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1.
Cancer Cell ; 29(1): 75-89, 2016 Jan 11.
Article in English | MEDLINE | ID: mdl-26725216

ABSTRACT

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System/genetics , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/genetics , Mice , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Pancreatic Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Time
2.
Mol Cancer Ther ; 13(1): 122-33, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24222664

ABSTRACT

The high prevalence of KRAS mutations and importance of the RalGEF-Ral pathway downstream of activated K-ras in pancreatic ductal adenocarcinoma (PDAC) emphasize the importance of identifying novel methods by which to therapeutically target these pathways. It was recently demonstrated that phosphorylation of RalA S194 by Aurora A kinase (AAK) is critical for PDAC tumorigenesis. We sought to evaluate the AAK-selective inhibitor MLN8237 as a potential indirect anti-RalA-targeted therapy for PDAC. We used a site-specific phospho-S194 RalA antibody and determined that RalA S194 phosphorylation levels were elevated in a subset of PDAC cell lines and human tumors relative to unmatched normal controls. Effects of MLN8237 on anchorage-independent growth in PDAC cell lines and growth of patient-derived xenografts (PDX) were variable, with a subset of cell lines and PDX showing sensitivity. Surprisingly, RalA S194 phosphorylation levels in PDAC cell lines or PDX tumors did not correlate with MLN8237 responsiveness. However, we identified Ki67 as a possible early predictive biomarker for response to MLN8237 in PDAC. These results indicate that MLN8237 treatment may be effective for a subset of patients with PDAC independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment.


Subject(s)
Azepines/administration & dosage , Carcinogenesis/drug effects , Carcinoma, Pancreatic Ductal/drug therapy , Ki-67 Antigen/genetics , Pyrimidines/administration & dosage , ral GTP-Binding Proteins/genetics , Aurora Kinase A/metabolism , Biomarkers, Tumor/genetics , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Humans , Mutation , Phosphorylation/drug effects , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , ral GTP-Binding Proteins/metabolism , ras Proteins/genetics
3.
Mol Cell Biol ; 32(8): 1374-86, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22331470

ABSTRACT

Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling. We determined that a majority of KRAS mutant PDAC cell lines exhibited invadopodia and that expression of activated K-Ras is both necessary and sufficient for invadopodium formation. Invadopodium formation was not dependent on the canonical Raf-MEK-ERK effector pathway and was instead dependent on the Ral effector pathway. However, this process was more dependent on RalB than on RalA. Surprisingly, RalB-mediated invadopodium formation was dependent on RalBP1/RLIP76 but not Sec5 and Exo84 exocyst effector function. Unexpectedly, the requirement for RalBP1 was independent of its best known function as a GTPase-activating protein for Rho small GTPases. Instead, disruption of the ATPase function of RalBP1 impaired invadopodium formation. Our results identify a novel RalB-mediated biochemical and signaling mechanism for invadopodium formation.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cell Surface Extensions/enzymology , GTPase-Activating Proteins/metabolism , ral GTP-Binding Proteins/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Surface Extensions/ultrastructure , Enzyme Activation , Humans , Neoplasm Invasiveness/ultrastructure , Pancreatic Neoplasms/pathology , Signal Transduction
4.
PLoS One ; 6(8): e23813, 2011.
Article in English | MEDLINE | ID: mdl-21876773

ABSTRACT

BACKGROUND: Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1) was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42. CONCLUSIONS: Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".


Subject(s)
Chemotaxis/physiology , Intercellular Junctions/metabolism , Receptors, Interleukin-8B/metabolism , ras GTPase-Activating Proteins/metabolism , Chemotaxis/drug effects , Chromatography, Liquid , HEK293 Cells , HL-60 Cells , Humans , Intercellular Junctions/drug effects , Interleukin-8/pharmacology , Mass Spectrometry , Protein Binding/drug effects , Proteomics , cdc42 GTP-Binding Protein/metabolism , ras GTPase-Activating Proteins/chemistry
5.
Genes Cancer ; 2(3): 275-87, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21779498

ABSTRACT

The high frequency of RAS mutations in human cancers (33%) has stimulated intense interest in the development of anti-Ras inhibitors for cancer therapy. Currently, the major focus of these efforts is centered on inhibitors of components involved in Ras downstream effector signaling. In particular, more than 40 inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade and phosphoinositide 3-kinase-AKT-mTOR effector signaling networks are currently under clinical evaluation. However, these efforts are complicated by the fact that Ras can utilize at least 9 additional functionally distinct effectors, with at least 3 additional effectors with validated roles in Ras-mediated oncogenesis. Of these, the guanine nucleotide exchange factors of the Ras-like (Ral) small GTPases (RalGEFs) have emerged as important effectors of mutant Ras in pancreatic, colon, and other cancers. In this review, we summarize the evidence for the importance of this effector pathway in cancer and discuss possible directions for therapeutic inhibition of aberrant Ral activation and signaling.

6.
Cancer Res ; 71(2): 603-13, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21123450

ABSTRACT

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1-CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1-CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1-mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Estradiol/analogs & derivatives , Receptors, CXCR4/biosynthesis , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Female , Fulvestrant , Humans , MAP Kinase Signaling System , Mice , Mice, SCID , Neoplasm Metastasis , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Receptors, CXCR4/metabolism , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/biosynthesis
7.
PLoS One ; 5(4): e10050, 2010 Apr 19.
Article in English | MEDLINE | ID: mdl-20419088

ABSTRACT

BACKGROUND: The chemokine receptor CXCR2 plays a pivotal role in migration of neutrophils, macrophages and endothelial cells, modulating several biological responses such as angiogenesis, wound healing and acute inflammation. CXCR2 is also involved in pathogenesis of chronic inflammation, sepsis and atherosclerosis. The ability of CXCR2 to associate with a variety of proteins dynamically is responsible for its effects on directed cell migration or chemotaxis. The dynamic network of such CXCR2 binding proteins is termed as "CXCR2 chemosynapse". Proteomic analysis of proteins that co-immunoprecipitated with CXCR2 in neutrophil-like dHL-60 cells revealed a novel protein, LIM and SH3 protein 1 (LASP-1), binds CXCR2 under both basal and ligand activated conditions. LASP-1 is an actin binding cytoskeletal protein, involved in the cell migration. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that CXCR2 and LASP-1 co-immunoprecipitate and co-localize at the leading edge of migrating cells. The LIM domain of LASP-1 directly binds to the carboxy-terminal domain (CTD) of CXCR2. Moreover, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4. Using a site-directed and deletion mutagenesis approach, Iso323-Leu324 of the conserved LKIL motif on CXCR2-CTD was identified as the binding site for LASP-1. Interruption of the interaction between CXCR2-CTD and LIM domain of LASP-1 by dominant negative and knock down approaches inhibited CXCR2-mediated chemotaxis. Analysis for the mechanism for inhibition of CXCR2-mediated chemotaxis indicated that LASP-1/CXCR2 interaction is essential for cell motility and focal adhesion turnover involving activation of Src, paxillin, PAK1, p130CAS and ERK1/2. CONCLUSIONS/SIGNIFICANCE: We demonstrate here for the first time that LASP-1 is a key component of the "CXCR2 chemosynapse" and LASP-1 interaction with CXCR2 is critical for CXCR2-mediated chemotaxis. Furthermore, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated chemotaxis. Thus, LASP-1 may serve as a new link coordinating the flow of information between chemokine receptors and nascent focal adhesions, especially at the leading edge. Thus the association between the chemokine receptors and LASP-1 suggests to the presence of a CXC chemokine receptor-LASP-1 pro-migratory module in cells governing the cell migration.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Chemotaxis , Cytoskeletal Proteins/metabolism , Receptors, Interleukin-8B/metabolism , Adaptor Proteins, Signal Transducing/physiology , Binding Sites , Cell Line , Cytoskeletal Proteins/physiology , Focal Adhesions , HL-60 Cells , Humans , Immunoprecipitation , LIM Domain Proteins , Protein Binding , Protein Interaction Mapping , Receptors, Interleukin-8B/physiology
8.
J Cell Sci ; 122(Pt 11): 1882-94, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19435808

ABSTRACT

Chemotaxis regulates the recruitment of leukocytes, which is integral for a number of biological processes and is mediated through the interaction of chemokines with seven transmembrane G-protein-coupled receptors. Several studies have indicated that chemotactic signaling pathways might be activated via G-protein-independent mechanisms, perhaps through novel receptor-interacting proteins. CXCR2 is a major chemokine receptor expressed on neutrophils. We used a proteomics approach to identify unique ligand-dependent CXCR2-interacting proteins in differentiated neutrophil-like HL-60 cells. Using this approach, vasodilator-stimulated phosphoprotein (VASP) was identified as a CXCR2-interacting protein. The interaction between CXCR2 and VASP is direct and enhanced by CXCL8 stimulation, which triggers VASP phosphorylation via PKA- and PKCdelta-mediated pathways. The interaction between CXCR2 and VASP requires free F-actin barbed ends to recruit VASP to the leading edge. Finally, knockdown of VASP in HL-60 cells results in severely impaired CXCR2-mediated chemotaxis and polarization. These data provide the first demonstration that direct interaction of VASP with CXCR2 is essential for proper CXCR2 function and demonstrate a crucial role for VASP in mediating chemotaxis in leukocytes.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Polarity , Chemotaxis/physiology , Leukocytes/physiology , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Receptors, Interleukin-8B/metabolism , Actins/metabolism , Animals , Cell Adhesion Molecules/genetics , Cell Membrane/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , HL-60 Cells , Humans , Interleukin-8/metabolism , Leukocytes/cytology , Microfilament Proteins/genetics , Phosphoproteins/genetics , Phosphorylation , Protein Kinase C/metabolism , Protein Structure, Tertiary , Receptors, Interleukin-8B/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine/metabolism , Signal Transduction/physiology
9.
Methods Enzymol ; 460: 315-30, 2009.
Article in English | MEDLINE | ID: mdl-19446732

ABSTRACT

Chemokine-receptor signaling is initiated upon ligand binding to the receptor and continues through the process of endocytic trafficking by the association of a variety of adaptor proteins with the chemokine receptor. In order to define the adaptor proteins that associate with CXCR2 before and after ligand activation, a protocol was developed using differentiated HL-60 cells transfected to express CXCR2 stimulated or not stimulated with ligand for one minute. CXCR2-associating proteins were isolated by immunoprecipitation with CXCR2 antibody and the eluted proteins were electrophoretically run into the separating gel directly without a stacking gel. The stained single band was subjected to in-gel trypsin digestion. The tryptic peptides were subjected to, LC/MS/MS proteomic analysis. Proteins identified in a minimum of three of four separate experiments with multiple peptides were then validated as CXCR2 adaptor proteins by coimmunoprecipitation, GST pull-down studies, and immunocytochemical CXCR2-colocalization experiments using dHL-60-CXCR2 cells. Subsequently, a functional analysis of the interaction between CXCR2 and CXCR2 interacting proteins was performed. This approach can be used to characterize chemokine receptor-associating proteins over time both before and after ligand stimulation, allowing definition of the dynamic spatial and temporal formation of a "chemosynapse."


Subject(s)
Proteins/metabolism , Proteomics/methods , Receptors, Interleukin-8B/metabolism , HL-60 Cells , Humans , Immunohistochemistry , Immunoprecipitation , Microscopy, Confocal , Protein Binding , Receptors, Interleukin-8B/genetics
10.
J Leukoc Biol ; 84(2): 406-19, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18505935

ABSTRACT

CXCR2 plays an important role during cutaneous wound healing. Transgenic mice were generated using the keratin-14 promoter/enhancer to direct expression of wild-type human CXCR2 (K14hCXCR2 WT) or mutant CXCR2, in which the carboxyl-terminal domain (CTD) was truncated at Ser 331 and the dileucine AP-2 binding motif was mutated to alanine (K14hCXCR2 331T/LL/AA/IL/AA). Our results indicate that K14hCXCR2WT transgenic mice exhibited a normal phenotype, while K14hCXCR2 331T/LL/AA/IL/AA transgenic mice were born with tails of normal length, but three to eight days after birth their tails degenerated, leaving only a short tail stub. The tissue degeneration in the tail started between caudal somites with degeneration of bone and connective tissue distal to the constriction, which was replaced with stromal tissue heavily infiltrated with inflammatory cells. The tail lesion site revealed coagulation in enlarged vessels and marked edema that eventually led to loss of the distal tail. Moreover, 66% of the mice exhibited focal skin blemishes and inflammation that exhibited an increase in the number of sebaceous glands and blood vessels, enlargement of the hair follicles due to increased number of keratinocytes, reduction in the connective tissue content, and a thickening of the epidermis. Furthermore, immunohistochemical staining of the epidermis from tail tissue in the transgenic mice indicated a loss of the cell adhesion markers E-cadherin and desmoplakin. These data suggest that keratinocyte expression of a CTD mutant of CXCR2 has effects on homeostasis of the connective tissue in the tail, as well as the maintenance of the epidermis and its appendages.


Subject(s)
Keratin-14/genetics , Promoter Regions, Genetic , Receptors, Interleukin-8B/genetics , Skin/pathology , Tail/abnormalities , Amino Acid Sequence , Animals , Enhancer Elements, Genetic , Humans , Mice , Mice, Mutant Strains , Mice, Transgenic , Molecular Sequence Data , Mutation , Sequence Deletion
11.
J Cell Sci ; 120(Pt 9): 1559-71, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17405813

ABSTRACT

The CXCR2 chemokine receptor is a G-protein-coupled receptor that undergoes clathrin-mediated endocytosis upon ligand binding. The trafficking of CXCR2 is crucial for cells to maintain a proper chemotactic response. The mechanisms that regulate the recycling/degradation sorting decision are unknown. In this study, we used dominant-negative (T19N) and GTPase-deficient activated (Q63L) RhoB mutants, as well as RhoB small interfering RNA (siRNA) to investigate the role of RhoB in CXCR2 trafficking. Expression of either of the RhoB mutants or transfection of RhoB siRNA impaired CXCR2-mediated chemotaxis. Expression of RhoB T19N and transfection of RhoB siRNA impaired sorting of CXCR2 to the lysosome after 3 hours of CXCL8 stimulation and impaired CXCL8-induced CXCR2 degradation. In cells expressing the RhoB Q63L mutant, CXCR2 recycling through the Rab11a recycling compartment was impaired after 30 minutes of CXCL8 stimulation as was CXCL8-induced CXCR2 degradation. For cells expressing activated RhoB, CXCR2 colocalized with Rab4, a marker for the rapid recycling pathway, and with the mannose-6-phosphate receptor, which traffics between the trans-Golgi network and endosomes. These data suggest that CXCR2 recycles through alternative pathways. We conclude that oscillation of RhoB GTPase activity is essential for appropriate sorting decisions, and for directing CXCR2 degradation and recycling--events that are required for optimal chemotaxis.


Subject(s)
Receptors, Interleukin-8B/metabolism , rhoB GTP-Binding Protein/physiology , Actins/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Chemotaxis/drug effects , Cycloheximide/pharmacology , Cytochalasin B/pharmacology , Endosomes/chemistry , Endosomes/metabolism , Guanosine Triphosphate/metabolism , Humans , Interleukin-8/pharmacology , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Lysosomes/chemistry , Lysosomes/metabolism , Models, Biological , Mutation , Protein Transport/drug effects , RNA, Small Interfering/genetics , Receptor, IGF Type 2/genetics , Receptor, IGF Type 2/metabolism , Receptors, Interleukin-8B/genetics , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Thiazolidines/pharmacology , Transfection , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab4 GTP-Binding Proteins/genetics , rab4 GTP-Binding Proteins/metabolism , rhoB GTP-Binding Protein/genetics , rhoB GTP-Binding Protein/metabolism
12.
Cancer Res ; 66(11): 5665-75, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16740704

ABSTRACT

The CXC chemokine receptor 4 (CXCR4) contributes to the metastasis of human breast cancer cells. The CXCR4 COOH-terminal domain (CTD) seems to play a major role in regulating receptor desensitization and down-regulation. We expressed either wild-type CXCR4 (CXCR4-WT) or CTD-truncated CXCR4 (CXCR4-DeltaCTD) in MCF-7 human mammary carcinoma cells to determine whether the CTD is involved in CXCR4-modulated proliferation of mammary carcinoma cells. CXCR4-WT-transduced MCF-7 cells (MCF-7/CXCR4-WT cells) do not differ from vector-transduced MCF-7 control cells in morphology or growth rate. However, CXCR4-DeltaCTD-transduced MCF-7 cells (MCF-7/CXCR4-DeltaCTD cells) exhibit a higher growth rate and altered morphology, potentially indicating an epithelial-to-mesenchymal transition. Furthermore, extracellular signal-regulated kinase (ERK) activation and cell motility are increased in these cells. Ligand induces receptor association with beta-arrestin for both CXCR4-WT and CXCR4-DeltaCTD in these MCF-7 cells. Overexpressed CXCR4-WT localizes predominantly to the cell surface in unstimulated cells, whereas a significant portion of overexpressed CXCR4-DeltaCTD resides intracellularly in recycling endosomes. Analysis with human oligomicroarray, Western blot, and immunohistochemistry showed that E-cadherin and Zonula occludens are down-regulated in MCF-7/CXCR4-DeltaCTD cells. The array analysis also indicates that mesenchymal marker proteins and certain growth factor receptors are up-regulated in MCF-7/CXCR4-DeltaCTD cells. These observations suggest that (a) the overexpression of CXCR4-DeltaCTD leads to a gain-of-function of CXCR4-mediated signaling and (b) the CTD of CXCR4-WT may perform a feedback repressor function in this signaling pathway. These data will contribute to our understanding of how CXCR4-DeltaCTD may promote progression of breast tumors to metastatic lesions.


Subject(s)
Breast Neoplasms/pathology , Cell Communication/physiology , Cell Movement/physiology , Receptors, CXCR4/physiology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Growth Processes/physiology , Cell Line, Tumor , Down-Regulation , Enzyme Activation , Epithelial Cells/physiology , Genetic Vectors/genetics , Humans , Mesoderm/physiology , Mitogen-Activated Protein Kinase Kinases/metabolism , Oligonucleotide Array Sequence Analysis , Protein Structure, Tertiary , Receptors, CXCR4/biosynthesis , Receptors, CXCR4/genetics , Retroviridae/genetics , Structure-Activity Relationship , Transduction, Genetic
13.
Cytokine Growth Factor Rev ; 16(6): 637-58, 2005 Dec.
Article in English | MEDLINE | ID: mdl-15998596

ABSTRACT

The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors.


Subject(s)
Cell Movement , Clathrin-Coated Vesicles/physiology , Endocytosis , Receptors, Chemokine/physiology , Animals , Humans , Membrane Microdomains/physiology , Protein Transport/physiology , Receptors, Chemokine/metabolism , rab GTP-Binding Proteins/physiology
14.
Immunol Lett ; 95(1): 37-44, 2004 Aug 15.
Article in English | MEDLINE | ID: mdl-15325796

ABSTRACT

Interleukin-3 (IL-3), which is derived from T cells and other sources, can promote the differentiation, proliferation, and migration of mast cells, basophils, and eosinophils. However, little is known about the ability of IL-3 to regulate the function of these cells in IgE-dependent and -independent allergic responses in vivo. Therefore, we sought to investigate the extent to which endogenously produced IL-3 can influence mast cell secretory function, the expression of local and systemic anaphylactic responses, and ragweed-induced eosinophilic peritonitis. We found that peritoneal mast cells from IL-3 deficient (IL-3 -/-) mice released less serotonin following challenge with low doses of anti-IgE antibody or antigen ex vivo than do cells isolated from corresponding wild-type (IL-3 +/+) mice. Both IL-3 -/- and +/+ mice expressed equivalent IgE-dependent passive cutaneous anaphylaxis responses following challenge with specific antigen and exhibited equivalent active systemic anaphylaxis responses to ovalbumin as assessed by changes in body temperature, death rates, total IgE production, and histamine release. In contrast, ragweed allergen immunization and peritoneal allergen challenge resulted in eosinophil recruitment that was greater in IL-3 -/- mice than in IL-3 +/+ mice. Our data demonstrates that IL-3 does not appear to be essential for local or systemic anaphylaxis. However, IL-3 production in vivo was found to enhance the mediator release from freshly isolated peritoneal mast cells stimulated ex vivo, and, unexpectedly, to inhibit the accumulation of eosinophils associated with a ragweed-induced allergic peritonitis model.


Subject(s)
Anaphylaxis/immunology , Eosinophils/immunology , Interleukin-3/physiology , Passive Cutaneous Anaphylaxis/immunology , Peritonitis/immunology , Rhinitis, Allergic, Seasonal/immunology , Allergens/immunology , Ambrosia/immunology , Anaphylaxis/etiology , Animals , Disease Models, Animal , Mast Cells/cytology , Mast Cells/metabolism , Mice , Peritoneal Cavity/cytology , Serotonin/biosynthesis
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