Hyaluronan and Its Receptors: Key Mediators of Immune Cell Entry and Trafficking in the Lymphatic System

Entry to the afferent lymphatics marks the first committed step for immune cell migration from tissues to draining lymph nodes both for the generation of immune responses and for timely resolution of tissue inflammation. This critical process occurs primarily at specialised discontinuous junctions in initial lymphatic capillaries, directed by chemokines released from lymphatic endothelium and orchestrated by adhesion between lymphatic receptors and their immune cell ligands. Prominent amongst the latter is the large glycosaminoglycan hyaluronan (HA) that can form a bulky glycocalyx on the surface of certain tissue-migrating leucocytes and whose engagement with its key lymphatic receptor LYVE-1 mediates docking and entry of dendritic cells to afferent lymphatics. Here we outline the latest insights into the molecular mechanisms by which the HA glycocalyx together with LYVE-1 and the related leucocyte receptor CD44 co-operate in immune cell entry, and how the process is facilitated by the unusual character of LYVE-1 • HA-binding interactions. In addition, we describe how pro-inflammatory breakdown products of HA may also contribute to lymphatic entry by transducing signals through LYVE-1 for lymphangiogenesis and increased junctional permeability. Lastly, we outline some future perspectives and highlight the LYVE-1 • HA axis as a potential target for immunotherapy.

23. Constitutively Active Signal Transducer and Activator of Transcription-3; an Oncogene that Increases Gap Junctional, Intercellular Communication

Gap junctions are specialized intercellular channels that connect the cytoplasm of adjacent cells and mediate the direct exchange of small ions and molecules between them. Gap junctional intercellular communication (GJIC) is usually blocked, or down regulated, in cells transformed by oncogenes, such as Src. One of the Src effector pathways leading to transformation and GJIC suppression is the Ras/Raf/MEK/ERK pathway that has a prominent etiological role in cell proliferation, differentiation and cell survival; inhibition of this pathway in vSrc transformed cells restores GJIC. In addition, the distinct downstream effector of Src that is obligatory for neoplasia is the signal transducer and activator of transcription-3 (Stat3). Stat3 is up regulated in a range of tumors, and a modified version, the constitutively active form of Stat3 (Stat3C), has been shown to function as an oncogene. To examine the role of Stat3 upon the Src-mediated, GJIC suppression, Stat3 was down regulated in rat liver epithelial T51B cells expressing activated Src. The extent of GJIC was determined by the migration of the fluorescent dye, Lucifer Yellow, through adherent cells subsequent to electroporation. The results demonstrate that, contrary to inhibition of the Ras pathway, Stat3 inhibition in cells expressing activated Src does not restore GJIC. On the contrary, Stat3 inhibition in normal cells with high GJIC levels eliminated junctional permeability. Interestingly, our results also demonstrate that expression of Stat3C T51B cells and human lung cancer SK-LuCi6 cells, which have extensive communication and low Src levels, increased GJIC. Therefore, Stat3 is actually required for and increases junctional permeability.

6. Effect of Erk vs. PI3 Kinase Activation by the Middle Tumour Antigen of Polyoma Virus Upon Gap Junctional, Intercellular Communication of Cultured Cells

Gap junctions are protein channels that permit the passage of small molecules and ions between adjacent cells. Gap junctional permeability is thought to lead to decreased cellular proliferation. In fact, a number of oncogenes, such as the middle tumour antigen of polyoma virus (mT), are known to interrupt gap junctional, intercellular communication (GJIC). The Ras/Raf/Mek/Erk and phosphoinositide 3‐kinase (PI3 kinase)/Akt signalling pathways, two commonly studied pathways in malignancy, are both activated by mT, and are both required for complete transformation by mT. This study is the first to investigate the effects of Erk vs. PI3 kinase activation by mT on GJIC in cultured cells. To this effect, two mT mutants,impaired in their ability to activate either the Erk or PI3 kinase pathway, were expressed through retroviral infection in rat liver epithelial T51B cells which have extensive GJIC. A novel in situ electroporation technique was used to quantitate the degree of GJIC in T51B cells expressing each mT mutant. The results showed that the Erk‐activating mutant exhibited the interrupted GJIC that is characteristic of mT expression, while the PI3 kinase‐activating mutant displayed levels of GJIC comparable to those observed in wild‐type T51B cells. This suggests that Erk activation by mT is sufficient to suppress GJIC, while PI3 kinase activation, despite its contribution to neoplastic transformation, is unable to interrupt GJIC. Given the importance of GJIC in cellular proliferation, apoptosis and differentiation, identifying the pathways affecting gap junctional permeability may yield novel targets for cancer therapy.

MarvelD3 couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1–c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival.