Shedding of collagen XVII ectodomain depends on plasma membrane microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.     

Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling

The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-alpha converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.     

HDLs activate ADAM17-dependent shedding

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high-density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs-stimulated TNF release occurred also on cell-free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1-dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17-dependent cleavage of TNF in the non-raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17-dependent processing of transmembrane substrates.     

Shedding of the p75NTR neurotrophin receptor is modulated by lipid rafts

Protein ectodomain shedding is the proteolytic release of the extracellular domain of membrane-bound proteins. Neurotrophin receptor p75(NTR) is known to be affected by shedding. The present work provides evidence, in rat brain synaptosomes, that p75(NTR) is present in detergent-resistant membranes (DRM), also known as lipid rafts, only in its full-length form. Disrupting the integrity of lipid rafts causes solubilization of p75(NTR) after detergent treatment and enhancement of the shedding. Analyses of the enzymes described as being responsible for p75(NTR) shedding, i.e. tumor necrosis factor alpha convertase (TACE) and presenilin-1 (PS1), revealed that TACE is absent in DRM, while variable proportions of the C-terminal and N-terminal fragments of PS1 are found. In summary, our results point to a role of lipid rafts in the modulation of the shedding of the p75(NTR) receptor.     

Increased membrane cholesterol in lymphocytes diverts T-cells toward an inflammatory response

Cell signaling for T-cell growth, differentiation, and apoptosis is initiated in the cholesterol-rich microdomains of the plasma membrane known as lipid rafts. Herein, we investigated whether enrichment of membrane cholesterol in lipid rafts affects antigen-specific CD4 T-helper cell functions. Enrichment of membrane cholesterol by 40-50% following squalene administration in mice was paralleled by an increased number of resting CD4 T helper cells in periphery. We also observed sensitization of the Th1 differentiation machinery through co-localization of IL-2Rα, IL-4Rα, and IL-12Rβ2 subunits with GM1 positive lipid rafts, and increased STAT-4 and STAT-5 phosphorylation following membrane cholesterol enrichment. Antigen stimulation or CD3/CD28 polyclonal stimulation of membrane cholesterol-enriched, resting CD4 T-cells followed a path of Th1 differentiation, which was more vigorous in the presence of increased IL-12 secretion by APCs enriched in membrane cholesterol. Enrichment of membrane cholesterol in antigen-specific, autoimmune Th1 cells fostered their organ-specific reactivity, as confirmed in an autoimmune mouse model for diabetes. However, membrane cholesterol enrichment in CD4(+)Foxp3(+) T-reg cells did not alter their suppressogenic function. These findings revealed a differential regulatory effect of membrane cholesterol on the function of CD4 T-cell subsets. This first suggests that membrane cholesterol could be a new therapeutic target to modulate the immune functions, and second that increased membrane cholesterol in various physiopathological conditions may bias the immune system toward an inflammatory Th1 type response.     

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.     

CHAPSTEROL. A novel cholesterol-based detergent

Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol-based detergent developed for functional solubilization of cholesterol-dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein-coupled receptor requiring cholesterol for its high-affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6-azocholestan-3beta-ol[3alphaH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol-binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins. Similar to Triton X-100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e. detergent-insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol-dependent membrane proteins and isolation of rafts.     

The shedding activity of ADAM17 is sequestered in lipid rafts

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.     

Lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle

Lipid rafts are involved in the life cycle of many viruses. In this study, we showed that lipid rafts also play an important role in the life cycle of severe acute respiratory syndrome (SARS)-coronavirus (CoV). Cholesterol depletion by pretreatment of Vero E6 cells with methyl-beta-cyclodextrin (MbetaCD) inhibited the production of SARS-CoV particles released from the infected cells. This inhibition was prevented by addition of cholesterol to the culture medium, indicating that the reduction of virus particle release was caused by the loss of cholesterol in the cell membrane. In contrast, cholesterol depletion at the post-entry stage (3h post-infection) caused only a limited effect on virus particle release. Northern blot analysis revealed that the levels of viral mRNAs were significantly affected by pretreatment with MbetaCD, but not by treatment at 3h post-infection. Interestingly, no apparent evidence for colocalization of angiotensin converting enzyme 2 with lipid rafts in the membrane of Vero E6 cells was obtained. These results suggest that lipid rafts could contribute to SARS-CoV infection in the early replication process in Vero E6 cells.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha (TNFalpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFalpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNFalpha trafficking and secretion. TNFalpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNFalpha surface distribution revealed these puncta to be the initial points of TNFalpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.     

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca²⁺-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.     

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.     

Crosslinking of the human Fc receptor for IgA (FcalphaRI/CD89) triggers FcR gamma-chain-dependent shedding of soluble CD89

CD89/FcalphaRI is a 55- to 75-kDa type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. At present, no information is available on the existence of soluble forms of this receptor. We developed an ELISA for the detection of soluble CD89 (sCD89) forms and investigated the regulation of sCD89 production. PMA/ionomycin stimulation of monocytic cell lines (U937, THP-1, and MM6), but not of neutrophils, resulted in release of sCD89. Crosslinking of CD89 either via its ligand IgA or with anti-CD89 mAbs similarly resulted in sCD89 release. Using CD89-transfected cells, we showed ligand-induced shedding to be dependent on coexpression of the FcR gamma-chain subunit. Shedding of sCD89 was dependent on signaling via the gamma-chain and prevented by addition of inhibitors of protein kinase C (staurosporine) or protein tyrosine kinases (genistein). Western blotting revealed sCD89 to have an apparent molecular mass of 30 kDa and to bind IgA in a dose-dependent fashion. In conclusion, the present data document a ligand-binding soluble form of CD89 that is released upon activation of CD89-expressing cells. Shedding of CD89 may play a role in fine-tuning CD89 immune effector functions.     

Mild cholesterol depletion reduces amyloid-β production by impairing APP trafficking to the cell surface

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ₄₀ and Aβ₄₂ in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP–Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP–PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.