Taurine enhances low density lipoprotein binding. Internalization and degradation by cultured Hep G2 cells

To evaluate the impact of taurine on hepatic cholesterol catabolism low density lipoprotein (LDL) binding, internalization and degradation were measured in cultured Hep G2 cells. Preincubation of cells with 0.1-10 mM taurine for 24 h stimulated LDL receptor activity by as much as 100%. Only the high affinity LDL receptor activity (specific) was increased by taurine preincubation, whereas the low affinity receptor activity (nonspecific) remained unchanged. Scatchard analysis of the binding data revealed that taurine doubled the number of LDL receptors without affecting receptor affinity. Taurine-enhanced LDL receptor activity was most pronounced when LDL concentrations exceeded 100 micrograms/ml, but was noted at taurine concentrations as low as 0.1 mM (plasma level). Interestingly, taurine had no effect on LDL receptor activity when it was added simultaneously with 125I-LDL to Hep G2 cells, or when non-bile acid-producing human skin fibroblasts were tested. Stimulation of LDL receptor activity was also obtained with 10 mM cysteine, a taurine precursor, but not with glycine. Increased cellular concentrations of taurine and cysteine were associated with an elevated rate of bile acid synthesis and a reduced cellular free cholesterol concentration. The data suggest that taurine enhanced LDL receptor activity by sparing cysteine, a known sulfhydryl group donor and stimulator of 7 alpha-hydroxylase activity, and that the latter stimulated bile acid production leading to increased utilization of cellular free cholesterol and enhanced LDL uptake.     

Enhancement of cholesteryl ester metabolism in cultured human monocyte-derived macrophages by verapamil

The effect of the Ca2+ entry blocker, verapamil, on the biosynthesis of cholesterol and the metabolism of low-density lipoprotein (LDL) was studied in cultured human monocyte-derived macrophages. Addition of verapamil (50 microM) of monocyte-derived macrophages enhanced 125I-LDL and 125I-labelled acetyl-LDL binding and internalization, and increased [2-14C]acetate incorporation into cholesterol. Since higher levels of LDL and modified lipoproteins may be implicated in atherogenesis, the more efficient processing of these lipoproteins by monocyte-derived macrophages in the presence of Ca2+ blocker warrants further assessment for its potential as an antiatherogenic agent.     

Free sterol metabolism and low density lipoprotein receptor expression as differentiation markers of cultured human keratinocytes

In contrast to most tissues, epidermis and its derivatives appear to lack low density lipoprotein (LDL) receptors and exhibit sterologenesis rates unaffected by circulating lipoprotein (LP) cholesterol content. Since LDL receptors have been demonstrated in both cultured squamous cell carcinoma cells and human foreskin keratinocytes, when maintained in low-calcium media, LDL receptor expression may be related to keratinocyte differentiation. We compared receptor binding and internalization of LDL-gold in normal keratinocytes at different stages of growth at physiological calcium concentrations (early, 3-5 days; preconfluent, 6-10 days; postconfluent, 12-17 days), and correlated receptor expression with sterologenesis in LP-replete vs.-depleted media. Whereas in early cultures about 60% of sterologenesis was LP dependent, this fraction declined in preconfluent and confluent cultures despite continued culture growth and little decline in total sterologenesis. Accordingly, LDL receptors were most evident in early cultures, declining in preconfluent cultures in parallel with the decrease in LP-dependent sterol synthesis. In contrast, sterologenesis in human foreskin fibroblasts was profoundly influenced by exogenous LP at all stages of confluence; total and LP-dependent sterologenesis declined in parallel with growth cessation. These studies represent the first demonstration that normal keratinocytes express functional LDL receptors at physiologic calcium concentrations. Moreover, they demonstrate that LDL receptor expression in keratinocytes, in contrast to fibroblasts, can only in part be attributed to growth requirements. Instead, loss of LDL receptor expression serves as a distinctive marker of keratinocyte differentiation and may reflect the specific functional requirements of the epidermis in vivo.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Hyperproliferation of normally quiescent keratinocytes in non-lesional psoriatic skin due to high calcium concentration (an organotypic culture model)

Calcium plays an important role in the regulation of different functions of keratinocytes. In the present work we studied the effect of different extracellular calcium concentrations (0.01 mM-2.0 mM) on the proliferation and differentiation of human keratinocytes in normal human and non-lesional psoriatic skin. Using explant culture model, the proliferative and differentiated subsets of keratinocytes were detected by specific antibodies related to cell proliferation [beta-1 integrin (CD29), proliferating cell antigen (Ki67), proliferating cell nuclear antigen (PCNA)] and differentiation [differentiated cell cytokeratins (K1/K10) and differentiating cell antigen (lectin Ulex europaius agglutinin, UEA-1)]. After 4 days of culturing at high Ca2+ (2.0 mM) we observed marked hyperproliferation among the normally quiescent keratinocytes of non-lesional psoriatic skin. In normal uncultured and cultured skin and in uncultured and two-day-cultured non-lesional psoriatic skin both at normal (1.2 mM) and at high (2.0 mM) Ca2+ concentration only one layer of basal CD29+/Ki67+/K1/K10-/UEA-1- cell was observed. In sections from non-lesional psoriatic skin cultured for 4 days in the presence of high Ca2+ (2.0 mM) this cell population has expanded from at least three layers above the basement membrane. This expanded cell population of the 4-day high Ca2+ cultured non-lesional skin showed clear PCNA positive staining on frozen sections with the strongest positivity among the most basal localized cells. These data suggest that (i) extracellular Ca2+ concentration can influence the proliferation of basal ("stem") keratinocytes, (ii) the proliferative response to high Ca2+ concentration of psoriatic non-lesional basal keratinocytes differs from that of normal basal keratinocytes, (iv) changes in the extracellular Ca2+ milieu might play a role in the induction of the hyperproliferative psoriatic lesion.     

Extracellular calcium affects the membrane currents of cultured human keratinocytes.

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage-clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of -24 mV. Voltage-clamp experiments showed that these cells had two membrane ionic currents: a large voltage-independent leak conductance, and a smaller voltage-dependent Cl- current that activated with depolarization. Increasing the extracellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage-gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2(+)-activated Cl- current in these cells suggests a role for this conductance in the initiation of differentiation.     

Specific epidermal protein markers are modulated during calcium-induced terminal differentiation

Extracellular calcium concentration has been shown to be an important determinant of proliferation rate in a number of cell culture models. Recently, the role of calcium as a regulator of cellular differentiation has also become apparent. This effect of calcium was exemplified by the discovery that keratinocytes of mouse or human origin grew as a proliferating monolayer in medium with a calcium concentration of 0.02-0.09 mM but that proliferation ceased and cells stratified and cornified when calcium was increased greater than 0.1 mM. While the morphological and biological effects of changes in calcium concentration are dramatic in keratinocyte cultures, it has been difficult to identify specific protein changes associated with the modulation of maturation. In vivo, however, several proteins that are markers for stratified squamous epithelia have been identified by specific autoimmune sera. Pemphigoid antigen is a 220-kdalton protein found in the basement membrane and closely associated with the plasma membrane of the basal cell. Pemphigus antigen is a 130-kdalton glycoprotein found on the cell surface of stratifying epithelial cells. Immunofluorescence staining of cells cultured in low Ca2+ or cells switched to high Ca2+ for 48 h before staining demonstrated that pemphigoid antigen was detected in low Ca2+ cultures but was diminished or absent in high Ca2+ cultures and that pemphigus antigen was seen only in high Ca2+ cultures. The synthesis of each antigen was studied in immunoprecipitates of cell lysates radiolabeled with 14C-amino acids or D-[1-14C]glucosamine. Pemphigoid antigen was synthesized mainly by proliferating cells in low Ca2+ medium and its synthesis was decreased by greater than 90% in cells switched to high Ca2+ medium. In contrast, synthesis of pemphigus antigen was detected only in stratifying cells cultured in high Ca2+ medium. These studies indicate that extracellular calcium concentrations which modulate the transition between proliferating and stratifying epidermal cells also modulate, in parallel, the synthesis of specific marker proteins for these cell types.     

The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was studied with respect to metabolism of human low-density lipoprotein (LDL). The Hep G2 cells bind, take up and degrade human LDL with a high-affinity saturable and with a low-affinity non-saturable component. The high-affinity binding possesses a KD of 25 nM-LDL and a maximal amount of binding of about 70 ng of LDL-apoprotein/mg of cell protein. The high-affinity binding, uptake and degradation of LDL by Hep G2 cells is dependent on the extracellular Ca2+ concentration and is down-regulated by the presence of fairly high concentrations of extracellular LDL. Incubation of the Hep G2 cells with LDL results in suppression of the intracellular cholesterol synthesis. It is concluded that the human hepatoma cell line Hep G2 possesses specific LDL receptors similar to the LDL receptors demonstrated on extrahepatic tissue cells.     

Phorbol ester- and calcium-induced reorganization of 180-kDa bullous pemphigoid antigen on the ventral surface of cultured human keratinocytes as studied by immunofluorescence and immunoelectron microscopy.

The hemidesmosome is an adhesion structure of the epidermal-dermal junction in keratinocytes. When keratinocytes migrate laterally or upward to differentiate, they must control the formation and disintegration of the hemidesmosomes. When keratinocytes are cultured in low-calcium (below 0.1 mM) medium, all cells behave like basal cells, adhere to the culture dish, and proliferate without differentiation. The calcium addition induces the differentiation. A bullous pemphigoid antigen, 180-kDa BPA, has been shown to be a component of the hemidesmosome. Using a monoclonal antibody to the 180-kDa BPA and a human squamous cell carcinoma cell line (DJM-1 cells), the fate of hemidesmosomes was studied after the addition of calcium to low-calcium-grown cells and 12-tetradecanoylphorbol-13-acetate (TPA) to high-calcium (1.87 mM) grown cells by immunofluorescence and immunoelectron microscopy. The antigen was distributed evenly as fine dots on the entire ventral surface of low-calcium cells, whereas they formed a peculiar, concentric ring or arch arrangement on the ventral surface of high-calcium cells. Immunoelectron microscopy revealed the deposits of gold particles at sites on the membrane surface, where some filamentous or electron-dense materials were associated, although the complete structure of hemidesmosomes was not formed. They deposited directly onto the membrane surface in low-calcium cells and with a distance of 20-50 nm from the membrane surface in high-calcium cells. The calcium addition caused a profound reduction of the 180-kDa BPA-positive area for 30 to 120 min and then formed the high-calcium-ring pattern after 4 to 6 h. A similar calcium response was seen in normal human keratinocytes. TPA (16 nM) treatment caused disintegration of the ring pattern in high-calcium DJM-1 cells. This was inhibited with a protein kinase C (PKC) inhibitor. H7 (20 microM). These results suggest that the hemidesmosome is a dynamic structure and PKC can be one of the major factors in controlling the hemidesmosome, since it is known that the low-high calcium shift induces a calcium influx and a PKC activation, and TPA activates PKC in keratinocytes.     

Regulation of the activity of the low density lipoprotein receptor in human fibroblasts.

A specific receptor on the surface of cultured human fibroblasts binds plasma low density lipoprotein (LDL) with high affinity, and thereby initiates a cellular process by which the LDL is internalized and degraded within lysosomes and its cholesterol component is made available for cellular membrane synthesis. Current studies demonstrate that the activity of this LDL receptor is under feedback regulation. Prior incubation of fibroblast monolayers with cholesterol, 25-hydroxycholesterol, or LDL progressively reduced the ability of the cells to bind 125I-labeled LDL at the high affinity site. A series of kinetic studies indicated that this reduction in binding was due to a decrease in the number of LDL receptors. From measurements of the rate of decline in 125I-LDL binding activity after administration of cycloheximide, the LDL receptor was calculated to have a half-life of about 25 hr. LDL appeared to reduce 125I-LDL-binding activity by suppressing the synthesis of receptor molecules. Thus cultured human fibroblasts regulate their intracellular cholesterol content by regulating the activity of the LDL receptor, which in turn controls the rate of cellular entry of cholesterol derived from plasma LDL contained within the culture medium.     

Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.     

A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography

Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.     

Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca++ medium (0.09 mM) as measured by incorporation of [3H]thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC50 of about 10 microM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 microM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number over control at 10 microM. These results are of importance since they suggest Ca++ may influence the effect of retinoids on keratinocytes.     

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

Regulation of lipid synthesis in relation to keratinocyte differentiation capacity

Cultured keratinocytes and squamous carcinoma cells provide a useful model system for studying the processes involved in the regulation of differentiation, as the differentiation capacity of the cells can be modulated experimentally by changing the extracellular calcium concentration. Furthermore, the squamous carcinoma cell lines exhibit a defect in their differentiation capacity which they express to different extents. In this paper, the effect of external lipoproteins has been studied on lipid synthesis in normal keratinocytes and three squamous carcinoma cell (SCC) lines which showed a decreasing capacity to differentiate in the order of normal keratinocytes greater than SCC-12F2 greater than SCC-15 greater than SCC-4. The ability of the cells to form cornified envelopes was taken as a measure of differentiation capacity. The rate of total lipid synthesis as well as the phospholipid-neutral lipid ratio decreased in the order SCC-4 greater than SCC-15 greater than SCC-12F2 greater than or equal to normal keratinocytes, clearly correlating with the differentiation capacity of the cells. Because of the high rate of phospholipid synthesis and the low rate of ceramide synthesis, it is concluded that, under these in vitro conditions used, the maturation of keratinocytes proceeds to a lesser extent than that seen under in vivo conditions. In proliferating cells, in which the low-density lipoprotein (LDL) receptor is operative to a high extent, the rate of lipogenesis, especially that of neutral lipids, responded dramatically to changes of extracellular lipoprotein concentration. In the presence of lipoproteins a marked decrease of cholesterol and triacylglycerol synthesis and an increase of cholesterol ester synthesis has been observed. On the other hand, in differentiating cells lipogenesis appeared to be independent of extracellular lipoproteins, due to the absence of the LDL uptake mechanism, the only exception being the synthesis of triacylglycerols, the rate of which could be modulated to a certain extent by extracellular lipoproteins. The results presented here demonstrate a close inverse relationship between the regulation of lipogenesis by extracellular lipoproteins and the ability of the cells to differentiate.     

Interaction of a high-affinity heparin subfraction with low-density lipoprotein stimulates cholesteryl ester accumulation in mouse macrophages

A high-affinity heparin subfraction accounting for 8% of whole heparin from bovine lung was isolated by low-density lipoprotein (LDL)-affinity chromatography. When compared to whole heparin, the high-affinity subfraction was relatively higher in molecular weight (11,000 vs. 17,000) and contained more iduronyl sulfate as hexuronic acid (76% vs. 86%), N-sulfate ester (0.75 vs. 0.96 mol/mol hexosamine), and O-sulfate ester (1.51 vs. 1.68 mol/mol hexosamine). Although both heparin preparations formed insoluble complexes with LDL quantitatively in the presence of 30 mM Ca2+, the concentrations of NaCl required for 50% reduction in maximal insoluble complex formation was markedly higher with high-affinity subfraction (0.55 M vs. 0.04 M). When compared to complex of 125I-LDL and whole heparin (H-125I-LDL), complex of 125I-LDL and high-affinity heparin subfraction (HAH-125I-LDL) produced marked increase in the degradation of lipoproteins by macrophages (7-fold vs. 1.4-fold over native LDL, after 5 h incubation) as well as cellular cholesteryl ester synthesis (16.7-fold vs. 2.2-fold over native LDL, after 18 h incubation) and content (36-fold vs. 2.7-fold over native LDL, after 48 h incubation). After a 5 h incubation, macrophages accumulated 2.3-fold more cell-associated radioactivity from HAH-125I-LDL complex than from [125I]acetyl-LDL. While unlabeled HAH-LDL complex produced a dose-dependent inhibition of the degradation of labeled complex, native unlabeled LDL did not elicit any effect even at a 20-fold excess concentration. Unlabeled particulate LDL aggregate competed for 33% of degradation of labeled complex; however, cytochalasin D, known inhibitor of phagocytosis, did not effectively inhibit the degradation of labeled complex. Unlabeled acetyl-LDL produced a partial (33%) inhibition of the degradation of labeled complex. These results indicate that (1) the interaction of high-affinity heparin subfraction with LDL leads to scavenger receptor mediated endocytosis of the lipoprotein, and stimulation of cholesteryl ester synthesis and accumulation in the macrophages; and (2) with respect to macrophage recognition and uptake, HAH-LDL complex was similar but not identical to acetyl-LDL. These observations may have implications for atherogenesis, because both mast cells and endothelial cells can synthesize heparin in the arterial wall.     

Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells

In cultured human keratinocytes (NHEK) maintained in medium containing low levels of Ca2+ (0.04 mM) desmoplakin is a component of certain electron-dense bodies in the cytoplasm. These bodies are associated with bundles of intermediate filaments. Upon elevation of the level of Ca2+ in the culture medium to 1.2 mM, desmoplakin first appears at sites of cell-cell contact in association with bundles of intermediate filaments. Subsequently, desmoplakin becomes incorporated into desmosomes in a manner comparable to that seen in mouse keratinocytes (Jones and Goldman: Journal of Cell Biology 101:506-517, 1985). NHEK cells maintained for 24 hr at Ca2+ concentrations between 0.04 mM and 0.18 mM were processed for immunofluorescence, immunoelectron, and conventional electron microscopical analysis. In NHEK cells grown at Ca2+ concentrations of 0.11 mM, desmoplakin appears to be localized in electron-dense bodies associated with intermediate filaments at sites of cell-cell contact in the absence of formed desmosomes. At a Ca2+ concentration of 0.13 mM desmoplakin is arrayed like beads on a "string" of intermediate filaments at areas of cell-cell association. At 0.15 mM, desmosome formation occurs, and desmoplakin is associated with the desmosomal plaque. In basal cell carcinoma cells desmoplakin is not restricted to desmosomes but also occurs in certain electron-dense bodies morphologically similar to those seen in NHEK maintained in low levels of Ca2+ and during early stages of desmosome assembly. We discuss the possibility of "cycling" of desmoplakin through these bodies in proliferative cells.     

Binding, internalization, and degradation of high density lipoprotein by cultured normal human fibroblasts.

Comparative studies were made of the metabolism of plasma high density lipoprotein (HDL) and low density lipoprotein (LDL) by cultured normal human fibroblasts. On a molar basis, the surface binding of (125)I-HDL was only slightly less than that of (125)I-LDL, whereas the rates of internalization and degradation of (125)I-HDL were very low relative to those of (125)I-LDL. The relationships of internalization and degradation to binding suggested the presence of a saturable uptake mechanism for LDL functionally related to high-affinity binding. This was confirmed by the finding that the total uptake of (125)I-LDL (internalized plus degraded) at 5 micro g LDL protein/ml was 100-fold greater than that attributable to fluid or bulk pinocytosis, quantified with [(14)C]sucrose, and 10-fold greater than that attributable to the sum of fluid endocytosis and adsorptive endocytosis. In contrast, (125)I-HDL uptake could be almost completely accounted for by the uptake of medium during pinocytosis and by invagination of surface membrane (bearing bound lipoprotein) during pinocytosis. These findings imply that, at most, only a small fraction of bound HDL binds to the high-affinity LDL receptor and/or that HDL binding there is internalized very slowly. The rate of (125)I-HDL degradation by cultured fibroblasts (per unit cell mass) exceeded an estimate of the turnover rate of HDL in vivo, suggesting that peripheral tissues may contribute to HDL catabolism. In accordance with their differing rates of uptake and cholesterol content, LDL increased the cholesterol content of fibroblasts and selectively inhibited sterol biosynthesis, whereas HDL had neither effect.     

Complexes of low-density lipoproteins and arterial proteoglycan aggregates promote cholesteryl ester accumulation in mouse macrophages

We studied the effect of complexes of low-density lipoproteins (LDL) and different proteoglycan preparations from bovine aorta on LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Native proteoglycan aggregate containing proteoglycan monomers, hyaluronic acid and link protein was isolated by associative extraction of aortic tissue, while proteoglycan monomer was obtained by dissociative isopycnic centrifugation of the native proteoglycan aggregate. In vitro proteoglycan aggregates were prepared by reaction of the proteoglycan monomer with exogenous hyaluronic acid. 125I-labeled LDL-proteoglycan complexes were formed in the presence of 30 mM Ca2+ and incubated with macrophages. At equivalent uronic acid levels in the proteoglycans the degradation of 125I-labeled LDL contained in the native proteoglycan aggregate complex was 3.7-7.5-fold greater than the degradation of the lipoprotein in the proteoglycan monomer complex. Degradation of 125I-LDL in the in vitro aggregate complex, while higher than that in the monomer complex, was markedly less than that in the native aggregate complex. The larger size and the greater complex-forming ability of the native proteoglycan aggregate might account for the greater capacity of the aggregate to promote LDL degradation in macrophages. The proteoglycan-stimulated degradation of LDL produced a marked increase in cholesteryl ester synthesis and content in macrophages. The LDL-proteoglycan complex was degraded with saturation kinetics, suggesting that these complexes are internalized through high-affinity receptors. Degradation was inhibited by the lysosomotropic agent, chloroquine. Acetyl-LDL, but not native LDL, competitively inhibited the degradation of the 125I-LDL component of the complex. Polyanionic compounds such as polyinosinic acid and fucoidin, while completely blocking the acetyl-LDL-stimulated cholesteryl ester formation, had no effect on the proteoglycan aggregate-stimulated cholesterol esterification. This suggests that LDL-proteoglycan complex and acetyl-LDL are not entering the cells through the same receptor pathway. These results demonstrate that the interaction of LDL with arterial wall proteoglycan aggregates results in marked cholesteryl ester accumulation in macrophages, a process likely to favor foam cell formation. A role for arterial proteoglycans in atherosclerosis is obvious.