Ultrastructure study of skin fibroblasts in patients with Ehlers-Danlos Syndrome (EDS): preliminary results.

AIM OF THE STUDY: To investigate, in vivo and in vitro, the fibroblast-to-myofibroblast transition in patients with hypermobile Ehlers-Danlos Syndrome (EDS). To analyze the dermis of patients with classical form of EDS (cEDS) and with hEDS, to identify qualitative and/or quantitative differences in ECM component and ultrastructural changes in collagen. MATERIALS AND METHODS: Seven subjects, aged over 18, two with cEDS and five with hEDS underwent two skin biopsy. One sample was prepared for transmission electron microscopy (TEM), the other for immunofluorescence. The diameter of collagen fibers was measured with TEM. Fibrils were analyzed in four patients: the two with cEDS and two with hEDS. For each patient, the diameter of n=250 collagen fibrils was measured. αSMA was used as specific marker for myofibroblast to highlight their presence in vivo in the skin of patients with hEDS. RESULT: IF observation could not assess an increased expression of αSMA in hEDS patients, which showed no statistical difference compared to classic form patients. The major result from the analysis of TEM images is the clear difference in ECM composition between the two forms of EDS: ECM in hEDS is optically more dense and more prominently composed of elastic fibers. CONCLUSION: Our study provides the following important evidence: 1) the absence in vivo of dermal fibroblasts in patients with hEDS, demonstrated by αSMA negativity; 2) the presence of statistically significant changes in the diameter of collagen fibrils between the classic and the hypermobile forms.

Free diffusion to and from the inner nuclear membrane of newly synthesized plasma membrane glycoproteins.

Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.

Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex.

We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.

Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization.

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

Effect of ATP depletion and DTT on the transport of membrane proteins from the endoplasmic reticulum and the intermediate compartment to the Golgi complex.

Newly synthesized membrane proteins are exported from the endoplasmic reticulum to the Golgi complex through an intermediate compartment. Incubation at low temperature (15 degrees C) arrests the proteins in the intermediate compartment and prevents the entry into the Golgi complex. We have studied, in living cells, the effect of dithiothreitol (DTT) and ATP depletion on the transport to the Golgi complex of proteins accumulated either in the endoplasmic reticulum or in the intermediate compartment after a temperature block. The morphological results obtained with vesicular stomatitis virus ts-O45 G glycoprotein and the biochemical analysis performed with human CD8 protein, an O-glycosylated protein, showed that: 1) ATP depletion blocks the export to the Golgi complex of proteins located either in the endoplasmic reticulum or in the intermediate compartment and ii) DTT interferes with the folding and export of proteins located in the endoplasmic reticulum, but it does not prevent the transfer from the intermediate compartment to the Golgi complex.

Human CD8 alpha glycoprotein is expressed at the apical plasma membrane domain in permanently transformed MDCK II clones.

Madin-Darby canine kidney cells (MDCK II) have been cotransfected with plasmids expressing the human CD8 alpha glycoprotein and the bacterial gene which confers resistance to neomycin. Stable transformants have been isolated in the presence of G-418 in the culture medium and screened for CD8 alpha expression by immunofluorescence. The three clones we have characterized showed: 1) high level of synthesis and efficient surface expression of glycosylated, homodimeric CD8 alpha and 2) preferential apical deposition of CD8 alpha in confluent monolayers. This polar distribution has been measured in cells grown on a plastic substratum as well as on nitrocellulose filter by means of EM immunocytochemistry and surface radioimmunoassay. CD8 alpha was 6 to 11-fold enriched on the apical membrane whereas the 58 kDa protein, a basolateral marker in MDCK II cells, resulted about 9-fold enriched on the basolateral membrane of the three clones. We believe these permanently transformed clones could prove to be a useful tool with which to study cell polarity.

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand.

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.

Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

Endocytosis of proteins by salivary gland duct cells.

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multivesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma.

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-µ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.

JNK2 is activated during ER stress and promotes cell survival.

Adaptation to endoplasmic reticulum (ER) stress relies on activation of the unfolded protein response (UPR) and induction of autophagy. Indeed, cells die if ER stress is not countered by the UPR. Here we show in U937 cells that the ER stressors tunicamycin and thapsigargin cause increased expression of c-Jun N-terminal kinase 2 (JNK2), which allows regulation of the UPR, whose silencing or pharmacological inhibition delays BiP (immunoglobulin heavy-chain binding protein) upregulation, and causes earlier and greater expression of CCAAT/enhancer-binding protein-homologous protein (CHOP). Furthermore, we show that pharmacological inhibition or silencing of JNK2 causes accumulation of both p62 and the acidic compartment, caspase 3 activation and apoptosis. Our results reveal that JNK2 prevents accumulation of the acidic compartment in U937 cells undergoing autophagic flux and, by this mechanism, it keeps stressed cells alive. Our findings highlight a potential role for JNK2 in tumor cell survival, senescence and neurodegenerative diseases, in which ER stress, autophagy and lysosome activity are known to interplay.

Celecoxib upregulates multidrug resistance proteins in colon cancer: lack of synergy with standard chemotherapy.

Recent phase II randomised trials in colorectal cancer failed to demonstrate any advantage of celecoxib combined with standard chemotherapy; some authors even reported that the addition of celecoxib to irinotecan and oxaliplatin in colon cancer results in an inferior response rate. This observation leads to the hypothesis that there are pharmacokinetic interactions between celecoxib and chemotherapeutic drugs. The aim of the study was to investigate the induction by celecoxib of some multidrug resistance proteins, MRP1, MRP2, MRP4 and MRP5, involved in the transport of irinotecan and 5-FU. WiDr and COLO-205 cells were treated with celecoxib at a clinically relevant concentration. A viability assay was performed by treating cells with chemotherapy alone and chemotherapy plus celecoxib. The expression of MRP1, MRP2, MRP4 and MRP5 was analysed by RT-PCR and Western blot analysis. The sub cellular localization of MRP4 and MRP5 was investigated by cryoimmunoelectron microscopy. In both cell lines celecoxib induced MRP4 and MRP5 over-expression at RNA and protein levels. No induction of MRP1 and MRP2 was observed in treated cells compared to controls. Cryoimmunoelectron microscopy showed increased MRP4 and MRP5 immunolabeling in celecoxib treated cells both at cytoplasmic level and along the plasma membrane. Our findings suggest that the low response rate observed in clinical trials using celecoxib added to 5-fluorouracil and irinotecan may reflect celecoxib-mediated extrusion of chemotherapeutic drugs from cancer cells through the up regulation of ATP-binding cassette proteins. Our findings, together with the results of clinical trials, may suggest that the combined use of celecoxib and drugs that are substrate for MRP4/MRP5 should be avoided.

Endocytosis of parotid salivary proteins by striated duct cells in streptozotocin-diabetic rats.

Salivary gland striated duct cells play an important role in the modification of primary saliva by secretion and reabsorption of electrolytes, and secretion of glycoproteins. Recent observations have shown that in the rat parotid gland these cells are able to internalize exogenous proteins, e.g., horseradish peroxidase and ferritin, from the ductal lumen. In rats made diabetic by injection of streptozotocin, dense vacuoles and crystalloids are present in the apical cytoplasm of parotid striated duct cells. In this study we utilized electron microscopic immunocytochemistry to determine if these vacuoles and crystalloids contain acinar secretory proteins. At various times after induction of diabetes by streptozotocin (65 mg/kg), the parotid glands were fixed in a glutaraldehyde-formaldehyde mixture, postfixed in OsO4, and embedded in epoxy resin. Thin sections were immunolabeled with antibodies to protein B1 (Ball et al., 1988) and alpha-amylase (Baum et al., 1982) using a modification of the Protein A-gold technique (Bendayan and Duhr, 1986). With antibody to B1, label was localized in the secretory granules of acinar and intercalated duct cells of both normal and diabetic rats. In striated duct cells of diabetic rats, label was present over the electron-dense vacuoles but not over the crystalloids. Since crystalloids appear to form within the vacuoles, their lack of reactivity may indicate degradation of the internalized protein. The same distribution of label was found with antibody to amylase except for the intercalated duct granules, which were unlabeled in both control and diabetic animals. These results demonstrate that striated duct cells take up salivary proteins from the lumen and that the endocytosis of some secretory proteins from the saliva may be a significant function of these cells in certain pathological conditions.

Endocytosis of native and glycosylated bovine serum albumin by duct cells of the rat parotid gland.

The ability of duct cells of the rat parotid gland to internalize bovine serum albumin (BSA) and several glycosylated albumins (glucosamide, galactosamide, fucosamide, lactosyl, p-aminophenyl-N-acetyl-D-glucosamide, p-aminophenyl-N-acetyl-D-mannopyranoside, p-aminophenyl-N-acetyl-D-galactosamide) was investigated. The various BSA preparations were infused into the gland via the main excretory duct, after which the tissues were fixed and prepared for light and electron microscopy. Immunolocalization of native BSA, as well as the glycosylated BSAs, was performed on thin sections, using an unlabeled antibody to BSA followed by protein A-colloidal gold. Gold particles were present over the lumina of both intercalated ducts and striated ducts, and over small endocytic structures and large vacuoles in the apical cytoplasm of both duct cell types. Endocytosis of the glycosylated BSAs by duct cells was greater than native BSA. Fucosylamide-BSA and mannopyranoside-BSA was taken up to a greater extent than the other glycosylated BSAs. Uptake by intercalated duct cells was greater than by striated duct cells, was independent of the concentration of the glycosylated BSA, and was reduced by an excess of the corresponding sugar. Striated duct cells showed some damage by the glycosylated BSAs that was concentration-dependent, and which was reduced in the presence of an excess of the corresponding sugar. These results suggest that endocytosis by salivary gland duct cells may involve specific recognition of carbohydrate residues and that the endocytosis of acinar secretory proteins observed in certain conditions may be due to increased and/or altered protein glycosylation.

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells.

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37 degrees C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37 degrees C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunolabelling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.

Regional distribution of Sindbis virus glycoproteins on the plasma membrane of infected baby hamster kidney cells.

Sindbis virus-infected baby hamster kidney (BHK) cells were analysed in surface replicas or conventional thin sections after specific immunolabelling with antiviral glycoprotein antibodies in conjunction with colloidal gold-conjugated protein A. Newly synthesized viral glycoproteins were detected, beginning 1 1/2 h after infection, while the virus maturation started 3 h after infection. The glycoproteins appeared to be inserted on the plasma membrane in large spots located mainly in the central area of the cells: no clustering of the labelling was detected. Later, the glycoproteins appeared to arrange linearly in regions in the medial portion of the cells. No labelling was found in the peripheral area or on the cell edges. A drastic change in the surface labelling was detected following the commencement of virus maturation: gold particles were organized mostly in small clusters, each labelling a budding virus. Very few glycoproteins appeared not to be involved in budding figures. The maturation of the virus was clearly regionalized, but during this time it also involved the peripheral area and the cell edges; preferential budding in narrow cellular processes was often observed. It appeared thus that either isolated glycoproteins soon after infection, or clustered glycoproteins at later times, are strictly regionalized on the plasma membrane: however, the early post-infection distribution is clearly different from that seen later during virus maturation. Our experiments support the concept of discrete plasma membrane domains even in cells that do not display distinct specialization of their surface.

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells.

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.

Nonrandom distribution of epidermal growth factor receptors on the plasma membrane of human A431 cells.

The localization of epidermal growth factor (EGF) receptors over the plasma membranes of human epidermoid carcinoma A431 cells was analyzed at the electron microscopic level using surface replica techniques and conventional thin sections, in combination with immunocytochemistry. Immunolabeling was performed using two distinct monoclonal antibodies directed against the extracellular portion of the receptor, followed by protein A-colloidal gold conjugates. Unexpectedly, with the first monoclonal antibody used, the distribution of the receptors in both unfixed and glutaraldehyde-fixed cells was clearly regionalized, showing a preferential localization of the immunolabeling at the cell periphery as well as over the areas rich in microvilli and in coated and uncoated pits. A similar pattern of distribution was observed also with the other monoclonal antibody, but only when the cells were fixed with glutaraldehyde before immunolabeling. Treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate modifies this distribution, inducing a more disperse pattern. Our observations suggest that a minor group of EGF receptors, which may represent the high-affinity receptors, presents a regional distribution, similar to that described for typical recycling receptors.

Surface distribution and internalization of erbB-2 proteins.

We report the localization over the cell surface and the early steps of antibody-induced internalization of the product of the erbB-2 proto-oncogene, structurally related to the epidermal growth factor receptor (EGFR). We show that erbB-2/p 185 is mostly excluded from endocytic pits on the cell surface. Incubation at 37 degrees C with an anti-erbB-2/p185 monoclonal antibody induces the rapid entry of the protein into the cell. Similar internalization is shown by a chimeric molecule EGFR/erbB-2 in response to EGF. Both the timing and the pathway of internalization followed by the erbB-2/p185 appear totally similar to those described for the EGFR. At variance with the normal erbB-2/p185, two mutant activated erbB-2 proteins are frequently localized within endocytic pits of the cell surface, indicating that mutations in the transmembrane regions may determine constitutive internalization of the protein.