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Cosimo Commisso - One of the best experts on this subject based on the ideXlab platform.
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EGFR-Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis.
Developmental cell, 2019Co-Authors: Szu-wei Lee, Yijuan Zhang, Michael Jung, Nathalia Cruz, Basheer Alas, Cosimo CommissoAbstract:Macropinocytosis has emerged as an important nutrient-scavenging pathway that supports tumor cell fitness. By internalizing extracellular protein and targeting it for lysosomal degradation, this endocytic pathway functions as an amino acid supply route, permitting tumor cell growth and survival despite the nutrient-poor conditions of the tumor microenvironment. Here, we provide evidence that a subset of pancreatic ductal adenocarcinoma (PDAC) tumors are wired to integrate contextual metabolic inputs to regulate Macropinocytosis, dialing up or down this uptake pathway depending on nutrient availability. We find that regional depletion of amino acids coincides with increased levels of Macropinocytosis and that the scarcity of glutamine uniquely drives this process. Mechanistically, this stimulation of Macropinocytosis depends on the nutrient stress-induced potentiation of epidermal growth factor receptor signaling that, through the activation of Pak, controls the extent of Macropinocytosis in these cells. These results provide a mechanistic understanding of how nutritional cues can control protein scavenging in PDAC tumors.
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Macropinocytosis in Cancer: A Complex Signaling Network.
Trends in cancer, 2019Co-Authors: Yijuan Zhang, Cosimo CommissoAbstract:Macropinocytosis is an important nutrient-scavenging pathway in numerous cancer types, including pancreatic, lung, prostate, and bladder. This Forum highlights recent work identifying the key regulators of Macropinocytosis that support tumor cell fitness in different contexts, providing a unique framework for strategies to target Macropinocytosis in the treatment of cancer.
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Macropinocytosis: A Metabolic Adaptation to Nutrient Stress in Cancer.
Frontiers in endocrinology, 2017Co-Authors: Maria Victoria Recouvreux, Cosimo CommissoAbstract:Oncogenic mutations, such as Ras mutations, drive not only enhanced proliferation but also the metabolic adaptations that confer to cancer cells the ability to sustain cell growth in a harsh tumor microenvironment. These adaptations might represent metabolic vulnerabilities that can be exploited to develop novel and more efficient cancer therapies. Macropinocytosis is an evolutionarily conserved endocytic pathway that permits the internalization of extracellular fluid via large endocytic vesicles known as macropinosomes. Recently, Macropinocytosis has been determined to function as a nutrient-scavenging pathway in Ras-driven cancer cells. Macropinocytic uptake of extracellular proteins, and their further degradation within endolysosomes, provides the much-needed amino acids that fuel cancer cell metabolism and tumor growth. Here, we review the molecular mechanisms that govern the process of Macropinocytosis, as well as discuss recent work that provides evidence of the important role of Macropinocytosis as a nutrient supply pathway in cancer cells.
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High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven Macropinocytosis.
Assay and drug development technologies, 2015Co-Authors: Myles Fennell, Cosimo Commisso, Craig Ramirez, Ralph Garippa, Dafna Bar-sagiAbstract:Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is Macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of Macropinocytosis. To measure oncogene-dependent Macropinocytosis, we used HeLa cells expressing oncogenic HRAS(G12D) driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of Macropinocytosis. We detected Macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70 kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent Macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of Macropinocytosis as well as novel targets.
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Abstract PR12: Bringing the outside in: Macropinocytosis and cancer therapeutics
Cancer Metabolism, 2013Co-Authors: Cosimo Commisso, Elda Grabocka, Craig Ramirez, Venugopal Chenna, Maitra Anirban, Dafna Bar-sagiAbstract:Oncogenic Ras mutations are prevalent in a variety of tumor types, including adenocarcinomas of the pancreas, colon, and lung. One of the most overt phenotypes associated with the expression of oncogenic Ras mutants is the stimulation of Macropinocytosis, an endocytic process that involves extensive membrane remodeling and the internalization of extracellular fluid via large membrane-bound vesicles called macropinosomes. The functional consequences of this stimulation in oncogenic Ras-expressing cancer cells were unknown prior to our recent work where we linked macropinocytic uptake to nutrient delivery and amino acid supply in tumor cells. We found that Ras-transformed cells utilize Macropinocytosis to internalize extracellular albumin, which is then lysosomally degraded releasing the constituent amino acids intracellularly. These protein-derived amino acids have the capacity to enter central carbon metabolism and fuel tumor cell proliferation even in a nutrient-depleted environment. Of particular relevance is the finding that the pharmacological inhibition of Macropinocytosis compromises the growth of Ras-driven pancreatic xenograft tumors. This discovery raises the question of whether the inhibition of Macropinocytosis can be utilized as a therapeutic intervention in a subset of cancers. Moreover, because Macropinocytosis is an established mechanism of drug delivery for nanoparticles, our work highlights the possibility of exploiting this feature of Ras-induced tumors for the effective delivery of nanoscale therapeutics. Our recent studies focused on identifying novel modulators of Macropinocytosis and examining Macropinocytosis as a delivery mechanism for nab-paclitaxel, an albumin-based nanoparticle therapy, will be discussed. Citation Format: Cosimo Commisso, Venugopal Chenna, Elda Grabocka, Craig Ramirez, Maitra Anirban, Dafna Bar-Sagi. Bringing the outside in: Macropinocytosis and cancer therapeutics. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR12.
Sergio Grinstein - One of the best experts on this subject based on the ideXlab platform.
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SLIT2/ROBO1-signaling inhibits Macropinocytosis by opposing cortical cytoskeletal remodeling
Nature communications, 2020Co-Authors: Vikrant K. Bhosle, Michael Glogauer, Tapas Mukherjee, Yi-wei Huang, Sajedabanu Patel, Bo Wen Frank Pang, Guang-ying Liu, Dana J. Philpott, Sergio GrinsteinAbstract:Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce Macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits Macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits Macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of Macropinocytosis and challenge the conventional notion that signals that enhance Macropinocytosis negatively regulate cell migration, and vice versa.
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constitutive and stimulated Macropinocytosis in macrophages roles in immunity and in the pathogenesis of atherosclerosis
Philosophical Transactions of the Royal Society B, 2019Co-Authors: Sergio Grinstein, Sasha A Doodnauth, Michelle E MaxsonAbstract:Macrophages respond to several stimuli by forming florid membrane ruffles that lead to fluid uptake by Macropinocytosis. This type of induced Macropinocytosis, executed by a variety of non-malignant and malignant cells, is initiated by transmembrane receptors and is involved in nutrient acquisition and mTOR signalling. However, macrophages also perform a unique type of constitutive ruffling and Macropinocytosis that is dependent on the presence of extracellular calcium. Calcium-sensing receptors are responsible for this activity. This distinct form of Macropinocytosis enables macrophages to continuously sample their microenvironment for antigenic molecules and for pathogen- and danger-associated molecular patterns, as part of their immune surveillance functions. Interestingly, even within the monocyte lineage, there are differences in macropinocytic ability that reflect the polarized functional roles of distinct macrophage subsets. This review discusses the shared and distinct features of both induced and constitutive Macropinocytosis displayed by the macrophage lineage and their roles in physiology, immunity and pathophysiology. In particular, we analyse the role of Macropinocytosis in the uptake of modified low-density lipoprotein (LDL) and its contribution to foam cell and atherosclerotic plaque formation. We propose a combined role of scavenger receptors and constitutive Macropinocytosis in oxidized LDL uptake, a process we have termed 'receptor-assisted Macropinocytosis'. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.
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Constitutive and stimulated Macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis.
Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2018Co-Authors: Sasha A Doodnauth, Sergio Grinstein, Michelle E MaxsonAbstract:Macrophages respond to several stimuli by forming florid membrane ruffles that lead to fluid uptake by Macropinocytosis. This type of induced Macropinocytosis, executed by a variety of non-malignan...
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Calcium-sensing receptors signal constitutive Macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages
Nature communications, 2016Co-Authors: Johnathan Canton, Daniel Schlam, Christian Breuer, Michael Gütschow, Michael Glogauer, Sergio GrinsteinAbstract:Macropinocytosis can be induced in several cell types by stimulation with growth factors. In selected cell types, notably macrophages and dendritic cells, Macropinocytosis occurs constitutively, supporting the uptake of antigens for subsequent presentation. Despite their different mode of initiation and contrasting physiological roles, it is tacitly assumed that both types of Macropinocytosis are mechanistically identical. We report that constitutive Macropinocytosis is stringently calcium dependent, while stimulus-induced Macropinocytosis is not. Extracellular calcium is sensed by G-protein-coupled calcium-sensing receptors (CaSR) that signal Macropinocytosis through Gα-, phosphatidylinositol 3-kinase and phospholipase C. These pathways promote the recruitment of exchange factors that stimulate Rac and/or Cdc42, driving actin-dependent formation of ruffles and macropinosomes. In addition, the heterologous expression of CaSR in HEK293 cells confers on them the ability to perform constitutive Macropinocytosis. Finally, we show that CaSR-induced constitutive Macropinocytosis facilitates the sentinel function of macrophages, promoting the efficient delivery of ligands to cytosolic pattern-recognition receptors.
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Amiloride inhibits Macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling
The Journal of cell biology, 2010Co-Authors: Mirkka Koivusalo, Christopher Welch, Hisayoshi Hayashi, Cameron C. Scott, Moshe Kim, R. Todd Alexander, Nicolas Touret, Klaus M. Hahn, Sergio GrinsteinAbstract:Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na+/H+ exchange. Yet, the functional relationship between Na+/H+ exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated Macropinocytosis and Na+/H+ exchange, elevating cytosolic pH and stimulating Na+ influx. Remarkably, although inhibition of Na+/H+ exchange by amiloride or HOE-694 obliterated Macropinocytosis, neither cytosolic alkalinization nor Na+ influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of Macropinocytosis, an effect counteracted by Na+/H+ exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to Macropinocytosis its unique susceptibility to inhibitors of Na+/H+ exchange.
Craig B Thompson - One of the best experts on this subject based on the ideXlab platform.
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The Canonical Wnt Pathway Drives Macropinocytosis in Cancer
Cancer research, 2018Co-Authors: Gil Redelman-sidi, Craig B Thompson, Anna Binyamin, Isabella M. Gaeta, Wilhelm Palm, Paul B. Romesser, Scott W. Lowe, Mukta Bagul, John G. Doench, David E. RootAbstract:Macropinocytosis has emerged as an important pathway of protein acquisition in cancer cells, particularly in tumors with activated Ras such as pancreatic and colon cancer. Macropinocytosis is also the route of entry of Bacillus Calmette-Guerin (BCG) and other microbial therapies of cancer. Despite this important role in tumor biology and therapy, the full mechanisms by which cancer cells can activate Macropinocytosis remain incompletely defined. Using BCG uptake to assay Macropinocytosis, we executed a genome-wide shRNA screen for Macropinocytosis activators and identified Wnt pathway activation as a strong driver of Macropinocytosis. Wnt-driven Macropinocytosis was downstream of the beta catenin-dependent canonical Wnt pathway, was Pak1 dependent, and supported albumin-dependent growth in Ras-WT cells. In cells with activated Ras-dependent Macropinocytosis, pharmacologic or genetic inhibition of Wnt signaling suppressed Macropinocytosis. In a mouse model of Wnt-driven colonic hyperplasia via APC silencing, Wnt-activated Macropinocytosis stimulated uptake of luminal microbiota, a process reversed by topical pharmacologic inhibition of Macropinocytosis. Our findings indicate that Wnt pathway activation drives Macropinocytosis in cancer, and its inhibition could provide a therapeutic vulnerability in Wnt-driven intestinal polyposis and cancers with Wnt activation.
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Macropinocytosis of protein is an amino acid supply route in ras transformed cells
Nature, 2013Co-Authors: Cosimo Commisso, Michel Nofal, Rengi G Soydanerazeloglu, Elda Grabocka, Shawn M Davidson, Seth J Parker, Jurre J Kamphorst, Sean R Hackett, Jeffrey A Drebin, Craig B ThompsonAbstract:Oncogenic Ras has previously been shown to promote Macropinocytosis; here it is demonstrated that this process allows tumour cells to use extracellular proteins to generate amino acids necessary to support tumour growth. This paper describes a previously unrecognized pathway of nutrient supply to cancer cells. Oncogenic Ras proteins are known to promote Macropinocytosis, an endocytic process by which extracellular fluid and its contents are internalized into cells through vesicles known as macropinosomes. Dafna Bar-Sagi and colleagues now demonstrate that Ras-transformed cells can use this process to 'feed' on extracellular protein. The macropinocytosed protein undergoes degradation, giving rise to free amino acids necessary to support tumour growth. This finding suggests that inhibition of Macropinocytosis may be effective against a subset of cancers. Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate Macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown1,2,3. Here we show that Ras-transformed cells use Macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with Macropinocytosis representing an important route of nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour xenografts. These results identify Macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer therapies.
Gabor Csanyi - One of the best experts on this subject based on the ideXlab platform.
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PKCδ stimulates Macropinocytosis via activation of SSH1-cofilin pathway.
Cellular signalling, 2018Co-Authors: Bhupesh Singla, Pushpankur Ghoshal, Hui Ping Lin, Mary Cherian-shaw, Gabor CsanyiAbstract:Macropinocytosis is an actin-dependent endocytic mechanism mediating internalization of extracellular fluid and associated solutes into cells. The present study was designed to identify the specific protein kinase C (PKC) isoform(s) and downstream effectors regulating actin dynamics during Macropinocytosis. We utilized various cellular and molecular biology techniques, pharmacological inhibitors and genetically modified mice to study the signaling mechanisms mediating Macropinocytosis in macrophages. The qRT-PCR experiments identified PKCδ as the predominant PKC isoform in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated the functional role of PKCδ in phorbol ester- and hepatocyte growth factor (HGF)-induced Macropinocytosis. Western blot analysis demonstrated that phorbol ester and HGF stimulate activation of slingshot phosphatase homolog 1 (SSH1) and induce cofilin Ser-3 dephosphorylation via PKCδ in macrophages. Silencing of SSH1 inhibited cofilin dephosphorylation and Macropinocytosis stimulation. Interestingly, we also found that incubation of macrophages with BMS-5, a potent inhibitor of LIM kinase, does not stimulate Macropinocytosis. In conclusion, the findings of the present study demonstrate a previously unidentified mechanism by which PKCδ via activation of SSH1 and cofilin dephosphorylation stimulates membrane ruffle formation and Macropinocytosis. The results of the present study may contribute to a better understanding of the regulatory mechanisms during macrophage Macropinocytosis.
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Identification of novel Macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs.
British journal of pharmacology, 2018Co-Authors: Hui Ping Lin, Bhupesh Singla, Pushpankur Ghoshal, Jessica L. Faulkner, Mary Cherian-shaw, Paul M. O'connor, Jin Xiong She, Eric J. Belin De Chantemèle, Gabor CsanyiAbstract:Background and purpose Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of Macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. Here we have sought to identify new, clinically relevant inhibitors of Macropinocytosis, using an FDA-approved drug library. Experimental approach In the present study, 640 FDA-approved compounds were tested for their ability to inhibit Macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC50 values and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis and clathrin-mediated and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to examine the mechanisms by which selected compounds inhibit Macropinocytosis. Key results The primary screen identified 14 compounds that at ~10 μM concentration inhibit >95% of macropinocytotic solute internalization. Three compounds - imipramine, phenoxybenzamine and vinblastine - potently inhibited (IC50 ≤ 131 nM) Macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Scanning electron microscopy imaging indicated that imipramine inhibits membrane ruffle formation, a critical early step leading to initiation of Macropinocytosis. Finally, imipramine has been shown to inhibit Macropinocytosis in several cell types, including cancer cells, dendritic cells and macrophages. Conclusions and implications Our results identify imipramine as a new pharmacological tool to study Macropinocytosis in cellular and biological systems. This study also suggests that imipramine could be a good candidate for repurposing as a therapeutic agent in pathological processes involving Macropinocytosis.
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pkcδ mediated nox2 activation promotes fluid phase pinocytosis of antigens by immature dendritic cells
Frontiers in Immunology, 2018Co-Authors: Bhupesh Singla, Pushpankur Ghoshal, Zheng Dong, Gabor CsanyiAbstract:Aims: Macropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC Macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC Macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved. Methods: Various cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC Macropinocytosis. Results: Confocal laser scanning microscopy confirmed that DCs internalize fluorescently-labeled antigens (ovalbumin) using Macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor (HGF)-induced antigen Macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC Macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-β. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC Macropinocytosis of antigens downstream of PKCδ activation. Conclusions: The findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC Macropinocytosis of antigens. PKCδ/Nox2-mediated antigen Macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC Macropinocytosis and downstream regulation of T cell-mediated responses.
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nox2 mediated pi3k and cofilin activation confers alternate redox control of macrophage pinocytosis
Antioxidants & Redox Signaling, 2017Co-Authors: Pushpankur Ghoshal, Bhupesh Singla, Douglas M Feck, Nadiezhda Cantumedellin, Eric E Kelley, Stephen Haigh, David Fulton, Gabor CsanyiAbstract:Abstract Aims: Internalization of extracellular fluid and its solute by Macropinocytosis requires dynamic reorganization of actin cytoskeleton, membrane ruffling, and formation of large endocytic vacuolar compartments, called macropinosomes, inside the cell. Although instigators of Macropinocytosis, such as growth factors and phorbol esters, stimulate NADPH oxidase (Nox) activation and signal transduction mediators upstream of Nox assembly, including Rac1 and protein kinase C (PKC), are involved in Macropinocytosis, the role of Nox enzymes in Macropinocytosis has never been investigated. This study was designed to examine the role of Nox2 and the potential downstream redox signaling involved in Macropinocytosis. Results: Phorbol myristate acetate activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of macromolecules by Macropinocytosis. Mechanistically, we found that pharmacological blockade of PKC, transcriptional knockdown of Nox2, and s...
Michael Glogauer - One of the best experts on this subject based on the ideXlab platform.
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slit2 robo1 signaling inhibits Macropinocytosis by opposing cortical cytoskeletal remodeling
Nature Communications, 2020Co-Authors: Vikrant K. Bhosle, Michael Glogauer, Tapas Mukherjee, Yi-wei Huang, Sajedabanu Patel, Bo Wen Frank Pang, Guang-ying Liu, Dana J. PhilpottAbstract:Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce Macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits Macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits Macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of Macropinocytosis and challenge the conventional notion that signals that enhance Macropinocytosis negatively regulate cell migration, and vice versa.
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SLIT2/ROBO1-signaling inhibits Macropinocytosis by opposing cortical cytoskeletal remodeling
Nature communications, 2020Co-Authors: Vikrant K. Bhosle, Michael Glogauer, Tapas Mukherjee, Yi-wei Huang, Sajedabanu Patel, Bo Wen Frank Pang, Guang-ying Liu, Dana J. Philpott, Sergio GrinsteinAbstract:Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce Macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits Macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits Macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of Macropinocytosis and challenge the conventional notion that signals that enhance Macropinocytosis negatively regulate cell migration, and vice versa.
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Calcium-sensing receptors signal constitutive Macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages
Nature communications, 2016Co-Authors: Johnathan Canton, Daniel Schlam, Christian Breuer, Michael Gütschow, Michael Glogauer, Sergio GrinsteinAbstract:Macropinocytosis can be induced in several cell types by stimulation with growth factors. In selected cell types, notably macrophages and dendritic cells, Macropinocytosis occurs constitutively, supporting the uptake of antigens for subsequent presentation. Despite their different mode of initiation and contrasting physiological roles, it is tacitly assumed that both types of Macropinocytosis are mechanistically identical. We report that constitutive Macropinocytosis is stringently calcium dependent, while stimulus-induced Macropinocytosis is not. Extracellular calcium is sensed by G-protein-coupled calcium-sensing receptors (CaSR) that signal Macropinocytosis through Gα-, phosphatidylinositol 3-kinase and phospholipase C. These pathways promote the recruitment of exchange factors that stimulate Rac and/or Cdc42, driving actin-dependent formation of ruffles and macropinosomes. In addition, the heterologous expression of CaSR in HEK293 cells confers on them the ability to perform constitutive Macropinocytosis. Finally, we show that CaSR-induced constitutive Macropinocytosis facilitates the sentinel function of macrophages, promoting the efficient delivery of ligands to cytosolic pattern-recognition receptors.
