Membrane associated proteoglycans in rat testicular peritubular cells

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG) Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (K_av=0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.Abbreviations PG Proteoglycans - CSPG Chondroitin Sulfate Proteoglycans - HSPG Heparan Sulfate Proteoglycans - HSCSPG Heparan and Chondroitin Sulfate Proteoglycans - DNAse I Deoxyribonuclease I - DMEM Dulbeccos modified Eagle's medium - H/D HAM F12/DMEM - ECM Extracellular Matrix - PBS Phosphate Buffered Saline - PI Phosphatidylinositol - GPI Glycosyl Phosphatidylinositol - PI-PLC Phosphatidylinositol Specific Phospholipase C - TBS Tris Buffered Saline - STI Soybean Trypsin Inhibitor - GAG Glycosaminoglycans - HA Hyaluronic Acid     

Biology of cell surface heparan sulfate proteoglycans

The central question in cell biology is how cells detect, interact and respond to extracellular matrix. The cell surface molecules, which mediate this recognition, consist of a lipophilic membrane domain and an ectodomain binding matrix materials. One group of this kind of molecules is the cell surface heparan sulfate proteoglycans (HSPG). This review summarizes recent information obtained on the cell surface PG of mouse mammary epithelial cells. The glycosaminoglycan containing ectodomain of this PG binds with high affinity Type I, III and V collagen fibrils and the C-terminal heparin binding domain of fibronectin. The PG is mobile on the cell surface, but can be immobilised by ligand binding. At the same time the PG associates with cytoskeleton and links the epithelial cytoskeleton to extracellular matrix. Thus the PG can mediate the changes in the matrix into changes in cellular behaviour, often seen during the regulation of cell shape, proliferation and differentiation. The cell surface PG is also released from the cell surface by cleaving the matrix-binding ectodomain from the membrane domain. Because of the binding properties of the ectodomain, this shedding may provide a means by which epithelial cells loosen their association with the matrix and with other cells, e.g., during normal epithelial development and the invasion of carcinomas.     

Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta

We have studied the interaction of 125I-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.     

Biochemical characterization of integral membrane heparan sulfate proteoglycans in Sertoli cells from immature rat testis

(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.     

Degradation of heparan sulfate proteoglycans enhances oxidized-LDL-mediated autophagy and apoptosis in human endothelial cells

BackgroundCell surface heparan sulfate proteoglycans (HSPG) play an important role in atherogenesis. We hypothesized that degradation of HSPG may increase the binding of atherogenic oxidized low density lipoprotein (ox-LDL) to endothelial cells, and result in extensive HSPG degradation as well as autophagy and apoptosis.MethodsPrimary human umbilical vein endothelial cells (HUVECs) were used to study the expression of lectin-like ox-LDL receptor-1 (LOX-1), HSPG, autophagy and apoptosis in response to ox-LDL and heparinase III (Hep III).ResultsAs expected, ox-LDL treatment resulted in LOX-1 expression, ox-LDL uptake and reactive oxygen species (ROS) generation. Ox-LDL treatment also resulted in a modest degradation of HSPG and increase in autophagy (expression of LC3, beclin-1 and Atg5) and apoptosis (enhanced expression of caspases and Bax, and reduced expression of Bcl-2 and Bcl-xL). The effects of ox-LDL were blocked by pretreatment of cells with LOX-1 antibody or apocynin, an NADPH oxidase inhibitor. Hep III alone caused HSPG degradation and slightly, but significantly, increased ROS generation, and induced autophagy and caspase expression. However, autophagy and apoptosis induced by Hep III were not affected by apocynin or LOX-1 antibody. Importantly, Hep III pretreatment of cells significantly enhanced ox-LDL-induced HSPG degradation, LOX-1 expression, ox-LDL uptake and ROS generation as well as autophagy and apoptosis.ConclusionThese data demonstrate that Hep III enhances the pro-atherosclerotic characteristics in HUVECs induced by ox-LDL.     

The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling

Binding of growth factors to specific cell surface receptors is the first step in initiating cell signaling cascades that ultimately result in diverse activities such as proliferation, differentiation, and apoptosis. Dimerization and phosphorylation of tyrosine kinase transmembrane receptors is the typical paradigm for this activation but, for many growth factors, cell surface interactions are not limited to a single receptor type. In particular, heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG) on the cell surface and within the extracellular matrix (ECM), and these molecules have been viewed as accessory co-receptors serving to facilitate tyrosine kinase receptor binding. Recent studies, however, have indicated that HSPG can directly participate in signal transduction in response to FGF-2 binding. Thus, in the present study, we used mathematical modeling to examine whether the kinetics of formation of the various FGF-2 bound complexes on the cell surface correlate with the activation of the downstream mediators of FGF-2 response, Erk1/2. We find that FGF-2 binding to its receptor correlates well with Erk1/2 activation and that HSPG can modulate this response through its ability to stabilize these ligand receptor complexes. Moreover, we also observed that FGF-2 binding to HSPG correlates strongly with Erk1/2 activation under conditions where there is a loss of receptor activity, and we demonstrate that the relative amounts of signaling and non-signaling HSPG on the cell surface, as well as the presence of competing HSPG in the ECM, can impact the signal potential via this pathway. Thus, the selective regulation of specific HSPG might provide a mechanism for fine tuned modulation of heparin-binding growth factor signaling in cells where signal intensity and duration could direct cellular response toward growth, migration or differentiation.     

Apolipoprotein A-I regulates lipid hydrolysis by hepatic lipase

Association of hepatic lipase (HL) with pure heparan sulfate proteoglycans (HSPG) has little effect on hydrolysis of high density lipoprotein (HDL) particles, but significantly inhibits (>80%) the hydrolysis of low (LDL) and very low density lipoproteins (VLDL). Lipolytic inhibition is associated with a differential ability of the lipoproteins to remove HL from the HSPG. LDL and VLDL are unable to displace HL, whereas HDL readily displaces HL from the HSPG. These data show that HSPG-bound HL is inactive. Purified apolipoprotein (apo) A-I is more efficient than HDL at liberating HL from HSPG, and HL displacement is associated with the direct binding of apoA-I to HSPG. However, displacement of HL by apoA-I does not enhance hydrolysis of VLDL particles. This appears due to the direct inhibition of HL by apoA-I. Both apoA-I and HDL are able to inhibit VLDL lipid hydrolysis by up to 60%. Inhibition of VLDL hydrolysis is associated with the binding of apoA-I to the surface of the VLDL particle and a concomitant decreased affinity for HL. These data show that apoA-I can regulate lipid hydrolysis by HL by liberating/activating the enzyme from cell surface proteoglycans and by directly modulating lipoprotein binding and hydrolysis.     

Mechanisms of fibroblast growth factor 2 intracellular processing: a kinetic analysis of the role of heparan sulfate proteoglycans

The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 degrees C for prolonged periods (0-48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4-10 kDa) were detected that accumulated to much higher ( approximately 10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse-chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native ( approximately 18 h) compared to HSPG-deficient cells ( approximately 4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.     

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.     

Severe hypoalphalipoproteinemia in mice expressing human hepatic lipase deficient in binding to heparan sulfate proteoglycan

Unlike human hepatic lipase (hHL) that is mainly cell surface-anchored via binding to heparan sulfate proteoglycans (HSPG), mouse HL (mHL) has a low affinity to HSPG and thus is largely blood-borne. The reduced HSPG binding of mHL is attributable to the C-terminal amino acids. To determine the functions of HSPG binding of hHL in vivo, we created adenovirus vectors encoding hHL or a chimeric protein (designated hHLmt) in which the C-terminal HSPG-binding sequences were replaced with the corresponding mouse sequences. Injecting hHLmt-expressing virus into C57BL/6J mice (1.8 x 10(10) virus particles/mouse) resulted in a 3-fold increase in pre-heparin HL activity, whereas infection with an identical dose of hHL virus did not change pre-heparin HL activity. In hHLmt-expressing mice, the concentration of total cholesterol and phospholipids was inversely related to the hHL activity in pre-heparin plasma in a dose- and time-dependent manner, and the decrease was mainly attributable to high density lipoproteins (HDL) cholesterol and HDL phospholipids. The expression of hHL exhibited no change in plasma total cholesterol or phospholipid levels as compared with control mice infected with luciferase or injected with saline. The reduced HDL lipids in the hHLmt-expressing mice were accompanied by markedly decreased plasma and hepatic apolipoprotein (apo) A-I. In primary hepatocytes isolated from hHLmt-expressing mice, the concentration of cell-associated and secreted apoA-I was decreased by 2-3-fold as compared with hepatocytes isolated from control mice, whereas the levels of apoB and apoE were unaltered. Infection of primary hepatocytes with hHLmt virus ex vivo also resulted in reduced apoA-I secretion but had no effect on cell-associated apoA-I. These results suggest that expression of HSPG binding-deficient hHL has a profound HDL-lowering effect.     

Characterization of proteoglycans and glycosaminoglycans in bovine renal AA-type amyloidosis.

Highly sulfated glycosaminoglycans (GAG) or proteoglycans (PG), especially heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG), are considered to be intimately associated with amyloid deposits in different types of amyloidosis. Based on this relationship an important role for HS has been suggested in amyloidogenesis. The present immunohistological and ultrastructural study shows that in bovine renal AA-amyloidosis, sulfated GAG/PG was not restricted to amyloid deposits proper and that areas without GAP/PG were also present within the amyloid. Both glomerular and papillary amyloid contained HS (PG), and the latter also contained chondroitin sulfate (CS) and dermatan sulfate (DS), suggesting a correlation between the location of the amyloid and the type of GAG/PG deposited. Amyloid P component (AP) had a distribution similar to that of HSPG, confirming their affinity-based relationship. The GAG types found ultrastructurally in amyloid fibril preparations of glomerular and papillary amyloid isolated from the same kidney, reflected the immunohistological findings. HS was shown to be the predominant GAG in all papillary amyloid fibril extracts. Taking into account the chemico-physical properties of HS, it cannot be excluded that this predominance is introduced by the purification procedure. These results suggest that the association of GAG/PG and amyloid is not necessarily mutually obligatory and that the proposed importance of GAG in amyloidogenesis is disputable.     

The low density lipoprotein receptor-related protein complexes with cell surface heparan sulfate proteoglycans to regulate proteoglycan-mediated lipoprotein catabolism

It has been proposed that clearance of cholesterol-enriched very low density lipoprotein (VLDL) particles occurs through a multistep process beginning with their initial binding to cell-surface heparan sulfate proteoglycans (HSPG), followed by their uptake into cells by a receptor-mediated process that utilizes members of the low density lipoprotein receptor (LDLR) family, including the low density lipoprotein receptor-related protein (LRP). We have further explored the relationship between HSPG binding of VLDL and its subsequent internalization by focusing on the LRP pathway using a cell line deficient in LDLR. In this study, we show that LRP and HSPG are part of a co-immunoprecipitable complex at the cell surface demonstrating a novel association for these two cell surface receptors. Cell surface binding assays show that this complex can be disrupted by an LRP-specific ligand binding antagonist, which in turn leads to increased VLDL binding and degradation. The increase in VLDL binding results from an increase in the availability of HSPG sites as treatment with heparinase or competitors of glycosaminoglycan chain addition eliminated the augmented binding. From these results we propose a model whereby LRP regulates the availability of VLDL binding sites at the cell surface by complexing with HSPG. Once HSPG dissociates from LRP, it is then able to bind and internalize VLDL independent of LRP endocytic activity. We conclude that HSPG and LRP together participate in VLDL clearance by means of a synergistic relationship.     

Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apparatus and the plasma membrane

Heparan sulfate proteoglycans (HSPG) of rat liver are associated with the plasma membrane in a hydrophobic intrinsic and a hydrophilic extrinsic form. We were interested in determining whether or not these two forms could be detected in the Golgi apparatus, the subcellular site of addition of oligosaccharides and sulfate to HSPG. In vivo and in vitro radiolabeled HSPG from rat liver Golgi apparatus membranes could only be solubilized with detergents that disrupt the membrane lipid bilayer, suggesting that they are solely associated via hydrophobic interactions. Both forms of HSPG were detected in plasma membranes of rat liver and isolated rat hepatocytes. The detergent-solubilized HSPG bound to octyl-Sepharose columns, whereas the hydrophilic form did not; this latter form, however, was released from the membrane by heparin. The hydrophobic anchor of HSPG in the Golgi and plasma membranes was insensitive to treatment with phosphatidylinositol-specific phospholipase C under conditions in which alkaline phosphatase was sensitive; this suggests that the hydrophobic anchor of HSPG is the core protein itself. Preliminary experiments suggest that the subcellular site of processing of the hydrophobic to the hydrophilic form of HSPG is the plasma membrane. A specific processing activity, probably a protease of the plasma membrane not present in serum or the endoplasmic reticulum membrane, converted hydrophobic HSPG of the Golgi membrane to the hydrophilic form. In addition, pulse-chase experiments with [35S]Na2SO4 in rats demonstrated that at short times, the bulk of the radiolabeled cellular HSPG was in the Golgi apparatus; later on, the bulk of the radioactivity was found in the plasma membrane, the only subcellular site where the hydrophilic form of HSPG was detected.     

Heparan sulfate-independent cell binding and infection with furin-precleaved papillomavirus capsids

Papillomavirus infection normally involves virion binding to cell surface heparan sulfate proteoglycans (HSPGs). However, we found that human papillomavirus type 16 pseudovirions efficiently bound and infected cells lacking HSPGs if their L2 capsid protein was precleaved by furin, a cellular protease required for infection. The inability of pseudovirions to efficiently bind and infect cultured primary keratinocytes was also overcome by furin precleavage, suggesting that the defect involves altered HSPG modification. We conclude that the primary function of HSPG binding is to enable cell surface furin cleavage of L2 and that binding to a distinct cell surface receptor(s) is a subsequent step of papillomavirus infection.     

Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.     

Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins

The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Lipoprotein lipase-dependent binding and uptake of low density lipoproteins by THP-1 monocytes and macrophages: possible involvement of lipid rafts

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 (125)I-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.     

Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein alpha

Mammalian phosphatidylinositol transfer protein alpha (PITP) is an intracellular lipid transporter with a binding site that can accommodate a single molecule of phosphatidylinositol (PI) or phosphatidylcholine (PC). Phospholipids are a heterogeneous population of molecular species that can be distinguished by their characteristic headgroups as well as their acyl chains at the sn-1 and sn-2 position. In this study, we have defined the acyl chain preference for PITPalpha when presented with a total population of cellular lipids. Recombinant PITPalpha loaded with bacterial lipid, phosphatidylglycerol (PG), was incubated with permeabilised HL60 cells, followed by recovery of PITPalpha by affinity chromatography. Lipids extracted from the PITPalpha were analysed by tandem electrospray ionisation mass spectrometry (ESI-MS) and showed total exchange of acquired bacterial lipids for HL60 cellular PI and PC. Detailed comparison of the molecular species composition of bound phospholipids with those in whole cells permitted the assessment of selectivity of acyl chain binding. For both phospholipid classes, progressive fractional enrichments in bound species possessing shorter acyl chains were apparent with a preference order: 16:1>16:0>18:1>18:0>20:4. A recapitulation of this specificity order was also seen from a dramatically altered range of molecular species present in HL60 cells enriched with arachidonate over many weeks of culture. We speculate that short-chain, saturate-binding preferences under both conditions may reflect properties in vivo. This is consistent with target cell membranes actively remodelling newly delivered phospholipids after transport rather than relying on the transport of the specific molecular species conventionally found in mammalian membranes.