of both Rab GTPases and phosphoinositide-binding proteins. This multicomponent process is enhanced and skewed in cancer cells; we review mechanisms enabling both major drivers of cancer, p53 and Ras, to bias recycling of integrins and receptor tyrosine kinases (RTKs). Likewise, cadherins and other junctional proteins of cancer cells are constantly removed from the cell surface, thereby disrupting tissue polarity and instigating motile phenotypes. Mutant forms of RTKs able to evade Cbl-mediated ubiquitination, along with overexpression of the wild-type forms and a variety of defective feedback regulatory loops, are frequently detected in tumors. Finally, we describe pharmacological attempts to harness the peculiar endocytic system of cancer, in favor of effective patient treatment.

Autophagy modulates cell migration and β1 integrin membrane recycling

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3−/− and atg5−/− MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.

Abstract B090: Collagen organization implicated in tumor dormancy

Abstract It is known that tumor cells can reside in a dormant state for decades. Data from numerous studies strongly suggest that extracellular matrix (ECM) proteins can impart a dormant phenotype, however little is known regarding how ECM regulates tumor cell dormancy. Given the technical difficulties in studying tumor cell dormancy in the context of secondary lesions (i.e., the searching for a needle in a haystack problem), here we use tumor suppression at the primary site as a model to investigate the role of ECM in breast cancer dormancy. Specifically, we hypothesize that insight into tumor dormancy can be obtained by studying the response of the mammary ECM to tamoxifen treatment and parity, two conditions that protect the mammary epithelium from tumorigenesis. Utilizing breast cancer cell lines capable of transitioning from indolent to proliferative/invasive states, we demonstrate that ECM isolated from tamoxifen treated or parous rat mammary glands decreases tumor growth and impedes invasive phenotypes compared to nulliparous mammary matrix, using both in vitro and in vivo models. Collagen stained tissues and proteomic analysis identified increased abundance of collagen I in tamoxifen treated and parous mammary stroma, an observation extended to breast tissue of parous women. These results are unexpected given the known pro-tumorigenic attributes of fibrillar collagen. Second harmonic generation imaging and atomic force microscopy reveals that the abundant collagen observed in the mammary glands of parous rats is less linearized and has decreased stromal stiffness compared to nulliparous mammary glands. These data implicate collagen organization rather than density in tumor suppression. Using 3D cell culture models, we demonstrate that linearized fibrillar collagen I induces an invasive phenotype in mammary tumor cells through a mechanism dependent on β1 integrin endocytic recycling. Conversely, high density, non-fibrillar collagen I decreases β1 endocytic integrin recycling, increases junctional E-cadherin staining, upregulates adherens and tight junction genes, and downregulates EMT transcription factors and metalloproteinase genes, all attributes consistent with tumor suppression. These data show that collagen organization, rather than collagen density, is a key contributor to the tumor cell invasive phenotype. In conclusion, we demonstrate that collagen | organization is dominant over density in driving tumor cell invasion, an observation that may have implications in understanding how mammographic breast density determines breast cancer risk, as well as how collagen organization contributes to tumor cell dormancy at distant sites. Citation Format: Ori Maller, Kirk C. Hansen, Valerie Weaver, Rytis Prekeris, Pepper Schedin. Collagen organization implicated in tumor dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B090.

RCP-driven α5β1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1–IQGAP1 complex

Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)-dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

Cyclin D1 localizes in the cytoplasm of keratinocytes during skin differentiation and regulates cell–matrix adhesion

The function of Cyclin D1 (CycD1) has been widely studied in the cell nucleus as a regulatory subunit of the cyclin-dependent kinases Cdk4/6 involved in the control of proliferation and development in mammals. CycD1 has been also localized in the cytoplasm, where its function nevertheless is poorly characterized. In this work we have observed that in normal skin as well as in primary cultures of human keratinocytes, cytoplasmic localization of CycD1 correlated with the degree of differentiation of the keratinocyte. In these conditions, CycD1 co-localized in cytoplasmic foci with exocyst components (Sec6) and regulators (RalA), and with β1 integrin, suggesting a role for CycD1 in the regulation of keratinocyte adhesion during differentiation. Consistent with this hypothesis, CycD1 overexpression increased β1 integrin recycling and drastically reduced the ability of keratinocytes to adhere to the extracellular matrix. We propose that localization of CycD1 in the cytoplasm during skin differentiation could be related to the changes in detachment ability of keratinocytes committed to differentiation.

Functional proteomics, human genetics and cancer biology of GIPC family members.

GIPC1, GIPC2 and GIPC3 consist of GIPC homology 1 (GH1) domain, PDZ domain and GH2 domain. The regions around the GH1 and GH2 domains of GIPC1 are involved in dimerization and interaction with myosin VI (MYO6), respectively. The PDZ domain of GIPC1 is involved in interactions with transmembrane proteins [IGF1R, NTRK1, ADRB1, DRD2, TGFβR3 (transforming growth factorβ receptor type III), SDC4, SEMA4C, LRP1, NRP1, GLUT1, integrin α5 and VANGL2], cytosolic signaling regulators (APPL1 and RGS19) and viral proteins (HBc and HPV-18 E6). GIPC1 is an adaptor protein with dimerizing ability that loads PDZ ligands as cargoes for MYO6-dependent endosomal trafficking. GIPC1 is required for cell-surface expression of IGF1R and TGFβR3. GIPC1 is also required for integrin recycling during cell migration, angiogenesis and cytokinesis. On early endosomes, GIPC1 assembles receptor tyrosine kinases (RTKs) and APPL1 for activation of PI3K–AKT signaling, and G protein-coupled receptors (GPCRs) and RGS19 for attenuation of inhibitory Gα signaling. GIPC1 upregulation in breast, ovarian and pancreatic cancers promotes tumor proliferation and invasion, whereas GIPC1 downregulation in cervical cancer with human papillomavirus type 18 infection leads to resistance to cytostatic transforming growth factorβ signaling. GIPC2 is downregulated in acute lymphocytic leukemia owing to epigenetic silencing, while Gipc2 is upregulated in estrogen-induced mammary tumors. Somatic mutations of GIPC2 occur in malignant melanoma, and colorectal and ovarian cancers. Germ-line mutations of the GIPC3 or MYO6 gene cause nonsyndromic hearing loss. As GIPC proteins are involved in trafficking, signaling and recycling of RTKs, GPCRs, integrins and other transmembrane proteins, dysregulation of GIPCs results in human pathologies, such as cancer and hereditary deafness.

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

SummaryPrecise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy.

Thrombosis and Inflammation Are Coupled by a Common Thioredoxin Reductase Pathway Downstream of the P2×7 Receptor.

AbstractAbstract 2218The purinergic P2X7 receptor contributes to thrombosis by promoting tissue factor (TF) activation and the release of prothrombotic microparticles (MP). On primed macrophages, P2X7 stimulation induces the procoagulant activity of cell surface TF and the release of MP that carry TF, integrin β1 and protein disulfide isomerase (PDI). The generation of TF+ MP is dependent on extracellular thiol-disulfide exchange, but intermediates leading to MP generation downstream of P2X7 signaling are incompletely defined. Tracking of cell surface TF by confocal microscopy shows that constitutive internalization of TF is prevented by P2X7 activation. In non-stimulated cells, inhibition of dynamin-dependent endocytosis retains TF on the cell surface, rapidly upregulates TF activity, and releases procoagulant MP carrying TF and integrin β1. Integrin recycling is dependent on the small GTPase ARF6 that is found incorporated into MP released when internalization is blocked. In contrast, activation of P2X7 releases ARF6 into the MP-depleted supernatant rather than associated with MP. Decreased ARF6 GTP loading in P2X7-stimulated cells and additional data in integrin α4 S988A mutant mice with reduced ARF6 activity support the conclusion that P2X7 interrupts constitutive ARF6 recycling to increase cell surface availability of TF-integrin complexes for incorporation into MP. Confocal imaging shows that P2X7 stimulation promotes filopodia formation and the peripheral transport of TF to the tips of filipodia. Proteomics identifies γ-actin as a thiol-regulated MP protein that is released by P2X7 signaling. We show that specific inhibition of thioredoxin reductase attenuates P2X7-induced actin remodeling and filopodia formation, the generation of highly procoagulant MP carrying γ-actin and PDI, and the increased availability of extracellular thioredoxin and free thiols. Importantly, inhibition of thioredoxin reductase also prevented the processing and release of the pro-inflammatory cytokine IL1β. Thus, the thioredoxin reductase system is a crucial intermediate downstream of P2X7 activation that may couple coagulation and inflammation in cardiovascular diseases. Disclosures:No relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Regulates Macrophage Migration Through Control Recycling of alpha5beta1 Integrin in Endosome

AbstractAbstract 3273Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. Macrophage migration is a critical step in the regulation of immune response and is also associated with many chronic inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling macrophage migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of macrophages to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate macrophage motility is therefore important for gaining a better understanding of macrophage function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on macrophage and the cellular mechanism for these effects by using small-interfering RNA technic to knockdown GMFG in human macrophage like THP-1cells. Knockdown of endogenous GMFG results in significantly reduced chemotactic migration toward fMLP and enhanced adhesion on fibronectin compared with control siRNA transfected cells. Flow cytometry analysis shows that GMFG-knockdown cells expresses significant amount of α5β1 integrin on the surface compared with control cells. Confocal microscopy analysis exhibited that GMFG was distributed throughout the cytosol and colocalized with the multiple endocytic compartment such as early endosome marker EEA1, Rab5, Rab4 and Rab11. Importantly, we found that most of the α5β1 integrins accumulated in clusters at the rear of the GMFG-knockdown cells, along with colocalization with endosome marker EEA1, rather than localization toward the leading edge in response to fMLP stimulation in control cells. These results suggest that impaired efficient α5β1 integrin recycling cause to increased adhesion, which is responsible for reduced macrophage migration on fibronectin in GMFG depletion macrophages. Accordingly, GMFG is required for proper trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-mediated macrophage motility. Disclosures:No relevant conflicts of interest to declare.

Rab25 Is a Tumor Suppressor Gene with Antiangiogenic and Anti-Invasive Activities in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK-Raf-MEK1/2-ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment.