Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains.

The plasma membrane pits known as caveolae have been implicated both in cholesterol homeostasis and in signal transduction. CavDGV and CavKSY, two dominant-negative amino-terminal truncation mutants of caveolin, the major structural protein of caveolae, significantly inhibited caveola-mediated SV40 infection, and were assayed for effects on Ras function. We find that CavDGV completely blocked Raf activation mediated by H-Ras, but not that mediated by K-Ras. Strikingly, the inhibitory effect of CavDGV on H-Ras signalling was completely reversed by replenishing cell membranes with cholesterol and was mimicked by cyclodextrin treatment, which depletes membrane cholesterol. These results provide a crucial link between the cholesterol-trafficking role of caveolin and its postulated role in signal transduction through cholesterol-rich surface domains. They also provide direct evidence that H-Ras and K-Ras, which are targeted to the plasma membrane by different carboxy-terminal anchors, operate in functionally distinct microdomains of the plasma membrane.

Surveillance of female patients with inherited bleeding disorders in United States Haemophilia Treatment Centres.

Inherited bleeding disorders are especially problematic for affected girls and women due to the monthly occurrence of menstrual periods and the effects on reproductive health. Although heavy menstrual bleeding (HMB) is the most common manifestation, females with inherited bleeding disorders (FBD) experience other bleeding symptoms throughout the lifespan that can lead to increased morbidity and impairment of daily activities. The purpose of this article is to describe the utility of a female-focused surveillance effort [female Universal Data Collection (UDC) project] in the United States Haemophilia Treatment Centres (HTCs) and to describe the baseline frequency and spectrum of diagnoses and outcomes. All FBD aged 2 years and older receiving care at selected HTCs were eligible for enrollment. Demographic data, diagnoses and historical data regarding bleeding symptoms, treatments, gynaecological abnormalities and obstetrical outcomes were analysed. Analyses represent data collected from 2009 to 2010. The most frequent diagnoses were type 1 von Willebrand's disease (VWD) (195/319; 61.1%), VWD type unknown (49/319; 15.4%) and factor VIII deficiency (40/319; 12.5%). HMB was the most common bleeding symptom (198/253; 78.3%); however, 157 (49.2%) participants reported greater than four symptoms. Oral contraceptives were used most frequently to treat HMB (90/165; 54.5%), followed by desmopressin [1-8 deamino-D-arginine vasopressin (DDAVP)] (56/165; 33.9%). Various pregnancy and childbirth complications were reported, including bleeding during miscarriage (33/43; 76.7%) and postpartum haemorrhage (PPH) (41/109; 37.6%). FBD experience multiple bleeding symptoms and obstetrical-gynaecological morbidity. The female UDC is the first prospective, longitudinal surveillance in the US focusing on FBD and has the potential to further identify complications and reduce adverse outcomes in this population.

Caveolin-3 associates with developing T-tubules during muscle differentiation.

Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the alpha 1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3-labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.

Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule.

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.

Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Major histocompatibility complex class I molecules mediate association of SV40 with caveolae.

Simian virus 40 (SV40) has been shown to enter mammalian cells via uncoated plasma membrane invaginations. Viral particles subsequently appear within the endoplasmic reticulum. In the present study, we have examined the surface binding and internalization of SV40 by immunoelectron microscopy. We show that SV40 associates with surface pits which have the characteristics of caveolae and are labeled with antibodies to the caveolar marker protein, caveolin-1. SV40 is believed to use major histocompatibility complex (MHC) class I molecules as cell surface receptors. Using a number of MHC class I-specific monoclonal antibodies, we found that both viral infection and association of virus with caveolae were strongly reduced by preincubation with anti-MHC class I antibodies. Because binding of SV40 to MHC class I molecules may induce clustering, we investigated whether antibody cross-linked class I molecules also redistributed to caveolae. Clusters of MHC class I molecules were indeed shown to be specifically associated with caveolin-labeled surface pits. Taken together, the results suggest that SV40 may make use of MHC class I molecule clustering and the caveolae pathway to enter mammalian cells.

Receptor-mediated endocytosis of ricin in rat liver endothelial cells. An immunocytochemical study.

The endocytic pathway of ricin in sinusoidal liver endothelial cells (EC) was traced by means of immunocytochemical labeling of ultrathin cryosections. Ricin, a highly mannosylated glycoprotein, is internalized mainly by receptor-mediated endocytosis via the mannose receptor in the EC. Labeling of specimens fixed at different time points after injection of ligand showed that several subcellular compartments are involved in processing of endocytosed ricin. One minute after injection ricin is seen in coated pits, coated vesicles and cisternal-shaped endosomes. After 6 min, the ligand associates with electron-dense, spherical vesicles and electron-lucent vesicles, presumably representing late endosomes. In the same time period we observed labeling in the vicinity of the Golgi stack. At later time points, ricin is increasingly localized in lysosomes. Both late endosomes and lysosomes showed labeling for Igp120, the lysosomal membrane glycoprotein. To compare uptake of ricin with another mannosylated ligand, we coinjected ricin and mannosylated colloidal gold particles (Man-Aun). Man-Au20, injected 24 h before fixation as a marker for late endocytic compartments, was found in two distinct compartments, presumably representing late endosomes and lysosomes. The distribution of ricin and Man-Au10, the latter injected 15 min before fixation, in early endosomes was strikingly different, indicating that the structure of this compartment is important in the process of sorting of ligand and receptor.

Endocytosis mediated by the mannose receptor in liver endothelial cells. An immunocytochemical study.

Immunocytochemical labeling of ultrathin cryosections from rat liver showed that mannose-terminated glycoproteins are removed rapidly from the blood stream mainly by the sinusoidal endothelial cells. The mannose-terminated glycoprotein ovalbumin was injected intravenously into rats 1 min, 6 min, and 24 min before perfusion fixation of the liver. Several minor and at least three major subcellular compartments were shown to be involved in the endocytic process. One minute after injection, ovalbumin was found at the cell surface, in coated pits, in coated vesicles, in tubular structures, and bound to the membrane of large early endosomes of which some showed a cisternal structure. After 6 min, ovalbumin was found in the lumen of large electron-lucent late endosomes and after 24 min in electron-dense structures, presumably lysosomes. The early endosomes have an ultrastructure which, together with the labeling pattern, indicates that this compartment has the same function as the CURL identified in parenchymal liver cells. The results are in accordance with recent biochemical findings indicating that ovalbumin endocytosed by endothelial cells is found sequentially in three different subcellular fractions depending on the time between injection and cooling for fractionation (G. M. Kindberg, T. Berg: Intracellular transport of endocytosed mannose terminated glycoproteins in rat liver endothelial cells. In: E. Wisse, D. L. Knook, K. Decker (eds.): Cells of the Hepatic Sinusoid. Vol. 2. pp. 120-124. Kupffer Cell Foundation. Rijswijk The Netherlands 1989).

Characterization of a peptide-loading compartment by monoclonal antibodies.

Whether or not peptide-loading compartments are classical or specialized compartments of the endocytic pathway of antigen presenting cells is still a matter of debate. One way to solve this discrepancy would be to characterize specific markers for the peptide-loading compartment. We chose to generate monoclonal antibodies against the peptide-loading compartment that we previously characterized as lysozyme loading compartment (LLC) [Escola, J.M., Grivel, J.C., Chavrier, P., Gorvel, J.P., 1995. Different endocytic compartments are involved in the tight association of class II molecules with processed hen egg lysozyme and ribonuclease A in B cells. J. Cell Sci. 108, 2337; Escola, J.M., Deleuil, F., Stang, E., Boretto, J., Chavrier, P., Gorvel, J.P., 1996. Characterization of a lysozyme-major histocompatibility complex class II molecule-loading compartment as a specialized recycling endosome in murine B lymphocytes. J. Biol Chem. 271, 27360]. A preliminary screening by dot blot enabled us to identify several monoclonal antibodies recognizing the LLC and not early and late endosomes. One of these antibodies, the 20C4, was then characterized. It is directed against mature class II molecules of all murine haplotypes. By electron microscopy, 20C4 labeling was restricted to both the plasma membrane and the LLC. These reagents may be useful in the further characterization of the specialized function of these intracellular organelles.

MHC class II-associated invariant chain-induced enlarged endosomal structures: a morphological study.

The major histocompatibility complex class II-associated invariant chain is believed to direct newly synthesized class II to endocytic compartments. Invariant chain synthesized at high levels in transiently transfected cells induces formation of large vesicular structures. We have examined the effect of stable expression of invariant chain in human fibroblasts by light and electron microscopy. Invariant chain expression dramatically modified endocytic compartments and induced the formation of greatly enlarged structures. These modifications were not lethal. Ultrastructurally, at least three morphologically distinct enlarged compartments could be discerned in the cells. These three compartments may represent early and late endosomes and lysosomes. Internalization of anti-invariant chain antibodies shows that invariant chain may reach the large endosomes via rapid internalization from the plasma membrane. Internalized protein remained in the enlarged vesicles for 4-6 h, indicating an invariant chain-induced delay in the pathway to lysosomes. Although the large invariant chain-induced vesicles have not yet been seen in professional antigen-presenting cells, the invariant chain-induced effects may play a role in regulating the endocytic pathway, creating a special environment for MHC class II to bind antigen.

The ultrastructural localization of metallothionein in cadmium exposed rat liver.

The ultrastructural localization of metallothionein (MT) was investigated in the liver of male Wistar rats by a cryo-immunocytochemical technique. The liver parenchymal and sinusoidal cells were studied in both cadmium-exposed (3 x 1.2 mg kg-1 as cadmium chloride) and non-treated animals. Treatment with cadmium induced the synthesis of MT yet differences in the distribution were evident amongst the various types of liver cell. MT was found most abundantly in the parenchymal and endothelial cells, yet was absent in the stellate cell and sparsely distributed in the Kupffer cell. In the cells where MT gene expression was induced, the metalloprotein was distributed within both the nuclear and cytoplasmic compartments. The significance of the nuclear localization of MT is discussed.

Endocytosis of the recombinant tissue plasminogen activator alteplase by hepatic endothelial cells.

The glycoprotein tissue-type plasminogen activator (t-PA, alteplase, CAS 105857-23-6) is a serine protease consisting of 527 amino acids and can activate plasminogen to plasmin, which subsequently dissolves the fibrin network of a thrombus. This activation occurs selectively on the thrombus, making recombinant t-PA a very effective agent in the treatment of thromboembolic disorders. t-PA has a short in vivo half-life and is rapidly removed from the circulation by the liver. The catabolism of t-PA involves receptor-mediated endocytosis and intracellular degradation in several cell types of the liver namely hepatic endothelial, parenchymal and Kupffer cells. Liver endothelial cells have been reported to possess a specific uptake system for t-PA based on the recognition of the high mannose carbohydrate structures on Asn117. To further elucidate the involvement of the mannose receptor on sinusoidal endothelial cells in the hepatic catabolism of t-PA and to identify the mechanisms involved, biochemical as well as electron microscopic studies were performed. The biochemical studies revealed that the removal of the mannose side chain in t-PA significantly reduced its clearance and degradation in isolated perfused livers. The binding of t-PA to preparations of primary hepatocytes and liver cell membranes could not be competed for by various sugars and glycoproteins, and was not dependent on the presence of carbohydrates on the molecule. This ruled out a major relevance of the sugar moieties of t-PA in its recognition by liver cells that were not of endothelial origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and hepatic catabolism of tissue-type plasminogen activator.

Recombinant tissue-type plasminogen activator (rt-PA, alteplase, Actilyse, Activase; CAS 105857-23-6) is the most effective agent currently available for thrombolytic therapy of life-threatening diseases such as acute myocardial infarction. It acts by rapid, clot-specific lysis of pathological thrombi, with only limited effects on systemic hemostasis. Pharmacokinetics of rt-PA have been extensively characterized in animal species and man, and can be generally described by a 3-compartment model. Preferred analytical methods for rt-PA in plasma are ELISA and chromogenic activity assays. The dominant plasma half-life of rt-PA in myocardial infarction patients is short (3.6 min), which allows excellent control of plasma levels during therapy. Steady-state plasma concentrations effecting coronary thrombolysis using the current dosage regimen are 2.2 micrograms/ml. A deep compartment results in elevated rt-PA concentrations several hours after termination of infusions, which may contribute to short-term maintenance of patency of reperfused blood vessels. Clearance of rt-PA can be saturated in animals at very high plasma concentrations (Km = 12-15 micrograms/ml), however, pharmacokinetics in clinical settings are linear. Clearance occurs via hepatic receptor mediated endocytosis and intracellular degradation in liver parenchymal, endothelial and Kupffer cells. The catabolism involves coated pits, coated vesicles, endosomes, and finally degradation in lysosomes. Current evidence supports the existence of hepatic receptors recognizing carbohydrate as well as polypeptide determinants in rt-PA. In conclusion, increasing knowledge of rt-PA pharmacokinetics will contribute to the optimization of new clinical dosage regimens, such as front-loaded infusions and boluses, and to the identification of novel molecular targets for pharmacologic control of rt-PA catabolism and of circulating fibrinolytic activity.

Invariant chain induces a delayed transport from early to late endosomes.

Invariant chain associated with class II molecules is proteolytically processed in several distinct intermediates during its transport through the endocytic pathway. Using subcellular fractionation, early and late endosomal compartments were separated in human fibroblasts transfected with HLA-DR (4N5 cells) and supertransfected with invariant chain (4N5Ii cells) or invariant chain lacking most of the cytoplasmic tail (4N5 delta 20Ii cells). Early and late endosome membrane fractions were characterized by morphology and by analyzing the presence of the Rab5 and Rab7 GTPases as markers of early and late endosomes, respectively. The transfer of endocytosed horseradish peroxidase from early to late endosomes proceeded relatively rapid both in 4N5 and 4N5 delta 20Ii cells (t1/2 = 25 min), whereas this transfer was significantly delayed (t1/2 = 2 h) in 4N5Ii cells. Pulse-chase experiments showed that invariant chain and its degradation products were first observed in early endosomes and thereafter in late endosomes. Our results strongly suggest that invariant chain induces a retention mechanism in the endocytic pathway.

Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization.

Class II molecules of the major histocompatibility complex (MHC) bind peptides derived from protein antigens delivered into endocytic compartments and present these peptides to CD4+ T cells. The precursors to functional MHC class II molecules loaded with peptides are complexes of the invariant chain associated with class II alpha beta heterodimers. Targeting of newly synthesized MHC class II molecules to endosomes is mediated by the invariant chain, but the intracellular transport route is not known. This study demonstrates that in a human B-cell line a large population of MHC class II-invariant chain complexes reaches endosomes by rapid internalization from the cell surface. Quantitation of cell surface MHC class II-invariant chain complexes and of their surface half-life revealed that 3000 complexes internalized per minute into endosomes. This highly efficient endocytosis was mediated by the cytoplasmic tail of the invariant chain. After internalization, the invariant chain dissociated from the MHC class II-invariant chain complexes. This pathway may represent an important mechanism for loading class II molecules with immunogenic peptides from several endocytic compartments.

Endocytosis and intracellular processing of tissue-type plasminogen activator by rat liver cells in vivo.

Endocytosis of tissue-type plasminogen activator (t-PA) by different types of rat liver cells was studied in immunocytochemically labelled cryosections as well as in biochemical experiments. For morphological localization of the ligand in different endocytic compartments involved in its catabolism, rat livers were fixed at various times (1-24 min) after injection of t-PA. Late-endosomal and lysosomal compartments were identified by double-labelling the sections with antibodies to the lysosomal proteins glycoprotein Igp 120 and cathepsin D. In liver t-PA was localized in sinusoidal endothelial cells (EC), parenchymal cells (PC) and to some extent in Kupffer cells (KC), indicating that it is internalized and degraded in all three cell types. In specimens fixed 6 min after injection PC, EC and KC were found to contribute to 69, 24 and 7% respectively of total t-PA endocytosed. The transfer from late endosomes to lysosomes was found to be faster in EC than in PC. The morphological findings were supported by studies of the endocytic mechanisms employing isolated perfused livers and primary hepatocytes. The presence of monensin, an inhibitor of lysosomal protein degradation, reduced the amount of t-PA degraded to about 50% of the control values. The catalytic site seems not to be required for the catabolism of t-PA in hepatic cells. The inhibition of t-PA by D-phenylalanyl-L-prolylarginyl-chloromethane did not influence receptor recognition and catabolic processing, as determined in morphological studies using labelled cryosections, in binding studies employing liver cell membranes and primary hepatocytes, as well as in liver-perfusion experiments.

Evidence for carbohydrate-independent endocytosis of tissue-type plasminogen activator by liver cells.

In the liver, tissue-type plasminogen activator (t-PA) is endocytosed by hepatic parenchymal (PC), endothelial (EC) and Kupffer (KC) cells. Although the endocytosis is receptor-mediated, it remains a matter of discussion which receptors are involved in this catabolic process. To evaluate the role of a protein-specific receptor, as well as the possible involvement of the galactose receptor on PC and the mannose receptor on EC, we have employed different glycosylation variants of t-PA in biochemical and immunocytochemical studies. Partial or total removal of carbohydrate side-chains by endoglycosidases did not prevent clearance and hepatic endocytosis of t-PA by either of the liver cell types. Blockade of the galactose and mannose receptors by co-application of a large excess of the glycoprotein ovalbumin remained without effect on the binding and uptake of t-PA by hepatic cells. However, the contribution of different liver cell types to the hepatic clearance of t-PA was to a certain extent dependent on the type of oligosaccharide chains removed. The mannose receptor on EC is partially responsible for the clearance of t-PA by this cell type, whereas the galactose receptor does not seem to be involved in this process. The results obtained in this study further demonstrate that the major portion of the hepatic catabolism of t-PA is independent of its carbohydrate side-chains.

Sorting of MHC class II molecules and the associated invariant chain (Ii) in polarized MDCK cells.

Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the basolateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells.