Cell Biology

The syndecans, a family of transmembrane heparan sulfate proteoglycans, are ubiquitous molecules whose intracellular function is still unknown. To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycan in fibroblasts, we performed transfection studies in COS-1 and Swiss 3T3 cells. Endogenous syndecan-2 colocalized with F-actin in cortical structures. Overexpression of full-length syndecan-2 induced the formation of long filopodia-like structures. These changes correlated with a rearrangement of the actin cytoskeleton, which strongly colocalized with syndecan-2. Overexpression of syndecan-2 lacking the extracellular domain increased the number of microspikes on the cell surface but failed to induce filopodia. Addition of heparin blocked the effect of full-length syndecan-2, suggesting that heparan sulfate chains in the extracellular domain are necessary to induce filopodia. Coexpression of cdc42Hs negative-dominant N17 blocked syndecan-2-induced filopodia and cdc42Hs positive-dominant V12 had a synergic effect. This indicates that active cdc42Hs is necessary for syndecan-2 induction of filopodia. These results provide a link between syndecan-2, actin cytoskeleton, and cdc42Hs.

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.

In recent years, several antagonists of αvβ3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-αv-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of αv-integrin and the molecular organization of focal structures. Anti-αv-integrin-blocking antibodies 17E6 and LM609, and an anti-αvβ3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. αv-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of αv-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with αv-integrin antagonists disrupts the actin cytoskeleton, redistributes αv-integrin and induces molecular disassembly of focal contacts.

Lipoprotein lipase (LPL) is produced by cells in the artery wall and can mediate binding of lipoproteins to cell surface heparan sulfate proteoglycans (HSPG), resulting in endocytosis (the bridging function). Active, dimeric LPL may dissociate to inactive monomers, the main form found in plasma. We have studied binding/internalization of human low density lipoprotein (LDL), mediated by bovine LPL, using THP-1 monocytes and macrophages. Uptake of 125I-LDL was similar in monocytes and macrophages and was not affected by the LDL-receptor family antagonist receptor-associated protein (RAP) or by the phagocytosis inhibitor cytochalasin D. In contrast, uptake depended on HSPG and on membrane cholesterol. Incubation in the presence of dexamethasone increased the endogenous production of LPL by the cells and also increased LPL-mediated binding of LDL to the cell surfaces. Monomeric LPL was bound to the cells mostly in a heparin-resistant fashion. We conclude that the uptake of LDL mediated by LPL dimers is receptor-independent and involves cholesterol-enriched membrane areas (lipid rafts). Dimeric and monomeric LPL differ in their ability to mediate binding/uptake of LDL, probably due to different mechanisms for binding/internalization.

Platelet-derived growth factor (PDGF) has been implicated in vascular smooth muscle cell proliferation and migration, a key process in vascular disease. PDGF is a family of dimeric isoforms of structurally related A-, B-, C- and D-chains that bind to PDGF receptors. PDGF A- and B-chains occur with and without basic C-terminal amino acid extensions as long (AL and BL) and short (AS and BS) isoforms. This basic sequence has been implicated as a cell retention signal through binding to glycosaminoglycans, especially to heparan sulfate. The aim of this study was to evaluate the biological relevance of PDGF interaction with glycosaminoglycans on the PDGF function in human arterial smooth muscle cells (hASMC). Here, we show that long PDGF isoforms showed greater affinity for hASMC cell surface and that they also presented more colocalization with heparan and chondroitin sulfates present on hASMC cell membrane than did short isoforms. Furthermore, all PDGF isoforms colocalized more with heparan sulfate than with chondroitin sulfate and there was little colocalization between heparan and chondroitin sulfate. PDGF-stimulated hASMC activation of DNA synthesis and directed migration (chemotaxis) was also examined. The isoform PDGF-BBS induced maximal proliferation and migration of hASMC. Collagen-I coating significantly increased hASMC motility towards PDGF isoforms, and particularly toward PDGF-BBS. These results strongly support the notion that cell surface glycosaminoglycans are not essential for receptor-mediated activity of PDGF and may contribute basically to the retention and accumulation of long PDGF isoforms.