Intracellular recycling pathways play critical roles in internalizing membrane and fluid phase cargo and in balancing the inflow and outflow of membrane and cell surface molecules. To identify proteins involved in the regulation of endocytic recycling, we used an shRNA trafficking library and screened for changes in the surface expression of CD1a antigen-presenting molecules that follow an endocytic recycling route. We found that silencing of the ADP-ribosylation factor (Arf)-like small GTPase Arl13b led to a decrease in CD1a surface expression, diminished CD1a function, and delayed CD1a recycling, suggesting that Arl13b is involved in the regulation of endocytic recycling traffic. Arl13b appears to be required for the major route of endocytic trafficking, causing clustering of early endosomes and leading to the accumulation of endocytic cargo. Moreover, Arl13b colocalized with markers of the endocytic recycling pathway followed by CD1a, namely Arf6 and Rab22a. We also detected an interaction between Arl13b and the actin cytoskeleton. Arl13b was previously implicated in cilia formation and function. Our present results indicate a previously unidentified role for Arl13b in endocytic recycling traffic and suggest a link between Arl13b function and the actin cytoskeleton.immune response | lipid antigens | ciliary traffic | vesicle traffic E ukaryotic cells sample the extracellular milieu to take up fluid and receptor-bound ligands. Many receptors release their ligands in the mildly acidic environment of early endosomes. From this compartment, also known as the sorting endosome, membrane-bound receptors and other plasma membrane proteins can recycle back to the plasma membrane, whereas fluid phase cargo and membrane proteins destined for utilization or degradation follow the endocytic pathway to late endosomes and lysosomes. Recycling back to the plasma membrane can occur via a fast recycling pathway or, alternatively, a slow recycling pathway through the perinuclear endocytic recycling compartment (ERC). Cells take up considerable amounts of membrane from the cell surface; thus, recycling pathways are essential to return membrane, membrane proteins, and lipids to the cell surface, balancing the internalized flow. Along with this function, regulated recycling has been shown to be important for cytokinesis, cell adhesion, morphogenesis, cell fusion, and even learning and memory (1).However, the machinery involved in the sorting and trafficking of recycling cargo through the ERC remains incompletely understood. We previously reported that CD1a, an MHC class I-like antigen-presenting molecule that presents lipids rather than peptides to T cells, follows an endocytic recycling pathway similar to that used by MHC class I and other cargo internalized independent of clathrin (2). This pathway has been shown to depend on the small GTPases Rab22a and Arf6 (3, 4).To uncover previously unknown proteins involved in regulation of the endocytic recycling pathway followed by CD1a and other clathrin-independent cargo, we developed ...

Objective: The aim of this study was to clarify the role of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) pathways in thyroid tumourigenesis. Methods: We examined VEGF, VEGFR-1 and VEGFR-2 expression on 34 papillary thyroid carcinomas (PTCs), 18 follicular thyroid carcinomas (FTCs), eight poorly differentiated thyroid carcinomas (PDTCs) and on a thyroid tumour-derived cell line (NPA 0 87) by immunohistochemistry, reverse transcriptase PCR, immunofluorescence and Western blotting. Results: We have demonstrated that VEGF expression was significantly (P , 0.05) more prevalent in PTCs (79%) than in FTCs (50%) or PDTCs (37%). Similarly, 76% of PTCs, 83% of FTCs and 25% of PDTCs expressed VEGFR-1, whereas 68% of PTCs, 56% of FTCs and 37% of PDTCs expressed VEGFR-2. Coexpression of VEGF and its receptors was observed in 50% of PTCs, 39% of FTCs and 12% of PDTCs, raising the possibility that VEGF may signal in an autocrine loop in these neoplasias, as observed previously for other types of cancer. In agreement with the idea that autocrine VEGF signalling plays an important role in thyroid carcinogenesis, the blockade of either VEGF or its receptors with neutralizing antibodies significantly reduced cell viability and increased apoptosis levels of the VEGFR-positive thyroid tumour cell line NPA 0 87. Conclusions: Our results highlight a previously undefined VEGF autocrine action in thyroid carcinomas which could play a crucial role in tumour cell survival and could represent a useful therapeutic target for thyroid tumours.European Journal of Endocrinology 153 701-709

The chemokine receptor CCR7 plays a critical role in lymphocyte and dendritic cell trafficking into and within lymph nodes, the preferential metastatic site for papillary (PTC) and medullary (MTC) thyroid carcinomas. In order to determine a possible role for CCR7 in mediating the metastatic behaviour of thyroid carcinomas, we analysed its expression in normal and tumoral thyroid tissues of different histotypes and studied the in vitro effects of its activation by the CCR7 ligand, CCL21. Using real-time quantitative-PCR, we observed that CCR7 expression was higher in PTCs and MTCs than in follicular and poorly differentiated thyroid carcinomas. CCR7 expression was ninefold higher in classic compared with follicular variants of PTCs, and its expression in MTCs was significantly correlated with lymph node metastases. Immunohistochemical staining for CCR7 showed protein expression in neoplastic thyroid cells, with higher intensity in PTCs, MTCs and their lymph node metastases (LNMs). We further showed that CCL21 stimulation of a CCR7-expressing thyroid tumour cell line (TPC-1) promotes cell proliferation and migration, and the chemotactic effect of CCL21 in these cells involves actin polymerization, increased b1-integrin expression and increased matrix metalloproteinase secretion. Taken together, our results demonstrate that CCR7 activation on thyroid carcinoma cells by CCL21 -a chemokine abundantly expressed in lymph nodes -favours tissue invasion and cell proliferation, and therefore may promote thyroid carcinoma growth and LNM.

Members of the ADP-ribosylation factor (Arf) family of small GTP-binding (G) proteins regulate several aspects of membrane trafficking, such as vesicle budding, tethering and cytoskeleton organization. Arf family members, including Arf-like (Arl) proteins have been implicated in several essential cellular functions, like cell spreading and migration. These functions are used by cancer cells to disseminate and invade the tissues surrounding the primary tumor, leading to the formation of metastases. Indeed, Arf and Arl proteins, as well as their guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) have been found to be abnormally expressed in different cancer cell types and human cancers. Here, we review the current evidence supporting the involvement of Arf family proteins and their GEFs and GAPs in cancer progression, focusing on 3 different mechanisms: cell-cell adhesion, integrin internalization and recycling, and actin cytoskeleton remodeling.