M-sec regulates polarized secretion of inflammatory endothelial chemokines and facilitates CCL2-mediated lymphocyte transendothelial migration.

Activation of endothelial cells by IL-1ß triggers the expression of multiple inflammatory cytokines and leukocyte-attracting chemokines. The machineries involved in the secretion of these inducible proteins are poorly understood. With the use of genome-wide transcriptional analysis of inflamed human dermal microvascular endothelial cells, we identified several IL-1ß-induced candidate regulators of these machineries and chose to focus our study on TNF-α-induced protein 2 (myeloid-secretory). The silencing of myeloid-secretory did not affect the ability of inflamed endothelial cells to support the adhesion and crawling of effector T lymphocytes. However, the ability of these lymphocytes to complete transendothelial migration across myeloid-secretory-silenced human dermal microvascular endothelial cells was inhibited significantly. These observed effects on lymphocyte transendothelial migration were recovered completely when exogenous promigratory chemokine CXCL12 was overlaid on the endothelial barrier. A polarized secretion assay suggested that the silencing of endothelial myeloid-secretory impairs T effector transendothelial migration by reducing the preferential secretion of endothelial-produced CCL2, a key transendothelial migration-promoting chemokine for these lymphocytes, into the basolateral endothelial compartment. Myeloid-secretory silencing also impaired the preferential secretion of other endothelial-produced inflammatory chemokines, as well as cytokines, such as IL-6 and GM-CSF, into the basolateral endothelial compartment. This is the first evidence of a novel inflammation-inducible machinery that regulates polarized secretion of endothelial CCL2 and other inflammatory chemokines and cytokines into basolateral endothelial compartments and facilitates the ability of endothelial CCL2 to promote T cell transendothelial migration.

Breast cancer: coordinated regulation of CCL2 secretion by intracellular glycosaminoglycans and chemokine motifs.

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (=GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 ((45)TIVA(48)). We have identified partial roles for R18+K19, H66, and the (45)TIVA(48) motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19+(45)TIVA(48), the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, (45)TIVA(48) and R18+K19+(45)TIVA(48) domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).

Mechanisms regulating the secretion of the promalignancy chemokine CCL5 by breast tumor cells: CCL5's 40s loop and intracellular glycosaminoglycans.

The chemokine CCL5 (RANTES) plays active promalignancy roles in breast malignancy. The secretion of CCL5 by breast tumor cells is an important step in its tumor-promoting activities; therefore, inhibition of CCL5 secretion may have antitumorigenic effects. We demonstrate that, in breast tumor cells, CCL5 secretion necessitated the trafficking of CCL5-containing vesicles on microtubules from the endoplasmic reticulum (ER) to the post-Golgi stage, and CCL5 release was regulated by the rigidity of the actin cytoskeleton. Focusing on the 40s loop of CCL5, we found that the (43)TRKN(46) sequence of CCL5 was indispensable for its inclusion in motile vesicles, and for its secretion. The TRKN-mutated chemokine reached the Golgi, but trafficked along the ER-to-post-Golgi route differently than the wild-type (WT) chemokine. Based on the studies showing that the 40s loop of CCL5 mediates its binding to glycosaminoglycans (GAG), we analyzed the roles of GAG in regulating CCL5 secretion. TRKN-mutated CCL5 had lower propensity for colocalization with GAG in the Golgi compared to the WT chemokine. Secretion of WT CCL5 was significantly reduced in CHO mutant cells deficient in GAG synthesis, and the WT chemokine acquired an ER-like distribution in these cells, similar to that of TRKN-mutated CCL5 in GAG-expressing cells. The release of WT CCL5 was also reduced after inhibition of GAG presence/synthesis by intracellular expression of heparanase, inhibition of GAG sulfation, and sulfate deprivation. The need for a (43)TRKN(46) motif and for a GAG-mediated process in CCL5 secretion may enable the future design of modalities that prevent CCL5 release by breast tumor cells.