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Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages.

ABSTRACT: 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 G?-, 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.

SUBMITTER: Canton J

PROVIDER: S-EPMC4823870 | biostudies-other | 2016 Apr

REPOSITORIES: biostudies-other

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Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages.

Canton Johnathan J Schlam Daniel D Breuer Christian C Gütschow Michael M Glogauer Michael M Grinstein Sergio S

Nature communications 20160406

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 depen ...[more]

PMID: 27050483

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Project description:Chronic low-grade inflammation is a hallmark of aging, but its etiology is not well understood. Mitochondrial dysfunction has been linked to both cellular senescence and age-related inflammation or inflammaging but fundamental mechanisms underlying these links are unclear. We analyzed age-related gene expression in different human tissues and discovered that in blood, the mRNA expression of Mitochondrial Calcium Uniporter (MCU) and its regulatory subunit MICU1 correlated inversely with age. MCU is the pore-forming subunit of a Ca2+-selective ion channel complex residing in the mitochondrial inner membrane and is the main conduit for Ca2+-influx into the mitochondrial matrix. Based on these findings we tested the simple hypothesis that mitochondrial Ca2+ uptake capacity of macrophages decreases with age. We found a significant reduction in the mitochondrial Ca2+-uptake capacity of macrophages derived from aged (80-90 weeks) mice. Notably, these macrophages display amplified cytosolic Ca2+ elevations and increased inflammatory output. To test the salience of mitochondrial calcium uptake in regulating macrophage inflammatory output, we deleted Mcu selectively in macrophages. The Mcu-/- macrophages of young mice (<25 weeks) express more inflammatory genes at baseline and show a hyper-inflammatory response when stimulated. We show that reduced mitochondrial Ca2+ amplifies cytosolic Ca2+ oscillations and potentiates downstream NFkB activation, which is central to inflammation. The regulation of inflammatory response by mitochondrial Ca2+ uptake is also seen readily in human macrophages. The siRNA-mediated knockdown of MCU in human blood derived macrophages recapitulates the phenomenon – the mitochondrial Ca2+-uptake is greatly reduced, cytosolic Ca2+ elevations are more pronounced, and the macrophages are hyper-inflammatory. Our findings pinpoint the MCU complex as a keystone molecular apparatus that links age-related changes in mitochondrial physiology to macrophage-mediated inflammation. Resident macrophages are intrinsic to every organ system and the steady erosion of their mitochondrial Ca2+-uptake capacity may play a germinal or exacerbating role in many age-related neurodegenerative, cardiometabolic, renal and musculoskeletal diseases that afflict us.

Project description:Background & aimsNucleotide oligomerization domain 2 (Nod2) polymorphisms increase the risk of developing Crohn's disease, which is characterized by chronic intestinal inflammation. Bacterial peptidoglycan products chronically stimulate Nod2 in the intestine. Recent studies found that chronic Nod2 stimulation in human macrophages down-regulates proinflammatory cytokines on Nod2 or Toll-like receptor (TLR) restimulation. Therefore, an emerging hypothesis is that Nod2-mediated cytokine down-regulation is required for intestinal homeostasis, but the mechanisms mediating this down-regulation are incompletely understood.MethodsUtilizing primary human macrophages, we examined secretory mediators as a mechanism of Nod2-mediated tolerance by inhibiting their function and assessing tolerance reversal through cytokine secretion. Signaling pathways contributing to secretory mediator induction and Nod2-mediated tolerance were identified through pathway inhibition.ResultsWe found that chronic Nod2 stimulation cross-tolerizes not only to TLRs but also to the interleukin (IL)-1 receptor. Moreover, chronic IL-1β stimulation down-regulates Nod2 responses. Accordingly, IL-1β blockade partially reverses Nod2-mediated tolerance. We found that an additional essential mechanism for Nod2-mediated tolerance is the early secretion of the anti-inflammatory mediators IL-10, transforming growth factor β, and IL-1Ra. Importantly, the mammalian target of rapamycin (mTOR) pathway, involved in cell growth, differentiation, and activation, significantly contributes to Nod2-induced anti-inflammatory as opposed to proinflammatory cytokines and to Nod2-mediated tolerance.ConclusionsInflammatory responses through the IL-1R are down-regulated upon chronic Nod2 stimulation, secretory mediators are a critical mechanism for Nod2-mediated cytokine down-regulation, and the mTOR pathway is crucial for Nod2-mediated tolerance. These results further contribute to our understanding of the mechanisms through which Nod2, a protein critical to intestinal homeostasis, down-regulates cytokine responses.

Dataset Information

Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages.

Publications

Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages.

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