ß-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. ß-Catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. ...However, in some developmental contexts and cell systems ß-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of ß-Catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous ß-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase¿synchronized epithelial cells. ß-Catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of ß-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not ß-catenin, is detected in M phase. Interestingly, overexpression of a stable form of ß-catenin, or inhibition of endogenous ß-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for ß-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.
The platelet-derived growth factor (PDGF) family comprises important mitogens for mesenchymal cells. The active dimeric form of PDGF consists of four structurally related A, B, C, and D chains. All ...PDGF-variants bind to PDGF-receptors. The A and B chains occur with and without basic C-terminal amino acid extensions as long (A(L) and B(L)) and short (A(S) and B(S)) isoforms. PDGF-A and -B form homo- or heterodimers. The biological relevance of short and long isoforms is unknown, although it may relate to different affinities for glycosaminoglycans of the cell glycocalix and intercellular matrix. Commercially available anti-PDGF-A and anti-PDGF-B antibodies cannot discriminate between the short and the long isoforms. Thus, to investigate the function of the long and short isoforms, we raised antibodies specific for the long A and B chain isoforms. The antibodies were affinity-purified and their properties analysed by surface plasmon resonance. Inhibition studies with different PDGF homodimers and dot-blot studies proved their high specificity for the respective isoforms. Both antibodies recognised the target PDGF homodimers complexed to the glycocalix of human arterial smooth muscle cells and human monocyte-derived macrophages. By using these specific antibodies, we were able to confirm at the protein level the synthesis of PDGF-A and -B during differentiation of human monocyte-derived macrophages and to demonstrate the presence of the PDGF-A(L) and PDGF-B(L) isoforms in human arterial tissue.
Syndecans, transmembrane heparan sulfate proteoglycans (HSPG), mediate cell–cell and cell–matrix adhesion thereby controlling cell movement and shape. Syndecan cytoplasmic domains are very short (ca. ...30 amino acids) and divided into two constant regions (C1 and C2) separated by one variable (V) region. Here we attempted to map the cytoplasmic region responsible for the filopodia-inducing effect of syndecan-3. We found that only the C1-region was necessary for this effect. In addition, the deletion of the C2-region led to extensive membrane blebbing. Nevertheless, the elimination of the entire cytoplasmic region did not affect delivery of syndecan-3 to the plasma membrane. These results indicate that the different regions of syndecan-3 cytoplasmic domain have different functions probably by binding to distinct proteins.
Syndecans (heparan sulfate proteoglycans) participate in cell–cell and cell–matrix adhesion and are co- and low-affinity receptors for growth factors and enzymes, respectively. We examined the ...influence of stable syndecan-2 expression in Swiss 3T3 cells on cell-adhesion and proliferation. Higher syndecan-2 expression changed cell morphology and increased spreading and adhesion in these cells and proliferation induced by FCS and FGF-2. This emphasizes the role of syndecan-2 in the integration of signals from soluble and insoluble factors.
Neonatal rat hepatocytes cultured in the absence of added growth factors dedifferentiate by epithelial‐mesenchymal transition (EMT). This involves the loss of their typical differentiation markers, ...the acquisition of a migrating morphology, and a change in the expression of the intermediate filament (IF) proteins. We attempted to determine whether the EMT of cultured neonatal rat hepatocytes could be modulated by factors that maintain and promote the differentiation state of adult and fetal hepatocytes such as epidermal growth factor (EGF) and dimethyl sulfoxide (DMSO). By (3H)‐thymidine incorporation, Western blotting analysis, flow cytometry, and double‐immunofluorescence studies, we found that both factors have marked but opposite effects on the EMT and on proliferation of neonatal liver cells. In DMSO treatment, albumin levels were higher than in the nontreated cells at all days studied. Moreover, DMSO reduced cytokeratin levels and inhibited cell proliferation, acquisition of the fibroblast‐like morphology, and vimentin expression typical of the EMT. The increase in vimentin‐positive cells in serum‐free medium was not observed in DMSO cultures. EGF also increased albumin levels at all days studied. In contrast, EGF treatment induced hepatocyte proliferation and enhanced vimentin and cytokeratin expression. However, the increase in vimentin levels did not correlate with a significant increase in the number of vimentin‐positive cells. Moreover, vimentin‐positive cells in EGF treatment were also cytokeratin‐positive and albumin‐positive, and they maintained epithelioid morphology in spite of the vimentin network. These results indicate that EMT of cultured rat neonatal hepatocytes is differentially regulated in response to EGF and DMSO.
HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, ...we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF
4
−) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF
4
−, in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions.
When hepatocyte‐enriched fractions from neonatal rat livers were cultured for different times in the absence of added growth factors, a population of highly proliferating and migrating fibroblastlike ...cells appeared. Double immunofluorescence with antibodies to cytokeratin and to vimentin showed a progressive reduction in the number of cytokeratin‐positive cells parallel to an increase in the vimentin‐positive cells. Some cells with transitional epithelial or migrating morphology coexpressed both intermediate filament proteins. Immunofluorescence with antibodies against hepatocyte differentiation markers showed that shortly after seeding most of the cells were positive to anti‐albumin antibodies, but after 1 week in culture, only 10% were positive. Cells presenting albumin and cytokeratin appeared morphologically epithelial. Fibroblastlike cells were not positive for albumin, but some cells with transitional epithelial morphology presented some labels for albumin and for vimentin. Immunofluorescence with antibodies to glutathione‐S‐transferase subunit Pi and vimentin showed that many fibroblastlike cells were positive for both markers, some of them binucleate. Cultures performed in the presence of dexamethasone, absence of arginine, or on collagen type I matrix had no effect on the behavior of neonatal hepatocytes. The appearance of fibroblastlike cells was ontogenically regulated because the highest increase in the percentage of vimentin‐positive cells was observed in cell cultures from livers of 7‐and 15‐day‐old animals. These data provide evidence that neonatal hepatocytes in culture have the potential to dedifferentiate by epithelial‐mesenchymal transition and contribute to an understanding of hepatic growth development.
Lipoprotein lipase (LPL), a key enzyme in lipoprotein triglyceride metabolism, produces a marked increase in the retention and uptake of all classes of lipoproteins by cultured cells. It was ...previously shown that two different receptors are involved in mediating the LPL effects: heparan sulfate proteoglycans (HSPG) and the low density lipoprotein (LDL) receptor-related protein/alpha 2 macroglobulin receptor (LRP). By immunofluorescence we show here that cell surface-bound LPL displays a pattern that corresponds to the previously described distribution of cell surface HSPG. No evident relation to the distribution of bound activated alpha 2-macroglobulin (alpha 2M*) or to LRP was observed. By immunoelectron microscopy we found that after 30 min at 37 degrees C most of the detected alpha 2M* (70% of the total gold particles) was inside the cells and associated with endosomal vesicles. However, at the same time, 76% of the LPL remained at the cell surface, suggesting that, LPL is internalized by a slow endocytic process. Binding of triglyceride-rich lipoproteins (TRL) or LDL together with LPL led to a spectacular increase in bound lipoproteins, which completely colocalized with LPL. After incubation at 37 degrees C, LPL and 1,1'-dioctadecyl-3,3,3,'3'-tetramethylindocarbocyanine (DiI)-TRL formed large clusters on the cell surface. Immunofluorescene and quantitative immunoelectron microscopy provided evidence of co-internalization of LPL and apoE-containing TRL by a slow endocytic process. In the absence of LPL, the fibroblasts rapidly internalized DiI-LDL and showed fluorescence in central, lysosome-like vesicles. In contrast, when LPL was present, internalization of DiI-LDL involved small, widely distributed vesicles. This pattern slowly changed to one consisting of large perinuclear vesicles. LDL receptor-deficient fibroblasts internalized DiI-LDL, either with or without LPL, into small widely distributed vesicles and no central vesicles were seen. Chloroquine-treated normal fibroblasts internalized DiI-LDL in a pattern similar to that of receptor-deficient fibroblasts. Taken together our results suggest an alternative receptor-independent endocytosis pathway for LDL. This pathway is potentiated by LPL and is characterized by a slow uptake involving small vesicles that gradually reach lysosomes. We suggest that, through its interaction with HSPG, LPL provides high capacity binding sites for lipoproteins and a independent internalization pathway.
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