Endothelial Rbpj is essential for the education of tumour-associated macrophages

Epithelial ovarian cancer (EOC) is one of the most lethal gynaecological cancers worldwide. EOC cells educate tumour-associated macrophages (TAMs) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumour microenvironment (TME). In addition, tumour cells frequently activate Notch1 receptors on endothelial cells (ECs) to facilitate metastasis. However, little is known whether the endothelium would also influence the education of recruited monocytes. Here, we report that canonical Notch signalling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature. This was associated with stronger cytotoxic activity of T cells and decreased tumour burden. Mechanistically, we identified CXCL2 as a novel Notch/RBPJ target gene. This angiocrine factor regulates the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. As such, EOC cells employ the tumour endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment.

Impaired Retromer Function in Niemann-Pick Type C Disease Is Dependent on Intracellular Cholesterol Accumulation

Niemann-Pick type C disease (NPC) is a rare inherited neurodegenerative disorder characterized by an accumulation of intracellular cholesterol within late endosomes and lysosomes due to NPC1 or NPC2 dysfunction. In this work, we tested the hypothesis that retromer impairment may be involved in the pathogenesis of NPC and may contribute to increased amyloidogenic processing of APP and enhanced BACE1-mediated proteolysis observed in NPC disease. Using NPC1-null cells, primary mouse NPC1-deficient neurons and NPC1-deficient mice (BALB/cNctr-Npc1m1N), we show that retromer function is impaired in NPC. This is manifested by altered transport of the retromer core components Vps26, Vps35 and/or retromer receptor sorLA and by retromer accumulation in neuronal processes, such as within axonal swellings. Changes in retromer distribution in NPC1 mouse brains were observed already at the presymptomatic stage (at 4-weeks of age), indicating that the retromer defect occurs early in the course of NPC disease and may contribute to downstream pathological processes. Furthermore, we show that cholesterol depletion in NPC1-null cells and in NPC1 mouse brains reverts retromer dysfunction, suggesting that retromer impairment in NPC is mechanistically dependent on cholesterol accumulation. Thus, we characterized retromer dysfunction in NPC and propose that the rescue of retromer impairment may represent a novel therapeutic approach against NPC.

The amount range of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition.

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we develop a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated well (called as “DA-X condition”) showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.

Cytokine receptor cluster size impacts its endocytosis and signaling

The interleukin-2 receptor (IL-2R) is a cytokine receptor essential for immunity that transduces proliferative signals regulated by its uptake and degradation. IL-2R is a well-known marker of clathrin-independent endocytosis (CIE), a process devoid of any coat protein, raising the question of how the CIE vesicle is generated. Here, we investigated the impact of IL-2Rγ clustering in its endocytosis. Combining total internal reflection fluorescence (TIRF) live imaging of a CRISPR-edited T cell line endogenously expressing IL-2Rγ tagged with green fluorescent protein (GFP), with multichannel imaging, single-molecule tracking, and quantitative analysis, we were able to decipher IL-2Rγ stoichiometry at the plasma membrane in real time. We identified three distinct IL-2Rγ cluster populations. IL-2Rγ is secreted to the cell surface as a preassembled small cluster of three molecules maximum, rapidly diffusing at the plasma membrane. A medium-sized cluster composed of four to six molecules is key for IL-2R internalization and is promoted by interleukin 2 (IL-2) binding, while larger clusters (more than six molecules) are static and inefficiently internalized. Moreover, we identified membrane cholesterol and the branched actin cytoskeleton as key regulators of IL-2Rγ clustering and IL-2–induced signaling. Both cholesterol depletion and Arp2/3 inhibition lead to the assembly of large IL-2Rγ clusters, arising from the stochastic interaction of receptor molecules in close correlation with their enhanced lateral diffusion at the membrane, thus resulting in a default in IL-2R endocytosis. Despite similar clustering outcomes, while cholesterol depletion leads to a sustained IL-2–dependent signaling, Arp2/3 inhibition prevents signal initiation. Taken together, our results reveal the importance of cytokine receptor clustering for CIE initiation and signal transduction.

Abstract P368: Caveolin-3 Prevents Swelling-induced Cell Lysis Via Regulation Of I Cl,swell Expression And Activity

Background: Caveolae membrane structures harbor mechanosensitive chloride channels (MCCs) which form a swelling-activated chloride current ( I Cl,swell ) and play an important role in cell volume regulation and mechano-electrical signal transduction. However, the role of muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCCs expression, activity, and contribution to cell viability in response to mechanical stress remains unclear. We hypothesized that Cav3-based mechano-protection is enabled by complimentary expression of MCCs. Methods and Results: Experiments were performed on native (Cav3-) and Cav3-transfected (Cav3+) HEK-293 cells. Cell stretch was mimicked by light (220 mOsM) or extreme hypoosmotic swelling (<20mOsM). Cav3+ HEK-293 cells were significantly resistant to extreme hypotonic solutions (15 minute incubation) compared to Cav3- HEK-293 cells, and this mechano-protection was significantly reduced when exposed to I Cl,swell selective inhibitor DCPIB (1 μM). We found that three MCCs (ClC-2, ClC-3, and SWELL1, also known as LRRC8A) contain caveolin-binding motifs in their structure, indicating their possible localization in caveolae structures. Co-IP analysis confirmed association of SWELL1 with Cav3. Interestingly, Cav3+ HEK-293 cells showed a significant (by 2-fold) increase of SWELL1 protein level, while ClC-2/3 protein levels remained unchanged. This was accompanied by a 2-fold increase of I Cl,swell , but no change in mRNA expression levels. FRET analysis showed a <10 nm membrane and intracellular association between Cav3 and tested MCCs. Furthermore, Cav3/SWELL1 membrane FRET efficiency was halved in light hypoosmotic solution, as well as after disruption of caveolae structures via cholesterol depletion by 1-hour treatment with 10 μM methyl-β-cyclodextrin. Cav3/ClC-2/3 average membrane FRET efficiency remained unchanged in hypotonic solution. Conclusions: We concluded that of MCCs tested, SWELL1 abundance and activity is regulated by Cav3 and that their association relies on membrane tension and caveolae integrity. The present study highlights the mechano-protective properties of Cav3 which are partially facilitated by complimentary SWELL1 expression and activity.

Abstract P413: Stretch-induced Caveolae-mediated Disruption Of Cyclic AMP Microdomains Leads To Arrhythmogenic Ca 2+ Mishandling In Mouse Atrial Myocytes

Background: Atrial fibrillation (AF) often occurs during hypertension and is associated with an increase in cardiomyocyte stretch. Mechanism of ectopic beats, that trigger AF, has been linked to Ca 2+ mishandling and leaky hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain elusive. Caveolae membrane structures are involved in cell mechanosensing processes and control the cAMP signaling pathway. We hypothesized that mechanical stretch disrupts caveolae, promoting cAMP production and sarcoplasmic reticulum Ca 2+ leak via augmentation of RyRs phosphorylation. Methods and Results: Cell size analysis and Ca 2+ dynamics measurements were performed by confocal imaging of isolated mouse atrial myocytes. Cell stretch was modeled by hypoosmotic swelling (from 310 mOsM to 220 mOsM to flatten caveolae structures) resulting in a ~30% increase in cell width (p<0.05) with no changes in cell length. Swelling resulted in a biphasic effect on Ca 2+ spark activity: a fast (<10 min of exposure) ~50% increase (p<0.001) followed by a slow decrease to the level observed in isotonic conditions (>30 min of exposure). Similarly, caveolae disruption via cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) led to 2-fold increase in Ca 2+ sparks frequency (p<0.001). Swelling- and MβCD-induced increases in atrial Ca 2+ spark activity were prevented via inhibition of cAMP production by adenylyl cyclases by 0.1mM SQ22536 or cAMP-dependent protein kinase A (PKA) by 1μM H-89. Then, we tested if this mechanism is present in atrial myocytes from pressure-overloaded (4-weeks transaortic constriction, TAC) mice. Atrial myocytes from TAC mice showed a 1.6 times higher Ca 2+ sparks frequency than wild-type myocytes (p<0.01), which was significantly reduced (p<0.01) to wild-type level after incubation with SQ22536. Conclusions: Our findings suggest that cell stretch increases spontaneous Ca 2+ spark activity through the disruption of caveolae and cAMP-mediated augmentation of PKA activity. This mechanism could be involved in the Ca 2+ mishandling and AF in pressure overloaded hearts.