The Atypical Receptor CCRL2 (C-C Chemokine Receptor-Like 2) Does Not Act As a Decoy Receptor in Endothelial Cells.

C-C chemokine receptor-like 2 (CCRL2) is a non-signaling seven-transmembrane domain (7-TMD) receptor related to the atypical chemokine receptor (ACKR) family. ACKRs bind chemokines but do not activate G protein-dependent signaling or cell functions. ACKRs were shown to regulate immune functions in vivo by their ability to scavenge chemokines from the local environment. This study was performed to investigate whether CCRL2 shares two of the main characteristics of ACKRs, namely the ability to internalize and scavenge the ligands. Cell membrane analysis of CCRL2-transfected cells revealed a weak, constitutive, ligand-independent internalization, and recycling of CCRL2, with a kinetics that was slower than those observed with ACKR3, a prototypic ACKR, or other chemotactic signaling receptors [i.e., chemokine-like receptor 1 and C-X-C motif chemokine receptor 2]. Intracellularly, CCRL2 colocalized with early endosome antigen 1-positive and Rab5-positive vesicles and with recycling compartments mainly characterized by Rab11-positive vesicles. CCRL2-transfected cells and activated mouse blood endothelial cells, that endogenously express CCRL2, were used to investigate the scavenging ability of CCRL2. These experiments confirmed the ability of CCRL2 to bind chemerin, the only recognized ligand, but excluded the ability of CCRL2 to perform scavenging. Collectively, these results identify unique functional properties for this member of the non-signaling 7-TMD receptor family.

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate.

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

Dendritic cell-derived VEGF-A plays a role in inflammatory angiogenesis of human secondary lymphoid organs and is driven by the coordinated activation of multiple transcription factors.

Lymph node expansion during inflammation is essential to establish immune responses and relies on the development of blood and lymph vessels. Previous work in mice has shown that this process depends on the presence of VEGF-A produced by B cells, macrophages and stromal cells. In humans, however, the cell types and the mechanisms regulating the intranodal production of VEGF-A remain elusive. Here we show that CD11c+ cells represent the main VEGF-A-producing cell population in human reactive secondary lymphoid organs. In addition we find that three transcription factors, namely CREB, HIF-1α and STAT3, regulate the expression of VEGF-A in inflamed DCs. Both HIF-1α and STAT3 are activated by inflammatory agonists. Conversely, CREB phosphorylation represents the critical contribution of endogenous or exogenous PGE2. Taken together, these results propose a crucial role for DCs in lymph node inflammatory angiogenesis and identify novel potential cellular and molecular targets to limit inflammation in chronic diseases and tumors.

Pro-lymphangiogenic properties of IFN-γ-activated human dendritic cells.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses. In addition, through the release of pro- and anti-angiogenic mediators, DCs are key regulators of blood vessel remodeling, a process that characterizes inflammation. Less information is available on the role of DCs in lymphangiogenesis. This study reports that human DCs produce VEGF-C, a cytokine with potent pro-lymphangiogenic activity when stimulated with IFN-γ. DC-derived VEGF-C was biologically active, being able to promote tube-like structure formation in cultures of human lymphatic endothelial cells (LECs). DCs co-cultured with IL-15-activated NK cells produced high levels of VEGF-C, suggesting a role for NK-DC cross-talk in peripheral lymphoid and non-lymphoid tissues in inflammation-associated lymphangiogenesis. Induction of VEGF-C by IFN-γ was detected also in other myeloid cells, such as blood-purified CD1c(+) DCs, CD14(+) monocytes and in monocyte-derived macrophages. In all these cell types, VEGF-C was found associated with the cell membrane by low affinity, heparan sulphate-mediated, interactions. Therefore, human DCs should be considered as new players in inflammation-associated lymphangiogenesis.

Angiogenic and antiangiogenic chemokines.

Chemokines are a family of vertebrate-specific, small-secreted molecules that were originally identified as mediators of leukocyte migration and tissue positioning during the immune response. Subsequently, chemokines were discovered to control movement also of endothelial cells and other cell types in many different contexts. The human chemokine system comprises about 50 chemokines and more than 20 receptors belonging to the seven-transmembrane receptor family. In the present chapter, we review the literature supporting a role for chemokines in angiogenesis and lymphangiogenesis. We highlight that chemokines exert both pro- and antiangiogenic roles either by acting directly on endothelial cells or by recruiting leukocytes that, in turn, secrete angiogenic mediators. This latter mode of action is possibly the most relevant in tumor angiogenesis. Finally, we explore the angiogenic properties of nonchemokine chemoattractant molecules.

Dihydroceramide delays cell cycle G1/S transition via activation of ER stress and induction of autophagy.

Dihydroceramides, the precursors of ceramides in the de novo sphingolipid synthesis, have been recently implicated in active signalling. We previously demonstrated that dihydroceramide accumulation, in response to treatment with the dihydroceramide desaturase inhibitor XM462, induced autophagy with no sign of cell death in the gastric carcinoma HCG27 cell line. Here we show that XM462 treatment induces a transient early increase in dihydroceramides that are successively metabolized into other sphingolipids. Dihydroceramides accumulation is associated with cyclin D1 expression modulation, delayed G1/S transition of cell cycle and increased autophagy. Moreover, XM462 treatment induces ER stress via the activation of the translation inhibitor eIF2α and the pro-survival transcriptional factor Xbp1. Exogenous addition of a short chain dihydroceramide analog reproduces the effects of endogenous accumulation of dihydroceramides, causing cell cycle delay of the G1/S transition, autophagy enhancement, eIF2α activation and Xbp1 splicing. Blocking autophagy with 3-methyladenine abrogates the effect of XM462 on cell cycle and reduces cell survival to XM462 treatment. Furthermore, the XM462-induced survival response is able to reduce etoposide toxicity in HCG27 and HCT116 cancer cells. Our data suggest a role of dihydroceramide in regulating cell proliferation and survival.

Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena.

Sphingolipids are a wide family of lipids that share common sphingoid backbones, including (2S,3R)-2-amino-4-octadecane-1,3-diol (dihydrosphingosine) and (2S,3R,4E)-2-amino-4-octadecene-1,3-diol (sphingosine). The metabolism and biological functions of sphingolipids derived from sphingosine have been the subject of many reviews. In contrast, dihydrosphingolipids have received poor attention, mainly due to their supposed lack of biological activity. However, the reported biological effects of active site directed dihydroceramide desaturase inhibitors and the involvement of dihydrosphingolipids in the response of cells to known therapeutic agents support that dihydrosphingolipids are not inert but are in fact biologically active and underscore the importance of elucidating further the metabolic pathways and cell signaling networks involved in the biological activities of dihydrosphingolipids. Dihydroceramide desaturase is the enzyme involved in the conversion of dihydroceramide into ceramide and it is crucial in the regulation of the balance between sphingolipids and dihydrosphingolipids. Furthermore, given the enzyme requirement for O2 and the NAD(P)H cofactor, the cellular redox balance and dihydroceramide desaturase activity may reciprocally influence each other. In this review both dihydroceramide desaturase and the biological functions of dihydrosphingolipids are addressed and perspectives on this field are discussed.

Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells.

Resveratrol has both apoptosis and autophagy-promoting activities in different cancer cells. Dihydroceramide is the immediate precursor of the apoptotic mediator ceramide in the de novo sphingolipid synthesis pathway. Here we demonstrate that resveratrol induces autophagy in HGC-27 cells, with no sign of cell death. Autophagy occurs after an increase in dihydroceramides by inhibition of dihydroceramide desaturase. The effects of resveratrol are mimicked by a dihydroceramide desaturase inhibitor. These results demonstrate that resveratrol-induced autophagy occurs with a rise in intracellular dihydroceramide levels as the result of inhibition of dihydroceramide desaturases activity and that dihydroceramide accumulation is responsible for autophagy promotion.