Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells.

Microglia are phagocytic cells that are the main inflammatory response cells of the central nervous system. In Alzheimer's disease brain, activated microglia are concentrated in regions of compact amyloid deposits that contain the 39-43-amino acid Abeta peptide. We examined the uptake, degradation, and release of small aggregates of fibrillar Abeta (fAbeta) or soluble Abeta (sAbeta) by microglia. We found that although some degradation of fAbeta was observed over 3 days, no further degradation was observed over the next 9 days. Instead, there was a slow release of intact Abeta. The poor degradation was not due to inhibition of lysosomal function, since the rate of alpha2-macroglobulin degradation was not affected by the presence of fAbeta in the late endosomes/lysosomes. In contrast to fAbeta, internalization of sAbeta was not saturable. After internalization, sAbeta was released rapidly from microglia, and very little was degraded. These data show that fAbeta and sAbeta interact differently with microglia but that after internalization a large fraction of both are released without degradation.     

Antibody-mediated phagocytosis of the amyloid beta-peptide in microglia is differentially modulated by C1q

Microglial ingestion of the amyloid beta-peptide (Abeta) has been viewed as a therapeutic target in Alzheimer's disease, in that approaches that enhance clearance of Abeta relative to its production are predicted to result in decreased senile plaque formation, a proposed contributor to neuropathology. In vitro, scavenger receptors mediate ingestion of fibrillar Abeta (fAbeta) by microglia. However, the finding that cerebral amyloid deposition in a transgenic mouse model of Alzheimer's disease was diminished by inoculation with synthetic Abeta has suggested a possible therapeutic role for anti-Abeta Ab-mediated phagocytosis. Microglia also express C1qR(P), a receptor for complement protein C1q, ligation of which in vitro enhances phagocytosis of immune complexes formed with IgG levels below that required for optimal FcR-mediated phagocytosis. The data presented here demonstrate FcR-dependent ingestion of Abeta-anti-Abeta complexes (IgG-fAbeta) by microglia that is a function of the amount of Ab used to form immune complexes. In addition, C1q incorporated into IgG-fAbeta enhanced microglial uptake of these complexes when they contained suboptimal levels of anti-Abeta Ab. Mannose binding lectin and lung surfactant protein A, other ligands of C1qR(P), also enhanced ingestion of suboptimally opsonized IgG-fAbeta, whereas control proteins did not. Our data suggest that C1qR(P)-mediated events may promote efficient ingestion of Abeta at low Ab titers, and this may be beneficial in paradigms that seek to clear amyloid via FcR-mediated mechanisms by minimizing the potential for destructive Ab-induced complement-mediated processes.     

Activation of microglia acidifies lysosomes and leads to degradation of Alzheimer amyloid fibrils

Microglia are the main immune cells of the brain, and under some circumstances they can play an important role in removal of fibrillar Alzheimer amyloid beta peptide (fAbeta). Primary mouse microglia can internalize fAbeta, but they do not degrade it efficiently. We compared the level of lysosomal proteases in microglia and J774 macrophages, which can degrade fAbeta efficiently, and we found that microglia actually contain higher levels of many lysosomal proteases than macrophages. However, the microglial lysosomes are less acidic (average pH of approximately 6), reducing the activity of lysosomal enzymes in the cells. Proinflammatory treatments with macrophage colony-stimulating factor (MCSF) or interleukin-6 acidify the lysosomes of microglia and enable them to degrade fAbeta. After treatment with MCSF, the pH of microglial lysosomes is similar to J774 macrophages (pH of approximately 5), and the MCSF-induced acidification can be partially reversed upon treatment with an inhibitor of protein kinase A or with an anion transport inhibitor. Microglia also degrade fAbeta if lysosomes are acidified by an ammonia pulse-wash or by treatment with forskolin, which activates protein kinase A. Our results indicate that regulated lysosomal acidification can potentiate fAbeta degradation by microglia.     

Complement opsonization of HIV-1 enhances the uptake by dendritic cells and involves the endocytic lectin and integrin receptor families

Interaction with the complement system is an underappreciated aspect of HIV-1 infection; even in primary infection, complement fragments are found on virions with potential to affect the interplay between the virus and dendritic cells (DC). Since opsonization may affect the efficiency of uptake and the type of receptors utilized, we compared the interactions of DC with free HIV-1 (F-HIV) and complement opsonized HIV-1 (C-HIV). We demonstrate that C-HIV significantly enhanced the uptake by immature DC (IDC) and mature DC (MDC) and that the internalization rate was dependent on both opsonization of the virus and DC maturation state. Increased DC uptake of C-HIV was not due to opsonization related increased binding of virus to the surface of DC but rather increased internalization of C-HIV despite utilizing a similar repertoire of receptors as F-HIV. Both F-HIV and C-HIV interacted with C-type lectins, integrins, and CD4 and blocking these receptor families prevented HIV-1 from binding to DC at 4°C. Blocking integrins significantly reduced the binding and uptake of F-HIV and C-HIV implicating the involvement of several integrins such as β1-integrin, CR3, LFA-1, and α4β7. Distinctive for C-HIV was usage of β1-integrin and for F-HIV, usage of β7-integrin, whereas both F-HIV and C-HIV utilized both integrin chains of CR3. We have in this study identified the receptor types used by both F-HIV and C-HIV to bind to DC. Noteworthy, C-HIV was internalized more efficiently by DC than F-HIV, probably via receptor mediated endocytosis, which may entail different intracellular processing of the virus leading to both elevated infection and altered activation of HIV specific immune responses.     

Matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-beta fibrils (fAbeta), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Abeta (sAbeta) could degrade fAbeta as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAbeta and that this ability is not shared by other sAbeta-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAbeta was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAbeta breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAbeta were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Abeta that are cleaved during its degradation. Only MMP-9 digests contained fragments (Abeta(1-20) and Abeta(1-30)) from fAbeta(1-42) substrate; the corresponding cleavage sites are thought to be important for beta-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAbeta and may contribute to ongoing clearance of plaques from amyloid-laden brains.     

Estrogen enhances uptake of amyloid beta-protein by microglia derived from the human cortex

In recent years, inflammatory mechanisms have been increasingly appreciated as important steps in the pathology of Alzheimer's disease (AD). There are two pathological defects in AD: chronic inflammation and impaired clearance of amyloid beta-peptide (Abeta). In the periphery, estrogen both increases macrophage phagocytosis and has antiinflammatory effects. If estrogen had a similar effect in the CNS, it could reverse inflammatory defects in AD. Although microglia are a key component of the immune system and help clear Abeta deposits in the AD brain, little is known about the effects of estrogen on CNS microglia. Therefore, we sought to determine the relationship between estrogen treatment and internalization of Abeta by microglia by quantifying the internalization of aggregated Abeta by human cortical microglia. Abeta uptake was found to be dose- and time-dependent in cultured microglia. Increased Abeta uptake was observed at 1.5 and 24 h after addition of aggregated Abeta (50, 100, or 1,000 nM: Abeta), and this uptake was enhanced by pretreatment with estrogen. The expression of estrogen receptor (ER) beta (ER-beta) was also up-regulated by estrogen treatment. Cells cotreated with ICI 182,780, an ER antagonist, showed significantly reduced internalization of Abeta in cultured microglia. These results indicate that microglia express an ER-beta but that the effect of estrogen on enhancing clearance of Abeta may be related to the receptor-independent action of estrogen or to nonclassical ER effects of estrogen. Thus, stimulation of the ER might contribute to the therapeutic action of estrogen in the treatment of AD.     

Fibrillar beta-amyloid-stimulated intracellular signaling cascades require Vav for induction of respiratory burst and phagocytosis in monocytes and microglia

Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the alpha(6)beta(1)-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav(-/-) mice. Stimulation of Vav(-/-) microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.     

Macrophage colony-stimulating factor differentially regulates low density lipoprotein and transferrin receptors

Endocytosis mediated by both LDL receptors (LDLRs) and transferrin receptors (TfRs) occurs in clathrin-coated pits and requires specific tyrosine-based internalization sequences located in the cytoplasmic domain of these receptors. Internalization of these receptors is mediated by endocytic proteins that interact with the internalization domains. We previously showed that macrophage colony-stimulating factor (M-CSF) rapidly increases LDLR-dependent uptake and metabolism of LDL. To study the mechanism by which M-CSF regulates LDL uptake, we compared the effect of M-CSF on the internalization of LDL and transferrin (Tf). Our results show that M-CSF substantially increased the rate of LDLR internalization without increasing LDLR localization on the cell surface. In contrast, M-CSF treatment of macrophages rapidly increased the localization of TfR to the cell surface but did not alter the relative rate of Tf internalization. Moreover, M-CSF regulated TfR and LDLR via the activation of distinct signaling pathways. Recruitment of TfR to the cell surface was attenuated by phosphatidylinositol 3-kinase inhibitors, whereas stimulated LDL uptake was inhibited by the serine/threonine phosphatase inhibitor okadaic acid. Taken together, our results indicate that M-CSF differentially regulates receptors that undergo endocytosis and that increased LDL uptake results from a selective increase in the rate of LDLR internalization.     

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.     

Mannose 6-phosphate receptor-mediated uptake is defective in acid sphingomyelinase-deficient macrophages: implications for Niemann-Pick disease enzyme replacement therapy

Progressive accumulation of lipid-laden macrophages is a hallmark of the acid sphingomyelinase (ASM)-deficient forms of Niemann-Pick disease (i.e. Types A and B NPD). To investigate the mechanisms underlying enzyme replacement therapy for this disorder, we studied the uptake of recombinant, human ASM (rhASM) by alveolar macrophages from ASM knock-out (ASMKO) mice. The recombinant enzyme used for these studies was produced in Chinese hamster ovary cells and contained complex type, N-linked oligosaccharides. Binding of radiolabeled, rhASM to the ASMKO macrophages was enhanced as compared with normal macrophages, consistent with their larger size and increased surface area. However, internalization of the enzyme by the ASMKO cells was markedly reduced when compared with normal cells. Studies using receptor-specific ligands to inhibit enzyme uptake revealed that in normal cells rhASM was taken up by a combination of mannose and mannose 6-phosphate receptors (MR and M6PR, respectively), whereas in the ASMKO cells the M6PR had a minimal role in rhASM uptake. Expression of M6PR mRNA was normal in the ASMKO cells, although Western blotting revealed more receptors in these cells when compared with normal. We therefore hypothesized that lipid accumulation in ASMKO macrophages led to abnormalities in M6PR trafficking and/or degradation, resulting in reduced enzyme uptake. Consistent with this hypothesis, we also found that, when rhASM was modified to expose terminal mannose residues and target mannose receptors, the uptake of this modified enzyme form by ASMKO cells was approximately 10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.     

Hepatic triglyceride lipase promotes low density lipoprotein receptor-mediated catabolism of very low density lipoproteins in vitro.

We demonstrate here that hepatic triglyceride lipase (HTGL) enhances VLDL degradation in cultured cells by a LDL receptor-mediated mechanism. VLDL binding at 4 degrees C and degradation at 37 degrees C by normal fibroblasts was stimulated by HTGL in a dose-dependent manner. A maximum increase of up to 7-fold was seen at 10 microg/ml HTGL. Both VLDL binding and degradation were significantly increased (4-fold) when LDL receptors were up-regulated by treatment with lovastatin. HTGL also stimulated VLDL degradation by LDL receptor-deficient FH fibroblasts but the level of maximal degradation was 40-fold lower than in lovastatin-treated normal fibroblasts. A prominent role for LDL receptors was confirmed by demonstration of similar HTGL-promoted VLDL degradation by normal and LRP-deficient murine embryonic fibroblasts. HTGL enhanced binding and internalization of apoprotein-free triglyceride emulsions, however, this was LDL receptor-independent. HTGL-stimulated binding and internalization of apoprotein-free emulsions was totally abolished by heparinase indicating that it was mediated by HSPG. In a cell-free assay HTGL competitively inhibited the binding of VLDL to immobilized LDL receptors at 4 degrees C suggesting that it may directly bind to LDL receptors but may not bind VLDL particles at the same time.We conclude that the ability of HTGL to enhance VLDL degradation is due to its ability to concentrate lipoprotein particles on HSPG sites on the cell surface leading to LDL receptor-mediated endocytosis and degradation.     

Cellular Uptake of α-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of α-Synuclein and Mediated via Fcγ Receptors

Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson’s disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen–antibody complexes.     

Prolactin receptors in cultured rat mammary tumor cells. Energy-dependent uptake and degradation of hormone receptors

After energy depletion by uncouplers of oxidative phosphorylation or inhibitors of electron transport, primary cultures of carcinogen-induced rat mammary tumors have a 2- to 20-fold increase in the number of cell surface prolactin receptors. When energy-depleted cells were treated with 0.15 M NaCl plus 50 mM glycine pH 3, for 1 min at 4 degrees C, 75% of the specific surface-bound 125I-labeled ovine prolactin was removed, but prolactin and its receptor were not destroyed. Using this technique, we found that receptor-bound prolactin can be internalized (becomes resistant to pH 3.0 treatment) and then degraded. The internalization of occupied receptors required energy, was completed 30-60 min before degradation, and was independent of protein synthesis. Hormone degradation (t1/2, 42 min) but not uptake was prevented by NH4Cl or lysosomotropic amines. In the presence of cycloheximide, receptors were lost (t1/2, 62 min) unless such loss was prevented by KCN. After unoccupied receptors were activated by energy depletion, surface receptors were lost when inhibitor was removed and glucose was added. Thus, both occupied and unoccupied prolactin receptors are constantly removed from the cell surface via an energy-dependent uptake mechanism. If the receptor levels are first increased by energy depletion (with or without bound ligand) or if protein synthesis is inhibited, there is a net loss of surface binding sites. Since the receptors reappeared with 15 h after cycloheximide removal, some of the receptors probably are recycled under normal steady state conditions.     

Modulation of macrophage mannose receptor affects the uptake of virulent and avirulent Leishmania donovani promastigotes.

The effect of oxidants and the anti-inflammatory steroid dexamethasone on the attachment and internalization of virulent and avirulent Leishmania donovani promastigotes by the macrophage mannosyl fucosyl receptor was examined. Oxidants and dexamethasone are known to down- and upregulate the expression of the mannose receptor. Macrophages, when treated with 500 microM H2O2 at 37 C for 30 min, stimulate about 45% inhibition in uptake of an avirulent strain (UR6), and 30 and 25% inhibition for virulent strains AG-83 and GE-I, respectively. Treatment of macrophages with dexamethasone for 20 hr resulted in a stimulation in uptake of the parasite. When UR6 was used, a 3-fold increase in uptake was observed compared with the controls. Parasite uptake was also inhibited by the H2O2-generating system, glucose/glucose oxidase; inhibition was blocked by catalase. Treatment of macrophages either with H2O2 or dexamethasone did not affect the binding of the advanced glycosylation end product-bovine serum albumin (AGE-BSA), the ligand for AGE receptor of macrophages. Similarly, indirect evidence also shows that both types 1 and 3 complement receptors (CR1, CR3) are not affected by these treatments, indicating that, besides the mannosyl fucosyl receptor, other receptors are minimally altered in the identified condition. These results suggest that the up- and downregulation of the mannose receptor of macrophages may play a role in affecting L. donovani infection.     

Binding, internalization, and intracellular localization of interleukin-1 beta in human diploid fibroblasts

This study demonstrates internalization of interleukin-1 (IL-1) via its cell surface receptor on human diploid fibroblasts and shows intracellular localization of IL-1 beta. Binding experiments at 8 degrees C using confluent fibroblast monolayers revealed 5,000-15,000 IL-1 receptors/cell that bound both IL-1 alpha and IL-1 beta. Incubation of monolayers with 125I-IL-1 beta (10(-9) M) at 8 degrees C and then at 37 degrees C for various times up to 8 h revealed a t1/2 for internalization of receptor-bound IL-1 beta of about 1.5 h. In addition, it was shown that IL-1 beta internalized via receptors was undegraded and retained binding activity. Electron microscopic autoradiography of monolayers incubated with 125I-IL-1 beta, as above, showed a progressive increase in the ratio of cytoplasmic to cell surface-associated grains. Grains at the cell surface were primarily localized at cell processes or attachment sites, frequently close to intra- and extracellular filamentous material. During incubation at 37 degrees C, most grains were free in the cytoplasm, with few present in lysosomes or vesicles. After 1 h, approximately 15% of the grains were over nuclei. Control cultures incubated at 37 degrees C with 125I-IL-1 beta and 100-fold excess unlabeled IL-1 beta showed increased uptake of label into lysosomes and little into nuclei. This study shows that IL-1 receptors are primarily located at fibroblast processes and that receptor-mediated internalization of the ligand is slow. Nuclear localization apparently requires IL-1 receptor-specific internalization of IL-1 beta, suggesting a possible role for this process in eliciting the IL-1 signal.     

Insulin receptor recycling in vascular endothelial cells. Regulation by insulin and phorbol ester

Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t1/2 = 18 +/- 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of 125I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by 125I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.     

Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation.     

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.     

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.     

The non-phagocytic route of collagen uptake: a distinct degradation pathway

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.