Pneumocystis species are opportunistic fungal organisms that cause severe pneumonia in immune-compromised hosts, with resultant high morbidity and mortality. Recent work indicates that IL-17 responses are important components of host defense against fungal pathogens. In the present study, we demonstrate that cell-surface β-glucan components of Pneumocystis (PCBG) stimulate human dendritic cells (DCs) to secrete IL-23 and IL-6. These cytokines are well established to stimulate a T helper–17 (Th17) phenotype. Accordingly, we further observe that PCBG-stimulated human DCs interact with lymphocytes to drive the secretion of IL-17 and IL-22, both Th17-produced cytokines. The activation of DCs was shown to involve the dectin-1 receptor with a downstream activation of the Syk kinase and subsequent translocation of both the canonical and noncanonical components of the NF-κB transcription factor family. Finally, we demonstrate that glycosphingolipid-rich microdomains of the plasma membrane participate in the activation of DCs by PCBG through the accumulation of lactosylceramide at the cell surface during stimulation with PCBG. These data strongly support the idea that the β-glucan surface components of Pneumocystis drive the activation of the IL-23/IL-17 axis during this infection, through a glycosphingolipid-initiated mechanism.
Pneumocystis; β-glucan; dendritic cells; IL-23; IL-17
Targeting; Epidermal Growth Factor Receptor (EGFR); Gold Nanoparticles; Pathway Switching; Lipid Microdomain; GTPases
Type Iγ phosphatidylinositol-4-phosphate 5-kinase and Exo70 cooperate in the directed targeting of E-cadherin on the plasma membrane to newly formed adherens junctions. This promotes the regional accumulation of E-cadherin, expansion and maturation of adherens junctions, and differentiation of the lateral membrane domain.
E-Cadherin–mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P2) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P2 is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P2 pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.
Cell wounding is an important driver of the innate immune response of ventilator-injured lungs. We had previously shown that the majority of wounded alveolus resident cells repair and survive deformation induced insults. This is important insofar as wounded and repaired cells may contribute to injurious deformation responses commonly referred to as biotrauma. The central hypothesis of this communication states that extracellular adenosine-5′ triphosphate (ATP) promotes the repair of wounded alveolus resident cells by a P2Y2-Receptor dependent mechanism. Using primary type 1 alveolar epithelial rat cell models subjected to micropuncture injury and/or deforming stress we show that 1) stretch causes a dose dependent increase in cell injury and ATP media concentrations; 2) enzymatic depletion of extracellular ATP reduces the probability of stretch induced wound repair; 3) enriching extracellular ATP concentrations facilitates wound repair; 4) purinergic effects on cell repair are mediated by ATP and not by one of its metabolites; and 5) ATP mediated cell salvage depends at least in part on P2Y2-R activation. While rescuing cells from wounding induced death may seem appealing, it is possible that survivors of membrane wounding become governors of a sustained pro-inflammatory state and thereby perpetuate and worsen organ function in the early stages of lung injury syndromes. Means to uncouple P2Y2-R mediated cytoprotection from P2Y2-R mediated inflammation and to test the preclinical efficacy of such an undertaking deserve to be explored.
Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of the glucose transporter isoform 4 (GLUT4) to the plasma membrane (PM) from specialized GLUT4 storage vesicles (GSVs). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. Here we study the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs, and fluorescence recovery after photobleaching (FRAP) of GLUT4-GFP in intracellular structures. Glycosphingolipids were located in endosomal vesicles in preadipocytes and redistributed to the PM with decreased expression at day two after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. While insulin-induced phosphorylation of IRS and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4-GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2, but not the transferrin receptor or cellubrevin, indicating the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycemic control and enhances insulin sensitivity in animal models of type 2 diabetes.
GLUT4; GLUT4 storage vesicles; VAMP2; Insulin-stimulated GLUT4 transport; 3T3-L1 adipocytes; glycosphingolipids
Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin-mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo-glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (“lipid rafts”), reduced caveolae and caveolin-1 at the plasma membrane by ~80%, and blunted activation of β1-integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo-gangliosides or other sphingolipids) at 10°C, suggesting that sialo-lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin-1 could be recapitulated by β1-integrin knockdown. These results suggest that both gangliosides and β1-integrin are required for maintenance of caveolae and plasma membrane domains.
caveolar endocytosis; glycosphingolipids; caveolin-1; sialidase; endoglycoceramidase; focal adhesions
Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and β1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, β-d-lactosyl-N-octanoyl-l-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits β1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of β1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify β1 integrin as a potential mediator of signaling by GSLs.
Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.
We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs
in various cell types using pathway-specific inhibitors and colocalization
studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on
inhibitor studies, all GSLs tested were internalized predominantly (>80%)
by a clathrin-independent, caveolar-related mechanism, regardless of cell
type. In addition, fluorescent lactosylceramide (LacCer) colocalized with
DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The
internalization mechanism for GSLs was unaffected by varying the carbohydrate
headgroup or sphingosine backbone chain length; however, a fluorescent
phosphatidylcholine analog was not internalized via caveolae, suggesting that
the GSL ceramide core may be important for caveolar uptake. Internalization of
fluorescent LacCer was reduced 80–90% in cell types with low cav-1, but
was dramatically stimulated by cav-1 overexpression. However, even in cells
with low levels of cav-1, residual LacCer internalization was clathrin
independent. In contrast, cholera toxin B subunit (CtxB), which binds
endogenous GM1, was internalized via clathrin-independent
endocytosis in cells with high cav-1 expression, whereas significant
clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent
GM1, normally internalized by clathrin-independent endocytosis in
HeLa cells with low cav-1, was induced to partially internalize via the
clathrin pathway in the presence of CtxB. These results suggest that GSL
analogs are selectively internalized via a caveolar-related mechanism in most
cell types, whereas CtxB may undergo “pathway switching” when
cav-1 levels are low.
Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.
endocytosis; caveolae; cholesterol; Eps15; lipid storage diseases